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Molecular characterization of the rabbit HK[alpha]2 gene

University of Florida Institutional Repository
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PAGE 1

MOLECULAR CHARACTERIZATION OF THE RABBIT HK 2 GENE By DEBORAH MILON ZIES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

PAGE 2

This dissertation is dedicated to the memory of James Byrne McCracken, Jr. (July 19, 1967 to January 13, 1999). Jims all-too-shortof-a-life has been, and always will be, an inspiration to continue on, even in the toughest of times.

PAGE 3

ACKNOWLEDGEMENTS The work described in this dissertation could not have been completed without the support that I have received on both a professional and a personal level. I would therefore like to acknowledge specific individuals who have been there for me over the past five years. Professionally, the most significant support came from my mentor, Dr. Brian Cain. He taught me to be an excellent scientist, an outstanding writer, and a supportive colleague and friend. I thank my committee members, Drs. Charles Wingo, Thomas Yang, Peggy Wallace, Sarah Chesrown, and Bruce Stevens, for their suggestions and support throughout this process. Their contributions are evident in both this dissertation and the scientist I have become. I thank the past and present members of the Cain laboratory. I owe a great deal to those people who were members of the lab when I joined, Drs. Paul Sorgen, James Gardner and Tamara Caviston Otto. They all helped make the transition into a new laboratory smooth and fun. In particular, Tammy remained in the the lab long enough to become a true friend. To this day, she continues to offer understanding and encouragement when times are tough. I owe even more to the current members of the Cain laboratory, Michelle Gumz and Tammy Bohannon Grabar. We three have been through so much on every level that there are no words to express my gratitude. I would also like to thank past and present members of our neighbors, the Yang laboratory. These people have made the work environment both stimulating and enjoyable. They are Christine Mione, Sara Jato-Rodriguez, Chien Chen and Sue Lee iii

PAGE 4

Kang. Specifically, I would like to thank Sue for all her help on completing the DNaseI hypersensitivity assay and Christine for her unending patience and guidance while teaching so many things about the computer. Finally, on a professional level, I thank the rest of the Department of Biochemistry and Molecular Biology. The entire group has made my experience as a graduate student one that I will never forget. On a personal level, the first people I would like to thank have already been mentioned. In addition to all they provided on a professional level, Michelle and Tammy have also provided emotional support and friendship. The rest of my thanks goes to my family. I thank my dad, Frank Milon III, for setting a great example for me by going to law school, and becoming an attorney, later in his life. I thank my mom, Barbara Milon, for being a mom, a cheerleader, a babysitter, a friend, and so much more. The rest of my family, Patricia Milon, Frank Milon IV, Christina Milon, Rebecca Milon, and Joshua Milon, I thank for all of their encouragement, help, and reminders of what is really most important in life. And finally, because she is the most important, I would like to thank my daughter, Lee Ann Zies, just for being her. As a single parent with a five-year-old daughter, it may not have been possible to attend graduate school and make such a big change in my own life. Lee Ann, however, has not only made it possible, she has made it all worthwhile. She is a blessing beyond her years. She has been happy to go the lab, understanding when we have had to be apart, considerate when I have had to study, and loving in every difficult moment. I could not have asked for a better companion. She makes everything worthwhile. I love her and thank her with all my heart. iv

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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS...............................................................................................iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Physiological Significance............................................................................................2 Membrane Potential...............................................................................................2 Hypokalemia..........................................................................................................3 Hyperkalemia........................................................................................................4 Mammalian Kidney......................................................................................................6 Evidence for H+, K+ ATPase Expression in the Collecting Duct...............................8 Protein....................................................................................................................9 mRNA....................................................................................................................9 Regulation of the Renal H+, K+ -ATPases..................................................................10 Potassium.............................................................................................................11 Sodium.................................................................................................................12 Acid-Base............................................................................................................14 Structure of the H+, K+ ATPase Complex................................................................14 P-Type ATPases..................................................................................................15 The H+, K+ ATPase Subunits............................................................................15 High Resolution Model of Rabbit HK2a............................................................17 The Reaction Cycle.............................................................................................19 Structure of the HK2 Gene.......................................................................................21 Eukaryotic Gene Organization............................................................................21 HK2 cDNAs......................................................................................................23 Human HK2 Gene.............................................................................................27 Mouse HK2 Gene.............................................................................................28 Summary.....................................................................................................................28 v

PAGE 6

2 CLONING OF THE HK2 GENE............................................................................29 Materials and Methods...............................................................................................30 Screening the Lamda Genomic Library..............................................................30 HK2.1 Sequence.............................................................................................34 HK2.5 Sequence.............................................................................................35 HK2.8 Sequence.............................................................................................38 PCR Amplification of the Missing Fragment......................................................38 Results.........................................................................................................................39 Screening the Genomic Library.......................................................................39 Determination of Overlapping Clones.................................................................43 HK2.1 Sequence.............................................................................................43 HK2.5 Sequence.............................................................................................44 HK2.8 Sequence.............................................................................................45 Completion of the HK2 Gene Sequence...........................................................47 Discussion...................................................................................................................48 3 MAPPING THE TRANSCRIPTION START SITES FOR THE HK2 GENE.......51 Materials and Methods...............................................................................................52 Analysis of Clone HK2.1...............................................................................52 RNase Protection Assay......................................................................................52 Results.........................................................................................................................57 Analysis of Clone HK2.1..................................................................................57 Transcription Start Sites......................................................................................57 Discussion...................................................................................................................59 4 REPORTER GENE ANALYSIS OF THE HK2 GENE PROMOTER...................64 Materials and Methods...............................................................................................65 Reporter Gene Constructs....................................................................................66 Tissue Culture Cells............................................................................................72 Transfection and Reporter Gene Activity............................................................73 Normalization of the Luciferase Data.................................................................74 Results.........................................................................................................................74 HK2a and HK2c Reporter Gene Activity.........................................................74 HK2a Reporter Gene Activity............................................................................79 Putative Repressor Mutations..............................................................................81 Putative TATA Element Mutations.....................................................................82 Effect of Cell Type on Reporter Gene Activity...................................................83 Discussion...................................................................................................................85 5 CELL DIFFERENTIATION AND HK2 GENE EXPRESSION............................92 Materials and Methods...............................................................................................93 Detection of HK2 mRNAs................................................................................93 vi

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Detection of HK2 Protein by Immunocytochemistry.......................................95 DNaseI Hypersensitivity.....................................................................................96 Results.........................................................................................................................97 RT-PCR...............................................................................................................97 Northern Analysis................................................................................................98 Immunocytochemistry.........................................................................................99 DNaseI Hypersensitivity...................................................................................100 Discussion.................................................................................................................102 6 CONCLUSIONS AND FUTURE DIRECTIONS...................................................107 Cloning the HK2 Gene...........................................................................................107 Transcription Start Sites for HK2a and HK2c........................................................110 Reporter Gene Analysis of the Region 5 of the HK2 Gene..................................111 Cell Differentiation and HK2 Gene Expression.....................................................112 Future Directions......................................................................................................113 Summary...................................................................................................................115 APPENDIX A RABBIT HK2 GENE SEQUENCE.......................................................................118 Sequence Data from Clone HK2.1 and HK2.5 Exons 1-11.............................118 Sequence Data from Products Exons 12, 13 and 14.................................................123 Sequence Data from Clone HK2.8 Exons 15-23................................................124 B TFSEARCH RESULTS............................................................................................127 LIST OF REFERENCES.................................................................................................158 BIOGRAPHICAL SKETCH...........................................................................................163 vii

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LIST OF TABLES Table page 1-1 cDNAs for mammalian HK2 transcripts................................................................25 2-1 Primers used for pDZ10 sequencing..........................................................................36 2-2 Primers used for HK2.8 sequencing........................................................................38 2-3 Primers for genomic PCR and sequencing.................................................................39 2-4 Exon and intron sizes for the known HK2 genes.....................................................49 viii

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LIST OF FIGURES Figure page 1-1 Mammalian kidney.......................................................................................................7 1-2 Schematic representation of the rabbit H+, K+ ATPase..........................................16 1-3 High resolution model of the rabbit HK2a subunit...................................................18 1-4 Reaction Cycle of the Ca++-ATPase as a representative of P-type ATPases............20 1-5 Common eukaryotic gene promoter elements............................................................23 1-6 Formation of the alternative transcripts from the rabbit and rat HK2 genes...........26 2-1 Location of Sequencing Primers on the Complete pDZ10 sequence.........................35 2-2 Southern analysis of PCR screen of the genomic library........................................41 2-3 Plaque lift screen of the genomic library.................................................................42 2-4 Southern analysis to determine overlapping clones...................................................44 2-5 Clone HK2.5 ..........................................................................................................46 2-6 Partial sequence from clone HK2.8......................................................................47 3-1 Construction of RNase protection probes for HK2a (A) and HK2c (B)..................54 3-2 CpG dinucleotide analysis of subclone pDZ10..........................................................58 3-3 Putative transcription factor binding sites determined by TFSearch..........................58 3-4 RNase protection assay for HK2a (A) and HK2c (B)..............................................60 4-1 Deletion constructs containing the HK2a and HK2c transcription start sites..........67 4-2 HK2a deletion constructs..........................................................................................70 4-3 Percent activity for reporter gene constructs that contain the HK2a and HK2c transcription and translation start sites.....................................................................76 ix

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4-4 Sequence analysis of plasmid pDZ15.........................................................................77 4-5 Sequence analysis of plasmid pDZ31.........................................................................78 4-7 Alignment of possible repressor sequences from human ATP1AL1 and rabbit HK2 genes.........................................................................................................................81 4-8 Percent luciferase activity in repressor mutation constructs......................................82 4-9 Alignment of the rabbit, human, rat and mouse DNA sequence upstream of the HK2 transcription start sites..................................................................................83 4-10 Percent activity in reporter gene constructs with mutations in the CATTTAA element.....................................................................................................................84 4-11 Reporter gene activity in various cell types..............................................................85 5-1 Micrographs of tissue culture cells.............................................................................93 5-2 RT-PCR products indicating the presence or absence of HK2 transcripts..............98 5-3 Northern blot of RCCT28A total RNA......................................................................99 5-4 Micrographs of the immunostaining of RCCT28A cells..........................................101 6-1. The HK2 gene .......................................................................................................109 6-2 Model for HK2 gene expression............................................................................117 x

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR CHARACTERIZATION OF THE RABBIT HK2 GENE By Deborah Milon Zies December 2003 Chair: Brian D. Cain Major Department: Biochemistry and Molecular Biology Blood potassium concentration is critical for normal cellular functions. Failure to maintain blood potassium levels within a very narrow range can lead to hypertension, cardiac arrest, respiratory failure and death. The kidney is the major organ responsible for maintaining a constant potassium level despite variations in dietary intake. One group of proteins the kidney employs to carry out this function is the H+, K+ ATPases. These ion transporters use the energy of ATP to absorb potassium ions from the nephron lumen in exchange for hydrogen ions. Evidence suggests that the levels and the activities of this group of transporters are responsive to potassium, sodium and possibly hormones. The rabbit HK2 gene produces two mRNAs encoding the HK2a and HK2c isoforms of the alpha subunit of the renal H+, K+ ATPase. In order to study the regulation of these isoforms, it was first necessary to clone the gene. Three genomic clones (HK2.1, HK2.5, and HK2.8) were identified by screening a lambda genomic library. These three clones span a majority of the HK2 gene. Subcloning and high-throughput xi

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sequencing of the clone HK2.5 provided information regarding gene structure and its similarities to the human, mouse, and rat homologs. RNase protection assays mapped two distinct transcription start sites that correspond to HK2a and HK2c transcripts. Sequence analysis of the 5 clone, HK2.1, suggested that this clone contained the gene promoter and 5 regulatory elements. Subsequently, a luciferase reporter gene approach was used to perform a deletion analysis of the region upstream of the transcription start sites. The promoter and two putative negative regulatory elements were identified. Furthermore, sequence alignments and mutational analysis provide evidence for a functional TATA-like element within the promoter region. Finally, RNA and protein analyses suggested that RCCT28A tissue culture cells must be differentiated in order to express the HK2 gene. Taken together, these data constitute the genomic organization of the rabbit HK2 gene, the initial characterization of the promoter, the first evidence of cell differentiation as a signal for HK2 gene expression,and a system for future studies of regulation of the HK2 gene at the molecular level. xii

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CHAPTER 1 INTRODUCTION The maintenance of extracellular and intracellular potassium ion (K+) concentrations is critical for normal cell functioning. The difference between these two concentrations, along with the concentrations of other ions, creates an electrochemical gradient that is known as membrane potential. When the extracellular concentration becomes too low, or too high, the membrane potential is altered and serious side effects occur. In order to maintain a constant blood K+ level the major organ employed by the body is the kidney. The H+, K+ ATPases comprise one class of proteins found in the collecting duct of the kidney. These proteins use the energy of ATP to bring K+ from the nephron filtrate into the cell in exchange for hydrogen ions (H+). This function is thought to play a key role in K+ conservation. In the rabbit kidney, three isoforms of the alpha () subunit of the H+, K+ ATPase have been identified. They are HK1, the gastric isoform, HK2a, the colonic isoform, and HK2c, a novel splice variant of HK2a (53). Evidence suggests that when blood K+ is low, there is little to no change in transcription from the HK1 gene. On the other hand, transcription from the HK2 gene appears increase under the same conditions (3). The purpose of our study was to characterize the rabbit HK2 gene and initiate studies on its regulation. The specific aims were (1) to clone the HK2 gene from rabbit, (2) to map the transcription start sites for HK2a and HK2c, (3) to perform promoter deletion analysis of the region 5 of the transcription start sites and (4) to determine the effect of cell differentiation of HK2 gene expression. 1

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2 This chapter will review background information on the physiological significance of the H+, K+ ATPases, the evidence for the presence of the H+, K+ ATPases in the kidney, the evidence for regulation of the HK2 gene products under a variety of cellular conditions, the structure and function of H+, K+ ATPases, and the organization of the known HK2 cDNAs and genes. Physiological Significance Healthy individuals maintain a blood K+ level between 3.5 and 5.5 milliequivalents per liter (mE/L) (1). If blood K+ concentration drops below 3.5mE/L (hypokalemia) or if blood K+ concentration increases above 5.5mE/L (hyperkalemia), serious side effects, and even death, can occur. This section discusses the importance of K+ concentration and the problems that are associated with low and high blood potassium levels. Membrane Potential In a typical cell, the concentration of K+ inside the cell is much greater (approximately 150 mE/L) than the concentration in the extracellular space and in the blood (approximately 5 mE/L). This concentration difference, along with that of other ions, such as sodium and chloride, creates a slight charge to the plasma membrane known as membrane potential. The charge in an animal cell typically ranges from to millivolts (4). This charge is important for a variety of cell functions including ion transport and cell signaling. In excitable cells, muscle cells and neurons, the membrane potential is absolutely required for stimulation. In a resting state, sodium ion concentration inside the cell is low (15mE/L) while potassium ion concentration is high (150mE/L). When the cell receives a signal, Na+ channels open and allow Na+ into the

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3 cell. The influx of Na+ depolarizes the membrane and the membrane potential becomes temporarily positive (+50mV). The change in membrane potential triggers K+ channels to open and allow K+ to exit the cell. The efflux of K+ causes the membrane to repolarize and the membrane potential once again becomes negative. These local changes in membrane potential are propogated across the entire cell and cause a muscle cell to contract or a nerve cell to fire a signal. K+ concentration plays a key role in this process, and an imbalance of K+ concentration leads to some of the side effects associated with hypokalemia and hyperkalemia. Hypokalemia Most individuals are capable of maintaining proper K+ levels. In fact, hypokalemia occurs in less than 1% of the healthy population (1). There are two main groups of people, however, that are very susceptible to developing hypokalemia. There are individuals that suffer primarily from other disease states and acquire hypokalemia as a secondary effect, and there are individuals who acquire hypokalemia as a side effect of taking medications. The most common occurrence of hypokalemia is among patients receiving diuretics, as many as 50% of these patients develop low blood K+. The second largest group of individuals that develop hypokalemia suffer from hyperaldosteronism associated with heart failure and hepatic insufficiencies. A third significant group of individuals that suffer from hypokalemia are those with renal diseases that effect potassium uptake. The most predominant effects of hypokalemia are hypertension, muscle weakness, and metabolic alkalosis. Blood volume, and therefore blood pressure, is associated with ion balance in the blood. It appears that low blood K+ levels lead to increased sodium retention thereby upsetting the normal blood ion balance and leading to

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4 hypertension. Even very mild hypokalemia, a serum potassium level of 3.4 mEq/L, can lead to increased blood pressure and risk for cardiovascular disease (50). When blood K+ is low, muscle cells can become hyperpolarized, or more negative. The hyperpolarized cell is difficult to depolarize, interfering with the ability of the cell to contract. In cardiac muscle this condition can lead to ventricular arrhythmias. In skeletal muscle, hypokalemia frequently leads to muscle weakness, cramping, and in severe cases, paralysis (1). Hypokalemia also has a profound effect of blood acid/base balance. Alkalosis, high blood pH, results from two main kidney activities. The first is the stimulation of proximal tubule bicarbonate absorption and proton excretion. The second is the increased activity of the H+, K+ ATPases. These complexes exchange K+ for H+ eventually leading to acidification of the urine and alkalosis in the blood. A failure to correct metabolic alkalosis can lead to additional cardiac and skeletal muscle weakness as well as liver and brain damage. Currently, the treatment for hypokalemia consists of oral or intravenous replacement of lost K+. Unfortunately compliance with potassium supplements is low due to its disagreeable taste and/or the inconvenience of intravenous replacement (50). Additionally, there are cases where potassium supplementation does not result in the desired increase in blood potassium levels (Wingo, personal communication). Finally, overaggressive potassium replacement therapy can lead to hyperkalemia and its severe side effects (see below). An understanding of the regulation of the H+, K+ ATPases may lead to better mechanisms for controlling blood K+ levels. Hyperkalemia Hyperkalemia can result from as a disruption of the normal intracellular and extracellular K+ concentrations or from a disruption in the balance between K+ intake and

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5 K+ excretion (49). The kidney usually has an extraordinary capacity for excreting excess K+. Therefore, hyperkalemia usually only occurs when there is a combination of increased K+ uptake or a redistribution of cellular K+ along with decreased renal efficiency. The most common cause for excess intake of K+ is its presence in supplements and salt substitutes. The most common causes for a redistribution of K+ include acidosis, anesthetics and hypertonicity. Interestingly, in the case of acidosis, one of the contributing factors to increased blood K+ is the H+, K+ ATPase found in the collecting duct of the kidney. The H+, K+ ATPase functions in combination with other cellular proteins to remove H+ from the blood. As part of this process, however, the H+ is exchanged for K+, causing an increase blood K+. Reduced renal efficiency can be caused by a variety of factors including reduction in the number of nephrons due to renal failure, medications, and hormone imbalance (43). The major adverse effect of hyperkalemia is on the cellular membrane potential. Increased extracellular K+ makes the resting membrane potential more positive than normal. The result is a decrease in the abililty of the cell to propogate a signal and an increase in the rate at which the cell repolarizes. The most severe effect is in cardiac muscle where there is a delay in conduction of the muscle contraction which can lead to fatal heart arrhythmias. In skeletal and smooth muscle, increased weakness and fatigue are common. Additionally, the weakened muscles in the respiratory tract can lead to severe respiratory depression and respiratory failure. Currently there are two approaches to treating hyperkalemia. Calcium supplements can be given as a treatment for the myocardial effects of hyperkalemia. This treatment is very rapid, and relieves the major concern associated with hyperkalemia, but

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6 it is not a long term solution to increased blood K+. Long term treatment of hyperkalemia can be attained by stimulation of cellular uptake of K+ and stimulation of renal excretion of K+. An understanding of the regulation of the H+, K+ ATPase may lead to a mechanism for its inhibition thereby decreasing the uptake of K+ in the nephron and reducing blood K+. Mammalian Kidney The mammalian kidney is an important regulatory organ. By excreting or retaining water and solutes, the kidney can maintain proper blood volume and composition despite changes in diet and activity (25). Figure 1-1A is a diagram of the mammalian kidney. The kidney is divided into three sections: the cortex, the medulla and the pelvis. Running through all three regions are millions of microscopic tubules called nephrons. The nephrons are the functional units of the kidney and are depicted in Figure 1-1B. As blood capillaries enter the nephron they form the glomerulus and come in contact with the nephron at the Bowmans capsule. Blood pressure promotes the free passage of water, urea, ions and solutes from the blood into the lumen of the nephron where they become part of the filtrate. Throughout the nephron there are a variety of proteins that function to pass additional waste products into the filtrate and recover needed materials from the filtrate. The collecting duct of the nephron is the primary location for the reabsorption of K+ when blood K+ concentration is low. Figure 1-1C is a diagram of the cells that make up the cortical collecting duct. The three types of cells are principal cells, type A intercalated cells and type B intercalated cells (30). In all three cases, the apical membrane faces the lumen of the nephron and contains a different constellation of proteins than the basolateral membrane, which faces the blood. There is evidence to suggest that all three cell types have the H+, K+ ATPase present on their

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7 apical membrane. Principal cells make up approximately 65% of the cells of the cortical collecting duct. They appear lighter in color under the microscope because they (A) Kidne y (B) Nephron(C) Cells of the Collecting Duct Cortex MedullaCortexMedulla GlomerulusBowmans Capsule Collecting Duct Blood Vessel Pelvis Principal IntercalatedB Type IntercalatedA Type Figure 1-1. Mammalian kidney. A) Cross-section of the mammalian kidney B) The nephron C) Cortical collecting duct cells. contain fewer mitochondria than either intercalated cell type. They have a highly folded basolateral membrane and a very smooth apical membrane. Type B intercalated cells are the second most abundant cell type, comprising about 25% of the population of cells in the cortical collecting duct. Their basolateral surface is highly folded, similar to the

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8 principal cells. Their apical membrane, however, is more extensive, containing several microplicae. Additionally, tubulovesicular structures can be seen scattered throughout the cell. Type A intercalated cells are the least abundant, making up the last 10% of the cell population. They have extensive microplicae on the apical surface and a numerous tubulovesicular structures just below the apical surface. Their basolateral membrane, in contrast to the principal cells and B type intercalated cells, is smooth. It has been suggested that the three cell types are capable of inter-converting in response to stimuli. In fact, there is a progressive change in cell type along the collecting duct that includes cell types that are intermediate to the three main types. Evidence for H+, K+ ATPase Expression in the Collecting Duct. The earliest evidence for the existence of H+, K+ ATPase activity in the collecting duct came from studies involving the perfusion of microdissected rabbit collecting duct tissue (51). By setting up a perfused tubule system, Dr. Wingo was able to measure ion flux under a variety of controlled conditions. In this way, it became clear that the collecting duct contained an apical ATPase that had properties similar to the previously identified gastric H+, K+ ATPase (HK1). Pharmacologically, this ATPase was sensitive to the gastric H+, K+ ATPase inhibitor omeprazole and insensitive to the Na+, K+ ATPase inhibitor ouabain. It differed from the HK1 H+, K+ ATPase in that it was insensitive to the compound Schering 28080. Furthermore, removal of luminal K+ had a profound effect on the proton secretion by the collecting duct segment of the kidney. This section discusses the molecular evidence that supports these early findings.

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9 Protein Immunohistochemistry has been used to localize the various isoforms of the H+, K+ ATPase in human (27) and rabbit (48). Kraut et al. (27) used antibodies raised against the human HK1, the rat HK2, and the human ATP1AL1 (HK2) proteins to probe human cortical and medullary collecting duct tissue. Using the HK1 antibody, they observed a darker staining in the intercalated cells and a lighter, but consistent staining of the principal cells in both the cortical and medullary collecting ducts. With the rat HK2 antibody, this group observed no staining. This result is not surprising because although the rat and human proteins (HK2 and ATP1AL1 respectively) are considered homologous proteins (see below), they share only about 87% amino acid identity. It is therefore likely that the antibody against rat HK2 was unable to recognize the human ATP1AL1 protein. Finally, with the antibody to ATP1AL1, light staining of the intercalated cells and occasional staining of the principal cells in both the cortex and the medulla was observed. In rabbits, Verlander et al. (48) used an antibody raised against the HK2c isoform of the H+, K+ ATPase and observed intense staining of the apical membrane of both A and B type intercalated cells and a lighter staining of apical membrane of the principal cells in both the corical and medullay collecting ducts. These two reports are consistent and support the localization of the H+, K+ ATPase activity to all the cell types present in the collecting duct. mRNA In rat, Ahn et al. (1) used in situ hybridization to show that the mRNA for HK2 was present in the connecting tubule and intercalated cells throughout the collecting duct. These experiments were not designed to distinguish between the two alternative

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10 transcripts found in rat, HK2a and HK2b. A contrasting study by Jassier et al. (22), detected HK1, but not HK2, in the cortex and the medulla. Although both authors used in situ hybridization for the detection of HK2, Ahn et al. suggest that the digoxigenin method used in their experiments is more sensitive technique than the 35S labeling method used by Jassier et al. In a third report, Marsy et al. (33) consistently found HK2 mRNA in the cortical collecting duct of rats using quantitative RT-PCR. The use of a different method in this report supports the presence of HK2 in the collecting duct in rat. Furthermore, two independent groups (6, 12) were able to use 5 rapid amplification of cDNA ends (RACE) to detect HK2 mRNA in samples from the cortical collecting duct of rabbit. The cloning of cDNAs for the HK subunits from human (17, 35), rat (9, 26) and rabbit (6, 12) kidney provided the strongest evidence for their expression in the kidney. Additionally, the cDNAs from rat and rabbit were the first indications that there were splice variants of the HK2 mRNAs. The characteristics of the cDNAs indentified to date are listed in Table 1 and are discussed below. Regulation of the Renal H+, K+ -ATPases The microperfusion assays performed on rabbit kidney nephrons that led to the discovery of renal expression of the H+, K+ ATPases also provided the first evidence suggesting that the activity of the enzyme was regulated under certain cellular conditions (51). Regulation by ion concentration, acid-base balance, and hormones has since been confirmed predominantly by in vivo studies with rat. In considering the earlier work as well as the work discussed below, is important to note that the studies measuring ATPase activity were not designed to distinguish between the activity of pumps containing HK1

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11 and HK2 subunits. Furthermore, the molecular mechanisms responsible for controlling these observed changes in activity have not been studied. Potassium In the cortical collecting duct, there is some controversy in the literature regarding the regulation of HK2 by low K+. Several investigators (26, 33) have been able to show that K+ depletion results in an increase in the HK2 message in the cortical collecting duct. In contrast, Sangan et al. (40) saw a decrease in the amount of HK2 mRNA with K+-depletion. The rat model in these experiments, however, was exposed to a low K+ diet for a longer period of time than previous studies and the effects of chronic hypokalemia may differ from those of acute hypokalemia. Furthermore, Ahn et al. (1) did not observe a change in HK2a message in the cortical collecting duct. More studies must be carried out in order to clarify the regulation of HK2 in the cortical collecting duct. HK2 transcripts are also present in the outer medullary collecting duct (OMCD) and appear to be subject to upregulation during K+ restriction. RT-PCR (33), Northern analysis (26), and in situ hybridization (1) have all been used to demonstrate increased HK2 gene activity. H+, K+ ATPase activity measurements reported also indicated increased activity in both rat and rabbit (19, 28, 36). Studies of the type A intercalated cells (23, 1), most abundant in the medullary collecting duct, showed that HK2 mRNA was present and that low K+ resulted in an increase in the message. A greater controversy centers on the presence of H+, K+-ATPase activity and the induction of the HK2 gene in the inner medullary collecting duct (IMCD). HK2 mRNA was not always detectable (23). Marsy et al. (33) reported the presence of the mRNA by RT-PCR. but she did not observe an upregulation of the message with

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12 potassium restriction. In contrast, Kone and coworkers were able to detect HK2 message and show an increase in the message during K+ restriction by both in situ hybridization and Northern analysis (1, 26). Moreover, the Northern blot experiments were reproduced independently by Nakamura et al. (36). These investigators were also able to show an increase H+,K+-ATPase activity. Taken together, these data suggest that HK2 ATPase is present in the IMCD and is likely responsive to K+ restriction. The observation that HK2 gene products are likely to play a role in K+ conservation led to the creation of an HK2 gene knockout mouse by Meneton et al. (34). The mouse knockout had no observable defects when fed a K+ replete diet. On the other hand, these animals developed severe hypokalemia when the animals were fed a K+ free diet. Interestingly, the kidney of the knockout mouse was still able to reduce K+ loss by 100-fold suggesting that other kidney proteins are capable of compensating for a loss of the HK2 protein. The knockout mouse maintained on a K+ free diet, however, developed a more severe case of hypokalemia than the normal mouse on the same diet. Additionally, the bulk of the in vivo data indicates that the HK2 gene is upregulated when K+ is restricted. It is therefore very likely that the colonic H+, K+-ATPase (HK2) is regulated by low K+ and plays a role in K+ conservation. Sodium A reduction in dietary sodium leads to several alterations including hyperaldosteronism and increased activity of the Na+, K+-ATPase. The Na+, K+-ATPase is present on the basolateral membranes of principal cells in the cortical collecting duct and functions by bringing K+ into the cell in exchange for Na+. As a result of this action, the principal cells must possess mechanisms to remove additional cellular potassium.

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13 The most likely mechanism for the removal of K+ is the opening of K+ specific channels and/or increased activity of KCl cotransporters present on the apical membrane. It has been suggested that these conditions stimulate the intercalated cells of the collecting tubule to reabsorb potassium by use of the H+, K+-ATPase. Silver et al. (41) identified intercalated cells of the cortical collecting duct by BCECF fluorescence and measured their ability to recover from acid load under sodium depleted conditions. Increases in H+ and K+ exchange were observed that could be attributed to either HK1 or HK2 containing ATPases. Sangan et al. (40) used a cDNA probe and a polyclonal antibody specific for HK2 to detect mRNA and protein from rats fed a low sodium diet. Northern and Western analyses of kidney cortex and kidney outer medulla revealed that sodium may have a slight effect on mRNA levels in the kidney cortex but had no apparent influence on the protein level. It is possible that the increase in H+, K+-ATPase activity that was observes may be a result of post-translational modification of the pump. The major hormone released during sodium restriction is aldosterone. It follows, therefore, that if low sodium increases the activity of H+, K+-ATPase in the cortical collecting duct, aldosterone could do the same. However, aldosterone levels apparently do affect HK2 H+, K+-ATPase activity. Eiam-Ong et al. (11) used adrenalectomized rats in which aldosterone was replaced at either physiological or pharmacological levels. When H+, K+-ATPase activity was measured in microdissected tubules there was no apparent difference between rats that had no aldosterone and those that had either physiological or pharmacological doses of aldosterone. In a similar set of experiments using adrenalectomized rats, Jaisser et al. (23) directly measured HK2 mRNA. In situ hybridization demonstrated that HK2 mRNA levels were very low in normokalemic rats

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14 and did not increase significantly with the addition of aldosterone or dexamethasone. Interestingly, experiments by Silver et al. (41) showed that when rats on a normal diet were injected with aldosterone in order to simulate levels found during sodium restriction, H+, K+-ATPase activity was not increased suggesting that the low sodium induction of H+, K+-ATPase activity was not mediated by aldosterone. Acid-Base One might expect that blood pH would have a profound effect on H+, K+-ATPase activity in the kidney and indeed the evidence for an increase in H+, K+ ATPase activity from alkalosis seems clear. In the rabbit cortical collecting duct, Northern analysis of mRNA derived from rabbits subjected to metabolic alkalosis generated a greater than four-fold increase in HK2 mRNA (13). On the other hand, metabolic acidosis decreased (13) or had no effect (10) on the level of HK2 mRNA. Collecting duct tubules taken from animals fed a K+-depleted diet have 50% less bicarbonate absorption when compared with the tubules from animals fed a K+-replete diet. At the same time, the K+depleted animals have a net increase in K+ absorption suggesting that an H+, K+ ATPase pump is upregulated under low K+ conditions (30). Komatsu and Garg also reported that metabolic acidosis increases in H+, K+ ATPase activity and metabolic alkalosis suppresses the same activity (14). Structure of the H+, K+ ATPase Complex The H+, K+ ATPases are considered P-type ATPases because they form a phosphorylated intermediate during the reaction cycle. Based on the information known about the structure and function of other P-type ATPases, models for the structure and

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15 function of the HK2 ATPases have been made. This section describes the P-type ATPases, their reaction cycle, and the models of the HK2 H+, K+ ATPases. P-Type ATPases The H+, K+ ATPases belong to a family of proteins known as the P-type ATPases (32). This family uses the energy of ATP hydrolysis to translocate ions against their electrochemical gradient. They are distinguished from other families of ATPases by forming a phosphorylated intermediate during the reaction cycle. This phosphorylation occurs at a highly conserved aspartate within the amino acid sequence DKTG. All P-type ATPases share a core structure with highly conserved domains known as the ATP binding domain (N), the phosphorylation domain (P) and the transmembrane domain. Outside of these core domains there are several regions that define subtypes of the family. In the P2-type ATPases, those that translocate non-heavy cations, the non-conserved domains appear to be responsible for activities such as the regulation of ATPase activity, cation specific conformational changes in the protein and proper insertion of the protein into the plasma membrane. One subclass of P2-type ATPases is the X+, K+-ATPases. This family, which includes the H+, K+ ATPases, contains ATPases that are made up of more that one protein subunit (32). The H+, K+ ATPase Subunits The H+, K+ ATPase complex is a heterotetramer of two alpha ( and two beta ( subunits (8). One of each of the subunits is diagrammed in Figure 1-2. The subunit is approximately 115 Kilodaltons (Kd) in size and contains 10 transmembrane segments responsible for forming the channel for the translocation of ions. The subunit also houses the ATP hydrolysis activity located in the large intracellular domain between

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16 transmembrane segments four and five. There are four possible subunits that may pair with HK2 gene products. They are the gastric H+, K+ ATPase HK subunit and the Na+,K+ ATPase subunits 1, 2, and 3. It is unclear whether the HK2 proteins pair with a specific subunit or if they can pair with any of the four. All of the subunits, however, share several characteristics. They are approximately 30 Kd in size and contain one transmembrane domain. They contain one large intracellular domain that has a varying number of potential glycolyslation sites. The subunit has no catalytic activity, but appears to be important for proper positioning and insertion of the subunit into the plasma membrane (37). ExtracellularIntracellularPlasma Membrane ExtracellularIntracellularPlasma Membrane Figure 1-2. Schematic representation of the rabbit H+, K+ ATPase. Arrows indicate direction of ion transport.

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17 High Resolution Model of Rabbit HK2a Recently, the high resolution structure of the E1 state of the Ca++-ATPase from rabbit sarcoplasmic reticulum was determined (46). Although this enzyme shares only 29% amino acid identity and 47% amino acid similarity with the rabbit HK2a subunit, the two enzymes are related. It was therefore possible to use the atomic coordinates for the Ca++-ATPase to create a high resolution model of the H+, K+ ATPase 2 subunit (18) (Figure 1-3). In the transmembrane portion of the protein, the ten transmembrane segments are shown to form a channel for the translocation of ions. The Ca++-ATPase does not have a subunit. It was therefore not possible to model the subunit into the H+, K+ ATPase structure. Biochemical and low resolution structural data for the Na+, K+-ATPase suggested that the subunit would be positioned near membrane spanning domains M7 and M10. In the model, there is a space behind M7 that could hold the HK subunit (18). In the cytoplasmic portion of the protein, three clear domains are represented: the nucleotide binding domain (N), the phosphorylation domain (P) and the actuator domain (A). The N domain is responsible for binding and hydrolysis of ATP. In Figure 1-3, lysine 517 is highlighted in green. This amino acid forms a portion of the ATP binding pocket. The P domain contains aspartic acid 385, shown in red. This amino acid becomes phosphorylated during the reaction cycle. These two amino acids are far apart in this model because the model represents the E1 state of the enzyme. When ions bind to the channel, the enzyme goes through a conformational change that brings these amino acids closer together so that the phosphate of ATP can be transferred to the aspartate.

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18 The A domain contains the conserved TEGS loop that appears to play a role in catalysis of the ATP. The A domain interacts with the P domain in the E1 state and appears to modulate the ability of the P domain to interact with the N domain. Extracellula r Intracellular 13425678910Plasma membrane Figure 1-3. High resolution model of the rabbit HK2a subunit. Courtesy of Michelle Gumz (18). This model is a representation of the E1 state of the reaction cycle. Each transmembrane domain is numbered. Highlighted in green is lysine 517, part of the ATP binding pocket. Highlighted in red is aspartic acid 385, the amino acid that becomes phosphorylated during the reaction cycle. The arrow indicates the direction of movement of the P domain during the conformational change to the E2 state.

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19 The Reaction Cycle It has long been established that the P-type ATPases go through significant conformational changes while translocating ions (44). Biochemical data suggested that the enzymes exist in two main conformational states. The E1 state binds ions and transfers phosphate from ATP to an amino acid residue within the enzyme. The hydrolysis of ATP causes a conformational change in the enzyme to the E2 state. This confirmation has low affinity for ions and releases them to the opposite side of the membrane. The recent crystal structures that represent the E1 (46) and E2 (47) states of the Ca++ ATPase taken together with the biochemical data and lower resolution structures of several P-type ATPases suggest that the reaction cycle is much more complicated, consisting of a series of stable intermediate steps (44). The reaction cycle described below and depicted in Figure 1-4 is for the Ca++ ATPase, but is likely representative of the P-type ATPases as a whole. The starting conformation is the closed (E2H) state (Figure 1-4A). The Ca++ binding sites were protonated during the previous reaction cycle and face the lumen of the sarcoplasmic reticulum. Deprotonation of these sites is accompanied by a rotation in the P domain and a reorientation of the Ca++ binding sites towards the cytoplasm of the cell. The P domain and the A domain are now able to interact with each other. This is the state is the open conformation (E1) (Figure 1-4B). Once Ca++ is bound to the channel, the N domain binds ATP (E1MgATPCa2) and the P domain loses its interactions with the A domain (Figure 1-4C). The P domain is then ready to accept a phosphate from an ATP that is hydrolyzed in the N domain (E1MgP(Ca2)ADP) (Figure 1-4D). The release of ADP from the enzyme causes a major conformation shift that orients the Ca++ ions to the lumen (E2MgPCa2) (Figure 1-4E).

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20 N A P ++ N A P-ATPCa++Ca++ N A PCa++ATP N A P-ADPCa++Ca++-P N A P-ADPCa++Ca++-P H+ ADCBE54321 N A P ++ N A P ++ N A P ++ N A P-ATPCa++Ca++ N A P-ATPCa++Ca++ N A PCa++ATP N A PCa++ATP N A P-ADPCa++Ca++-P N A P-ADPCa++Ca++-P N A P-ADPCa++Ca++-P H+ N A P-ADPCa++Ca++-P H+ ADCBE54321 Figure 1-4. Reaction Cycle of the Ca++-ATPase as a representative of P-type ATPases. The reaction intermediates are (A) E2H (B) E1 (C) E1MgATPCa2 (D) E1MgP(Ca2)ADP (E) E2MgP. Numbers represent transitions between states as described in the text. This conformation has a low affinity for Ca++ and it is released (E2MgP). The sequential release of H20, Mg++, and inorganic phosphate returns the enzyme in the E2 state ready for protonation and the beginning of another reaction cycle (Figure 1-4A).

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21 Structure of the HK2 Gene The gene that encodes the rabbit HK2 subunit was previously unknown. One of the aims of our study was to clone the gene from a rabbit genomic library. The genes for the human and mouse gene, however, have been cloned. This section will review general characteristics of eukaryotic genes and the genomic organization for the human and mouse HK2 genes. Eukaryotic Gene Organization By definition, a gene is considered to be the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide or RNA (31). According to this definition, the nucleotide sequence that codes for the protein or functional RNA is only a portion of the entire gene. The rest of the gene consists of non-coding sequences that play a role in creating the final product. This section describes common features of eukaryotic genes such as intron/exon organization, core promoter elements and transcriptional control elements. The nucleotide sequence that codes for a protein is not found as a continuous sequence. Instead, the coding regions (exons) are interrupted by non-coding regions (introns). Once the entire region is transcribed, RNA splicing machinery recognizes specific splice junction sequences, removes the introns and joins together the exons creating a complete mRNA sequence. Importantly, this arrangement allows for alternative splicing. It is possible for the RNA splicing machinery to join some exons and remove others creating alternative RNA transcripts from the same gene. The production of alternative transcripts may be regulated by different stimuli or in a tissue specific manner.

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22 The core promoter elements are those that are necessary for basal transcription (7). They are located near the transcription start site. Common core promoter elements are diagramed in Figure 1-5. They include the TATA box, the initiator element (Inr), the downstream promoter element (DPE), the TFIIB recognition element (BRE), and the CpG island. The TATA element, with the consensus sequence TATAAA, is usually located about 25-30 bases upstream of the transcription start site. This element is the binding site for the TATA binding protein (TBP). TBP and its associated factors (TAFs) make up the general transcription factor TFIID. Once bound, TFIID is capable of recruiting and/or interacting with other general transcription factors and RNA polymerase. The entire complex is then capable of determining the specific transcription start site and initiating basal level transcription. In the absence of a TATA box, an initiator element may be present and act to initiate transcription. This element has a very loose consensus sequence of PyPyA+1NT/APyPy and usually overlaps with the transcription start site. It is thought that the consensus sequence is recognized by TBP associated factors and directs TBP to bind upstream in a TATA box independent manner. The rest of transcription initiation occurs similarly to TATA box containing promoters. The downstream promoter element is found about 30 bases downstream of the transcription start site. It has a consensus sequence of RGA/TCGTG. Similarly to the initiator, the DPE is thought to bind a TBP associated factor and direct specific initiation of transcription. It is known to work in conjuction with the initiator sequence at TATA-less promoters, but may also function to stabilize core promoters with weak TATA elements. The TFIIB recognition sequence, G/CG/CG/ACGCC, is located immediately upstream of the TATA box (29). As its name implies, this sequence binds the general

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23 transcription factor TFIIB. This element is present at many, but not all, eukaryotic promoters. Finally, some promoters do not have TATA box or an initiator element. Instead these promoters contain a region of high GC content upstream of the coding region. This GC rich region, known as a CpG island, can form multiple binding sites for SP family members. The stimulatory protein family can bind and direct the formation of preinitiation complexes. This process, however, is often imprecise and allows for multiple transcription start sites. In addition to basal level transcription, many eukaryotic genes are activated and repressed by environmental stimuli. This change in regulation is modulated by the binding of sequence-specific DNA-binding proteins that can interact with the core promoter proteins and either activate or repress transcription. DPE INR RNA Transcript General Transcription FactorsRNAPolymerase Regulatory Elements(activators, repressors) CpG Island TATA DPE INR RNA Transcript General Transcription FactorsRNAPolymerase Regulatory Elements(activators, repressors) CpG Island TATA Figure 1-5. Common eukaryotic gene promoter elements. TATA represents the TATA element. INR represents the initiator element. DPE represents the downstream promoter element. Adapted from Lodish et al. (31). HK2 cDNAs Table 1-1 lists all known HK2 cDNAs along with the sizes of their 5 untranslated regions (UTR), open reading frames and the 3UTRs. The HK2a cDNA

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24 sequences are very similar. The first rat HK2a cDNA was obtained by screening a colonic cDNA library (9). Subsequently, Kone and coworkers (26) reported a cDNA with an identical open reading frame that they cloned from kidney. The 3 UTRs were also identical, but Kone obtained a longer 5UTR by using primer extension analysis. In addition to the HK2a sequence, a second HK2 cDNA termed HK2b was found (see below). Two groups independently cloned the HK2a cDNA from rabbit kidney and performed 5 and 3 Rapid Amplification of cDNA Ends (RACE) to determine the length of the HK2a cDNA (6, 12). RACE has the capacity to produce a full-length cDNA, but the length of the PCR product is dependent upon the efficiency of the reverse transcriptase reaction. Therefore, a full-length cDNA is not necessarily produced. For the rabbit HK2a cDNAs, Fejes-Toth et al. (12) acquired more 5 UTR sequence while Campbell et al. (6) obtained the longer 3 UTR sequence. Our study used the RNase protection assay to determine the true 5 end of the HK2a and HK2c transcripts. The 3UTR obtained by Campbell et al. extended to the likely poly A signal, and was viewed as complete. It is notable that the complete 3 UTR of the human ATP1AL1 transcript was apparently much shorter than that of other mammalian species. Whether this effects mRNA stability or has some other regulatory significance has not been studied. cDNA clones representing splice variants of HK2 gene transcripts have been obtained for rat (HK2b) (26) and rabbit (HK2c) (6). HK2b and HK2c cDNAs differ from the HK2a cDNA only at the extreme 5 end but the deduced protein products differ significantly.

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25 Table 1-1. cDNAs for mammalian HK2 transcripts. Figure 1-6 is a schematic representation of the likely mechanism for the formation of the alternative transcripts. In both species, HK2a transcription initiates at exon 1 and the 5 end of the mRNA is produced by splicing exon 1 to exon 2. The rat HK2b and the rabbit HK2c transcripts arise from transcription initiation within what had been designated as intron 1 of the HK2 gene. The rat HK2b cDNA had a peculiar organization. The 5 UTR was longer than any of the other HK2 5 UTRs (739bp) and is in fact much longer than a typical mammalian 5UTR. In addition, it contained eight short open reading frames prior to the HK2b translation start site that may offer a mechanism for Organism Subunit Apparent 5UTR Open Reading Frame/ Amino acids Probable3UTR Genebank Accession Number Reference Human ATP1AL1 168 3117 / 1039 290 U02076 [Grishin, 1999 #8] Rat HK2a 202 3108 / 1036 650 M90398 [Crowson, 1992 #1] HK2a 275 3108 / 1036 650 U94912 [Kone, 1998 #66] HK2b 739 2787 / 929 348 U94913 [Kone, 1998 #66] Rabbit HK2a 190 3101 / 1034 875 AF106063 [Fejes-Toth, 1999 #5] HK2a 39 3101 / 1034 939 AF023128 [Campbell, 1999 #3] HK2c 198 3285 / 1095 939 AF023129 [Campbell, 1999 #3] Guinea Pig HK2a 145 3101 / 1034 996 D21854 Genbank Database Mouse HK2a 253 3108/1036 629 AF350499 [Zhang, 2001 #31]].

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26 HKa2aHK2c RatRabbitmRNAProteinGenomic DNA Exon1Intron1Exon2OtherExons ATG ATG ATGHK2aHK2bHK2cHK2a ATG HK2aHK2b ATGATGATGATGHKa2aHK2c RatRabbitmRNAProteinGenomic DNA Exon1Intron1Exon2OtherExons ATG ATG ATGHK2aHK2bHK2cHK2a ATG ATG ATG ATGHK2aHK2bHK2cHK2a ATG HK2aHK2b ATGATGATGATG Figure 1-6. Formation of the alternative transcripts from the rabbit and rat HK2 genes. translational regulation (26). Northern analysis of total RNA from various rat tissues shows that the HK2b transcript was present in vivo. The HK2b protein appeared to be truncated by 108 amino acids, but the presence of the protein in vivo has not been reported. In rabbit, the HK2c 5 UTR contains only two short open reading frames prior to the translation start and is of similar length to other HK2 transcripts. Unlike the truncated HK2b of rat, the HK2c protein contains a 61 amino acid extension on the N-terminus of HK2a. Fejes-Toth et al. (12) failed to detect HK2c in their 5 RACE experiments. However, Campbell et al. (6) demonstrated by both Northern and Western analyses that HK2c mRNA and protein were present in rabbit kidney tissue as well as in tissue culture cells derived from the rabbit cortical collecting duct (RCCT28A). The

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27 cloning of homologous cDNAs from other species will be necessary to determine if the formation of splice variants is common among mammalian species or whether it is unique to rat and rabbit. Human HK2 Gene The human HK2 gene was originally identified as the ATP1AL1 gene (45). It was initially unclear as to whether or not this H+, K+ ATPase should be considered homologous to the rat HK2a (8). The amino acid identity for these two proteins was much lower (87%) than that of the HK1 proteins from human, rat and rabbit (97%). The cloning of the rabbit HK2a cDNA (6) suggests that all three proteins are homologous as it had approximately 87% amino acid identity with both rat HK2a and human ATP1AL1 sequences (8). The genomic structure of the mouse HK2 gene (below), the rat gene (NCBI database), and the rabbit HK2 gene (this study) confirm that these genes are homologous genes. The complete genomic organization of the human ATP1AL1 gene was reported by Sverdlov et al. in 1996 (45). The gene is approximately 32Kb in length and contains 23 exons and 22 introns. The sizes of the exons and introns are included in Table 2-1. The transcription start site was mapped using S1 nuclease protection and primer extension. The S1 nuclease protection assay produced 4 clustered bands from to 188. The primer extension produced a single band marking the transcription start site at 187 with respect to the ATG start codon. Analysis of the region immediately 5 of the transcription start site identified possible regulatory elements including a TATA box, SP family binding sites, AP-2 binding sites and NF-kB. Additionally, the region from to +369 met the criteria for a CpG island. Analysis of the 3 region of the gene identified

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28 3 possible polyadenylation sites. The authors suggest that these sites may be used in a tissue specific manner. Mouse HK2 Gene Recently, the complete sequence and structure of the murine HK2 gene was reported (52). Similarly to the human gene, the murine gene spans 23.5Kb and contains 23 exons. The transcription start site was mapped using primer extension. It is located at with respect to the ATG start codon. These authors did not observe an alternative transcription start site as seen in rat (26) and rabbit (6). Computer analysis of 7.2Kb of sequence immediately 5 of the start site identified many possible transcription factor binding sites including a TATA box, CEBP, NF-kB, cAMP and glucocorticoids. There appears to be one poly A signal designating the 3 end of this gene. Summary Intracellular and extracellular K+ concentrations play a critical role in normal cell functioning. The collecting duct segment of the nephron is the major location where K+ ions are reabsorbed when blood K+ becomes too low. The HK2 subtype of the H+, K+ ATPase, located on the apical membrane of collecting duct cells, appears to play a major role in K+ reabsorption since its activity and its expression appear to be increased when blood K+ concentration is low. There is additional evidence that the expression of the HK2 gene products are also regulated by Na+ levels, acid/base balance, and hormones. All of the studies on HK2 gene expression, however, have been performed in vivo. There is nothing known about the molecular mechanisms responsible for the change in gene expression. The goal of our study was to characterize the rabbit HK2 gene and initiate studies on the molecular regulation of the gene.

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CHAPTER 2 CLONING OF THE HK2 GENE The first specific aim of this dissertation was to clone the rabbit HK2 gene. There were two important reasons for carrying out this goal. First, cloning the gene would provide sequence information essential for the future experiments designed to study the genes promoter and regulatory elements. The cDNAs for HK2a and HK2c have been previously identified (6) and shown to be products of the same gene (5). However, there is currently nothing known about the genomic sequence that is 5 of the cDNA ends. This dissertation, therefore, provides the first data regarding the upstream sequence that contains the genes promoter and its regulatory elements. The second purpose for cloning the rabbit HK2 gene was to determine its genomic organization. There has been some controversy over whether or not the HK2 proteins identified from several species were in fact homologous proteins (8). The subunit proteins from rabbit (HK2a), rat (HK2a), mouse (HK2), guinea pig (HK2) and human (ATP1AL1) share an amino acid identity of 87%. This percentage is much lower than the amino acid identity that the same species share for the HK1 subunit (97%). One method for resolving this controversy is to compare the intron and exon sizes of the genes that encode the proteins since genes that were derived from common ancestors should maintain a consistent organization. The genomic organization of the human ATP1AL (45) and mouse HK2 (52) genes have been determined independently from this dissertation. The genomic organization of the rat gene was determined from the rat 29

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30 genome database at the National Center for Biotechnology Information (NCBI). The cloning and sequencing of the rabbit gene is the subject of this chapter. The comparison of the organization of the four genes supports the conclusion that these genes are homologous and have been derived from a common ancestor. The rabbit HK2 gene was cloned from a bacteriophage library of the rabbit genome. Three clones were identified that span a majority of the rabbit HK2 gene. The polymerase chain reaction (PCR) was used on rabbit genomic DNA in order to amplify the remaining portion of the gene. This chapter discusses the cloning, sequencing and analysis of the bacteriophage clones and the PCR products that contain the HK2 gene. Materials and Methods Screening the Lamda Genomic Library A bacteriophage library containing 15Kilobase pair (Kbp) inserts of rabbit genomic DNA was purchased from Clontech, Inc. (Catalog #TL1008j). Two approaches were used to screen the library. The first method was a PCR-based screen (21) and the second was a more conventional plaque lift/hybridization screen (Clontech, Inc). The PCR approach used primers designed to the HK2a cDNA to identify aliquots of the library that contained bacteriophage with inserts that corresponded to the HK2 gene. The primers BC334 (5-TATCTGTAGCTGCATGGTGCTCCAC-3) and BC386 (5-ACCCGCGCGCTCCAGCGCGACAT-3) were used in the PCR reaction. These primers correspond to base pairs 69-93 and base pairs 16-40 the HK2a cDNA and are known to amplify a 647 base pair fragment from rabbit genomic DNA (5). The amplified fragment is larger than expected because it includes intron sequence that is not present in the cDNA. This reaction was repeated as a positive control for the PCR approach to

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31 screening the library. Additionally, 2l of the PCR reaction was ligated into the TOPO cloning vector (Invitrogen, Inc. Cat# K4574-J10). This construct was designated pDZ6 and is referred to as the 5 probe for all future experiments. To screen the library, 500l of E.coli strain K802 (Clontech, Inc.) was infected with 1 X 106 plaque forming units (pfu) of the genomic library and incubated at 37C for 15 minutes. The infected bacteria were brought to a volume of 10mL with LB broth (tryptone, yeast extract, NaCl) supplemented with 10mM MgSO4. l of infected bacteria were placed into each well of a 96 well plate and amplified by growth at 37C for 5 hours. 25l from each row and each column were pooled to form 16 samples representing the 96 wells. 10l of the pooled samples were used in a PCR reaction (250pm each primer, 250M dNTP mix, 1X PCR buffer, 5U Taq polymerase, 10l of pooled template, dH2O to 40l). The PCR conditions were 94C for 1 minute 30 seconds; 94C for 15 seconds + 72C for 2 minutes times 5 cycles; 94C for 15 seconds + 70C for 2 minutes times 5 cycles; 94C for 15 seconds + 68C for 2 minutes times 25 cycles; 68C for 8 minutes. Southern analysis of the PCR products was carried out as described in Maniatis et al. (39). The PCR reactions were run on a 1% agarose gel and visualized with ethidium bromide stain and UV light. The gel was soaked gel in denaturation solution (1.5M NaCl, 0.5M NaOH) for 30 minutes, neutralizing solution (1.5M NaCl, 1.0M Tris-Cl pH 8.0) for 30 minutes, and equilibrated in 10X SSC (1.5M NaCl, .15M sodium citrate) for 30 minutes. The DNA was transferred to a nylon membrane by capillary action overnight. The DNA was UV crosslinked to the nylon membrane, placed in a hybridization tube and incubated with 5mL of buffer (.25M Na2HPO4 pH 7.2, 1mM EDTA, 1% BSA, 7% SDS) at 65C for at least 15 minutes. A radioactive probe was prepared by digesting pDZ6 with EcoRI and

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32 running the digestion on a 1% agarose gel. The 647bp band was cut from the gel and extracted using the Gel Extraction Kit from Qiagen, Inc.(Catolog #28706). 25ng of the DNA fragment were used to create a 32P labeled probe. The method used was the Prime-It RmT Random Primer Labeling Kit from Stratagene, Inc. (Catolog #300392). The probe was added directly to the prehybridization solution and incubated with the nylon membrane overnight at 65C. The next day, the membrane was washed three times with 300mL of wash buffer (20mM Na2HPO4 pH 7.2, 1% SDS, 1mM EDTA) at 65C, wrapped in saran wrap and exposed to autoradiograph film for an appropriate length of time. Positive rows and columns were aligned to determine possible positive wells. The PCR was repeated on samples from individual wells. The contents of positive wells were used to create plaques on agar plates. Plaque lift hybridizations were performed to purify positive plaques (see below). The second method for screening the library was to perform a series of plaque lift hybridizations. The protocol was modified from the procedure recommended by the rabbit genomic library manufacturer, Clontech, Inc. The initial screens were performed with approximately 20,000 pfu per 150mm agar plate. The library was diluted 1/500 with dilution buffer (1M NaCl, 0.1M MgSO4, 1M Tris pH 7.5) and 100l was used to infect 600l of E. coli strain K802 at 37C for 15 min. 7mL of warmed soft agar (LB broth + 7g/L agar) was added to infection and poured over a 150mm LB agar plate. After overnight incubation at 37C, plaque lifts were performed by placing circular nylon membranes on top of each plate for 2 minutes, the filter was removed placed plaque side up on top of Whatman paper #3 soaked in denaturing solution for (1.5M NaCl, .5N NaOH) for 5 minutes, neutralizing solution (1.5M NaCl, 1M Tris pH 8.0) for 5 minutes

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33 and 2X SSC (.3M NaCl, .03M Sodium Citrate) for 5 minutes. The filters were baked dry at 80C for 1 hour. The filters were probed with either a 5 probe (pDZ6), a mid probe (HK2a cDNA bases 1264 1569) or a 3 probe (HK2a cDNA bases 3265 4073) as described for the Southern blot filters. Positive plaques were pulled from the agar plates with a Pasteur pipette and diluted into 1mL of dilution buffer. Serial dilutions of the plaque were used to infect E.coli strain K802, mixed with 3mL of soft agar and poured over 100mm agar plates. The plaque lift procedure was repeated. Isolated positive plaques were picked and the procedure was repeated until all plaques on a plate were positive and each plaque was considered pure. Pure plaques that were isolated by both screening methods were grown in large scale and DNA was isolated using the Qiagen Lambda Maxi Kit (Cat# 12562). A 5mL overnight culture of E.coli strain K802 was pelleted and resuspended in 1.5mL of bacteriophage dilution buffer. Approximately 1 X 107 bacteriophage were added to the bacterial cells and incubated at 37C for 20 minutes. The infected bacteria were added to 250mL of LB supplemented with 10mM MgSO4 and 0.2% maltose. The culture was allowed to grow at 37C until the bacteria lysed (approximately 4 hours). The bacterial debris was pelleted while the bacteriophage that remained in the supernatant was used for DNA isolation. 400l of buffer L1 (300mM NaCl, 100mM Tris-Cl pH 7.5, 10mM EDTA, .2mg BSA, .2mg/mL RNaseA) was added to the lysate and incubated at 37C. This step digests away any bacterial RNA. In order to precipitate and pellet the bacteriophage, 50mL of buffer L2 (30% polyethylene glycol, 3M NaCl) was added and the mixture was incubated on ice for 60 minutes and centrifuged at 10,000 rpm for 10 minutes. The pellet was resuspended in 9mL of buffer L3 (100mM NaCl, 100mM Tris

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34 Cl pH 7.5, 25mM EDTA) and 9mL of buffer L4 (4% sodium dodecyl sulfate (SDS) and the mixture was heated to 70C for 10 minutes. This step denatured the bacteriophage proteins and released the bacteriophage DNA. After cooling on ice, 9mL of buffer L5 (3M potassium acetate) was added and mixture was centrifuged for 30 minutes at 15,000 rpm in order to pellet bacteriophage proteins. The supernatant that contained the bacteriophage DNA was poured over a Qiagen column that was equilibrated with buffer QBT (750mM NaCl, 50mM MOPS, 15% isopropanol, .15% triton X-100). The DNA bound to the column. The column was washed with 60mL of buffer QC (1M NaCl, 50mM MOPS, 15% isopropanol) and then the DNA was eluted off the column with 15mL of buffer QF (1.25M NaCl, 50mM Tris-Cl pH 8.5, 15% isopropanol). 10.5mL of isopropanol was added to the eluate and centrifuged at 15,000 rpm for 30 minutes to precipitate and pellet the bacteriophage DNA. The DNA was washed with 70% ethanol and resuspended in TE (10mM Tris pH 7.5, 1mM EDTA). The DNA from each clone was digested and used in Southern analysis in order to identify overlapping clones that span the entire HK2 gene. HK2.1 Sequence Clone HK2.1 was the 5 most clone. Southern analysis showed that the EcoRI fragment that hybridized to the 5 probe was attached to the SP6 arm and sequencing with the SP6 promoter primer (5-ATTTAGGTGACACTATAG-3) indicated that the orientation of the insert was such that the rest of the clone contained sequence 5 of the HK2 gene. In order to obtain the sequence of the region immediately 5 of the cDNA, a 6.3Kbp XhoI fragment was subcloned into pBluescript (pBS, Stratagene, Inc.) creating plasmid pDZ10. The sequence of the entire fragment was determined by walking along

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35 the sequence in both 5 and 3 directions. Sequencing was carried out by the University of Florida Interdisciplinary Center for Biomedical Research (ICBR) sequencing core facility. Complete sequences from both directions were obtained by compiling sequences from individual primers. The two complete sequences were compared and any base pair mismatches were resolved by additional sequencing through the region. The location of each primer on the complete pDZ10 sequence and the sequence of primers can be found in Figure 2-1 and Table 2-1 respectively. T7 DZ12DZ14DZ15DZ18DZ25DZ20DZ26DZ27 T7 DZ12DZ14DZ15DZ18DZ25DZ20DZ26DZ27 T7 DZ12DZ14DZ15DZ18DZ25DZ20DZ26DZ27 SP6DZ6DZ16DZ19DZ21DZ23DZ24DZ5DZ4 SP6DZ6DZ16DZ19DZ21DZ23DZ24DZ5DZ4 SP6DZ6DZ16DZ19DZ21DZ23DZ24DZ5DZ4 Figure 2-1. Location of Sequencing Primers on the Complete pDZ10 sequence. Black represents the 6.3Kbp XhoI fragment subcloned from HK2.1. Orange represents the ends of pBS (Stratagene, Inc.). Blue represents primers used for sequencing in the 5 to 3 direction. Red represents primers used for sequencing in the 3 to 5 direction. The star indicates the location of the 5 probe. HK2.5 Sequence Clone HK2.5 was the middle clone. It hybridized to both the 5 and the mid probes (HK2a cDNA base pairs 16-93 and 1264-1569 respectively). Since this clone was being used to determine the intron/exon boundaries within the rabbit HK2 gene, it was necessary to obtain the sequence of the entire clone. The DNA arms were

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36 removed by digestion of the clone with XhoI. The digestion was run on a 1% agarose gel and visualized with ethidium bromide. The genomic DNA insert was cut from the gel Table 2-1. Primers used for pDZ10 sequencing. Name Primer Sequence 5 to 3 Location T7 CCCTATAGTGAGTCGTATTA arm DZ12 CAATCCACGTTGCCCGCATGGG 784-805 DZ14 CCAGTCCGGATACGGAGCAGG 1437-1457 DZ15 CCCCACCAACAGCCCAGACG 2228-2247 DZ18 CCTCCAGGTGAGGACTACTCC 2974-2994 DZ25 CTCTCCCCTCCAACTCTGAAGG 3689-3710 DZ20 GAACGGCCGGCGCTGCGG 3774-3791 DZ26 GTGTCCCATGTGGGAAGCCAGG 4378-4399 DZ27 CTTGGGGGCTCCGGATCCTGG 5064-5083 SP6 ATTTAGGTGACACTATAG arm DZ4 CGCATGTCGCGCTGGAGG 5587-5570 DZ5 CTGCACTCTCAGAGTGAAGG 4889-4870 DZ6 GGCTATGGGACAGGGATGACG 4165-4145 DZ16 GGCACAGAGAAGTAGTGCCC 3469-3450 DZ19 GAAACCTACTCATGCCAGGCTC 2752-2731 DZ21 GATGAGTTCTCAGGACTCTGAC 1977-1956 DZ23 GCTGCAGCCTAGCACAC 1237-1221 DZ24 GGGGAGTAAACCTCAGGATGGG 568-547 Blue represents primers used for sequencing in the 5 to 3 direction. Red represents primers used for sequencing in the 3 to 5 direction. and extracted from the agarose using the QIAquick Gel Extraction Kit (Qiagen, Inc. Cat # 28706). This procedure was repeated until 25g of insert DNA was obtained. The insert fragment was then sheared and shotgun subcloned into the TOPO cloning vector according to Invitrogen, Inc. (Cat.# K7000-01). 25g of DNA was added to 750l shearing buffer (TE, 20% glycerol) and placed in a nebulizer attached to a compressed air pump. The DNA was sheared twice at 10psi for 90 seconds. The sheared DNA was precipitated (80l 3M NaOAc, 4l glycogen, 700l 100% isopropanol) on dry ice for 15 minutes, pelleted by centrifugation at 12,000 rpm for 15 minutes, washed with 80% ethanol and resuspended in 200l of sterile dH2O. In order to repair the sheared ends for

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37 cloning, 2g of DNA was added to a blunt-end repair reaction (20l DNA, 5l 10X blunting buffer, 1l BSA, 5l dNTP mix, 2l T4 DNA polymerase, 2l Klenow DNA polymerase) and incubated at room temperature for 30 minutes. The enzymes were deactivated by heating the reaction mix to 75C for 20 minutes. Dephosphorylation of the repaired ends was carried out by adding 35l sterile dH2O, 10l 10X dephosphorylation buffer, and 5l calf intestine alkaline phosphatase (CIP) to the blunt end repair reaction and incubating the reaction at 37C for 60 minutes. The reaction was phenol/chloroform extracted, precipitated and resuspended in 20l of sterile dH2O. Shotgun cloning of the DNA was carried out with 3 concentrations of DNA (60ng, 20ng, 5ng), 1l salt solution, and 1l pCR4-blunt TOPO vector (Invitrogen, Inc.). The ligations were incubated at room temperature for 5 minutes and then transformed into chemically competent E. coli strain DH5. The resulting bacterial colonies were screened to identify 48 plasmids containing inserts of approximately 1500bp. Each colony was grown overnight in 3mL of LB broth and then miniprep DNA was isolated using the QIAprep Spin Miniprep Kit from Qiagen, Inc. (Catolog # 27106). The DNA was digested with EcoRI, run on a 1% agarose gel and visualized with ethidium bromide stain. When 48 bacterial colonies that contained plasmids of the appropriate size were identified, 200l of an overnight culture of each colony was placed in an ELISA plate and taken to ICBR for high throughput sequencing. Approximately 250bp of sequence was obtained from each end of the plasmid inserts. These sequences were assembled into ten contiguous fragments (contigs) by ICBR. The high-throughput sequencing core used the Helix Finch program distributed by Giospiza, Inc. in orger to assemble the sequences. The order of the contigs was determined by alignment of the contigs with the HK2a

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38 cDNA and by determination of plasmids that contained sequence in two contigs. The gaps between the fragments were closed by additional sequence from the plasmids that spanned the gaps as well as sequence from the original clone HK2.5. The primers used for the additional sequencing are listed in Table 2-2. Table 2-2. Primers used for HK2.8 sequencing Name Sequence 5 to 3 Location DZ83 CCCCGCTCTAAAGAAGGCCG 2418 DZ94 GGGCTTTCGGCCGACCTCACTG 2873 TC4 CCTGGAATGGACAGGCT 2983 DZ93 GCCTTCTGCCTCCAGGGC 3181 DZ96 GCCCCCGTTTTGACTCCC 3815 DZ95 GAGCGGGGGTGTCATTCACTCCG 2190 MG45 CGTCCATTCCTGTCCATAGCTATCTTCCAAGTCGTTCAGGTG 2897 MG49 CATCGTATACCCAGATCAGGATGGCATGGGGTACGGCCAC 3188 Blue represents primers used for sequencing in the 5 to 3 direction. Red represents primers used for sequencing in the 3 to 5 direction. Location indicates the position of the primer on the HK2a cDNA. HK2.8 Sequence Clone HK2.8 was the 3 clone. It hybridized to the 3 probe (HK2a cDNA base pairs 3265-4073). The approximate intron/exon boundaries were determined by alignment of the cDNA from rabbit HK2a to the exon sizes of human ATP1AL1 gene. Primers were then designed near the expected end of each exon (Table 2-3) and were used for partial sequencing of clone HK2.8. The sequencing was carried out by the ICBR sequencing core. PCR Amplification of the Missing Fragment In order to obtain the exon boundaries for the portion of the HK2 gene that was missing from the three clones, PCR primers were designed to the approximate ends of the exons. The sequences of the primers, their orientations, and their location along the

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39 HK2a cDNA are listed in Table 2-3. Proofstart DNA polymerase (Qiagen, Inc. Cat# 202203) and RCCT28A genomic DNA were used for the PCR reaction. The PCR reaction mix consisted of 1X Proofstart PCR buffer containing 15mM MgSO4, 300M each dNTP, 1M each primer, 2.5U ProofStart DNA polymerase, 0.5g DNA template, and dH2O up to 50l. The PCR cycle conditions were 95C for 5 minutes times one cycle and 94C for 1 minute, 60C for 1 minute, 72C for 2 minutes times 40 cycles. The PCR products were run on a 1% agarose gel and visualized with ethidium bromide. The most intense band was cut from the gel and extracted using the QIAquick Gel Extraction Kit (Qiagen, Inc. Cat# 28706) as described by the manufacturer. In order to obtain the sequence of the exon boundary, 50ng of DNA was sent to ICBR sequencing core along with the primers used to create the PCR product. Table 2-3. Primers for genomic PCR and sequencing Name Sequence 5 to 3 Location DZ98 CCTTGGGTGCGGGGGGACAG Intron11 DZ99 GGGCGGCCTGGGCGAGC 1852 TC80 GGCTCTCTTATCAATGATTCATCC 1979 DZ81 GCCATTGCCAAGAGTGTAGGG 2093 BC231 GCTTGTCATTGGGATCTTCC 1702 DZ100 CTGAGTCAAATGAGTAGGTCTCTGG 1913 DZ101 CCCTACACTCTTGGCAATGGC 2093 DZ97 CTGGGGAAACTTTGCCCTCC Intron14 Blue represents primers used for sequencing in the 5 to 3 direction. Red represents primers used for sequencing in the 3 to 5 direction. Location indicates the position of the primer on the HK2a cDNA. Results Screening the Genomic Library A majority of the rabbit HK2 gene was cloned by screening a rabbit genomic library using PCR and plaque lift hybridization methods. These two techniques identified nine clones. Two of the clones were identified by the PCR method (HK2.2, HK2.4)

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40 and seven of the clones were identified by traditional plaque lift hybridization (HK2.1, HK2.3, HK2.5, HK2.6, HK2.7, and HK2.8). Figure 2-2 is an example of the PCR screen that identified clone HK2.2. Figure 2-2A shows a Southern analysis of the initial screen. Samples were pooled across rows and down columns and PCR was performed on 25l of the pooled sample. The PCR products were run on a 1% agarose gel and Southern analysis was performed with the 5 probe. In this example, rows F and H and columns 6, 7, 8 and 11 contain positive clones. Figure 2-2B is the Southern blot that was performed on PCR products from the screen of individual wells in row F. Well F7 was identified as a well containing a positive clone. The sample in well F7 was diluted and used in plaque lift experiment to purify the positive clone that was designated HK2.2. Additional PCR screening with the 5 probe identified clone HK2.4. PCR amplification of positive fragments of DNA quickly caused cross-sample contamination problem that was difficult to overcome. This method was therefore abandoned and the remainder of the clones were isolated using standard plaque lift techniques. Figure 2-3 is example of the plaque lift and purification procedure that used the 5 probe to identify clone HK2.1. In the first screen (Figure 2-3A) approximately 20,000 plaques were plated on a 150mm dish. After transfer to nylon and hybridization to the 5 probe, only one positive plaque was identified. An agar plug was taken from that region of the plate, diluted, and used to create a new plate with approximately 200 plaques. About one half of the plaques on this plate hybridized to the 5 probe (Figure 2-3B). An agar plug of an isolated plaque was taken from that plate and used to create a new plate with a similar number of plaques. All of the plaques on the new plate hybridized to the

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41 CDEFGH1567891011 F4F6F7F8F11(A) First screen of PCR product pools(B) Second screen of PCR products from individual wells CDEFGH1567891011 CDEFGH1567891011 F4F6F7F8F11 F4F6F7F8F11(A) First screen of PCR product pools(B) Second screen of PCR products from individual wells Figure 2-2. Southern analysis of PCR screen of the genomic library. A) First screen 1kb represents the 1kb ladder, GD represents genomic DNA, A-H represent samples pooled across the rows of an ELISA plate, 1-12 represent samples pooled down the columns of the same ELISA plate. Samples 2 and 10 were cut of before DNA transfer. B) Second screen. Individual wells from positive pools were screened for positive clones. probe (Figure 2-3C). Any plaque from this plate was considered pure and could be used for a DNA maxiprep. This plaque lift method was used to identify seven clones; two hybridized to the 5 probe (HK2.1, HK2.3), three hybridized to the mid probe (HK2.5, HK2.6, 17-1), and two hybridized to the 3 probe (HK2.7, HK2.8).

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42 A.B. C. A.B. C. C. Figure 2-3. Plaque lift screen of the genomic library. A) First screen. 20,000 plaques per plate, one hybridizes to the 5 probe. B) Second screen. Plug from first screen is diluted to 200 plaques per plate, the plaques hybridize to the 5 probe. C) Third screen. Individual plaque picked from second screen diluted to 200 plaques per plate, all plaques hybridize to the 5 probe

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43 Determination of Overlapping Clones In order to determine which clones overlapped and spanned the HK2 gene, Southern analysis was performed on digested DNA from each clone. Figure 2-4 is an example of a Southern analysis showing that HK2.1 and HK2.5 overlap in the region of the 5 probe. HK2.1, HK2.5 and HK2.6 DNA were digested with XhoI and HindIII individually and in combination. The digests were run on a 1% agarose gel and visualized with ethidium bromide (Figure 2-4A). The DNA was transferred to nylon membrane and hybridized to the 5 probe. The membrane was washed and exposed to autoradiograph film (Figure 2-4B). In the lanes representing HK2.1 and HK2.5 a single band appears that hybridizes to the 5 probe while in the lanes representing HK2.6 no band appears. These data clearly show that HK2.1 that was isolated using the 5 probe and HK2.5 that was isolated using the mid probe overlap in the region of the 5 probe. HK2.6, that was isolated using the mid probe, does not extend to the 5 probe. A similar analysis was carried out for the remainder of the clones and with all three probes. It was determined that three clones, HK2.1, HK2.5 and HK2.8 spanned a majority of the HK2 gene, but a gap existed in between HK2.5 and HK2.8. Genomic PCR was carried out in order to obtain the missing portion of the gene (see below). HK2.1 Sequence Clone HK2.1 hybridized to the 5 probe and contains approximately 14Kbp of sequence upstream of the HK2 gene. Appendix A contains all of the known sequence from the rabbit HK2 gene. The 6300bp XhoI fragment subcloned from HK2.1 is represented in base pairs 1 6298. This sequence was used to determine potential

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44 promoter and regulatory elements. The complete analysis of this sequence is discussed in Chapter three of this dissertation. hoIhoI 2.1 uncut2.5 uncut2.6 uncut2.1HindIIII2.1HXhoI2.1 double2.5HindIIII2.6HindIIII2.5HXhoI2.5 double2.6 double2.6HXhoI2.1 uncut2.5 uncut2.6 uncut2.1HindIIII2.1HX2.1 double2.5HindIIII2.6HindIIII2.5HXhoI2.5 double2.6 double2.6HXhoI2.1 uncut2.5 uncut2.6 uncut2.1HindIIII2.1HXhoI2.1 double2.5HindIIII2.6HindIIII2.5HXhoI2.5 double2.6 double2.6HXhoI2.1 uncut2.5 uncut2.6 uncut2.1HindIIII2.1HX2.1 double2.5HindIIII2.6HindIIII2.5HXhoI2.5 double2.6 double2.6HXhoI (A) Ethidium bromide stainedAgarose gel(B)Autoradiographof gel probedWith the 5 probe(A) Ethidium bromide stainedAgarose gel(B)Autoradiographof gel probedWith the 5 probe Figure 2-4. Southern analysis to determine overlapping clones. (A) Each clone was digested as indicated and run on a 1% agarose gel. The DNA was visualized by staining the gel with ethidium bromide. (B) The DNA was transferred to nylon membrane and probed with the 5 probe (B). HK2.5 Sequence Clone HK2.5 hybridized to both the 5 and the mid probes suggesting that it contained many of the 5 exons for the HK2 gene. ICBR high through-put sequencing of the ends of 48 plasmids that were subcloned from HK2.5 yielded 144 sequences.

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45 The M13 forward primer (5-GTAAAACGACGGCCAG-3) sequencing reaction was performed twice and the M13 reverse primer (5-ACAGGAAACAGCTATGAC-3) sequencing reaction was performed once. The average read-length for the reactions was 307 bases. ICBR used a computer alignment algorithm to assemble the sequences into 10 contiguous fragments labeled 1-10 based upon size. Using the HK2a cDNA and subclones in which sequence from opposite ends mapped into different contigs, nine of the ten sequences were placed in order. Figure 2-5 depicts the nine sequences and the subclones that spanned the gaps. In order to obtain the remainder of the HK2.5 sequence, the gaps between the contigs were filled with additional sequence from the indicated plasmids as well as with sequence directly from the HK2.5 clone. Table 2-3 lists the DNA template and the primers that were used to complete the sequence. Appendix A contains the known sequence of the rabbit HK2 gene. The sequence determined from HK2.5 overlaps with the sequence from pDZ10 and is represented in base pairs 4616 to 19766. HK2.8 Sequence Clone HK2.8 hybridized to the 3 probe. Partial sequences were determined for this clone. The purpose was to determine the precise intron/exon boundaries of the remainder of the gene as well as the 3 end of the gene. The partial sequence revealed that HK2.8 contained sequence from exons 15-23. The sizes of the exons and introns identified are listed in Table 2-5. A portion of the sequence of exon 25 is shown in Figure 2-7. The red bases represent the 3 end of the HK2a cDNA cloned by Fejes-Toth et al. (12). Just upstream of the cDNA end there is a poly A signal sequence (blue) and just downstream there is a T-rich region of DNA. It seems likely that this poly A signal

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46 10 7 8 1 9 6 5 2 4 H6 H3 E4 H4 B6 G3 A2 D4T7DZ85DZ88DZ91T3T3T3 DZ84T7T7T7ABCDEFGH DZ74 DZ89 10 10 7 7 8 8 1 1 9 9 6 6 5 5 2 2 4 4 H6 H3 H3 E4 H4 B6 G3 A2 D4T7DZ85DZ88DZ91T3T3T3 DZ84T7T7T7ABCDEFGH DZ74 DZ89 Figure 2-5. Clone HK2.5. Pink boxes represent 9 continuous sequences numbered according to size. Letter A-H represent the gaps between the continuous sequences. Lines below represent plasmid used to fill the gaps. Perpendicular lines represent primers used for sequencing.

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47 represents one true end to the HK2 mRNAs. Campbell et al. (6), however, cloned a cDNA of HK2a and HK2c that was slightly longer at the 3 end (green). Just downstream of this cDNA end there are two possible poly A signals and T-rich region of DNA. Therefore it seems likely that one of these two poly A signals, or both represent alternative ends to the HK2a and HK2c mRNAs. The partial sequence obtained from HK2.8 is located in Appendix A. 301 AGGTTTTTTT TTTTAAATAA AAGATGTTTT TAAGTAAAAT GTTTTATGAA 351 ACAAAATCTA ATTGTGATGT TTTACTTAAT TCAAGTTTTT CCAGAGGCAG 401 GCACGGAAAA TACCAAAAAA ATAAAATAAA ATAAGATTCT GGGTTTTTTT 451 TCTTTTTTGC TCCTTCTGGT CATTTTCTTT ACACACAGAG TGTCTGGAAA 501 TACAGGCTTT TCCTCGTGAG TGCTTCCCGC ACCTGTGCCC CCTCCCCCCC Figure 2-6. Partial sequence from clone HK2.8. Blue represents 3 possible poly A signals. Red represents the last three bases of the Fejes-Toth cDNA for HK2a. Green represents the last three bases of the Campbell cDNA for both HK2a and HK2c. Completion of the HK2 Gene Sequence The complete sequencing of clone HK2.5 and the partial sequence of clone HK2.8 revealed that three HK2 exons were not contained in either clone. Therefore, genomic PCR was performed on RCCT28A cell DNA in order to amplify four DNA fragments that contained the missing exons. Primer set DZ98/BC231 amplified a band of approximately 4000 base pairs. This fragment contained a portion of intron 11 and the 5 boundary of exon 12. Primer set DZ99/DZ100 amplified a band of approximately 1500 base pairs. This fragment contained the 3 boundary of exon 12, intron 12 and the 5

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48 boundary of exon 13. Primer set DZ80/DZ101 amplified a band of approximately 700 base pairs. This fragment contained the 3 boundary of exon 13, intron 13 and the 5 boundary of exon 14. Primer set DZ81/DZ95 amplified a band of approximately 4000 base pairs. This fragment contained the 3 boundary of exon 14 and a portion on intron 15. The sequences obtained from these PCR products are located in Appendix A. Discussion The screening of the library generated three clones that contained 87% of the HK2 exons and 65% of the HK2 gene. Most importantly, clone HK2.1 hybridized to the 5 probe and contains approximately 14kbp of sequence upstream of the gene. Obtaining this clone was a necessary first step in the study of the regulation of the HK2 gene, which is the subject of the remaining chapters of this dissertation. Additionally, the genomic organization of the rabbit HK2 gene was determined using the complete sequence of HK2.5, the partial sequence of HK2.8 and the genomic PCR fragments that spanned the gap between the two clones. HK2.5 contained 15150bp of gene sequence including the HK2 exons 1-11, HK2.8 contained exons 15 23, and the PCR fragments contained exons 12, 13, and 14. The entire gene spanned approximately 30Kbp of genomic DNA. It is notable that intron 11 and intron 14 are approximately 4200bp each. The mid probe hybridizes to exon nine and the 3 probe hybridized to the 3 UTR. The size of the intervening DNA (18Kbp) is the likely reason why a clone containing these three exons was not obtained when the bacteriophage library was screened. Table 2-4 compares the sizes of the exons and introns of the rabbit HK2 with those of the rat HK2 gene (ICBR database), the mouse HK2 gene (52), and the human

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49 ATP1AL1 gene (45). The exon sizes for the four genes are absolutely identical except for the three 5 exons one, two and four. It is not suprising that the 5 end of the gene is Table 2-4. Exon and intron sizes for the known HK2 genes. Exon Rabbita Ratb Mousec Humand Intron Rabbita Ratb Mousec Humand 1 208 287 262 195 1 567 677 658 (700) 2 141 153 150 159 2 2702 2133 2286 (2300) 3 60 60 60 60 3 3061 2511 2321 (2900) 4 204 201 201 204 4 1049 746 762 738 5 114 114 114 114 5 1032 742 726 937 6 135 135 135 135 6 113 124 129 123 7 118 118 118 118 7 173 233 227 258 8 269 269 269 269 8 854 939 970 1187 9 199 199 199 200 9 141 144 134 157 10 110 110 110 110 10 2282 1324 1269 (1700) 11 135 135 135 135 11 (4200) 4231 2010 (4200) 12 135 193 193 193 12 1600 1484 1471 (1600) 13 176 176 176 176 13 600 834 639 (900) 14 137 137 137 137 14 4200 1599 1597 (4200) 15 151 151 151 151 15 365 1419 557 16 169 169 169 16 88 90 87 17 155 155 155 17 (700) 1425 1311 (1900) 18 124 124 124 124 18 172 184 195 193 19 146 145 146 146 19 445 594 590 (600) 20 134 134 134 134 20 167 174 168 195 21 103 102 102 102 21 (1155) 434 388 431 22 92 92 92 92 22 87 137 161 83 23 658 658 658 905 23 Sources: a this dissertation, b NCBI database, c (52), d (45).Notes: () indicates introns with sizes determined by estimating the size of restriction fragments. indicates sized that could not be determined due to incomplete database sequence. the most variable since it is in this region where the rabbit and rat genes can undergo alternative splicing to create HK2c and HK2b while the mouse and human gene apparently do not. The controversy over whether or not these genes are homologous was partially resolved by a distance analysis of the HK and NaK subunit proteins (8). The analysis showed that the three HK2 proteins were more closely related to each other than to any of the other X+,K+ ATPase subunits suggesting that they are homologous.

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50 The exon/intron sizes, compared in Table 2-4, support the exisiting evidence and confirm that these four genes are homologous and were derived from a common ancestor.

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CHAPTER 3 MAPPING THE TRANSCRIPTION START SITES FOR THE HK2 GENE The second specific aim of this dissertation was to map the transcription start sites for the two alternative mRNAs produced by the HK2 gene. The determination of the transcription start sites was an important step in characterizing the HK2 gene for two reasons. The main goal of our study was to initiate an investigation of the regulation of the rabbit HK2 gene. The core promoter and regulatory elements directing transcription from the gene are likely to be found just 5 of the transcription start sites. Additionally, there was some controversy over the existence of HK2c. Identification of the transcription start site for HK2c would resolve this controversy. The first experimental goal was to identify potential promoter and regulatory elements 5 of the transcription start sites. The 5 ends of the cDNAs for HK2a and HK2c were previously identified by studies that used 5 Rapid Amplification of cDNA Ends (RACE) (5). This method, however, is not likely to determine the true transcription start site. 5RACE uses a reverse transcription reaction to extend a primer annealed near the 5 end of the mRNA to the true end of the mRNA. The reverse transcriptase reaction often terminates before reaching the absolute end of the RNA and the cloned cDNA will therefore end 3 of the true transcription start site. In fact, the HK2a 5 RACE performed by two independent groups produced two different 5 ends. Campbell et al. (6) obtained a 5UTR of 39 bp, while Fejes-toth et al. (12) obtained a 5UTR of 190 bp. Furthermore, Campbell et al. was able to obtain a 5 cDNA end corresponding to the 51

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52 splice variant HK2c while Fejes-toth et al. did not. The RNase protection assay used in this dissertation more accurately determines the transcription start sites because it does not rely on primer extension. This chapter describes the construction of RNase protection probes using the analysis of the sequence from clone HK2.1 and the use of the probes in mapping the transcription start sites the for HK2 gene. The second experimental goal in mapping the transcription start sites for the HK gene was to confirm the existence of the HK2c transcript. Our laboratory previously showed that the HK2c transcript and protein were present in both rabbit kidney and colon (6). A second laboratory, however, was unable to detect the HK2c transcript while using similar detection techniques (12). This chapter describes the successful mapping of the transcription start sites for both the rabbit HK2a and HK2c transcripts. Materials and Methods Analysis of Clone HK2.1 Clone HK2.1 was identified by hybridization to the 5 probe (see Chapter 2). A 6.3Kbp XhoI fragment from HK2.1 was subcloned into pBS (creating pDZ10) and completely sequenced. The sequence was analyzed for elements commonly found at eukaryotic promoters. RNase Protection Assay The RNase protection assay was used to map the transcription start sites for HK2a and HK2c. This assay was performed in three steps. First, an antisense radioactive RNA probe was created from a fragment of genomic DNA likely to contain the transcription start sites as well as sequence 5 of the start sites. Second, the

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53 radioactive probe was annealed to the specific mRNA thereby protecting it from RNase digestion. And third, the protected fragment was run on an acrylamide gel along with a sequencing ladder of known size. Once the size of the protected fragment was determined, the transcription start site could be mapped on the genomic DNA sequence. In order to create the RNA probe, a 1.1Kbp HincII fragment likely to contain both transcription start sites was cloned into the pGEM vector (Promega, Inc) creating plasmid pDZ12. The pGEM vector contained the SP6 polymerase promoter that was used in an in vitro transcription reaction to create a radioactive RNA probe. Preliminary experiments showed that one probe could not be used to map both start sites. Therefore, pDZ12 was modified to create one plasmid with the region likely to contain the start site for HK2a (pDZ44) and a second plasmid with the region likely to contain the start site for HK2c (pDZ43). Figure 3-1 depicts the construction of these two plasmids, the expected sizes of the full-length probes, and the predicted size of the protected fragment based on the 5 end of the cDNAs. Plasmid pDZ44 was created by digesting pDZ12 with SacII and SphI, filling in the vector ends with Klenow DNA polymerase, and religating the vector. A 450bp fragment containing the HK2c start site was removed from the vector creating a shorter HK2a RNA probe (Figure 3-1A). The full-length probe was 235 base pairs and the protected fragment was expected to be approximately 87 base pairs. Plasmid pDZ43 was created by digesting pDZ12 with XmnI and HincII. The resulting 168bp fragment contained the HK2c transcription start site and had blunt ends. The fragment was cloned directly into the HincII site of pGEM vector (Figure 3-1B). This plasmid produced a 182 base pair full-length probe and an expected 105 base pair protected fragment.

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54 T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271 FspI 5271 2a 5393 Protected fragment estimated 87bpT7SP6A. T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271B. T7 HincII 4840 SP6 HincII5923 EcoRI 2c 5771 Protected fragmentEstimated 105bp T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271 FspI 5271 2a 5393 Protected fragment estimated 87bpT7SP6A. T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271 FspI 5271 2a 5393 Protected fragment estimated 87bpT7SP6A. T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271B. T7 HincII 4840 SP6 HincII5923 EcoRI 2c 5771 Protected fragmentEstimated 105bp T7 SP6 HincII 4840HincII5923SacII 5480SacII 5565SacII 5919SphI XmnI 57552a 5393ApaI5876 2c 5771FspI 5271B. T7 HincII 4840 SP6 HincII5923 EcoRI 2c 5771 Protected fragmentEstimated 105bp Full length probe 235bp Full length probe 182bp Full length probe 235bp Full length probe 235bp Full length probe 182bp Full length probe 182bp Figure 3-1. Construction of RNase protection probes for HK2a (A) and HK2c (B). Orange represents the cloning region of pGEM vector (Promega, Inc.) Black represents 1.2Kbp fragment of rabbit genomic DNA subcloned into the pGEM vector. Brown represents pertinent restriction sites. Blue represents the 5 ends of the HK2a and HK2c cDNAs. Purple represents the binding sites for T7 and SP6 polymerase. Red represents the expected sizes of the in vitro transcription products. Green represents the sized of the protected fragments estimated based on the end of the cDNAs.

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55 Each plasmid was used in the MAXIscript in vitro transcription kit (Ambion, Inc. Cat. # 1308) in order to create radioactive RNA probes. 1g of the plasmid DNA was added to the in vitro transcription reaction (2l 10X transcription buffer, 1l 10mM each ATP, CTP and GTP, 2.5l 10mCi/ml [32-P] UTP, 2l SP6 polymerase, and dH2O up to 20l). The reaction was incubated at 37C for 10 minutes. 1l of DNaseI was added and the reaction was incubated at 37C for an additional 15 minutes. After incubation, the entire reaction was loaded on a 5% acrylamide gel and run at 300 volts for 30 minutes. The probe fragment was visualized by wrapping the gel in saran wrap and laying down a piece of Polaroid type 57 high-speed film. When developed, a white band appeared on the film at the position of the probe. The film was aligned with the gel, the band was excised, and gel fragment was pressed through a 1mm syringe containing 250l of elution buffer (.5M NH4Acetate, 1mM EDTA, 0.2% SDS). The probe was eluted from the gel fragments by incubation of the mixture at 37C for one hour. The specific activity of the probe was measured on using a Beckman LS3801 scintillation counter. An aliquot of probe containing a specific activity of 8 x 104cpm was used in the ribonuclease protection assay. The RPAIII kit from Ambion, Inc. (Cat # 1414) was used for the ribonuclease protection assay. The probe was co-precipitated with 10g of rabbit colon total RNA by bringing the volume of the probe and RNA to 100l, adding 10l NH4OAc and 250l 100% ethanol, incubating at -20C for 15 minutes and centrifuging at 15,000 rpm for 15 minutes. The pellet was air dried, resuspended in hybridization solution (Ambion, Inc.), heated to 95C for 5 minutes and incubated at 42C overnight. During this time, the probe annealed to its specific mRNA. The next day 1.5l of RNaseA/RNase T1 cocktail

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56 was diluted 1:100 in RNase Digestion buffer (Ambion, Inc.), added to the hybridization reaction and incubated at 37C for one hour. During this incubation, all the single stranded nucleic acids were degraded and only the double stranded protected fragment remained intact. After the incubation, the protected fragment was precipitated by adding 225l of RNase inactivation/precipitation buffer (Ambion, Inc.), incubating the tube at -20C for 15 minutes and centrifuging the tube at 15,000 rpm for 15 minutes. The pellet was air dried and resuspended in 5l of gel loading buffer (95% formamide, .025% xylene cyanol and bromophenol blue, 18mM EDTA, .025% SDS). In order to visualize the protected RNA fragment, the each sample was loaded onto a 6% polyacrylamide sequencing gel along with a sequencing reaction of a known size. The sequencing reaction was carried out using Sequenase 7-deaza-dGTP DNA Sequencing Kit (USB, Cat # 70990) with the control M13 single stranded DNA provided with the kit. The M13 single stranded template (1.0g) was annealed to the primer (0.5pM) by mixing with 2l of sequenase reaction buffer (200mM Tris HCl pH 7.5, 2mM DTT, 0.1mM EDTA, 50% glycerol) and dH2O up to 10l and then heating to 65C for two minutes. After cooling to room temperature, the labeling reaction (annealed DNA, 0.1M DTT, 2l labeling mix (1.5mM 7-deaza-dGTP, 1.5M dCTP, 1.5M dTTP), 0.5l [-32P]dATP, 2l Sequenase polymerase (1U/l)) was incubated at room temperature for five minutes. The reaction was terminated by adding 3.5l of the labeling mixture to each of four pre-warmed termination tubes. All termination tubes contained 80mM of each 7-deaza-dGTP, dCTP, dATP, dTTP, and 50mM NaCl. Additionally, each tube contained 80M of either ddGTP, ddATP, ddTTP, or ddCTP. The termination reaction was incubated at 37C for five minutes. The termination reaction was stopped with 4l

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57 of stop solution (95% dien, 20mM EDTA, 0.05% xylene cyanol). The sequencing reactions were run on a 5% polyacrylamide gel along with the protected fragments from the RNase protection assay. The gel was run at 65 volts for approximately five hours, dried for two hours and exposed to autoradiograph film overnight at -80C. Results Analysis of Clone HK2.1 In order to determine the region most likely to contain the HK2 gene promoter, the sequence of the 6.3Kbp XhoI fragment was analyzed for characteristics common to eukaryotic promoters. First, it was determined that the 3 end of the sequence contains a CpG island. Figure 3-2 is a graph that shows the number of CpG dinucleotides found in 50 base pair windows of the sequence. Most of the sequence contained very few CpG dinucleotides, but there was a clear peak in the number of CpGs at the 3 end. Next, the computer program TFSearch was used to determine if any possible transcription factor binding sites were present along the sequence. The results showed a wide variety of possible binding sites. Appendix B contains the entire search results. Figure 3-3 is a cartoon depicting the possible transcription factor binding sites that seemed most rational based on previously known data about the regulation of the HK2 gene (see discussion). These include a TATA-like element, five SP family member binding sites, a downstream promoter element, a cyclic-AMP response element (CRE) and a steroid response element (SRE). Transcription Start Sites The transcription start sites for HK2a and HK2c were determined using the RNase protection assay. In each case two protected fragments were observed. Figure 3-4

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58 01234567850300550800105013001550180020502300255028003050330035503800405043004550480050505300555058006050Number ofCpG dinucleotidesThe sequence of the 6.3kbp insert of pDZ10 01234567850300550800105013001550180020502300255028003050330035503800405043004550480050505300555058006050Number ofCpG dinucleotidesThe sequence of the 6.3kbp insert of pDZ10 Figure 3-2. CpG dinucleotide analysis of subclone pDZ10. HK2aHK2c CRESP1TATACAATSP1SP1SP1/ SREHK2aHK2c CRESP1TATACAATSP1SP1SP1/ SRE Figure 3-3. Putative transcription factor binding sites determined by TFSearch. The 5 ends of the HK2a and HK2c cDNAs are indicated. Blue represents a CAAT box. Pink represents a sequence with weak homology to the TATA box. Green represents possible binding sites for SP family members. Red represents a cyclic AMP response element. Orange represents a sterol response element. Slashes indicate a break in the sequence of approximately 500bp.

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59 is an example of a polyacrylamide sequencing gel in which the protected fragments for HK2a and HK2c were run next to a sequence of known size (M13 single stranded DNA). By comparison to the known sequence, it was determined that the two protected fragments for HK2a were 94 and 95bp. These fragments correspond to the bases of genomic DNA indicated in Figure 3-4. They are 10 and 11 bases upstream of the cDNA end obtained by Fejes-toth et al. (12), making the 5UTR for HK2a 200 and 201 base pairs. Similarly the HK2c protected fragments were 116 and 118bp and correspond to the bases of genomic DNA indicated in Figure 3-4. They are five and seven bases upstream of the cDNA end obtained by Campbell et al. (6), making the 5 UTR for HK2c 203 and 205 base pairs. For the remainder of this dissertation, the first transcription start site for HK2a was designated as +1 and all other positions are designated relative to that transcription start site. The HK2c transcription start sites were therefore designated +382 and +384. Discussion Mapping the transcription start site was an important step in the characterization of the HK2 gene promoter. The RNase protection assay was used to map the transcription starts sites for HK2a and HK2c. The results of this assay yielded several interesting observations. First and foremost, the protected fragments observed with the HK2c probe confirmed the existence of the transcript in rabbit colon. Second, each probe yielded two protected fragments. Third, the sequence upstream of the two start sites contained a variety of possible core promoter elements. And finally, further upstream, the sequence revealed several possible cis acting regulatory elements.

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60 HK2a GATCRCRA. HK2cGATCRCRB.2a start site:CAGCATTTAACATTTAAGGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGAATCGGC TGCGGAGGTG 2c start site:GGTCAATCAATCCA GACACGCGGG GAAGGAGTTC CAGGGGTCAG CCTCCGCCCTC GCACCTGCGG HK2a GATCRCRHK2a GATCRCRA. HK2cGATCRCRB. HK2cGATCRCR HK2cGATCRCRB.2a start site:CAGCATTTAACATTTAAGGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGAATCGGC TGCGGAGGTG 2a start site:CAGCATTTAACATTTAAGGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGAATCGGC TGCGGAGGTG 2c start site:GGTCAATCAATCCA GACACGCGGG GAAGGAGTTC CAGGGGTCAG CCTCCGCCCTC GCACCTGCGG Figure 3-4. RNase protection assay for HK2a (A) and HK2c (B). GATC represents those nucleotides for the M13 control sequence. RCR represents the protected fragment from the RNase protection assay performed with rabbit colon RNA. A portion of the genomic sequence 5 of the HKa2 gene is shown below each figure. Arrows indicate the position of the transcription start sites. Pink represents putative core promoter elements and blue represents the 5 end of the respective cDNAs.

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61 The RNase protection assay was used to map two transcription start sites for HK2c. Previously, Fejes-Toth et al. (12) questioned the existence of the alternative transcript identified by Campbell et al. (6). Fejes-Toth states that their 5 RACE experiments generated one amplicon corresponding to the 5 end of HK2a. They go on to state that the convergence of their data from that of Campbell may be due to the fact that the 5 RACE of Campbell was carried out in tissue culture cells instead of rabbit tissue and furthermore that Campbell et al. was unable to detect HK2c mRNA in the renal cortex. Although these statements are true, Fejes-Toth failed to recognize that HK2c mRNA was detected in rabbit colon, and HK2c protein was detected in both rabbit renal cortex and colon. These facts alone substantiate that HK2c was not an artifact of working with tissue culture cells. The RNase protection assay performed in this dissertation, however, confirmed the existence of the HK2c transcript at least in rabbit colon. There are several explanations for the fact that each RNase protection probe yielded two protected fragments in very close proximity for both HK2a and HK2c. One possibility is that RNA polymerase had difficulty lying down in an exact position and starting transcription at a precise site because the GC content of a region is high. The genomic sequence surrounding the HK2a and HK2c start sites are 67% and 68% GC respectively (Figure 3-4). Additionally, the putative TATA box upstream of the HK2a start site (see below) had very weak homology to the consensus TATA box. It is therefore likely that the putatitive core promoter elements surrounding the TATA box play a role in transcription initiation and my not precisely position RNA polymerase. A second explanation for the two protected fragments comes from the RNase protection

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62 technique itself. It is possible to observe a fragment slighty longer than the true protected fragment because the RNases used in the assay (A and T1) are endonucleases and may leave bases on the end of a protected fragment. It is also possible to get a protected fragment shorter that the true fragment because the end of the RNA-RNA duplex may occasionally separate. It might be possible to distinguish between these possibilities with a primer extension assay. This assay, however, also has inherent problems with distinguishing one correct start site as it relies on reverse transcription similarly to the 5 RACE (see Chapter 3 Introduction). The putative core promoter elements found upstream of the HK2a transcription start site were a weak TATA box at -31, four SP family binding sites at -102, 154, and -170, a downstream promoter element (DPE) at +17, a TFIIB responsive element at -42, and a CpG island that extends from to +504. Additionally, directly upstream of the HK2c transcription start site a single CAAT box at +351 was observed (31 bases upstream). The fact that this is the only promoter-like element immediately upstream of the HK2c transcription start site suggests that the HK2 gene has one core promoter that is able to direct transcription from the two alternative starts. The CAAT box may be important in directing the initiation of transcription form HK2c. The weak TATA element (CATTTAA) may be serving as a binding site for the general transcription factor TFIID. Additionally, the other core promoter elements found surrounding this element may serve to stabilize the preinitiation complex at the weak element. The function of the TATA-like element is further investigated in Chapter 4 of this dissertation. Further upstream of the transcription start sites, a possible cyclic AMP response element (CRE) at and a possible sterol response element (SRE) at were

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63 identified. There is evidence in vivo that cyclic AMP is increased in hypokalemic rats (24). The CRE could provide a mechanism for upregulating the HK2 gene. Additionally, there is evidence from our laboratory that aldosterone may upregulate the HK2 gene (5). The SRE may provide a binding site for aldosterone and its hormone receptor. In summary, the transcription start sites for HK2a and HK2c were mapped using the RNase protection assay and rabbit colon total RNA. HK2c was confirmed as a transcript in rabbit colon. Upstream of the transcription start site several putative transcription factor binding sites were observed. This work is the first analysis of the rabbit HK2 gene 5 of the transcription start site. The sequence contains many putative transcription factor binding sites. Additionally, determination of the sequence allowed for the design of future experiments regarding the regulation of the HK2 gene.

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CHAPTER 4 REPORTER GENE ANALYSIS OF THE HK2 GENE PROMOTER The third specific aim of this dissertation was to analyze the HK2 gene promoter using a reporter gene system. At the time that this study was proposed, there was in vivo evidence that expression of HK2 gene products was regulated by a variety of cellular conditions including ion concentration, acid-base balance and hormones. There was, however, nothing known about the mechanisms by which the expression was altered. cDNAs for rabbit, rat, guinea pig and human had been identified, but only the human ATP1AL1 gene was known. There have been no studies undertaken to determine promoter elements for the human gene. Recently, the mouse gene was identified and a reporter gene analysis of its 5 flanking region was carried out in mouse inner medullary collecting duct cells (mIMCD3) (52). In their reporter gene experiments, Zhang et al. found that thier longest deletion construct had significant luciferase activity and the deletion of bases -177 had little to no effect on activity. The authors suggest that the core promoter elements as well as positive regulatory elements are located between bases +235 and Although putative regulatory elements were identified in a database search, there were no attempts made to determine the functionality of any specific core promoter or regulatory elements. Additionally, Zhang et al. tested their promoter constructs in outer medullary collecting ducts cells (mOMCD1) and medullary thick ascending limb cells (ST-1). All of the deletion constructs had significant activity in the second collecting duct cell type (OMCD) but little to no activity in the ST-1 cells. The results suggested that either positive regulatory elements are absent in ST-1 cells or 64

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65 that negative regulatory elements, including a closed chromatin structure, are present in ST-1 cells. The experiments described in this chapter used the luciferase reporter gene assay to analyze rabbit HK2 promoter constructs in a rabbit cortical collecting duct cell line (RCCT28A). It was determined that clone HK2.1 contained the HK2 gene promoter (see Chapter 2). Portions of the 6.3Kbp XhoI fragment from HK2.1 were cloned in front of the luciferase reporter gene in the pGL3 basic vector (Promega, Inc.). The constructs were transfected into RCCT28 cells and reporter gene activity was measured. Our goals were to provide the first data regarding the regulation of the rabbit HK2 gene, to identify possible regulatory elements, and to test the functionality of those elements by mutating specific bases within the identified elements. In this way, important regulatory regions would be identified for future studies. Materials and Methods The Promega dual luciferase reporter gene assay (Promega, Inc. Catalog # E1960) was chosen for the promoter analysis. Each promoter construct was cloned in front of the firefly luciferase reporter gene in the pGL3 reporter gene plasmid (Promega, Inc. Catalog # E1751). The plasmids were then transfected into RCCT28A tissue culture cells using the Superfect transfection reagent (Qiagen, Inc. Catalog #301305). The cells were simultaneously transfected with the pRL control plasmid which contained the Renilla luciferase reporter gene driven by the thymidine kinase promoter (Promega, Inc. Catalog # E2241). After 24 hours the cells were lysed and both the firefly luciferase activity and the Renilla luciferase activity were measured using a Berthold Sirius Luminometer. These data were normalized using the Renilla luciferase activity and represented as a

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66 percentage of the highest normalized activity observed. The results identified fragments of DNA 5 of the HK2 transcription start sites that may play a role in the regulation of HK2 gene transcription. Reporter Gene Constructs Four sets of reporter gene plasmids were constructed. The first two sets were promoter deletion plasmids and the second two sets were mutation plasmids. The first set of deletion constructs contained both the 2a and the 2c transcription start sites cloned into the pGL3 reporter vector (Figure 4-1). These constructs had little to no luciferase activity. Therefore, a second set of deletion plasmids that contained only the HK2a start site were created (Figure 4-2). These constructs had varying amounts of activity as expected in a promoter deletion experiment. Based on the data obtained from the deletion analysis, two sets of mutation constructs were created. The first set tested the functionality of two potential repressor elements (Figure 4-3) and the second set tested the functionality of a potential core promoter element (Figure 4-4). The plasmid pDZ10, which contained the .3Kbp XhoI fragment of clone HK2.1, was used to create the first set of deletion constructs (Figure 4-1). A 5259bp StuI/XhoI fragment was cloned into the pGL3 vector (pDZ15). This sequence extended from to +930 and contained the transcription start sites (+1 and +382) and the translation start sites (+200 and +585) for both 2a and HK2c. In order to create a plasmid that contained upstream DNA, but did not produce the HK2a protein, the Quikchange Mutagenesis Kit (Stratagene, Inc. Cat# 200-518-5) was used to mutate the ATG start codon at +200. The primers created for the mutation were DZ24 (5CTCCAGCGCGACACGTGCCAGGTGTGTGAGG3) and DZ25 (5CCTCA

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67 CACACCTGGCACGTGTCGCGCTGGAG3). This plasmid was designated pDZ28. Deletion plasmids were made from pDZ15 and pDZ28 by removing a 3463bp NheI/AatII fragment, filling in the ends using Klenow DNA polymerase, and religating the vector fragment using T4 DNA ligase. These plasmids were designated pDZ29 and pDZ30 respectively. The construction of pDZ29 and pDZ30 inadvertently placed a potential stop codon in the 5 UTR. In order to create a construct that removed the stop codon, two PacI sites were inserted into pDZ28 and pDZ29 by Quikchange with primer sets DZ41/42 (5CAGAGAAAGCTGTTAATTAACTCCGTGGAGCAC CATGCAGC3, 5GCTGCATGGTGCTCCACGGAGTTAATTAACAGCTTTCTCTG3) and DZ43/44 Luc GTG Luc GTG Luc GTG Luc PPATG-876AatII Luc PATG -876AatIIpDZ15pDZ28pDZ29pDZ30pDZ31pDZ32 Luc ATG +920XhoI-4339StuI Luc GTG Luc GTG Luc GTG Luc PPATG-876AatII Luc PATG -876AatIIpDZ15pDZ28pDZ29pDZ30pDZ31pDZ32 Luc ATG +920XhoI-4339StuI Figure 4-1. Deletion constructs containing the HK2a and HK2c transcription start sites. Lines represent the HK2 gene 5 DNA. Base pair numbers indicate the position of the restriction enzyme recognition site with respect to the HK2a transcription start site. ATG represent the HK2a translation start site. GTG represents the mutation created to abolish translation from the HK2a translation start site. P represents the PacI sites inserted by Quikchange. Luc represents the cDNA for the luciferase reporter gene.

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68 (5CAGCTTGGCATTCCGGTACTTTAATTAAAGCCACCATGGAAGACGCC3, 5GGCGTCTTCCATGGTGGCTTTAATTAAAGTACCGGAATGCCAAGCTG3). The 85bp fragment was removed by digestion with PacI, and the vectors were religated creating plasmids pDZ30 and pDZ31 (Figure 4-1). The second set of deletion constructs contained only the transcription start site for HK2a (Figure 4-2). Plasmid pDZ10 was used as a starting plasmid for these constructs as well. A 5459bp XhoI/SacII fragment was cloned into the pGL3 vector (pDZ11). This sequence extended from to +93. A series of plasmids were then constructed by digestion of pDZ11 with NheI for the 5end and a second enzyme for the 3 end. The overhangs on the vector ends were filled in using Klenow DNA polymerse and the vector was religated using T4 DNA ligase. These plasmids digested with the indicated enzymes were designated pDZ16 (StuI), pDZ20 (NdeI), pDZ22 (SpeI), pDZ21 (MscI), pDZ19 (AatII), pDZ18 (EcoRI), and pDZ23 (SmaI). There were no convenient restriction enzymes recognition sequences that could be used to make deletions intermediate to plasmids pDZ21 and pDZ22. Therefore, two plasmids of an intermediate size were created by introducing MluI sites into pDZ22 by Quikchange (Stratagene, Inc.). Primer set DZ31/32 (5GGGTAGGGGATGTCACGCGTGGCCAAATGAAGTTG3, 5CAACTTCATTTGGCCACGCGTGACATCCCCTACCC3) introduced an MluI site at position Plasmid pDZ26 was then created by digestion with MluI and NheI, filling in of the overhangs and religating the remaining vector fragment. Primer set DZ33/34 (5CTTCTCTGTGCCACGCGTGGCCCAAAAGTTGG3, 5CCAACTT TTGG GCCACGCGTGGCACAGAGAAG3) created an MluI site at postion

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69 Digestion and religation of this vector fragment created pDZ27. Furthermore, a plasmid intermediate to pDZ21 and pDZ19 was created by inserting an MluI site at position In this case, the MluI site was introduced as part of a primer set (DZ48 5ACGGCTCCCTGTCCCATAGCCAGAGAATCCC3) used for PCR. The PCR reaction contains10M of primers DZ48 and DZ5 (5CTGCACTCTCAGAGTGA AGG3), 10ng pDZ21, 50l of Qiagen PCR Master Mix (Taq DNA polymerase, Qiagen PCR Buffer with 3mM MgCl2, 400M each dNTP) and dH2O up to a volume of 100l. The PCR conditions were one cycle of 95C for 5 minutes, 30 cycles of 95 for 30 seconds, 68C for 30 seconds, 72C for 30 seconds, and one cycle of 72C for 5 minutes. The PCR reaction was run on a 1% agarose gel and visualized with ethidium bromide. The reaction produced a single 700bp band which was gel extracted (Qiagen, Inc.) and cloned into the TOPO cloning vector (Invitrogen, Inc.). In order to create the deletion construct, the TOPO clone was digested with EcoRI and MluI and the overhangs were filled in. The blunt ended fragment was then cloned into the SmaI site of pDZ23 in order to create plasmid pDZ36. The shortest construct (pDZ25) was created by performing Quikchange on plasmid pDZ18. Primer set DZ35A/35B (5CGCGCAGCATTTAACGC GTACAC CACCTCCCC3, 5GGGGAGGTGGTGTACGCGTTAAATGCTGCGCG3) inserted an MluI site at Digestion of the plasmid with MluI and NheI, filling in the overhangs and religation of the vector fragment completed pDZ25. One final construct was made to ensure that the size of the deletion plasmid was not having and effect on reporter gene activity. A 4.2Kbp HindIII fragment of non-specific DNA (from E. coli F1F0 plasmid pAES9) was ligated into the HindIII site of pDZ18 located at This

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70 fragment made the construct pDZ49 approximately the same size as the largest construct, pDZ11. The sizes of all of these deletion constructs are indicated in Figure 4-2. Luc -4339StuIpDZ16 Luc -3299NdeIpDZ20 Luc -2464MluIpDZ26 Luc -1241MluIpDZ36 Luc-631EcoRIpDZ18 Luc -295SmaIpDZ23 Luc -26MluIpDZ25 Luc -631EcoRIpDZ49 Non-specific DNA +93SacII-5367XhoI Luc pDZ11 Luc -2883SpeIpDZ22 Luc -1916MluIpDZ27 Luc -1485MscIpDZ21 Luc-AatIIpDZ19876AatII Luc -4339StuIpDZ16 Luc -3299NdeIpDZ20 Luc -2464MluIpDZ26 Luc -1241MluIpDZ36 Luc-631EcoRIpDZ18 Luc -295SmaIpDZ23 Luc -26MluIpDZ25 Luc -26MluIpDZ25 Luc -631EcoRIpDZ49 Non-specific DNA +93SacII-5367XhoI Luc pDZ11 +93SacII-5367XhoI Luc pDZ11 Luc -2883SpeIpDZ22 Luc -1916MluIpDZ27 Luc -1485MscIpDZ21 Luc -1485MscIpDZ21 Luc-AatIIpDZ19876AatII Figure 4-2. HK2a deletion constructs. Lines represent the HK2 gene 5 DNA inserted into the pGL3 reporter gene plasmid. Base pair numbers indicate the position of the restriction enzyme recognition site with respect to the HK2a transcription start site. Luc represents the cDNA for the luciferase reporter gene.

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71 The third set of luciferase reporter gene constructs were created to test the functionality of two putative repressor elements identified by the deletion analysis (see results). Three mutations were made using Quikchange. Primer set DZ55/56 (5GCAGCACCACGCAGCCCGGGACCATTAATTAAAAACGCTCACTGACCCAG ACCCTCC3, 5GGAGGGTCTGGGTCAGTGAGCGTTTTTAATTAATGGTCCC GGCTGCGTGGTGCTGC3) mutated the element located at Primer set DZ59/60 (5GCCCTCCACGCTCACTGACCATTTAAATACATCCCCACCCC TCTCTCC3, 5GGAGAGAGGGGTGGGGATGTATTTAAATGGTCAGTGAGCG TGGAGGGC3) mutated the element at The two putative elements were close together, so a third primer set, DZ63/64 (5GGACCATTAATTAAAAACGCTCACTGACCATTTAAA TACATCCCCACCCCTCCTCTCC3, 5GGAGAGAGGGGTGGGGATGTATTTAAA TGGTCAGTGAGCG TTTTTAATTAATGGTCC3) was used to mutate both elements. The fourth set of luciferase reporter gene constructs were created to test the functionality of the TATA-like element found at (see results). The second smallest deletion construct that contained the element (pDZ18) and the smallest deletion construct that did not contain the element (pDZ25) were used as positive controls for this set of experiments. Three mutation constructs were made using Quikchange with three primer sets. Primer set DZ53/54 (5GCGGGGCGCGCAGCGGCCGCGGCGGACACCACC3, 5GGTGGTGTCCGCCGCGGCCGCTGCGCGCCCCGC3) created a GC box in place of the TATA element (pDZ34). Primer set DZ86/87 (5GCGGGGCGCGCAGCGAT CGAGGCGGACACCACC3, 5GGTGGTGTCCGCCTCGATCGCTGCGCG CCCCGC3) created a random sequence in place of the TATA element (pDZ48). And finally, primer set DZ57/58 (5GCGGGGCGCGCATATAAAAGGCGGACACCACC3,

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72 5GGTGGTGTCCGCC TTTTATATGCGCGCCCCGC3) created a consensus TATA sequence at the same location (pDZ39). Tissue Culture Cells RCCT28A cells were chosen for the majority of the reporter gene analyses. These cells were isolated from rabbit cortical collecting duct and transformed with the SV40 virus by Arend et al. (2). They were characterized by their ability to bind several antibodies and by their response to specific hormones. It was determined that RCCT28A cells maintain characteristics most similar to the intercalated cells of the cortical collecting duct. Furthermore, Campbell et al. (6) showed that these cells express the HK2 mRNAs and proteins. RCCT28A cells were therefore known to contain factors required for expression of the reporter gene driven by the HK2 gene promoter. In addition to RCCT28A cells, three other cell types were used for one of the reporter gene assays. The activity of several of the deletion constructs were tested in HIG-82 cells, HEK293 cells and HK2 cells. HIG-82 cells were established by spontaneous transformation of fibroblasts from rabbit soft tissue lining the knee joint (15). They are not likely to express the HK2 gene products. HEK293 cells are human embryonic kidney cells transformed from sheared human adenovirus type 5 (42). They display general characteristics of renal tubular cells, but it is not possible to relate their characteristics to a specific renal segment. Grishin et al. (16) used these cells for the functional expression of cloned ATP1AL1 cDNAs. Antibodies raised against a portion of the ATP1AL1 protien did not react with untransfected HEK293 cells suggesting that these cells do not express the HK2 gene product. HK2 cells are human adult proximal tubule epithelial cells immortalized by transduction with human papiloma virus (38). It

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73 has been shown that rabbit proximal tubule cells do not express the HK2 gene products (12). It is therefore unlikely that HK2 cells express the ATP1AL1 gene product. Transfection and Reporter Gene Activity RCCT28A cells were grown in a 24 well plate format with each well containing 1mlL of media (Dulbeccos Modified Eagle Medium F12 (DMEM-F12), 10% Fetal Bovine Serum (FBS)) until they reached approximately 70% confluency (usually over one night). For each transfection, 250pmoles of the deletion construct to be tested, non-specific DNA to 1g and 0.2g of pRL control plasmid were mixed with 140l DMEM and 40l of Superfect reagent (Qiagen, Inc.) and incubated at room temperature for 10 minutes to allow for complex formation between the transfection reagent and the DNA. During this incubation, the RCCT28A cells were washed two times in PBS. The complexed solution was brought up to 400l with DMEM-F12 plus FBS and 200l was pipetted on top of each of two duplicate wells. The transfection proceeded at 37C for 2 hours. The transfection reagents were washed off the cells with PBS and 1mL of fresh media was added to each well. After 24 hours at 37C, the media was removed and the cells were lysed by adding 100l of lysis buffer (Promega, Inc.). Each well was scraped with a 20l pipette tip to facilitate lysis. Ten microliters of the lysed cells were added to 100l of firefly luciferase substrate and the raw firefly luciferase activity was measured for 10 seconds. One hundred microliters of quenching buffer plus Renilla luciferase substrate was added and the raw Renilla luciferase activity was measured for 10 seconds. Each construct was transfected into RCCT28A cells at least three times and in duplicate each time in order to obtain statistically relevant data. Each round of transfections included the plasmid that initially had the most activity (pDZ18) and the plasmid with the

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74 least activity (pGL3 empty vector). All the constructs in one transfection were normalized to the Renilla control data (see below). The pDZ18 activity was set to 100% and data for the other constructs were calculated as a value relative to 100%. Normalization of the Luciferase Data Table 4-1 is an example of the calculations required for normalization of the luciferase data. For each set of transfections, the raw firefly reading and the raw Renilla reading are taken directly from the luminometer (Column one and Column two respectively). The first Renilla reading was divided into each of the subsequent Renilla readings to create a normalizing factor for each sample (Column three). Each firefly reading was multiplied by its normalizing factor to obtain the normalized firefly reading (Column four). The average of the two readings for the plasmid that initially gave the highest activity reading (pDZ18) was divided into the normalized firefly reading for each sample and multiplied by 100 to obtain a percentage of the highest activity (Column five). The background activity observed for the empty vector (pGL3) was subtracted out (Column six) and the activity for the plasmid with the highest activity (pDZ18) was reset to 100% (Column seven). Each plasmid was transfected at least three time and in duplicate. The average percent relative activity for each plasmid was graphed and error bars were added to indicate plus and minus the standard of error. Results HK2a and HK2c Reporter Gene Activity Figure 4-3 is a graph of relative luciferase activity for the constructs that contain both HK2a and HK2c transcription and translation start sites. All constructs were transfected into RCCT28A cells in duplicate and at least three times. These data were normalized using the Renilla luciferase internal control. Plasmid pDZ18 contains only

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75 Table 4-1. Example of the normalization of raw luciferase data Vector Raw firefly Raw Renilla Normalizing factora Normalized fireflyb % activityc Subtract pGL3 Reset to 100% pGL3 616 13166 1.00 616 7.66 -0.45 -.050 623 11912 1.11 689 8.56 0.45 0.50 pDZ11 4540 34459 0.38 1735 21.56 13.45 14.66 3887 26942 0.49 1899 23.61 15.50 16.90 pDZ18 12923 20904 0.63 8139 101.16 93.05 101.42 10513 17404 0.76 7953 98.84 90.73 98.90 a) Normalizing factor equals first renilla reading/each raw renilla reading b) Normalized firefly equals raw firefly normalizing factor c) % activity equal average for(normalized firefly/pDZ18)*100 the HK2a transcription start site and contained the most firefly activity of all the constructs created. This reading was set to 100% activity and the relative activity for the remaining constructs was calculated. The original construct containing the HK2a and HK2c transcription and translation start sites (pDZ15) had no activity. Since the ATG start codons for HK2a and HK2c were part of this construct, it seemed possible that the HK2 amino acids added to the N-terminus of the luciferase protein could affect luciferase activity. Therefore, Quikchange was used to create a plasmid in which the ATG start codon for HK2a was mutated (pDZ28). This plasmid also had no luciferase activity. At this point, reporter gene activity data from the second set of constructs, those that only contained the HK2a transcription start site, had shown that shorter plasmids contained more activity (see below). Therefore, plasmids pDZ15 and pDZ28 were digested with AatII and NheI, the ends were filled in, and the vectors were religated. These two plasmids, pDZ30 and pDZ31 still had no activity. A portion of the sequence from plasmid pDZ30 is shown in Figure 4-4A. Analysis of the two alternative mRNA

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76 transcripts that would be produced by this sequence (Figures 4-4B and C) revealed a potential stop codon that was introduced by the ligation of the HK2 gene fragment into the pGL3 vector (shown in red). This stop codon would terminate translation from both -20020406080100 pDZ18pDZ15pDZ28pDZ29pDZ30pDZ31pDZ32% Luciferase Activity -20020406080100 pDZ18pDZ15pDZ28pDZ29pDZ30pDZ31pDZ32% Luciferase Activity -20020406080100 pDZ18pDZ15pDZ28pDZ29pDZ30pDZ31pDZ32% Luciferase Activity Percent of Relative Luciferase Activity Figure 4-3. Percent activity for reporter gene constructs that contain the HK2a and HK2c transcription and translation start sites. Error bars respresent the standard of error. the HK2a translation start and the HK2c translation start before reaching the ATG start codon for the luciferase gene. A new set of constructs were created to fix this problem and to remove some of the HK2 amino acids that were added to the N-terminus of the luciferase protein. PacI sites were introduced into pDZ29 and pDZ30 using Quikchange (Figure 4-1). Subsequent digestion with PacI and religation of the vector resulted in plasmids pDZ31 and pDZ32. The sequence for pDZ31 and the mRNA transcripts produced by this plasmid are shown in Figure 4-5. Although the potential stop codon was successfully removed, plasmid pDZ31 had no luciferase activity suggesting that the

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77 A. -44 GCGGGGCGCG CAGCATTTAA GGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGATCGGC 17 TGCGGAGGTG CGCGCAGGGC CCGCGTGGCT GTGGGTACCT CCTTCGCCAG CACCGTCGCC 77 ACTACCAACG CCGCCACCGC GGGACCCTAC CCCGCATCGG TCGCCGCCGC CACCGCAGGT 137 CCCACGACCC CTCCTGCCCT CCGCGCCCCC TGCCCGCCGA CCCGCGGCGC CTCCAGCGCG 197 ACATGCGCCA GGTGTGTGAG GAAGTGACGC GGTGCGGACT GGAGAGAAGT GCGGGAAAGG 257 GTGAAGGGCT CCGTCCGGGG GTCTTTACTC TGCAACCCTG TTCCAGCCGC CGAGCACCCG 317 TGTGTCACTC GGGAACTGGC TGGGTAAAGA GGTCAATCCA GACACGCGGG GAAGGAGTTC 377 CAGGGGTCAG CTCCGCCCTC GCACCTGCGG GCTCGGATTC GGAGAAAAGT GCTAGACTGG 437 AGCTACACGT ATGCGTAGCG GTCTGGAAAA TGCCCCAGGC TCGGGTCTGA GGGGCCCAAG 497 TCTATGCACC GCTGGTGTGA CCCCGCAGGG CAACCCCGCG GTTAACTTCT CTCCTGCCCA 557 CCCCTAGAGG TGTCTTCCTG GGAAGACGAT GGCAGGCGGT GCCCACCGAG CCGACCGTGC 617 AACAGGGGAA GAGAGGAAGG AGGGAGGTGG GAGGTGGCGC GCTCCCCACA GCCCTTCCCC 677 TCCTGGCCCG CGAGGGTGTC CGGTCCCACT CAAGGCAGCT GCGCAGAGCC TGTGCAGAAA 737 TACCACCTGG GGCCGGTATT GCACTCTGCT TCTCTTTCAG A797 CGTGGAGCAC CATGCAGCTA CAGATATCAA GAAGAAGGAG GGGCGAGATG GCAAGAAAGA 857 CAATGACTTG GAACTCAAAA GGAATCAGCA GAAAGAGGAG CTTAAGAAAG AACTTGATCC 917 TCGAGATCTG CGATCTAAGT AAGCTTGGCA TTCCGGTACT AG CCACCATGGA 977 AGACGCCAAA AACATAAAG GAAAGCTGG AAATTTACTC GTTGGTAA B. A TG CGC CAG AGA AAG CTG GAA ATT TAC TCC GTG GAG CAC CAT GCA GCT ACA GAT ATC AAG AAG AAG GAG GGG CGA GAT GGC AAG AAA GAC AAT GAC TTG GAA CTC AAA AGG AAT CAG CAG AAA GAG GAG CTT AAG AAA GAA CTT GAT CCT CGA GAT CTG CGA TCT AAG GCT TGG CAT TCC GGT ACT GTT GGT AAA GCC ACC ATG GAG ACG CCA TAA TAA A AA ACA TAA AG C. A TG GCA GGC GGT GCC CAC CGA GCC GAC CGT GCA ACA GGG GAA GAG AGG AAG GAG GGA GGT GGG AGG TGG CGC GCT CCC CAC AGC CCT TCC CCT CCT GGC CCG CGA GGG TGT CCG GTC CCA CTC AAG GCA GCT GCG CAG AGC CTG TGC AGA AAT ACC ACC TGG GGC CGG TAT TGC ACT CTG CTT CTC TTT CAG AGA AAG CTG GAA ATT TAC TCC GTG GAG CAC CAT GCA GCT ACA GAT ATC AAG AAG AAG GAG GGG CGA GAT GGC AAG AAA GAC AAT GAC TTG GAA CTC AAA AGG AAT CAG CAG AAA GAG GAG CTT AAG AAA GAA CTT GAT CCT CGA GAT CTG CGA TCT AAG GCT TGG CAT TCC GGT ACT GTT GGT A AA GCC ACC ATG GAA GAC GCC AAA AAC ATA AAG Figure 4-4. Sequence analysis of plasmid pDZ15. (A) Sequence of the plasmid. Black indicates HK2 sequence while green indicates pGL3 sequence. Dark purple represents the transcription start sites and light purple the translation start site for HK2a. Dark pink represents the transcription start site and dark pink the translation start site for HK2c. Blue represents the translation start site for the luciferase gene. Orange indicates the two sets of bases mutated to PacI sites for future experiments (see text). (B) mRNA transcribed by initiation from the HK2a start site. Red indicates stop codon introduced before the luciferase translation start site. (C) mRNA transcribed by initiation from the HK2c construct.

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78 A. -44 GCGGGGCGCG CAGCATTTAA GGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGATCGGC 17 TGCGGAGGTG CGCGCAGGGC CCGCGTGGCT GTGGGTACCT CCTTCGCCAG CACCGTCGCC 77 ACTACCAACG CCGCCACCGC GGGACCCTAC CCCGCATCGG TCGCCGCCGC CACCGCAGGT 137 CCCACGACCC CTCCTGCCCT CCGCGCCCCC TGCCCGCCGA CCCGCGGCGC CTCCAGCGCG 197 ACATGCGCCA GGTGTGTGAG GAAGTGACGC GGTGCGGACT GGAGAGAAGT GCGGGAAAGG 257 GTGAAGGGCT CCGTCCGGGG GTCTTTACTC TGCAACCCTG TTCCAGCCGC CGAGCACCCG 317 TGTGTCACTC GGGAACTGGC TGGGTAAAGA GGTCAATCCA GACACGCGGG GAAGGAGTTC 377 CAGGGGTCAG CTCCGCCCTC GCACCTGCGG GCTCGGATTC GGAGAAAAGT GCTAGACTGG 437 AGCTACACGT ATGCGTAGCG GTCTGGAAAA TGCCCCAGGC TCGGGTCTGA GGGGCCCAAG 497 TCTATGCACC GCTGGTGTGA CCCCGCAGGG CAACCCCGCG GTTAACTTCT CTCCTGCCCA 557 CCCCTAGAGG TGTCTTCCTG GGAAGACGAT GGCAGGCGGT GCCCACCGAG CCGACCGTGC 617 AACAGGGGAA GAGAGGAAGG AGGGAGGTGG GAGGTGGCGC GCTCCCCACA GCCCTTCCCC 677 TCCTGGCCCG CGAGGGTGTC CGGTCCCACT CAAGGCAGCT GCGCAGAGCC TGTGCAGAAA 737 TACCACCTGG GGCCGGTATT GCACTCTGCT TCTCTTTCAG AGAAAGCTGAG 797 CCACCATGGA AGACGCCAAA AACATAAAG T TAATTAA TTAATTAA TTA ATTAA B. A TG CGC CAG AGA AAG CTG A GCC ACC ATG GAA GAC GCC AAA AAC ATA A AG C. A TG GCA GGC GGT GCC CAC CGA GCC GAC CGT GCA ACA GGG GAA GAG AGG AAG GAG GGA GGT GGG AGG TGG CGC GCT CCC CAC AGC CCT TCC CCT CCT GGC CCG CGA GGG TGT CCG GTC CCA CTC AAG GCA GCT GCG CAG AGC CTG TGC AGA AAT ACC ACC TGG GGC CGG TAT TGC ACT CTG CTT CTC TTT CAG AGA AAG CTG A GCC ACC A TG GAA GAC GCC AAA AAC ATA AAG Figure 4-5. Sequence analysis of plasmid pDZ31. (A) Sequence of the plasmid. Black indicates HK2 sequence while green indicates pGL3 sequence. Dark purple represents the transcription start sites and light purple the translation start site for HK2a. Dark pink represents the transcription start site and dark pink the translation start site for HK2c. Blue represents the translation start site for the luciferase gene. Orange indicates the remaining PacI site after digestion and religation. (B) mRNA transcribed by initiation from the HK2a start site. (C) mRNA transcribed by initiation from the HK2c construct. HK2 amino acids added to the N-terminus of the luciferase protein were inactivating the enzyme. Plasmid pDZ32, which has a mutation at the HK2a ATG translation start was the first plasmid in this series to have luciferase activity. The amount of activity, however, was about 50% of that seen for the similar sized plasmid that contains only the

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79 HK2a transcription start site (pDZ19). At this point in the study, it became clear that the second set of deletion constructs, those that contain only the HK2a transcription start site had significant reporter gene activity and would produce the desired deletion data (see below). Therefore, attempts to restore luciferase activity to these constructs were terminated. HK2a Reporter Gene Activity Figure 4-6 is a graph of the relative reporter gene activity for the all of the plasmids in the second set of deletion constructs. These plasmids contain the HK2a transcription start site and varying amounts of 5 DNA. These constructs were transfected into RCCT28A cells and after 24 hours, the luciferase activity was measured. Once again the normalized activity from plasmid pDZ18 was set to 100% and the percent activity for the remaining constructs was calculated. The clear result was that progressively shorter promoter fragments contained progressively more luciferase activity. The one exception was that the shortest plasmid (pDZ25) contained activity similar to background (pGL3). It is notable that the luciferase activity increased gradually as the plasmid length decreased. In order to eliminate the possibility that the size of the plasmid was affecting luciferase activity, plasmid pDZ49 was created. This plasmid contained a random fragment of DNA placed in front of the pDZ18 fragment resulting in a plasmid the length of pDZ11. The luciferase activity of this construct was similar to pDZ18, not pDZ11, suggesting that plasmid size does not affect reporter gene activity. A one way ANOVA analysis was performed on the deletion data. The red stars in Figure 4-6 indicate plasmids with a statistically significant difference in the level of

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80 020406080100120pDZ25pDZ23pDZ18pDZ19pDZ36pDZ21pDZ27pDZ26pDZ22pDZ20pDZ16pDZ11 020406080100120pDZ25pDZ23pDZ18pDZ19pDZ36pDZ21pDZ27pDZ26pDZ22pDZ20pDZ16pDZ11 Figure 4-6. Percent activity for constructs that contain the HK2a transcription start site. Percent of Relative Luciferase Activit y

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81 luciferase activity when compared to the preceding plasmid. The differences between pDZ19 and pDZ18, and pDZ23 and pDZ25 were the most significant and therefore their sequences were analyzed for possible regulatory elements. Putative Repressor Mutations The difference in luciferase activity between plasmids pDZ19 and pDZ18 was examined. The sequence difference between these two plasmids came from a 245bp deletion that extended from bases to A transcription factor binding site database (TFSEARCH) analysis did not reveal any known repressor binding sites in this region. The human ATPAL1 gene was the only HK2 gene that also had known sequence in this 5 region. An alignment of the rabbit sequence and the human sequence in this region did show a short sequence that was well conserved between the two species (Figure 4-7). Quikchange mutagenesis was used to randomize the sequence at -713-700-680CCAGAGCCCT--CCAAHumanRabbit -713-700-680CCAGAGCCCT--CCAAHumanRabbit CAGA--CCTCTCCACCAGA-CCCT--CCA CAGA--CCTCTCCACCAGA-CCCT--CCA Figure 4-7. Alignment of possible repressor sequences from human ATP1AL1 and rabbit HK2 genes. (pDZ38), the sequence at (pDZ40), and both sequences together (pDZ41). These constructs were transfected into RCCT28A cells along with pDZ18 and pDZ19. After 24 hours the luciferase activity was measured. The activity for pDZ18 was set to 100% and the relative activity for the rest of the plasmids was calculated. Figure 4-8 is a graph of these data. Although there was a small increase in activity over that of pDZ19, it did not

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82 appear as though the conserved sequence that was analyzed has a major effect on HK2 repression. 020406080100120 Luc Luc Luc Luc Percent Luciferase Activity Luc 020406080100120 Luc Luc Luc Luc Luc Luc Luc Percent Luciferase Activity Luc Luc Percent of Relative Luciferase Activity XX XX XX XX Figure 4-8. Percent luciferase activity in repressor mutation constructs. Red X indicates the position of the putative repressor mutation. Putative TATA Element Mutations The dramatic drop in luciferase activity between plasmids pDZ23 and pDZ25 suggested that the core promoter for the HK2 gene was deleted. An alignment of the 269 bases deleted in pDZ25 with the sequences known for the human ATP1AL1 gene, the mouse HK2 gene, and the rat HK2 gene revealed a great deal of homology as indicated by the stars at the bottom of Figure 4-9. In particular, the CATTTAA (red lettering) element near the rabbit transcription start site was completely conserved in human, rat and mouse. In order to test if this element was functioning as a TATA element, pDZ18 was mutated in several ways. These data are represented graphically in Figure 4-10. The mutation constructs that destroyed the element (pDZ34 and pDZ49) show a drop, although not a complete loss, of reporter gene activity. It therefore appears

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83 as though the element is necessary for full activity, but the surrounding bases are capable of initiating an intermediate level of transcription despite the mutation to the TATA box. The mutation that created a consensus TATA box (pDZ39) had activity that was not significantly different from the wild type element, again suggesting that the native element was functioning well as a consensus TATA box. rabbit ATCCTCGGTTTCCCAGCCCCCTGGGGTGTCTGCAG-GCCGGGCTACTTGCACAGCAGCAG human GCCGGCGGGTTTCCTACCCTCCGAGGCGTCCGCTG-GCC--------TGCGCCCTGGCGG rat GCACTCGGGTTTCCTATCC-CAGGGGCGTCCCTGGTGCAGTG--AACTGTGTCGCCCCAG mouse GTCCTCGGGTTTCCTACCC-CAGGGGCGTCCCCAGTGCAGTG--AGCTGCGTCGCCCCAG *** ** ** ** * ** *** ** ** * rabbit GTGCGTAGGCGGGGCGCGCAGGCGGCTGG human GGACGTGGGCGGGGCGGGCGGCCCGGTGCAGCGGCTGG rat GTACGTGGGCGGAGCTCGCAGACAGTGGCTGG mouse GTACGTGGGCGGAGCGCGCGG *** ***** ** ** rabbit CGATCGGCTGCGGAGGTGCGCGCAGGGCCCGCGTGGCT human CGATCGGCCGCGGAGGTGCGTGCAGGGCCCGCGCCGCC rat TGATCGGCCGCGGAG-TGCTTG-TGCTTCCGG-TTGGC mouse CGGTCGGCCGCGGAGGTGCGTGCTGGGTCCGGGTTGGT ***** ****** *** * *** CATTTAAGGCGGACACCACCTCCCCTGGGCACATTTAAGGCTGGTGCCACCTCCATTTAAGGCGCGCTCCACCTCCCAGGGCATTTAAGGCGCGCTCCACCTCCCCAGGACAGCGGCTGG ********** ********* *** ****** Figure 4-9. Alignment of the rabbit, human, rat and mouse DNA sequence upstream of the HK2 transcription start sites. Stars represent completely conserved bases. Red indicates the completely conserved TATA-like element. Dark purple represents the transcription start sites for rabbit HK2a determined in Chapter 2, human ATP1AL1 (45), rat HK2a (26) and mouse HK2 (52). Effect of Cell Type on Reporter Gene Activity In order to test the effect of cell type on reporter gene activity, three deletion constructs were transfected into four cell types. Plasmids pDZ18, pDZ19 and pDZ11 were transfected into HEK293, HK2, RCCT28A and HIG-83 cells. Within each cell line, the normalized activity for pDZ18 was set at 100% and the other two constructs are

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84 Luc Luc XX XX XX 020406080100120140160 Luc Luc LucPercent Luciferase Activity Luc Luc 020406080100120140160 Luc Luc Luc Luc Luc 020406080100120140160 020406080100120140160 Luc Luc LucPercent Luciferase Activity Percent of Relative Luciferase Activity Figure 4-10. Percent activity in reporter gene constructs with mutations in the CATTTAA element. Red X represents mutations that randomize the CATTTAA element. Green triangle represents a mutation that converts the CATTTAA element into a consensus TATA element. represented relative to 100%. Figure 4-11 is a graph representing the results of the transfections. In the three adult cell lines (RCCT28A, HIG-82, HK2), the expression pattern for the three constructs was similar. The longest construct was repressed and the shorter constructs had increasing amounts of luciferase activity. The embryonic cell line (HEK293), however, had a different pattern of expression. The most dramatic difference was with the longest construct. Plasmid pDZ11 was repressed in all three adult cell lines, but was not repressed in this embryonic cell line. The activity was most similar to pDZ19 suggesting that the embryonic cell line did not contain some or all of the factors required for HK2 gene repression.

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85 0204060801001234 pGL3pDZ11pDZ19pDZ18 0204060801001234 pGL3pDZ11pDZ19pDZ18 0204060801001234 pGL3pDZ11pDZ19pDZ18 Percent of Relative Luciferase Activity Figure 4-11. Reporter gene activity in various cell types. Plasmid constructs are as indicated. Light pink represents HEK293 cells. Dark pink represents HK2 Cells. Light orange represents HIG-82 cells. Dark orange represents RCCT28A cells. Discussion The use of the luciferase reporter gene assay to evaluate the region of DNA 5 of the HK2 gene provided the first data regarding the regulation of the rabbit HK2 gene. Four sets of reporter gene constructs and four tissue culture cell types were used in this assay and each provided important information. The first set of constructs contained the transcription and translation start sites for HK2a and HK2c cloned in front of the luciferase reporter gene. These constructs had little to no reporter gene activity. The removal of the stop codon introduced during plasmid construction, and the mutation of the ATG start codon for HK2a resulted in one construct with significant luciferase activity (pDZ32). This activity, however, was still low when compared to the construct with same deletion (pDZ19) in the series that

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86 contained only the HK2a start site (60% for pDZ19 vs. 30% for pDZ32). This result was difficult to interpret. The mutation of the HK2a ATG should have removed the amino acids that were added to the N-terminus of the luciferase protein. The luciferase activity, however, was not restored to the expected level suggesting that there were negative regulatory elements that effect the HK2a transcription in the downstream region that was eliminated when the second set of constructs were made (+93 to +920). Plasmid pDZ31 removed the stop codon that would terminate translation from the HK2c transcript. This construct, however, had no luciferase activity. There are several possible explanations for the lack of activity. First, the 72 amino acids added to the N-terminus of the luciferase protein could cause a loss of function (Figure 4-5C). Second, the splicing machinery may not recognize the reporter gene construct and fail to produce the alternative HK2c transcript. And finally, the HK2c transcript may be regulated, and without the proper signal, it may completely repressed. As it would be difficult to distinguish between these possibilities, and more importantly, the second set of deletion constructs were producing results, this line of investigation was therefore abandoned. The second set of reporter gene constructs provided the most information regarding the regulation of the HK2 gene. The 3 end of these were shorter, ending at +92, and contained only the transcription start site for HK2a. Interestingly, the longest construct (pDZ11) contained the least reporter gene activity and progressively shorter constructs showed a gradual increase in reporter gene activity. These results suggested that under the conditions of the luciferase assay, the HK2 gene promoter was repressed and significant reporter gene activity was seen only after deleting the DNA responsible for binding repressor elements. The increase in luciferase activity, however, appeared as

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87 a gradual increase rather than as distinct jumps in activity that would be expected when repressor elements are deleted. It seemed possible, therefore, that the decrease in size of the constructs, rather than the removal of repressor elements, caused the change in luciferase activity. Plasmid pDZ49, however, eliminated this possibility. This construct was made by taking the plasmid with the most activity, pDZ18, and inserting a fragment of E. coli DNA into a HindIII site upstream of the putative promoter element. The result was a plasmid the size of pDZ11, but devoid of any additional eukaryotic transcription factors. Plasmid pDZ49 has luciferase activity similar to pDZ18, not pDZ11, meaning that the size of the plasmid was not having an effect on luciferase activity. Although the changes in luciferase activity were smaller than one might expect, the one way ANOVA analysis of these data did reveal several deletions that caused a statistically significant change in reporter gene activity. There are clear increases in luciferase activity between constructs pDZ20 and pDZ22, pDZ21 and pDZ36, and pDZ19 and pDZ18. Additionally, there is a dramatic decrease in luciferase activity between plasmids pDZ23 and pDZ25 (Figure 4-6). These changes in activity provided the basis for the construction of the mutation plasmids that are discussed below. The fact that the HK2 gene was repressed in these assays was a surprise because RCCT28A cells have been shown to express the HK2 mRNAs and proteins (6). Additionally, Zhang et al. (52) performed a promoter deletion analysis of the mouse HK2 gene and did not observe repression with their longest constructs. The discrepancy between the results of our study and the study of Zhang et al. may be explained by the differences in the cell types. The RCCT28A cells were derived from the cortical collecting duct while the cells used by Zhang et al. were derived from medullary collecting duct cells. To date, it has been unclear whether or not

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88 the cortical collecting duct normally expresses the HK2 gene products (see introduction) whereas the medullary collecting duct has been consistently shown to express the HK2 gene products. The reporter gene assay performed in our study suggested that there may be certain cellular conditions necessary for HK2 gene expression in the cortical collecting duct. This may, in part, explain the discrepancies present in the literature. One likely explanation for the repression observed in the deletion analysis is the condition under which the RCCT28A cells were grown for the assay. The transfection of the RCCT28A cells necessitated that they were grown to 70% confluency. At this level of confluency the RCCT28A cells are undifferentiated. It was hypothesized that the HK2 gene was repressed in this state and then when the cells reach 100% confluency and differentiate, they would begin to express the HK2 gene products. This hypothesis resulted in the experiments that are discussed in Chapter 5 of this dissertation. The third set of reporter gene constructs were designed to test the functionality of putative repressor elements identified in the DNA fragment between plasmids pDZ19 and pDZ18. The increase in luciferase activity between these two plasmids was the largest observed (40%) and seemed a good point to begin a search for novel repressor elements. A transcription factor database search did not identify any known binding sites for transcription factors. An alignment of the rabbit DNA sequence in this region with that of the human ATP1AL1 gene, however, did identify one sequence in human that was well conserved to two sequences in rabbit (Figure 4-7). Unfortunately, random mutation of these sequences both individually and combined did not have a significant effect on luciferase activity (Figure 4-8) suggesting that these sequences are not directly involved

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89 in the repression of the HK2 gene. In the future, it may be possible to make smaller deletions in this region and identify a short sequence that does play a role in repression. This information could be used to design new experiments, for example, gel shift assays or yeast two hybrid assays, that are aimed at the identification of novel repressor elements. The fourth set of reporter gene constructs were designed to test the functionality of the CATTTAA element upstream of the HK2a transcription start site. Two different mutations, both of which changed the element to random sequence, caused a drop in luciferase activity of greater than 50%. The mutation of the CATTTAA to a consensus TATA element (TTTTATAT) had very little, if any, effect. These data suggest that the CATTTAA element plays a necessary role in the initiation of transcription at the HK2a start site. There are, however, other sequences surrounding the element that also appear to be important for full activity. Additional core promoter elements include an initiator (Inr), a downstream promoter element (DPE), a TFIIB recognition element (BRE) and SP family member binding sites (GC boxes)(see introduction). The sequence surrounding the CATTTAA element contains DNA that has homology to all of these elements except for the BRE, and the alignment of the sequences surrounding the CATTTAA element for rabbit, human, mouse and rat (Figure 4-9) reveals that these putative core promoter elements are at least partially conserved. All four species contain a completely conserved sequence that matches the Inr. The sequence, however, does not overlap with the published the transcription start site for any of the four species. Since there does appear to be a discrepancy between the four species regarding the transcription start site, this sequence should not be discounted as a promoter element. Downstream of the four

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90 transcription start sites there is a completely conserved sequence that matches the consensus DPE in all but one base. Immediately upstream of the CATTTAA element, all four species contain sequence that is extremely GC rich. Although the GC boxes are not completely conserved, it seems likely that SP family member could bind and stabilize transcription initiation for all four species. In a fifth experiment, several of the HK2a deletion constructs were used to test the effect of cell type on reporter gene activity. The longest construct, pDZ11, a middle length construct, pDZ19, and the construct with the highest luciferase activity, pDZ18, were transfected into four cell types, RCCT28A, HIG-82, HK2, and HEK293. In the two rabbit cell lines, RCCT28A and HIG-82, the reporter gene activity followed the same pattern as seen in the deletion analysis. Initially, these results were unexpected because RCCT28A was considered an expressing cell line while HIG-82 was considered non-expressing. It later became clear, however, that under the conditions of this assay, the RCCT28A cells were not expressing the HK2 gene. Therefore, the similarity in the activity levels between RCCT28A and HIG-82 cells was not surprising. The same pattern of expression was also observed for the human adult kidney cell line HK2. This result was expected since HK2 cells were derived from proximal tubule cells that are considered a non-expressing section of the nephron (38). A dramatic difference in expression pattern was seen with the human embryonic cell line HEK293. The longest construct in this case was not repressed, but instead had activity similar to pDZ19. Apparently, the factors required for repression of the HK2 gene promoter are not present in this cell type. Although it is possible the lack of factors is due to a species difference, the fact that HK2 cells were capable of causing repression, makes it more

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91 likely that the developmental stage of the cell type is the important factor. HEK293 cells, however, do not appear to express the endogenous ATP1AL1 gene product (12). It may be that repressor elements are absent allowing for expression of the reporter gene from a plasmid, but that a repressive chromatin structure inhibits expression for the endogenous gene. In summary, the reporter gene analysis of the region 5 of the HK2a transcription start site provided the first information regarding the regulation of the rabbit HK2 gene. It was determined that under the necessary transfection conditions, the HK2 gene is repressed. The deletion analysis narrowed the location of two repressive elements to 365bp and 245bp respectively. Furthermore, the observed repression of the HK2 gene promoter was the driving force for the experiments carried out in Chapter 5 of this dissertation showing that RCCT28A cell differentiation plays an important role in the expression of the HK2 gene. Additionally, the DNA sequence between bases and as were identified as containing core promoter elements. The CATTTAA element that was completely conserved between the four known HK2 genes was mutated and found to be a functional TATA box. Additional core promoter elements were identified by alignment, but their functionality was not tested. In addition, the importance of cell differentiation and the developmental stage of the organism were also identified as a possible regulatory factor.

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CHAPTER 5 CELL DIFFERENTIATION AND HK2 GENE EXPRESSION During the course of our study, it became clear that RCCT28A cells undergo a change in cell morphology once the cells are grown past confluency. Figure 5-1 depicts the RCCT28A cells just as they reach confluency (A) and well after confluency (B). After reaching confluency, the RCCT28A cells appear to differentiate and form ring-like structures that resemble cross-sections of nephron collecting duct tubules. It seemed likely that the change in cell morphology might be accompanied by a change in gene expression. The results of the promoter deletion experiment led us suspect the HK2 gene may be one example of a gene that is altered in expression when RCCT28A cells differentiate. In the promoter deletion experiments (Chapter 4), the construct with the most 5 DNA (pDZ11) had the least reporter gene activity. This result suggests that under the conditions of the assay, the HK2 gene was repressed. The transfection of reporter gene constructs into RCCT28A cells necessitated that the cells be grown to about 70% confluency. At this level of confluency, many of the cells were not in contact and no ring structures were apparent. We hypothesized that under these conditions, similarly to the reporter gene driven by the HK2 gene promoter, the HK2 endogenous gene would also be repressed. Furthermore, we hypothesized that once the cells come in contact and begin to differentiate the HK2 gene would become transcriptionally active. In order to test this hypothesis, several experiments were performed using several cellular conditions. RT-PCR was performed in order to evaluate the level of HKa2 transcript in 92

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93 cells of different confluency. Northern blots were performed in to determine the level of HK2 transcript in confluent and past confluent cells. Immunocytochemistry was performed to determine the specific RCCT28A cells within a population of differentiating cells that were expressing the HK2 proteins. Finally, DNaseI hypersensitivity experiments were performed to determine if there was a difference in chromatin conformation at the HK2 gene promoter under the different cellular conditions. This is the first report of RCCT28A cell differentiation in tissue culture and HK2 is the first gene in which a change in expression appears to be correlated with RCCT28A cell differentiation. Figure 5-1. Micrographs of tissue culture cells. All cells are photographed at a magnification of 200X. (A) RCCT28A cells undifferentiated. (B) RCCT28A cells differentiated. Materials and Methods Detection of HK2 mRNAs Total RNA was isolated from RCCT28A tissue culture cells using the Trizol method (Invitrogen, Inc.). The cells were grown in a 60mm dish to 70% confluency, 100% confluency or past confluency until ring-like structures appeared. They were

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94 washed with PBS and 2mL of Trizol was added directly to the 60mm dish. After incubation for 5 minutes at room temperature the cells were pipetted up and down to facilitate lysis and transferred to a 15mL conical tube. 0.4mL of chloroform was added to the tube and then the tube was vigorously shaken for 15 seconds and allowed to incubate at room temperature for 5 minutes. The tube was centrifuged at 4000 rpm for 20 minutes and the aqueous phase was transferred to a new 15mL conical tube. The RNA was precipitated by adding 1.0mL of isopropanol to the tube, incubation at room temperature for 10 minutes and then centrifugation at 4000 rpm for 15 minutes. The pellet was washed with 2.0mL of 75% ethanol, dried briefly, and resuspended in 200l of RNase free dH2O. The RNA samples were used for both RT-PCR and Northern blot analysis. The RT-PCR was carried out with g of the indicated RNAs. The reverse transcriptase reaction (2l RT buffer, 2l dNTP, 2l random decamers, 1.5l RNase Inhibitor, either minus RT or plus 1l RT, and volume up to 20l) was incubated at 37C for 1 hour. The entire reaction was added to 50l PCR mastermix (Qiagen, Inc.), which included 26l dH2O, 2l of primer BC230 (5CCGACACGAGTGAAGACAAT3), and 2l of primer BC231 (5GCTTGTCATTGGGATCTTCC3). This primer set amplifies a 305 base pair band from the common region of the HK2 RNAs (HK2a 1264-1569). The PCR conditions were one cycle of 94C for 2 minutes, forty cycles of 94C for 30 seconds and 68C for 1 minute, and one cycle of 68C for 5 minutes. 30l of each PCR reaction was run on a 1% agarose gel and visualized with ethidium bromide. Northern blot analysis was carried out with 30g of the indicated RNAs. The RNA was brought to a volume of 50l with dH2O and 10l of dye (0.25% bromophenol

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95 blue, 0.25% xylene cyanol, 30% glycerol, 5% 1g/mL ethidium bromide) and run on a 1% agarose gel with 1X MOPS (20mM MOPS, 5mM NaOAc, 0.5mM EDTA) as running buffer. After visualizing the RNA under UV light, the gel was denatured in 50mM NaOH for 25 minutes, denatured with 200mM Tris-HCl for 25 minutes, equilibrated to 10X SSC (3M NaCl, 0.3M NaCitrate) for 25 minutes and set up to transfer the RNA to nylon membrane by capillary action. The blots were blocked and hybridized to the HK2 mid probe as described in Chapter 2. Detection of HK2 Protein by Immunocytochemistry Tissue culture cells were grown to 100% confluency and past confluency to ring-like structures in Nunc Lab-Tek II chamber slides (Fisher CAT# 12-565-7) that contained four wells. The cells were fixed in paraformaldehyde fixative (2.5mL paraformaldehyde, 7.5mL buffer (4.6mL Solution A (0.2M lysine-HCL pH to 7.4 with 0.1M Na2HPO4), 3.4mL Solution B (0.1M Na2HPO4 pH to 7.4 with 0.1M NaH2PO4)) for 30 minutes then washed three times with PBS (150mM NaCl, 3mM KCl, 8mM Na2HPO4, 2mM KH2PO4). In order to remove any endogenous peroxidase activity, the fixed cells were incubated in 3% H2O2 for 30 minutes and washed three times five minutes with PBS. The cells were then incubated in blocking serum (50l donkey serum in 1mL PBS) for 15 minutes and then incubated with LLC26 anti HK2c antibody (1:100 dilution in PBS) overnight at 4C. The next day, the cells were washed two times five minutes in PBS, and incubated for 30 minutes with rabbit chicken secondary antibody (1:500 dilution in PBS). After washing two times five minutes in PBS, the antibodies were detected using the ABC elite system (Vector Laboratories, Inc.).

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96 DNaseI Hypersensitivity DNaseI treatment was carried out on T-75 flasks grown until the cells were differentiated. Six flasks were washed with PBS and 1mL of trypsin was added. The cells from the T-75 flasks were combined into one conical tube and pelleted at 1000 rpm for 10 minutes. The pellet was washed with solution A (150mM sucrose, 80mM KCl, 35mM HEPES pH7.4, 5mM K2HPO4, 5mM MgCl2, 0.5mM CaCl2) and pelleted at 1000 rpm for 10 minutes. The cells were resuspended in solution B (150mM sucrose, 80mM KCl, 35mM HEPES pH7.4, 5mM K2HPO4, 5mM MgCl2, 2mM CaCl2) to a concentration of 107 cells/mL, aliquoted into 1mL fractions in 15mL conical tubes and incubated at 37C. An appropriate concentration of DNaseI (see results) was added to 0.4% NP-40/Solution B mixture and quickly added to the cells. The mixture was incubated at 37C for 5 minutes. Three milliliters of lysis buffer (50mM Tris-Cl pH 8.5, 150mM NaCl, 25mM EDTA, 0.5% SDS, 300ug/mL Proteinase K) was added to the DNaseI treated cells and incubated overnight at room temperature. Genomic DNA was isolated from each sample by phenol/chloroform extraction. 3mL of phenol was added to each sample and nutated at room temperature for 1 hour. The tubes were centrifuged at 3000 rpm for 10 minutes and the aqueous layer was transferred to a new 15mL conical tube. Three milliliters of phenol/chloroform/Isoamyl alcohol (IAA)(25:24:1) was added and nutated for 1 hour at room temperature. The tubes were centrifuged at 3000 rpm for 10 minutes and the aqueous layer was transferred to a new tube. Three milliliters of chloroform/IAA (24:1) was added and nutated for 1hour at room temperature. The tubes were centrifuged at 3000 rpm for 10 minutes and the aqueous layer was transferred to a new 15mL conical tube. Each sample was treated with

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97 5l RNase cocktail (Ambion, Inc.) at 37C for 1 hour. The samples were then phenol/chloroform extracted as before except that the incubation time was reduced to 30 minutes. Finally, the samples were precipitated with 2X volume of 100% ethanol, washed with 70% ethanol and resuspended in 500l of dH2O. Thirty micrograms of the genomic DNA from each DNaseI treated sample was digested with SpeI at 37C overnight and then run on a 0.7% agarose gel over the next night. The DNA was visualized with ethidium bromide and then the gel was acid washed (0.125M HCl) for 30 minutes, denatured for 30 minutes, neutralized for 30 minutes, soaked in 10X SSC and set up to transfer to nylon membrane by capillary action overnight. The DNA was UV crosslinked to the nylon membrane and hybridized to the SpeI 3 probe (see results) at 55C overnight. The membrane was washed three times at 55C in wash buffer and exposed to autoradiograph film at C for 5 days. Results RT-PCR RT-PCR was used to investigate the levels of expression of HK2 mRNAs in 70% confluent and 100% confluent RCCT28A cells. Total RNA was isolated from RCCT-28A cells grown under the two experimental conditions and 10g of the RNA was used for RT-PCR. The primers BC230 and BC231, used in the PCR reaction, should amplify a 305bp fragment. Figure 5-2 is an ethidium bromide stained gel of the RT-PCR products. A band of the expected size was only seen in the lane that contained the RNA isolated from differentiated cells. This result clearly indicated a relationship between cell differentiation and HK2 gene expression.

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98 12456789103 500bp350bp210bp305pb 12456789103 500bp350bp210bp305pb Figure 5-2. RT-PCR products indicating the presence or absence of HK2 transcripts. lanes are (1) 1Kbp ladder (2) blank (3) 70% confluent RCCT28A cells RT. (4) 70% confluent RCCT28A cells + RT. (5) 100% confluent RCCT28A cells RT. (6) 100% confluent RCCT28A cells + RT. (7) 100% confluent RCCT28A cells RT +Aldosterone. (8) 100% confluent RCCT28A cells + RT + Aldosterone (9) blank (10) plasmid control. Northern Analysis Northern analysis was pursued in an attempt to extend the RT-PCR data and test HK2 expression levels by a second method. Total RNA was isolated from the 100% confluent and the past confluent RCCT28A cells. The RNA was run on a 1% agarose gel and transferred to nylon membrane. The membrane was probed with the HK2 cDNA mid probe (cDNA base pairs 1264-1569). Figure 5-3 is an ethidium bromide stained agarose gel (A) and an autoradiograph film exposure of the hybridization (B). Although bands appear only in the differentiated cell lane, they are not of the expected 4000bp and 4400bp lengths. Figure 5-3C shows the same blot hybridized to the GAPDH probe. This figure, as well as the ethidium bromide stained gel (Figure 5-3A) show that the RNA preparation as a whole was not degraded and suggests that the HK2 mRNAs are very rapidly turned over and only degradation products are detectable by Northern analysis. This result is consistent with the RT-PCR and strengthens the argument that RCCT28A cells must be differentiated in order to express the HK2 gene.

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99 4800bp1900bp160bp 1282bp(A) Ethidium Bromide stained agarose gel(B)AutoradiographHK2 mid probe(C) AutoradiographGAPDH probe 4800bp1900bp160bp 1282bp(A) Ethidium Bromide stained agarose gel(B)AutoradiographHK2 mid probe(C) AutoradiographGAPDH probe Figure 5-3. Northern blot of RCCT28A total RNA. Lane (1) RCCT28A non-confluent cells (2) RCCT28A confluent cells. (A) Ethidium Bromide stained gel (B) HK2 mid probe (C) GAPDH probe. Immunocytochemistry In order to investigate the HK2 protein expression in RCCT28A cells in the differentiated state, immunocytochemistry was performed using the HK2c specific antibody LLC26. This antibody has previously been used to localize HK2c to the apical

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100 membrane of cortical collecting duct cells in the rabbit kidney (48), and therefore it seemed likely that it would also recognize HK2c in tissue culture cells. Figure 5-4A is an example of a well containing differentiated cells that were treated with 2 antibody only. There is no apparent background staining. Figure 5-4B is a similar well of RCCT28A cells that were stained with the HK2c specific 1 antibody as well as the donkey anti-chicken 2 antibody. Within this well, there appear to be two populations of cells. The cells that are differentiated into ring-like structures appear to be stained while and those cells between the structures appear to be undifferentiated and not stained. In Figure 5-4C, a well containing RCCT2A cells grown to 100% confluency, the there also appear to be a two populations of cells. One group of cells that are stained and a larger group of cells that were not stained. Although no ring-like structures were apparent, the cells in the photograph that appeared stained also appeared to be closer together and the staining pattern suggested that they were perhaps beginning to undergo differentiation. The majority of the cells, however, to appeared to be undifferentiated and unstained. DNaseI Hypersensitivity Although the immunocytochemistry experiments suggested that only a portion of the cells in a differentiated culture of RCCT28A cells were in fact expressing the HK2 gene, DNaseI hypersensitivity was attempted in order to distinguish between an inactive gene (undifferentiated RCCT28A cells) and an active gene (differentiated RCCT28A cells). Tissue culture cells were grown until the cells formed ring-like structures, and then individual aliquots were treated with increasing concentrations of DNaseI (0g to 120g). Genomic DNA was then isolated and 5mg of each sample was run on a 0.7% agarose gel to confirm DNaseI digestion (Figure 5-7A). Thirty micrograms of the same

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101 (A) Differentiated cells, secondary antibody only ( B ) Differentiated cells, H K 2cs p ecific antibod y ( C ) Undifferentiated cells, H K 2cs p ecific antibod y Figure 5-4. Micrographs of the immunostaining of RCCT28A cells. Conditions for each photograph are as indicated. Magnification is 200X.

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102 aliquots of DNAseI treated DNA were digested with SpeI and run overnight on a long 0.7% agarose gel (Figure 5-7B). The DNA was transferred to nylon membrane and probed with a fragment of DNA that corresponds to the 3 end of the SpeI fragment from pDZ10. This SpeI fragment contained the HK2 gene promoter. Figure 5-5C is an example of an autoradiograph film exposure of a DNaseI hypersensitive blot. The full length SpeI fragment (4700bp) can be seen in each lane. As the DNaseI was increased, the intensity of the full length band decreased. As the full length band decreased in intensity, however, smaller bands that correspond to hypersensitive sites were not apparent after several trials. Discussion The results of the promoter deletion experiment led us suspect that when the RCCT28A cells are undifferentiated the HK2 gene is repressed. Only when the cells are allowed to differentiate, do they begin to express the HK2 transcripts at a basal level. In order to test this hypothesis, several experiments were performed on differentiated an undifferentiated RCCT28A cells. The results of each individual experiment are open to several possible interpretations. When taken together, however, the evidence strongly suggests a correlation between RCCT28A cell differentiation and the expression of the HK2 gene. The RT-PCR data regarding the mRNA transcripts for the HK2 gene products was the most convincing evidence that cell differentiation is a factor that regulates HK2 gene expression. The promoter deletion analysis performed in Chapter 4 of this study suggested that in RCCT28A cells grown to 70% confluency the HKa2 gene promoter was repressed. The RT-PCR performed on RNA from cells grown to the same level of

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103 Southern Blot probedwithSpeI3probe(A) Ethidium bromide stained gel ofDNaseItreated samples(B) Ethidium Bromide stainedgel ofDNaseItreated andSpeIdigested samplesSouthern Blot probedwithSpeI3probe(A) Ethidium bromide stained gel ofDNaseItreated samples(B) Ethidium Bromide stainedgel ofDNaseItreated andSpeIdigested samples Figure 5-7. Genomic Southern of DNaseI treated differentiated RCCT28A cells. Treatments are as indicated.

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104 confluency produced no product. Only in the RT-PCR experiment that contained RNA from 100% confluent cells was a product obtained. This is the first report of a correlation between RCCT28A cell confluency and gene expression. The fact that so much RNA (10g) was needed to obtain a RT-PCR product led us to question whether or not allowing the cells to grow past confluency until they started to produce ring-like structures would increase the yield of HK2 mRNA. Northern blot analysis was performed on undifferentiated and differentiated RCCT28A cells. Although bands that hybridized to the HK2 mid probe were consistenly observed in the differentiated cell lane, they were not of the appropriate size. No bands were detected in the 100% confluent, but undifferentiated cell lanes. The mRNA that was detectable by RT-PCR was apparently below the dectable level of this assay even though 30g of total RNA were used to create the Northern blot. It seemed likely that the bands that were observed were degredation products of the full-length HK2 transcripts. The ethidium bromide stain of the agarose gel, the GAPDH probe, and the repeatability of the result all confirm that the RNA preparation as a whole was not degraded. It appears, therefore, that the HK2 transcripts in particular were unstable. It may be that once the RCCT28A cells become differentiated, the HK2 mRNAs are produced and rapidly turned over providing the cells with a very low steady state level of the HK2 proteins. An appropriate signal, such as ion imbalance or hormone activity, may be necessary to stabilize and/or upregulate the HK2 transcripts and provide the cells with an increased level of the HK2 proteins. Immunocytochemistry was performed on RCCT28A cells in order to specifically determine which cells in a culture of differentiating cells were expressing the HK2

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105 proteins. Our initial expectation was to find staining only in the cells that had differentiated into ring-like structures. Some of the most intense staining was indeed seen in these cells. There was, however, lighter staining in many of the cells that surrounded the rings. There were also cells within the rings that appeared undifferentiated and were not stained. The RCCT28A cells were derived from rabbit cortical collecting duct tissue, and most closely resemble the intercalated cells that make up the lining of the cortical collecting duct (2). These cells would not normally be found in the tissue surrounding the collecing duct tubule. It may be possible that in the artificial tissue culture environment, the cells that were surrounding the ring structures were also receiving the signal to differentiate and were therefore producing the HK2 protein. The most convincing evidence for this interpretation was the staining in the RCCT28A cells that were just at 100%confluency (Figure 5-5). In this case, all of the cells that had formed the closest contacts appeared to be expressing HK2c, not just the single layer of cells that was most likely to form a ring structure. Furthermore, the pattern of staining suggested that the cells expressing the HK2c protein were beginning to form rings. DNaseI hypersensitivity assays were carried out on the differentiated RCCT28A cells. The full length SpeI fragment (4.7Kbp) can be seen in each lane, and with increasing DNaseI, the intensity of the full-length band decreases. Unfortunately, DNaseI hypersensitive bands were unable to be detected. If the RNA and protein are present in the differentiated cells, it follows that there should be a DNaseI hypersensitive site present at the core promoter. As seen in the immunocytochemistry experiment, however, only a portion of the cells in tissue culture are actually differentiated and expressing the HK2 gene product. Additionally, the requirement for large amounts of

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106 RNA and protein for the other experiments suggests that the expression in the differentiated cells is very low. The occupancy of transcription factor binding sites at the core promoter is therefore also low. Considering a low percentage of cells expressing the HK2 gene products at a low levels, the DNaseI hypersensitive site present at the core promoter of expressing cells was probably below a detectable level. The inability to detect a hypesensitive site in a differentiated cell population made it unnecessary to perform the experiment on the undifferentiated cells. In summary, the experiments in this chapter were designed to test the hypothesis that cell differentiation led to the expression of the HK2 gene products. The reporter gene experiments carried out in Chapter 4 led to the conclusion that if RCCT28A cells were approximately 70% confluent, the HK2 gene was repressed. During the course of growing cells for these experiments is it was observed that if RCCT28A cells were allowed to grow past confluency, they began to differentiate and form ring-like structures. We hypothesized that cell differentiation may be the signal required to initiate transcription from the HK2 gene and the experiments presented in this chapter generally supported the hypothesis. The RT-PCR showed the requirement for above a 70% confluency for expression of the HK2 transcript. Northern data showed the presence of a transcript only in differentiated RCCT28A cells that hybridized to the HK2 mid probe. The immunocytochemistry data showed the presence of a protein that is recognized by an HK2c specific antibody. Taken together, these data strongly suggests a correlation between cell differentiation and HK2 gene expression.

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CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS The rabbit HK2 gene produces two splice variants of the alpha subunit of the colonic isoform of the H+, K+ ATPase (HK2a and HK2c). There is a great deal of in vivo evidence that the HK2 transcript, protein, and activity are increased by a variety of cellular conditions including low blood potassium and sodium, acid/base balance and hormones. The molecular mechanisms by which these levels are increased, however, have not been studied. The purpose of our study was to characterize the rabbit HK2 gene as a precursor to the initiation of studies of its regulation. To this end I have (1) cloned the HK2 gene from a rabbit genomic library, (2) mapped the transcription start sites for HK2a and HK2c, (3) performed a reporter gene analysis of the region 5 of the transcription start sites, and (4) determined the effect of cellular differentiation on HK2 gene expression. Cloning the HK2 Gene The first specific aim of this dissertation project was to clone the rabbit HK2 gene. At the time that this project began, the cDNAs for the rabbit and rat HK2 proteins and the human ATPAL1 protein were known. The only HKa2 genomic organization that was known was for the human ATPAL1 gene. It was unclear whether the rabbit, rat, and human proteins were homologous because the conserved amino acid similarity between the three was lower that that of other homologous P-type ATPases. One of our goals in cloning the rabbit HK2 gene was to determine its genomic 107

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108 organization and compare it to the human ATPAL1 gene. Our second goal in cloning the HK2 gene was to obtain sequence 5 of the previously determined HK2a and HK2c cDNA ends because the DNA elements responsible for regulating transcription from the gene would be found in this region. Three bacteriophage clones were identified that contain 20-of-23 exons and 65% of the entire HK2 gene. Four genomic PCR products that contained the missing exons were later obtained. The complete sequence of a 6.3Kbp fragment from clone HK2.1, the complete sequence of clone HK2.5 and partial sequences from clone HK2.8 and the PCR products are listed in Appendix A. The sequence obtained enabled us to determine the genomic organization of the rabbit HK2 gene. The organization is depicted in Figure 6-1. The HK2 gene spans 30kbp of genomic DNA. The 23 exons are shown in black while the 22 introns are shown in blue. The location of the three probes used to screen the bacteriophage rabbit genomic library, the three clones isolated in the screen, and the four PCR products that completed the HK2 gene are also shown. During the course of this study, the mouse HK2 genomic organization was published (52), and the rat HK2 genomic organization became available through the NCBI sequence database. The genomic organization for the rabbit HK2 gene was determined and found to be nearly identical to the human ATPAL1 gene, the mouse HK2 gene and the rat HK2 gene. These data, along with the distance analysis performed by Caviston et al. (8) confirm that these four genes are in fact homologous genes and were derived from a common ancestor (8). Furthermore, the sequence from the 6.3Kbp fragment of clone HK2.1 provided the necessary data for mapping the transcription start sites for HK2 gene and initiating studies on its regulation.

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109 Figure 6-1. The HK2 gene. The arrows represent the transcription start sites for HK2a and HK2c as indicated. The red stars mark the locations of the three cDNA probes used to screen the bacteriophage library. Black represents HKa2 exons 1-23. Blue represents HKa2 introns 1-22. Orange represents clones indicated. Green represents PCR products generated to complete the HK2 gene sequence. HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2aHK2c HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2aHK2c 1Kbp HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2aHK2c HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2 geneHK2.1HK2.5HK2.8PCR1PCR2PCR3PCR4 HK2aHK2c 1Kbp *** *** *** ***

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110 Transcription Start Sites for HK2a and HK2c The second specific aim of this dissertation was to map the transcription start sites for the two alternative transcripts produced by the HK2 gene (HK2a and HK2c). The determination of the transcription start site was an important step in characterizing the HK2 gene for two reasons. First, the region of DNA just upstream of the transcription start sites was likely to contain the core promoter elements and regulatory elements. And second, the existence of the alternative transcript (HK2c) was called into question by Fejes-Toth et al. (12). Mapping the transcription start site for HK2c would prove that it was an authentic rabbit transcript. A transcription factor binding site search of the 6.3Kbp of DNA immediately upstream of the cDNA end for HK2a and HK2c suggested that there were putative promoter and regulatory elements present. Upstream of HK2c there was one potential CAAT box. Upstream of HK2a there was a potential TATA box as well an initiator element, a downstream promoter element and multiple SP family member binding sites. Additionally, a CpG island spanned both cDNA ends. The RNase protection assay performed in our study revealed that the true transcription start sites were just upstream of the previously identified cDNA ends for HK2a (12) and HK2c (6). The HK2a transcription start site was ten to eleven bases upstream and the HK2c transcription start site was five to seven bases upstream. It seemed likely, therefore, that some of the putative elements identified in the search would be functional. This was the basis for some of the reporter gene assays performed in Chapter 4 of this dissertation. Furthermore, the use of an independent assay to determine the transcription start site for HK2c confirms that it is an authentic transcript found in rabbit tissue and not an artifact of tissue culture and/or 5 RACE.

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111 Reporter Gene Analysis of the Region 5 of the HK2 Gene The third specific aim of this dissertation was to perform a reporter gene analysis of the promoter activity present in DNA 5 of the HK2a transcription start site. An analysis of the reporter gene activity would provide the first data regarding the molecular regulation of the rabbit HK2 gene. The luciferase reporter gene assay was chosen to carry out this specific aim. Four sets of reporter gene constructs were made and transfected into the rabbit cortical collecting duct cell line RCCT28A. Two of the four sets of constructs provided significant data on the regulation of the HK2 gene promoter. The deletion constructs that contained the HK2a transcription start site and decreasing amounts of 5 DNA revealed that the HK2 gene was repressed under the conditions of the assay. The two largest increases in reporter gene activity were seen when the DNA between and and between and were deleted. A search of the transcription factor database, and the mutation of specific bases in those regions, did not identify any known repressor binding sites. The largest decrease in reporter gene activity was seen when the bases between and were deleted. An alignment of this region with the sequence for the human, mouse and rat HK2 genes showed conservation of several sequences that could act as core promoter elements. The most likely sequence to serve as a TATA box was a completely conserved CATTTAA element. Mutation of this sequence to a random sequence decreased the reporter gene activity by about 50%. Other putative core promoter elements that are conserved and may contribute to the remaining reporter gene activity include SP1 binding sites, an initiator sequence and a downstream promoter element. The functionality of these elements has not been tested. Several of the luciferase constructs were transfected into four different cell types, and it

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112 became apparent that the developmental stage of the cell type played a role in the ability of the cell type to repress the reporter gene activity. The three adult cell lines, from both rabbit and human, were able to repress reporter gene activity. The human embryonic cell line, however, was unable to repress activity from the same reporter gene constructs. These results suggested that the HKa2 gene may be active in early development. During the course of growing the RCCT28A cells for the reporter gene assays, it was noted that the cells undergo differentiation into ring structures when they are grown past confluency. For the transfection of the RCCT28A cells with the reporter gene constructs, however, the cells were only grown to 70% confluency. The fact that the HK2 gene was repressed under these conditions led to the hypothesis that the HK2 gene is only expressed when the RCCT28A cells undergo differentiation. This hypothesis was tested by the next set of experiments. Cell Differentiation and HK2 Gene Expression The fourth specific aim of this dissertation was to investigate the effect of cell differentiation on HK2 gene expression. In order to carry out this aim, several experiments were performed on RCCT28A cells at various levels of confluency. RT-PCR experiments were performed to determine the level of endogenous HKa2 transcript under the conditions used for the luciferase assays. Northern blots were performed on total RNA preparations in order to determine the level of HK2 gene transcripts present in the differentiated and undifferentiated cells, and immunocytochemistry was carried out in order to determine which cells in a culture of differentiating cells were expressing the HK2 protein. The RT-PCR experiment resulted in a band of the expected size only in the RNA samples grown to 100% confluency. The cells grown to the same level as used

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113 in the luciferase assay (70%) were not expressing the endogenous transcript. In the Northern blot experiments, the transcripts from the 100% confluents cells were below the level of detection for this method. In the differentiated cells, however, bands of a smaller size than the full-length HK2 transcripts were detected. The RNA samples were tested and found to contain full-length transcripts for GAPDH. Therefore, it appeared as though the HK2 mRNA was a particularly unstable transcript. The instability of the transcript in the cortical collecting duct cell line may explain why some investigators have been able to show HK2 expression in the cortical collecting duct tissue (6, 26, 33) and others have not (40). The immunocytochemistry performed with the HK2c specific antibody further substantiated that the HK2 gene was expressed in the RCCT28A cells that were differentiated into ring structures. Future Directions In our study, the rabbit HK2 gene was cloned, and initial studies on the regulation of the HK2 gene has generated many possible avenues for future studies. There are several experiments that can be done to support the findings of this dissertation and to expand upon the scientific data regarding gene expression as a whole. The identification of the transcription start sites for HK2a and HK2c, the reporter gene assay and the observation that RCCT28A cells undergo cellular differentiation are all discoveries that can be used as a basis for future experiments. Some of these represent immediate experimental opportunities to study regulation of the HK2 gene. For example, the identification of the transcription start sites allowed for a transcription factor database search of the DNA surrounding the start sites. The functionality of the TATA-like element was tested our study. The functionality of the

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114 other conserved elements (INR, DPE, SP1) could be tested in the future by using the same reporter gene assay. Additionally, chromatin immunoprecipitation (ChIP) can be used to determine the specific proteins that bind to functional core promoter elements. In our study, the reporter gene assay identified two regions of DNA likely to bind novel repressor elements. Smaller deletion construct through these two regions may identify the exact sequences to which a novel repressor protein binds. This experiment could be followed up with gel shift assays and/or yeast two hybrid assays in order to isolate and identify the specific proteins that bind to the sequences. Furthermore, the reporter gene assay can be modified in order to study the effect of cell differentiation on the HK2 gene promoter. Stable transfections of the reporter gene constructs could be made. The RCCT28A cells containing stable reporter gene constructs could be grown to 70% confluency and past confluency to cell differentiation and then the reporter gene activity can be compared between the two conditions. In this way, the effect of cell differentiation on the HK2 gene promoter can be studied in a system that may allow for the isolation of factors required for the change in gene expression. The work presented in this dissertation also generated some larger scientific question for future study. This is the first report of RCCT28A cell differentiation and it appears as though HK2 gene expression is dependent upon differentiation. It would be very interesting to determine the molecular mechanisms by which gene expression is altered by cell differentiation. Unfortunately, HK2 gene expression was found to be extremely low and/or the transcripts were unstable even in the differentiating cell population. It may be more advantageous to identify a gene with a higher basal level expression that is upregulated by cell differentiation. Perhaps the molecular mechanisms

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115 for regulating that gene would also apply to the HK2 gene. Furthermore, now that it has been established that cell differentiation is required for basal level expression of the HK2 gene, it is possible to attempt to identify a signal for the upregulation of the gene. RCCT28A cells that have differentiated, and also RCCT28A cells with stable transfections of reporter gene constructs, can be treated with a variety of substances that could potentially upregulate transcription from the HK2 gene promoter. The transcription factor database search carried out as part of this dissertation identified and SREBP and a CRE upstream of the HK2 core promoter. Steroid hormones and cyclic AMP are therefore two good choices for beginning such a screen. Another interesting mechanism for HK2 gene expression is related to tissue specificity. Although it is somewhat unclear as to which segments of the collecting duct express the HK2 gene products, it is clear that HK2 gene expression is limited to very few tissues beyond the kidney and colon. There have been reports of HK2 gene expression in uterus, brain and spleen (52) but all at extremely low, and not always detectable, levels. The reporter gene constructs designed in our study could be used in transfection with tissue culture cells derived from a variety of tissues. It could be determined if the same regulatory elements involved in repression in the collecting duct cell line are functional in other tissues, or if there are other elements, such as chromatin structure, responsible for the repression. Summary This dissertation project successfully cloned the rabbit HK2 gene and initiated studies on its regulation. The rabbit HK2 genomic organization is depicted in Figure 6-1. The data obtained regarding the regulation of the HK2 gene lead to the model for HK2 gene expression depicted in Figure 6-2. As seen in the reporter gene assay, the

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116 HK2 gene repressed when the RCCT28A cells are not confluent and therefore not differentiated (Figure 6-2A). There appeared to be at least two regions of DNA upstream of the HK2 gene promoter that were necessary for binding repressor proteins and inhibiting the formation of an initiation complex at the HK2 gene promoter. The mRNA and protein analyses indicated that when the RCCT28A cells were grown past confluency and began to differentiate into tubule-like structures, the HK2 mRNA and protein became detectable, although they were apparently at a low level and unstable (Figure 6-2B). Furthermore, we hypothesize that given the appropriate signal, perhaps low K+, activators may either bind to the promoter and increase expression from the gene and/or stabilize the message and protein resulting in increased levels of protein (Figure 6-2C).

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117 S S S S S S S SRepressed RR Basal RNAPol Activated A + RNAPol A S S S S S S S SRepressed RR Basal RNAPol Activated A + RNAPol A Figure 6-2. Model for HK2 gene expression.

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APPENDIX A RABBIT HK2 GENE SEQUENCE Appendix A contains all of the sequence compiled to determine the intron/exon boundaries for the 23 exons of the HK2 gene. In all cases, lower case letters indicate intron sequence and upper case letters indicated exon sequence. Sequence Data from Clone HK2.1 and HK2.5 Exons 1-11 -5384 tgggtaccgg gccccccctc gagttgtaat cttgtgtgat gctcttgaaa tttcctaagg gaatagattg ggggttgctt ttatcacaaa aaaagattgt gatgtaatga tgtgagagga tggataagtc aatttgctta actactttgt tatgaataag tatacatatt tattttattt tattttattt tattttagga accagcactg tggctcagca ggttaagcca ccatatgcga agatagcatc ccataggagt gctggtttgg gtcccagtac atgggcccat gccatctaca aaggagacct ggaattccag gcttctggtt tcagcctgac ccagtcctag ctattatggc catttggaga gtggaccaga ggatagacta tttctctcct ctctctctct ctctctctgt aactctgact ttcaaataaa taaattataa aaattataaa tatattagat aatgtataat agaatagaaa aataaataat aaaaattatt tcctaaaggc agagagagat agacaaagac agatttccca tcctgaggtt tactccccaa atggccacaa cagctgaacc aggagcctga aattctattc aggtctccca catggagtcc tttagaataa ataaacctat tttatttatt tgaaaggcag agttacagag tgagagtgac agagatggag agagattgcc catcctcagg tccactcacc aaaagcctgt aacaaccagg gctgggccag gccaaagtca ggagctggaa tctcaatcca cgttgcccgc atgggtgtca gggatcgatg tactttagtc aacacatatt acctcccagg gtgcacatta gtgggaaact gatgtagaga gtggatctgg gacccaaacc caggcaaact gataggggac acagccatcc caagcagtgg cttaaccact gggccaaaca cccaccctta tctgagcata ttaacacagc atgttgtaca cctcctatac agtcaacttt ttaaaggtct gtcttagccc cttgcaggcc ttcacctgcc tctgaaaaca ccacagctca cggtcctgga ggtctctaac agccccagga aagatcaaca cagcagatta ccagtgattt ttggaaagcg tgtttctctc ctgcaggatg tttacatgcc agatatccta taaacgaaga aatgaggaaa ccatataaaa gtgtgctagg ctgcagcttt gttttgcttt tggttctcat tctgactcca actccaggca gtccccattg gcagagaaat gccggcctag tcatagcctg gcacgctggg cagaagcagg tgtgcaggaa gggctccagg tggggatgtc cagtgtgtgg agaggaaccc agggagaggg agagggagag ggcaggtggg cagtagaggc accagccagt ccggatacgg agcaggaagc agagcttcct cttgtgctca tcacctggat cctcctccct gtatgtttgg atcatcagtc acaccccaag ccagctgggg gacagctcca agagggctcc cccaactaaa gccccacctc aacggccctt tttgcctctt gtccaccttc cttggagggt gcgaagggat agaaaagtat ctgaataata aatcctgaat ttggagatgg ggctgttctc cctgtcttct tctgtatttt gttgctgtaa cagaaagcta gaaactgggt gatttataaa gaagatggtt taaatcggct cgtggtccta gagactggga gatttgaggt taaggggcac ttcccgtgca ggtttcttgc ctgtggggac tttctgcaga gtcccaaggc agtgcagggt aagggaagga tacatccaac agagtcaaga ggacttttat attagacccg ctctctagat aattcactaa accattaatc caataatgat gaaagtcaga gtcctgagaa ctcatcaact attaaagtcc ctctccatgc ctctacaatg gggattacat ttcaatgtga gagctgcaga catttggcct aggagctaag atgccagttc cattaccctg tcccatatgg gattgcctgg attcaattct catctccagt tactgattcc tgccagtgca ggctctggga ggcagcaggg acggctcaag aagttgagtc tctgccaccc aataaggggg acctggatgg agttcccagc tcccagcccc accaacagcc cagacgtggc aggaattcta ggtatgagct agtgaatggg agctctcgtt ctctatcatc tatctatcta tctatctatc tatctatcta tctacctacc tacctaccta tctctaataa acaattttaa catgagattt ggtggggaca ttcaaaccat agcggtcccc ccaagcaatc tctttccctt aatttcttcc agcacttaca gcctgataat gacaactgca cactgtatta tttcttaccc tgccttattc actagtgctg cttggttaac 118

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119 ttggcctctc caactccagc ccaacttcct ggaggacaga gaatagcata gtttttggta ccatttctcc ccaccacccc caccaccccg acccattaac ctcaagttga gctccatgag ttgatgaaaa tgaactagga gcagtcacac acatgatgac aggctggccc agatatgtaa aatacttgcc caaaagaacc acagtaaaag agcctggcat gagtaggttt cttttgacag acagtaaaat tcagcctcaa gcggaagctg ctgctagagt gtgtgggctg gggaggtgtc gccagttgca gcctggcttc ctctgtggcc agtcttaaga gaggcattca aaataggtgg agtgcaaggt ttgttccctg gaacagggta ggggatgtcc cctgtggcca aatgaagttg aatcagtgtc tgaaaggaaa cccagattct tccctccagg tgaggactac tccaggtgct cccatcacag acagccagca gccactcagg cacaacagga catccaggcg actagaggca gcagggggct ggccccacgt tccctcttca gtacaggtca acgctccggg acacctgaag acaccaggga ctgccaaagg ccatggcagc agcagcagca gcagctaaca ctgatccctg gcaaaagctg gaaactgtac aaggccagag aacgtgacaa tagaaactct tttgtccagt ttgctggtaa gtctacttct tcacacttga caccagcaga gataagcagc ccacttccca agtgtagttg taattcacca atttttaaaa ttgcaccatc acttctccat gagtcccctc cctccacacc tgccaacaca ctacatccac ccccacctcc ctatattcca tcactaaagc ttgaatgggg cttcactctg ggcactctgg gcactacttc tctgtgccac acctggccca aaagttggaa caaggctaga aaggtggaaa gtgagcagca gccacctgta caacgactct caccagaggt ttccagtaaa cagtgaactg tcagtttcat aaaattgtgt atttcgttac ctctttctgc aaatctttct ttggagaaaa gatacaaagc agagctcctt ccgatgaccc tgctgcttca gtttagacta gaatctactc tcccctccaa ctctgaagga cctgtgatgt gtgatctctg cacaagctgt caatgccatc ttcttgtccc ttaagagtta atgaacggcc ggcgctgcgg ctcactaggc taatcctccg ccttgcagcg ccggcacact gggttctagt cctggtcagg gcactggatt ctgtcccggt tgcccctctt ccaggccagc tctctgctgt ggccagggag tgcagtggag gatggcccaa gcacttggga gaccaggata agtacctggc tcctgccatc ctatcagcac ggtgcgctgg ccgcagcacg ccagccatgg cggccattgg agggtgaacc aacggcaaag gaagaccttt ctctctgtct ctctctctca ctgtcctctc tgcctgtcaa aaaaaaaaaa aaaaaaaaaa aaaaaaagag ttaatgaaga agtaactcac tggcgtcatc cctgtcccat agccagagaa tcccagactt aaattctgct ctttggattt agggtgtttc taatggattt tctttttgta acttcaagta tttatctatt taatgtattt gaaagacaaa gacacagaga tgtcttcccc cttactggtt cactctccaa atgtccccaa agcaggggct gggccaagag ggagccaggg atccaagggc tgggagctca acctaggtgt cccatgtggg aagccaggac ccaaatactt gagcagtccc tgctgcctcc cagggcatgt attagcagga agtcagaatt gggaggaggg gcggtgcttg aatccaggta ctctgataag acaggcagac gtctccagcg ctgcaccaaa tggtcacccc agatcctggc cttgaggagc caagggaagg ggcgaaaact tgtgtgtgca gtactgtggg tcggggtctc catcgatccg gagtctgggg gtgtcatcct aaattctgct aacaactggg cgcagcacca cgcagcccgg gaccccagag ccctccacgc tcactgaccc agaccctcca tccccaaccc ctctctcctc caaaatggct tcagaattcc caaattctga tgtagatgct gcacagggta taccagcgtc tccttagagt ctctcgcggc tgggcccaca gtggcggtta acccagatcc gctccccaag cgacttgacc ttcactctga gagtgcagct gctactggaa ctgcaatttc ctctcctctg cttacatatc tgtataaacc cctttatggg ttagcaaatg aaattttata aagacaagtg tgtaggggtt cccacgaaag cttgaacagg gagtgggagc acccggagcg cggagcctca gcagccccgg ggcgcttggg gacttggggg ctccggatcc tggggccccg gggtgggggt gctgagcaca gagggctact gcggagctga aggcgttgtt ccaagcgcca aggatttggg acccggcccg gagacgcccc acgccgctgt gttcggctcc tggaaggaat tggggtcccc agccccggac tctccctgcc tcttgccata gccagcccgg tcccggactg cgcatcctcg gtttcccagc cccctggggt gtctgcaggc cgggctactt gcacagcagc aggtgcgtag -44 gcggggcgcg cagcatttaa ggcggacacc acctcccctg ggcaGCGGCT GGCGATCGGC 17 TGCGGAGGTG CGCGCAGGGC CCGCGTGGCT GTGGGTACCT CCTTCGCCAG CACCGTCGCC 77 ACTACCAACG CCGCCACCGC GGGACCCTAC CCCGCATCGG TCGCCGCCGC CACCGCAGGT 137 CCCACGACCC CTCCTGCCCT CCGCGCCCCC TGCCCGCCGA CCCGCGGCGC CTCCAGCGCG 197 ACATGCGCCA Ggtgtgtgag gaagtgacgc ggtgcggact ggagagaagt gcgggaaagg 257 gtgaagggct ccgtccgggg gtctttactc tgcaaccctg ttccagccgc cgagcacccg 317 tgtgtcactc gggaactggc tgggtaaaga ggtcaatcca gacacgcggg gaaggagttc 377 caggggtcag ctccgccctc gcacctgcgg gctcggattc ggagaaaagt gctagactgg 437 agctacacgt atgcgtagcg gtctggaaaa tgccccaggc tcgggtctga ggggcccaag 497 tctatgcacc gctggtgtga ccccgcaggg caaccccgcg gttaacttct ctcctgccca 557 cccctagagg tgtcttcctg ggaagacgat ggcaggcggt gcccaccgag ccgaccgtgc 617 aacaggggaa gagaggaagg agggaggtgg gaggtggcgc gctccccaca gcccttcccc 677 tcctggcccg cgagggtgtc cggtcccact caaggcagct gcgcagagcc tgtgcagaaa 737 taccacctgg ggccggtatt gcactctgct tctctttcag AGAAAGCTGG AAATTTACTC 797 CGTGGAGCAC CATGCAGCTA CAGATATCAA GAAGAAGGAG GGGCGAGATG GCAAGAAAGA 857 CAATGACTTG GAACTCAAAA GGAATCAGCA GAAAGAGGAG CTTAAGAAAG AACTTGATCT

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120 917 Ggtcagtagc cttggaaagg gctcccgcgg acttggtcat ttccctctgg gagttggacc 977 cttcaccggc ctcttacaaa agaaaaatca ggctcaaggc tttggaatcc ccctaacagt 1037 cgtttccttg tggattacta tctagatgtg ataatatgta tccgggtctc aggtcctctg 1097 caggatgcct gagctctggt tgaaggcagt cctggggaga atcgggaggc acaaatccct 1157 aactcccaaa ccacaggcat ggacaagagg tctccctgtc tgcttcaacg agatagaaat 1217 aaccagctgc ccgtttttag ttggaaaaac tatttgggct ggcacctgcc aggaagaaaa 1277 cccaagctac tggaccttgt taggtttggg gggtgtgtgc tgcctgggtg agaaggactc 1337 aggcagtgag aatgttgcac ttggaaccac ttaggttcct cacagcccat tctccatagt 1397 ctgagctgta aacgatctag cctcgtggaa gctcacagaa aatgggaaat aaaagaatct 1457 gtagggatct gtgaggagaa aagcgaggta caagtcttta gaaattaaga tgctttctag 1517 gagtgcaaca ttgttattat caaggatgaa ctctggagtt atttcaactg gggttattta 1577 ataagtatta ataaattcat tagattactt actgaataaa gagctgctgg ggatctgaaa 1637 ggtgacagat tgcatctcag gaatgtcagg gagggagaga gattttgctg gattgccctc 1697 cagcccctca cagacgttac ggctctatga ctggtaggca gccgctgggc tggcagaggc 1757 cagcatgctc aggctagaca actgattgtc aaatattcag gaacttggcg agccgactga 1817 gattctgttc acagctggaa atttggctct ggtcagaaat cagtggttgc cttgaaccag 1877 agccatttgt ttatcttgcc tggagttccc atgtgtgaat gacactcact agtttcctat 1937 tttgaaaaaa attgggatta aaaatggtgc ctgccaccag aggttattaa gaagattaag 1997 tgagataaag caggaaaaaa gcactagtca atagtcatgg ttatttccta tgtatctctg 2057 gcatttaaga ttaaaaacac atctgttctt ccttgaaatt gctcttgtac cattctctcg 2117 tatacctatt ggctagatga aaatccccct tttctgagca ctttatgagg atgtatgata 2177 caatatagaa gaatagcttc cattgtatta ggcaaaaagt tccccttcct ccttgctggg 2237 aaaaagagct cctttctttc cttccttgtt ctcaagctcc tcacaagaat tctgcagttt 2297 gcaagagtgc acttcaatac tgctgcctct ccacctttcc tgcaaatcag agccttgata 2357 actaagcacc cctggcttct gaataaagga ctccgctgca gggtgcttcc ctgtgctaag 2417 ccagctagct ggcagaaggc acccagacac ccagcactcc tcacaccact taactcgcag 2477 gctcccctcc aagtccaggc tggaaagaca agaaaggtag agtgctttgc ctcacagtgc 2537 taccaggtct gcggtccatc cacacaaatc tttactactg tgtctgccca gtgctcacac 2597 agtctgccct gtaaccttcc atgcccctag caacaacaca tcccaatgac actggcacaa 2657 tgcctgatga gcagagcaga atctgactga gaagagagcc agaaacaaaa taacaatacg 2717 tcatctttta ttggaaggct taatttccta atgcaacatg aactattact ttttagcatg 2777 catttaaaaa gagagatggt tctagtacca gtcacagcat aaatcttcat ttcagtgaat 2837 cagggtatga agaatcctga gtgctggggt tttcttgggg gtaggagggt gaggaagata 2897 tgccactgga ttttctgcta taggaagagg tttcttttca tccgttagcc ccgtggttct 2957 tgaaaggaga agagtgagtg gattttgtcc catccctttc ccccacaaaa acgcattggc 3017 ccaaacgtta ggaagtctgc attagtctaa ttagtctaat gattctgtgg caaaatctgc 3077 aagtgtgagc catactggta tggtttgttt gtcgggtttt tagtccctac cagtgaactt 3137 gttgcatttc atagggcaaa taagggaagg tgtgcttctt tctgccagag accacctagg 3197 agcaatgagg agttgtccag agaaagtggg tctggtttgt tttgctcctc agcctcaacc 3257 aatcctagaa ttcaggtctg aatgaagtat ccagcttgct ggtacttgga agatgtgcta 3317 gagatgagat tgacatgata tgtgacttcg ttgataaaaa ttacagatgc accaggtagc 3377 cagcagcagg tgtaacctga gctctttggg ctggccatgg tgcaggacag ggctctcctg 3437 tagcagtgtc ttccctggct gcacagctcc tggcaccaag ggtccctgtg ttcctttcct 3497 gcaccctcca gcccctccta gccacagtgc tactccagct gtcagggggt ggtcagagct 3557 tgggctttga gtcatttagc attatgtcac tcattctgct cctcttctga ttgtgcattt 3617 CAGGATGACC ACAAACTCAG CAATAAGGAG CTGGAAACGA AATATGGCAC AGACATCATT 3677 CGGgtaagtt ataagggaag tgggcatcag gcaaggaagc ctgcggacta caaaggggaa 3737 agctatatgc tctgcatgtg tggaaaagta agagggcacc tttggggcag gtggcctgcc 3797 acacgttgca gtcatgggca cacatgtttg gcttcccatg ctgcaccaag gctatggctg 3857 tgtccctaca cagagggcaa tgcttggttg tagagagttc ttccatctgt gactgcagga 3917 aggctgtggt gcctaggaga tatgcgattg gatgctttta atctctgcct cctgggagtg 3977 taatgaagga gaagctgggg gatccagtat tagaaggaaa agacccaggg gtgcggtgtt 4037 tggtgcaaca gttgagacac cacttggtac actctcctcc catattggag tgcctagatt 4097 tcatatccca ctctggttcc ccactccagc ttcatgacag cacagtccct ctgaggcaac 4157 aggaaatggc tccttaggtt cctaccacct gcgtgagagg cctggattga gttaccagct 4217 cccagtttct tcctggccca gtcttggcca tcatagtagc catttgggga gtgaaccaga 4277 agacaggagt ttctctctct ctctctccct ctccaccccc caccccgccc ccatcacatt 4337 ttttaaaatt acaaagaaga agacacagac ccagagcaac atctgaaacc ctgctggtca 4397 gccagacccc ctgagaaggg atgatagccc caggcacacc tctgtgattt ttctgcctct 4457 tacatattct ctccccactg gagcactgct tccctctggg aattcaagtt caaattacat 4517 ttccatagtt tttgtttatt gacatcagga ggtctggata ctgagtgtac atctgtgtaa 4577 atcaatacaa atccctacta tgtggttcac attggactgg cactggggca ggtgacctcc 4637 ctgaccttaa ggaggcagct gggtagggaa gtgaacatcc agcctatccc atgccaatgg

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121 4697 ccaatgaatg gttgagacca tctgttggga gaatgatgaa ttctgaccag agggaccagg 4757 gagcgcagag tttctaaaga gggaagcaac ttgatacaat tcactttcac caggcaattc 4817 tttaagtgga aggtggaagc ttctagaaaa atgaaataaa ccaggactgt gaaatgggga 4877 tcccaaagag gggagcccag taagatggga aggaagggga caggtttaag agggagttca 4937 cagaaataac cagtagctct tgggggatac tttttgagtg aatccctaac tcaagttaga 4997 gaagtaggag gcagagattt ggccaagttg gggcgggggg cagctgggtg aaggtgagag 5057 attagaggag gtggtaggga ggagatgatg gtgagtttta ttttaaatta atcagccctc 5117 aatatccata aggtccctcc ttgatagtaa aatccacagg tgctttagtc atttatataa 5177 aatggcatac tacttgtata taacttgtat gcatatcctc ccttatactt taaatcatct 5237 ctagattacc tataataact aatacaatgt gaatgctatg taaataggtg ttgtatggta 5297 ttatttacag tacaaagaaa aaaagtctgt acatgttcac taaatatgca accaccacag 5357 gatccaaggt tcattgaccc catgggcctg gaacccatgg atacaaaggg cagattatat 5417 ggttaggaat gactctgcaa tatctgtgca gaaatgacca acagttattg agaaggatga 5477 gccagggttc tagaaaagga ttgtggttag agggtgatgg catcaacttg gtgaccaaga 5537 atccagctac tcaataatta cgggagttca ctagtcactt aaaaataaaa aagcattaga 5597 ccctaaaaaa gacacagaaa acacagaaat gatcaagaca gagtccttgc cattatgtag 5657 ctggcaacat aattagggag aaaatgtgtg tacaaaacta gtcccataaa aggtagtatt 5715 tggtggggga agccctatgc aaatgcagga ttttggagtg cgggtatcct ctatgcacct 5777 gtgaatgtca ctctagatct tgggtgggat tggtaactgt taaaacaaac aagaactggg 5837 aacttagaat gcatatgtag aaaagtacca gagtaaagat ggaaaaatca gcccacatgg 5897 tctacagagt tccagaatca gttgtatttt cttcagggtc actgcattgt ccttaatcct 5957 gatgacaagc tcctttattt caaatactcc ttatccaagt tacttccttc tgtggatgaa 6017 tctctagaat gctaaaatgc ctaaacatgc agggggcttt gaaccggtca cctggctgag 6077 ttctggtgcc actttttaac attttttaag attttattta tttatttgag agctacagtt 6137 acagacagag agaaggcaag gagacagaga gaaaggtctt ccttctgctg gtttactccc 6197 cgaatgggtg caatggctgg agatgagcca atctgaagcc aggagccagg agcttattcc 6257 agggcttcca cgtggatgca ggggcacaag cacctaggcg atcttctaac tgctttccca 6317 ggccaccaca gagagctgga taggaagagg acatgaactg gcacccatat gggatgcctg 6377 caccacaggc aaaggcttag cctactacac cacagtgcca gccccctggt gccacttctt 6437 accagccagg tgaccttagg aaggtatgtc acctccgtgt gcctcacatt ccatgtctac 6497 agatgagaat aataatagta tcctcttcag agccattctg aagactaagt gagtaaactc 6557 atggggtata cttgctggct atcagtatta aatattcctt ccagaaaaat gggtattgcc 6617 catatttctt gggactttct ccagtgtgtt aatgcctgtg tgccacctgc agaaacagga 6677 agcacagcct caaagggagc aggatgtctc actccagttt gcttttgtgc tttttgtgtt 6737 tcagGGTCTC TCCAGCACCA GAGCTGCTGA GCTCCTGGCA CAGAACGGAC CCAACGCCCT 6797 CACCCCTCCC AAACAGACCC CAGAGATCAT CAAGTTCCTC AAGCAGATGG TGGGCGGCTT 6857 TTCCATCCTT CTGTGGGTAG GAGCTGTCCT GTGTTGGATC GCATTTGGGA TTCAGTATGT 6917 CAGCAATCCA TCTGCCTCCC TGGACAGCgt aaggctcctg gtgtcaactt cctggttttg 6977 ctctgcaagg ggtggggtcg gttatgggag gggagagcac agctaaaata aaggccatca 7037 gcgacatctg tgagagcagc catgggctgg aggcaggatt ctaccatgga tcaaccactg 7097 ccagggtgtg ggaaaatcag gacttttccc agatgtagaa caaacgctct gtgactttct 7157 gatgcctcag tgggtagagt aagcatggat tttagaagag ggagagaaag ccaaaacagt 7217 ttttaagttc ctcaaggagt cgacatccat gtgaagtgtc tagggccttc cctccttaaa 7277 gctgactttt cccttgaacc tggtaagcat ctgttcctgc tgggagccag cgaccaaccc 7337 cacaacacag agctggccgg catggtgctg ggaaaggagc cagatgcaag tgcctcaagt 7397 tgtgtggctc ctcttacaca gagcacccag atgggcatat ctgtggagac agggagctca 7457 ccagtgggga cagggatggg gcaggggcag gaggtgactg cctaggggcc tgatggccat 7517 gctgcagagc tgaatgacag ctgcacagct ctgcacattc aggagaaacc ggtgagttgt 7577 gcacttacaa tgagtacagg ctgggtatca ggatgcaaat gccagcagct catccctgtg 7637 gagagctgta gcccaggacc tggctcatac ttgacccagg ttgatgagcc gtcatctgca 7697 atgcttgggg ccagaatatt tgcacacatc cataacgaga tgtcttgggg atggaaccca 7757 agtctctaca tgaaatttat ccttcacaca cagcttacac acatagcctg taggtgattc 7817 ctgagccata tttttaatgc acttgtttta ttgtggttgt catgtgaggt caagtgtgag 7877 atttttcact tgtggtatca tgtcagtact caataagtgt caactctgga acctttcaga 7937 ttagggatgc acaacccaag catgtgtctc tgactgatgc ttttcttcct taaccagGTG 7997 TACCTGGGCA CTGTACTTGC CGTGGTTGTC ATTTTAACAG GAATCTTTGC CTATTACCAA 8057 GAGGCAAAAA GCACCAACAT CATGGCCAGC TTCTGCAAGA TGATCCCCCA Ggtgagcatc 8117 agttgccttt cctctggtct cagcacaact ttcaggtgag gaagcatcag acaggccctg 8177 ggggcagggt cgctgcgtag ccctgcccca tctgtgctgt ctgagaaatc ttgagcgggt 8237 tatccctgct ttctagaccc cagctcctca cagggtttag aggtgatcga actcagtaag 8297 atcgggcaaa atgcttttca aaacgtgagt tgtcacacaa catcttccat taccaaaaag 8357 tttgcagaaa ttgaattaaa agataagttt attttggtgc aaaaaagaaa ttggaatcca 8417 tgcatagttt tttttttcat aatccacatt ttccatgaac ttttgatggc ccctcgtact

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122 8477 gttccttggc atttctgggc cagctgggtg gtgacatggt ggctccaagg agggaaccta 8537 gaattccaag ggctgtttgt gcactggcag ccatggagga ccccttgtgc tcctctccat 8597 gggactgctt ttcctcctat ccctgagcta tagtagaagt tgcccccaaa tgctctgggt 8657 aaggggaccc agcattcccc tcttccccca agagccctgc cagctgcgca cagcagggag 8717 accagtgttc ccaccccaga caacagccag ctattagaag gggaaggctc tggctagtga 8777 agatgcctcc taagaagtgg gccatcaatc acctcatcaa agttccccca gccttatcag 8837 ctgcataagc tcctgggctc ctcccaggca cccagccatg taattgtccc cctctctggg 8897 agatccagag ctctcacttc tccacgtcca cctgacagca gcattttctg gttctgtgag 8957 cacaggaaaa gattagttct tggggtcttc agccccgccc gcggcggaag tcttccattt 9017 catcctcgct cttgggacgg ccatgggtac catacctctg ttctgggatt cggtgaggtg 9077 tggatggaga aggaggccag catcaccgcg ggtctgaggc tctgtcatgc tttgcttcca 9137 cagCAAGCTG TTGTCATCCG TGACTCGGAG AAAAAGGTTA TCCCTGCAGA GCAGCTGGTG 9197 GTGGGGGACA TCGTGGAGAT TAAAGGAGGT GACCAGATTC CTGCCGACAT CAGGCTGCTG 9257 TCTGCCCAGG GGTGTAAGgt aagggccggg ggaaccccga ggatcctgct tggagcgagc 9317 aaacattctg ctttttccct ggccgtggta ggcgaggcag ttccctctga gctgctgtca 9377 aactttttcc tagGTGGATA ACTCATCTCT TACTGGAGAG TCTGAGCCCC AGTCCCGCTC 9437 AAGTGAGTTC ACCCACGAAA ACCCCCTGGA AACAAAGAAC ATCACTTTCT ACTCCACGAC 9497 CTGCCTGGAA Ggtaagccca gcagctgtca gggtcgcaag ccccaccttg tcactgctga 9557 cccctaaggc cagagggttt ttcgctgcct tccctcgtta caaagctttc ccccacccca 9617 agaagcatac agtggaggga gccaccaagg agggccttga ataaaacgtc tacttctccc 9677 atagGCACGG CAACTGGCAT GGTCATCAAC ACGGGTGACC GGACCATCAT TGGCCGCATT 9737 GCCTCCTTGG CTTCAGGCGT CGGGAATGAG AAGACGCCCA TTGCCATTGA GATCGAACAT 9797 TTTGTGCACA TTGTGGCAGG AGTGGCCGTC TCCGTCGGCA TCCTGTTCTT CATCATCGCA 9857 GTGTGCATGA AGTACCACGT CCTGGACGCC ATCATCTTCC TCATTGCCAT CATTGTGGCC 9917 AACGTGCCTG AAGGCCTCCT GGCCACTGTC ACTgtgagtc tacactgtta ggcagcctgc 9977 agcccaactg tggctgctgg ggaggctcca catgcagacc acaagtctcc ctggcccctg 10037 tcttcagaac agggacaacc aaggaacatt ctggaaagac tgcttcctag agggacaaat 10097 ggaattgctt atttctaatg tttgaatgca acctccttga aaacatcaag atttcatgcg 10157 atatctctgt aattcacaga ggttacttag ctcccttaag atctagagag gacagacctc 10217 caaccaccac ttacagattt tctagaagcc tgacgcctcc ccaaagttaa aaaaaaaaaa 10277 aaaaaaaaag aaagagaaaa agatgtccag gtctcttctg actccaggag atagaatttg 10337 gagaagctgg agccaacaga acccttggag agctgagggc ttaaaagatc tttaaaatat 10397 ggggctcttg acatgaaata caaatgcttc tggcctcccc agagcagcca gtcagggtct 10457 ctctcacaca ttcatgctcc tggtgacagg atcagggtgg cccaggcacc ctaattggat 10517 gcaagcacca gccacagcaa tgtggctgtc tccactacct ataagccctc gggattatat 10577 tcttcaagaa ataaaggaac aagaatagtg tctcatttaa ttttttttaa aaaggattgt 10637 gacctgtaag acttttagtg gcttacacat tggcagttct gccttaatgc taaaacaaac 10697 ccagcccctc ctgaaattcc attgtagctc atcttagctg tttatacacc tgaactccag 10757 atcaaagtta ccttgcccaa agcttctttc cctctctctt ctgccagGTG GCCCTGTCGC 10817 TCACAGCCAA ACGGATGGCC AAGAAGAACT GCCTGGTGAA GAACTTGGAG GCAGTGGAGA 10877 CCCTCGGCTC CACCTCCATC ATCTGCTCTG ACAAGACTGG GACTCTGACG CAGAACAGGA 10937 TGACCGTGGC CCATCTGTGG TTTGACAATC AGATCTTCGT GGCCGACACG AGTGAAGACA 10997 ATTTAAGtaa gacttttagg gtggggcatt gagcctgggc tgaagctgtg gactcaagca 11057 gtggctaggc tgagcactgt gaccagcaca gtgcaggcat ggtgggccca gcacccatag 11117 cagccattct tctttctcct tgtttcagAC CAAGGCTTTG ACCAAAGCTC TGGAACCTGG 11177 ACCTCCTTGT CCAAGATAAT AGCATTGTGT AACCGAGCTG AGTTCAAGCC AGGAGAGGAG 11237 AGTGTCCCCA TCATGAAGgt aaaatttcca catgcttgtc ttaaatcact gctggtttta 11297 ggtcactttc tctgcccacc ttgggtgcgg ggggacagag aatctctctg catccaagca 11357 gagttggtac gagcaagagt aaatctgact tacaagacac atagcaagct ttctccactt 11417 tagttctgag aaatcacaaa gttgcttaaa agatcttggg cccttctgaa aattcacagt 11477 atgggagatg aatgtgcctt tttatttctt tcctaacttc tttaccagct aagtggagat 11537 agtcaaatgt tttgccattc actgaagttt aaaatataat tagatggaaa taggctggcg 11597 ccacagctca ctaggctaat cctccgcctt gcggcgccgg cacactgggt tctagtcccg 11657 gtcggcgcgc cagattctgt cccagttgcc ccttttccag gccagctctc tgctgtggcc 11717 agggagtgca gtggaggatg gcccaagtgc ttgggccctg cactccatgg gagaccagga 11777 gaagcacctg gctcctgcca tcggatcagc gcggtgcgcc ggccacggaa aggaagacct 11837 ttctctctgt ctctctctca ctgtccactc tgcctgtcaa aaaaaaaaaa tatatatata 11897 tatatatata attagatgga aataaaggac ttggtcttag ttcatcttcc tcttgactat 11957 aagatggtag taggtggaac actaaggggc cctgaaattt ggaaatatac aatattccaa 12017 aattccaaaa gtgcagtgac tcttttctct ctccctatct ctgtccctat ctctgtctct 12077 atctctgtct ctacctctac ctctacctct acctctacct ctacctctac ctcaacaagt 12137 caccaaatct gggttctcct tgcttttcca ttcaccatct gtttctttca ctgactcact 12197 ttctttctta tatttttaat ttgcttttcc ttattagaaa gctcatcaat gctccctttg

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123 12257 gccaattact tctccatccg taccttttga atctctatgt cctatcctga tttctcatct 12317 aaatcccaac ctggattccc agcaagctgc atgtgtccac tcagaagttc cactgagttc 12377 acctatcaac aaattacact tgtccagttc cacctgtatc taccttattt tgtcctgagt 12437 tttctattat actgtatcat aaccattcca catgtcatcc aggcaaatgc ttttagaagt 12497 cttctttgac tgtctccata aatctagtga atcaccaagt cctatcattc ttgcttcaaa 12557 atacacataa ttttatactt ctatgatcat taataccttg catctttttc aaagtttttt 12617 ttatttattt acttgagaag tagagttaca gacagtgaga ggaagagaga gataaaggcc 12677 ttccacctgt tggttcactc cccaaataga cataatggcc aaagctgagc tgatctgaag 12373 ccagcatcca ggagcttctt ccagatatcc caagtgggtg cagaggccca agcacttggg 12797 ccatctttta ctgccttccc aggccataac agagggctgg atcagaagta cagcagctgg 12857 gtctcaaact ggtgcacata cggaatgcca gcactgcagg tggcggcttt atctgctatg 12917 ccacagcact ggccctaata ccttgcatct taatagctgc accaacctcc ccagattcca 12977 gaatatgcct tccacagtct ttcttcaatt ttccttaaaa attgctttta tcttcaccta 13037 actgtgatca aaatttctcc tgcttcccta ttttctgctt cttcagtcta gacctcagcc 13097 tgtctttcag ggacctgtct aaccatactc ggtgtccacc tgtactccca gcactcacag 13157 tgcaatcttc cactccatgc tcggtgtgta gaatcctggc ttcttctgct cctgtgtctc 13217 tcggggaccc atggctatca agttctagtt catagctgct tttttccctg aagctctcca 13277 gtcttcacag agctgccacc catttgaact tccctagtac cggtaagtgg caccactgtt 13337 ttgcactctt gccatggtca tgactgggtc actgatggac aagtgagcac cttgtccttc 13397 tgaccaggta gcaagctgag ctaggagatg agtggcttgc ttctaaaata atgttggtcc 13457 attaaaaaca atcacaaaag aaggtgtggt atttgcaggt gatatactgc aaatatttgt 13517 ttctctcttt tgcttcccag AGAGTCGTGG TTGGAGATGC TTCAGAAACT GCTCTTCTGA 13577 AATTCTCAGA AGTCATTTTG GGTGACGTGA TGGAAATTAG AAAAAGAAAC CACAAAGTAG 13637 TCGAAATCCC TTTTAACTCA ACCAACAAAT TTCAGgtgag catttcctca tagtcgacaa 13697 tctctgttat cactagaaca acatttttac ttgtacacat tctttaatag cccattgttt 13575 gagtaataca tgacttcaaa agtggtcttc agggaacaga gccttggcac ctgttcaacc 13817 caggcaaggt ccattgcttt ccctgatgta acagagggaa aaaagaaaga atctatgcgg 13877 gcctggctgc tgctgtgaat gaccatctct attgcgtcca cactcactca tgctcacaca 13937 cactcataca tgcctccacc accaccacca ccaccaccat caggtaactt ttgcacaggt 13997 gaggagcagc agccctgttt cgtagggtaa atgagaggct ggggaagagg agagacctaa 14057 tttttatggc tcgctcagta cagttgcccc tctgaatctg tgcattcaat attagtgaga 14117 aaaaaatgtt gttgaatttg tgtagacttt tgttcttgtc catattccct aaacaataca 14177 gtacaacagc catcaagcat aacattgaca ttgtgtttgg tagtgtatgt aatctggaga 14237 ttttttaaat ctacaggatt acatgggctc tgcacaaaca ctatcccatt ttaggtgagg 14297 cacttgagca tccccagttc ttagtatctc aagagtcaag ggcgaattcg ttaaacctgc 14357 agatactcct tggatgacta ttttcc Sequence Data from Products Exons 12, 13 and 14 60 aagggtcgga actaagaatc aagcgtagcc aggtggtccg tgctggaaca taccaggtta 120 gatgcatctt ccaggcatga accagactcc gactcccgct gcagcgccat ctgggacagg 180 aaagggtctt cctgtcatgt gggcagtgaa agaancaggc cccatgagac ctgctgaatt 240 ctgactcagg gtgcccctgt nggctggggt gatgcccaga tgctcctagt tggccacaaa 300 aagaaaaggg ctagaacatt cctctgaatg ncgttcccct tcctccttta cacctcaagg 360 acagtcatga aaaagtactg tgtctccaag tcctgtccta ttgctaattg gaagggctgt 420 ggcgagcagg ctacatctaa cgtgccctat tcatgctctc tcAGCTCTCC ATACACCAGA 480 CGGAAGATCC CAATGACAAG CGCTTCCTGC TGGTGATGAA GGGGGCCCCC GAGAGGATCC 540 TAGAGAAGTG CAGCACCATC ATGATCAACG GCAAGGAGCA GCCACTGGAC AAGAGCATGG 600 CCCAGGCCTT CCACACGGCC TACATGGAGC TGGGCGGCCT GGGCGAGCGC GTGCTGGgtg 660 agtgtggggg cacagcccct gtctctcccc cagaggtgcc aaaccgaggc tcaggagctg 720 gtggagtagc tgcagtatcc acgcatcaca gaaagaagaa tatgatcaac agtatagcaa 780 cagataagag gaggcttcgt gaggggtctt caaaagggca tggaaatgca tattaggaaa 840 gaacaataca ggcatttaaa atttttttgc aacagaatga cttctttttt tgaaagattt 900 atttatttga aaggcaaaaa tatacagaga gaggaaaaag agagagtttc actccgctgt 960 ttcactcccc aaatggctgc aatagccaat gctgggccag gctgaagcca ggagccagga 1020 gcttcttctg ggtctcccat gtgggtgcag gggttcaagc ccttggccat cttcctctgc 1080 tttcccagac acattagcag ggagctcgat tggaagcaga gcagccagaa ctcaaagtga 1140 cacctatatg ggatgctggc atgcagacag aggcttaacc ttctgcacca cagcgccagc 1200 ccagaataac ttatttttta attccatttt caagggtctt ttgaagtcca ctaacataat 1260 ggcattgagg agtgtggggt cacttccaga caagcataat cagggaagac ttcctggagg 1320 gagttccgtt t Gap in intron 12

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124 60 gtcttttcca gGTTTCTGCC ATTTCTACCT GCCAGCAGAT GAGTTTCCAG AGACCTACTC 120 ATTTGACTCA GAATCCATGA ACTTCCCCAC CTCCAACTTA TGTTTTGTGG 60 GGCTCTTATC AATGATTGAT CCTCCTCGAT CCACTGTCCC AGATGCAGTC ACCAAATGCC 120 GGAGTGCAGG AATCAAGtgg ccactgagca gtgtgcccag agctcacggg gacag 60 ccttcagctt gcgcgtttta tggcgccggc tcagcacgct cagccccaaa cctctgctct 120 tggcccGTTA TCATGGTTAC AGGTGATCAT CCCATCACAG CCAAAGCCAT TGCCAAGAGT 180 GTAGGGATCA TTTCAGCCAA CAGTGAGACA GTGGAAGACA TTGCAA 60 AACGCTGCAA CATCGCCGTG GAGCAGGTTA ACAAACGgta agcacagacc caatactgcg 120 tacactcagg gtcttactca caggaaccat gctatttgga gcaattgaag caaattgaag 180 caaacctttt ttnaaaaatg tttatttatt tatttattta tttaaaaatc agagttacac 240 agagagagaa ggagaggcag agagagagaa agaagtcttc catccactgg tncactcccc 300 aattggccac aatggtggga gctatgc Gap in intron 14 and to HKa2.8 Sequence Data from Clone HK2.8 Exons 15-23 1 aatggctcta gtttaaactt tctgtttact gtcttgaaat ggagggcaaa gtttccccag 60 tagtcataac ccctgagata gccccagagt attcgcccat ctgggatatt aggttccaaa 120 tggagtcaaa aagaattgag gccacatcca gcctagaacc atttcaacca aagatctgcc 180 cttgggcctc actaggtttc ttctggccct tttagGGATG CCAAGGCCGC CGTGGTGACC 240 GGCATGGAGC TGAAGGACAT GAGCCCAGAA CAGCTGGATG AGCTCTTAGC CAACTACCCG 300 GAAATCGTGT TTGCACGGAC GTCCCCCCAG CAAAAGCTGA TCATCGTGGA GGGCTGTCAG 360 AGGCAGgtgg gtgacagcag cacctggaga atcaactacc ctcagccaca tgtccccagc 420 tctgcatttg atcatgggag tctggggttt cgacagcaac acatttgcca ctgtggcagt 480 gccactgtag aaagatgata aaatagtaag tttcaaaaca aagcttgcag agacatagga 540 tatccaacac ctccatttgc acgcacctta ttcggagctc cttgccttgc atctgagatt 600 tcctgatggt ctcttctccc aacgggacct catcctggcc ccagcaatcc ttgcagcccc 660 aaggacagca ggaggctgtg tgtggctttc gcccgctcac tcactcccct cttcccacgg 720 ggcccccgca gGACGCAGTT GTGGCCGTGA CGGGGGACGG AGTGAATGAC TCCCCCGCTC 780 TAAAGAAGGC CGACATTGGC GGTGCCATGG GGATAACGGG TTCTGACGCG GCCAAGAACG 840 CAGCCGACAT GATCCTGCTG GATGACAACT TCTCCTCTAT CGTCACAGGG GTGGAGGAAG 900 gtgagcgggg tccccaaggt ctgccggagg gccagggtcc acgggaggac tggggacaag 960 ccctctaagg agaaccatct ctgcctagGC CGCTTGATAT TTGACAACCT AAAGAAGACC 1020 ATCGCTTACA CCCTGACCAA GAACATTGCC GAGCTCTGCC CCTTTTTGAT TTACATCATT 1080 CTCGGGCTGC CCCTGCCCAT TGGCACCATC ACCCTCCTGT TCATCGACTT GGGCACAGAC 1140 ATAGtaagtg acactaaagg gaacagggtc cttctgcacc gcaggggagt gcacaggttg 1200 cggttgttac cttggagggc caggagatgg gctgaggccc acgctgctca gacttctctg 1260 caatggagtt caaagttttg cctttctgac aagctcccag gacatagcga ttctgctggg 1320 cccacagacc accacttcaa gggactagag acatttgtca agtgtttgga aattcaaact 1380 tcactgttta cggggatttt taaggcagga gcccctcaaa aaatatcttt tgccaatggt 1440 cagttttagc agccaggcag cccttttctt tggcgcagct gaagggccct gtgctactgg 1500 gggcttggtg gctaatgtga taaccccaca gcgccccctg tggagggaga agaatgggcg 1560 ctcttaaggg cgggccctag ggttctgctc t Gap in intron 17 1 agcatcccac atgggtgcca gttcatgacc cagctgttcc acctccgatc cagctctctg 60 ctatggcctg ggaaggcagt ggaagatggc ccaagtgctt gggcccctgc aactgtgtgg 120 gagacctgga ataagctcct ggctccaggt ttcatattag cccagctcca gccattgcgg 180 ccatttgggg agtgaaccag cggatggaag acctctctct ctgcctctgc ctctgcccct 240 ctgtaacact gcctttcaga taaataacta aatttaaaaa aaaaaaaaag acttcatggg 300 ctatctccct ctccagATCC CCTCCATTGC CTTGGCGTAT GAGAAAGCAG AAAGTGACAT 360 TATGAACAGG AAGCCTCGGC ACAAGAAAAA GGACAGACTG GTGAACCAGC AGCTTGCTGT 420 ATACTCGTAC CTGCACATTG gtacgcctgg gctctcctac tgtcactgga gggcctgtcc 480 caggcacagt cggagagtgt tttcctttgg ggctgaggtt gggatggtag gaggggctga 540 gaggagaaag gatgaggcca tcctctggag gcatcaggga gtctaagccc ttagctctct

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125 600 tgttctttca gGCCTCATGC AAGCCCTGGG AGCTTTCCTG GTGTACTTCA CTGTGTACGC 660 ACAGCAGGGC TTTCGGCCGA CCTCACTGTT TCACCTGCGG ATAGCGTGGG ACAGCGACCA 720 CCTGAACGAC TTGGAAGACA ACTATGGACA GGAATGGgta agcgctgggg cgctctgtgc 780 cagctttgcc tgcttcttct tccctcctct cgctgccctg ccaggactgg ggcagagcct 840 gcattcctcc acatggcctt gacaagaccg actgtggaag cttgctagtt gatagggctg 900 gggacccctg gaggacactg gtttggcctt tgccaggacc cagccttttg tcttgggcca 960 gaggtcaaga gctcaccttt taatgttagc tttgccattg gcgtggaaaa gtaatcatgt 1020 caacacgccc acagttcagc cagagacgtc agcatctttc tctcctcacc tggctgaaga 1080 ccaaagtttc tctctccatc tgggtcaggc agagatgaga ggaaagcttc ccagagatcc 1140 ccccggactc tacaactttc ctccaagctc tcactgtctc ctccttctct gcccatcgac 1200 agACGAGTTA TCAGAGGCAA TACCTGGAAT GGACAGGCTA CACGGCTTTC TTTGTTGGCA 1260 TCATGGTCCA GCAAATAGCA GATCTGATCA TCAGGAAGAC CCGCAAGAAC TCCATCTTCA 1320 AGCAGGGGCT CTTCAGgtat tgctgtatcg gcctcatggg tcagcctcag gcctgcgggc 1380 ttgctggagc ttcctccggc catgcctggc acacacaagg ccttgctcag ctggtggccg 1440 gttctgcgga gggcatgatc acagagccgt gggaactcag aatctgctta ctttgtcttg 1500 cagAAATAAA GTCATCTGGG TGGGGATCGC CTCCCAGATC ATCGTCGCCC TGCTCCTCTC 1560 TTACGGGCTC GGCAGTATCA CAGCCCTAAA TTTCACCATG CTCAAGtgag ttcgccttcc 1740 acagcagcaa ggaaactgac agccctgccc cgagctgtca cagcccaaat aataactagg 1800 atatcccaag gtccctctcc cacccacccc cccttttttt tttaaagatt tatttattta 1860 tttgaaagag ttacacagag aggcagagag agaaagagag tgagagagag aaagaggggt 1920 cttccatctg ctggttcatt ccccaattgg ccgcaatggc cagaggtggg ccaatcttca 1980 gaaaccagag ccaggagctt cttccaggtc tcccacacag ttgcaggggc ccaaggactt 2040 gggccatctt ctactgcttt cccaggccat agcagagagc tggattggaa gtgacacagc 2100 cgggactcca atcggcgtcc atatgggatg ctggcactac aggc Gap in intron 22 1 cctgcttcct gctaatgcag accctgaaag cagcagatca cagttcaagt atttcggtcc 60 ctgctaccca agtgggagac tcagattagc atctgagctc cagacttcag cttggcccag 120 ccctggctat tgcagacatt tagggtgtag accagaagat gggagggccc cctcttactt 180 ctctgtctgt ctctctctct ctctttcaaa taaataaaaa catagaagta ctttcttttt 240 ggacaggcag agttagccaa tgagagagag agacagagag aaaggtcttc cttccttcca 300 ttggttcacc ccccagaatg gccgctacgg ccggcacact gcgccaatcc gaagccagga 360 gtcaggtgct tcctcctggt ctcccatgcg ggtgcagggc ccaagcactt gggccatcct 420 ccactgcctt cctgggccac agcagagagc tggactggaa gaggagcagc cgggacagaa 480 tccgacaccc caaccaggac tagaacccgg ggtgccggtg ccgcaggcag aggattagcc 540 tagtgagcca cggtgccggc cagaagtact tattttaagt acaactaaaa taaggaactt 600 tctggtttga gtgtggtgtg acccagtgga tttggtgtgc ttatcagtaa agcagggcca 660 agggccaagg gcaggaggct ccctgcttgc tggcattcgc acgttccagc gctcatcatc 720 ctgccttctg cctccagGGC TCAGTACTGG TTTGTGGCCG TACCCCATGC CATCCTGATC 780 TGGGTATACG ATGAAATGCG GAAACTCTTC ATCAGGCTCT ACCCCGGAAg tgagtagtgc 840 agcggtatct tggaggttct gtgtgcttcc tctaccagac tctccatttc taatttacct 900 tcttctgact ctctagGCTG GTGGGATAAG AACATGTATT ACTGAGACCA GGTCTGTCTC 960 TGAGTCTCCC AGCGGCACCT GCCTGGTGGT CTTCGGCAAG ACCTCTGTGT AGTGTGGATG 1020 TTGCCAAGCT CCACTCGGGA GGAGACTCTC ATCTAGAACA CAGTGGTGAA GCTTCTTACT 1080 GATCTGTTGT ACTTCAAAGC TGAGATTCAG CTGGTTGTAT ATGATTTTCA TCTCTATCTC 1140 CATCTCCTTA CCTTAAAAGA TGTGGATGTC AAGGTCATGG TGTAGGGAAG GATGTGTTTA 1200 TCTGTATATG AAGCTCACTG ATGTCACACA GACTTGTGTA ACCCAGGTGG CTGCTGGAGT 1260 CTGCCATAAG TTGAGCTAGA ATTGCTCAGA TCTCCTTCCA CACCCTGTCA AAGGCCCGGT 1320 GAGCTCCATA GGATTTCTGT GAATCCCCCT GAAACATAAC TTTTGGGGTT TGCTTTGCTC 1380 AGCTGAGGGT GTGAGTTGGA AGTGTGGCAG CAGGAGCACC TCAGAACAGC AAAGACAGCC 1440 CCCGTTTTGA CTCCCAGACA CTTTGTTGCT GTGATGGGTT CCTGGCCATG CGGCCCCAGT 1500 CCGCCTCTCA CAGCACTCCA CCACCTGTTC CTGCAAAGCT GACCTCCAAG TCCATTCCAC 1560 AAACCTTAAC TCAAACATTC GTGGACCCAA AGGGGCTGTC ACTGACTGGG ACTCGGCCTC 1620 TCCGGAAAGC CACTGTGGTT TAGATAGCAC TATTTATTTC TTGTAGATAG GCTGCCAAGC 1680 ACTCTCCAGC AGCCATTTTA TGTCAATCAC ATTTTTGTAA CTTAGATATA TTTGTGTGGG 1740 ACACGAAACA CATACATCCA TGTTGACAGG TTTTTTTTTT TAAATAAAAG ATGTTTTTAA 1800 GTAAAATGTT TTATGAAACA AAATCTAATT GTGATGTTTT ACTTAATTCA AGTTTTTCCA 1860 GAGGCAGGCA CGGAAAATAC CAAAAAAATA AAATAAAATA Agattctggg ttttttttct 1920 tttttgctcc ttctggtcat tttctttaca cacagagtgt ctggaaatac aggcttttcc 1980 tcgtgagtgc ttcccgcacc tgtgccccct ccccccctca cctgactggg actcctgtgg 2040 gccagatcaa cctgctggca gcagcaccac aggcaaagtc attttcacag actttccatc 2100 aaccacacac ccacactaac tgatttcaca gactttaaaa gctgtacttg gttaaacttt

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126 2160 gcccattcag tggcctctcc acgccagcca cttgcagctg ctgctgcagg cttcagcttg 2220 gcatctctgg gacaggaaag ggagacgtag agtccat

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APPENDIX B TFSEARCH RESULTS ** TFSEARCH ver.1.3 ** (c)1995 Yutaka Akiyama (Kyoto Univ.) Scoring scheme is so straightforward in this version. score = 100.0 ('weighted sum' min) / (max min) The score does not properly reflect statistical significance! Database: TRANSFAC MATRIX TABLE, Rel.3.3 06-01-1998 Query: untitled (6300 bases) Taxonomy: Vertebrate Threshold: 80.0 point TFMATRIX entries with High-scoring: 1 TGGGTACCGG GCCCCCCCTC GAGTTGTAAT CTTGTGTGAT GCTCTTGAAA entry score <--------M00011 Evi-1 89.9 <---M00147 HSF2 86.5 -------------> M00137 Oct-1 84.7 ----M00147 HSF2 84.6 ----------> M00075 GATA-1 84.5 ----M00052 NF-kap 84.1 <-M00074 c-Ets83.8 <-----M00240 Nkx-2. 83.7 <---------M00008 Sp1 83.6 <---M00146 HSF1 82.6 <-M00109 C/EBPb 82.4 <--------M00008 Sp1 82.2 -----> M00271 AML-1a 82.1 ----M00053 c-Rel 81.8 <------M00083 MZF1 81.7 ----------> M00076 GATA-2 80.6 <--M00261 Olf-1 80.6 ----M00146 HSF1 80.4 <----------M00079 Evi-1 80.3 <------M00083 MZF1 80.0 51 TTTCCTAAGG GAATAGATTG GGGGTTGCTT TTATCACAAA AAAAGATTGT entry score ---------> M00077 GATA-3 89.4 -------M00077 GATA-3 88.4 --M00045 E4BP4 87.3 <----------M00203 GATA-X 86.6 ----M00147 HSF2 86.5 ----------> M00106 CDP CR 85.8 <------------M00128 GATA-1 85.7 -------M00162 Oct-1 85.7 M00147 HSF2 84.6 -------> M00148 SRY 84.5 ----> M00052 NF-kap 84.1 ---------M00074 c-Ets83.8 M00039 CREB 83.7 ---M00109 C/EBPb 83.6 <----------M00072 CP2 83.3 -------> M00100 CdxA 83.3 <-------------M00127 GATA-1 83.1 <-----M00148 SRY 82.7 ----M00146 HSF1 82.6 --------M00075 GATA-1 82.4 127

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128 --------------> M00126 GATA-1 82.4 -----------M00109 C/EBPb 82.4 ----------> M00104 CDP CR 82.1 <----M00271 AML-1a 82.1 --M00271 AML-1a 82.1 <------M00100 CdxA 82.1 ------------> M00087 Ik-2 82.0 ----> M00053 c-Rel 81.8 ---------> M00227 v-Myb 81.8 ----------> M00075 GATA-1 81.6 --------M00076 GATA-2 81.0 M00101 CdxA 80.7 <--M00116 C/EBPa 80.7 <--------M00077 GATA-3 80.6 ------------------M00261 Olf-1 80.6 ----> M00146 HSF1 80.4 101 GATGTAATGA TGTGAGAGGA TGGATAAGTC AATTTGCTTA ACTACTTTGT entry score -------------> M00137 Oct-1 93.8 -----------> M00203 GATA-X 93.6 > M00077 GATA-3 88.4 --------------> M00116 C/EBPa 87.3 --------> M00045 E4BP4 87.3 --------------> M00117 C/EBPb 87.2 -------> M00101 CdxA 87.1 <-----M00148 SRY 86.4 <---M00148 SRY 86.4 ----------> M00075 GATA-1 86.1 ----------> M00076 GATA-2 85.8 -----> M00162 Oct-1 85.7 -------M00228 VBP 85.6 -------------> M00159 C/EBP 84.6 ------> M00039 CREB 83.7 ---------> M00109 C/EBPb 83.6 <-------------M00109 C/EBPb 83.6 ----------------> M00099 S8 82.8 > M00075 GATA-1 82.4 ---------> M00199 AP-1 82.1 --> M00271 AML-1a 82.1 <-------------M00162 Oct-1 81.6 <------M00160 SRY 81.1 > M00076 GATA-2 81.0 -------M00260 HLF 80.9 --------> M00041 CRE-BP 80.8 <-----M00137 Oct-1 80.7 ------> M00101 CdxA 80.7 --M00101 CdxA 80.7 ---------M00116 C/EBPa 80.7 ----------> M00106 CDP CR 80.4 <---------M00260 HLF 80.1 151 TATGAATAAG TATACATATT TATTTTATTT TATTTTATTT TATTTTAGGA entry score ------------> M00131 HNF-3b 90.2 -------> M00101 CdxA 90.0 -------> M00100 CdxA 89.7 <-----M00101 CdxA 87.9 <------M00101 CdxA 87.1 -M00148 SRY 86.4 <--------------M00081 Evi-1 85.2 <--------------M00081 Evi-1 85.2 <--------------M00081 Evi-1 85.2 ------------> M00130 HFH-2 85.1 <------M00101 CdxA 85.0 <------M00148 SRY 84.5 <-----M00148 SRY 84.5 <------M00148 SRY 84.5 <-----M00148 SRY 84.5 <------M00148 SRY 84.5 --------> M00199 AP-1 83.0

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129 ------> M00101 CdxA 82.9 <-----M00101 CdxA 82.9 -------> M00101 CdxA 82.9 <------M00101 CdxA 82.9 ------> M00101 CdxA 82.9 <-----M00101 CdxA 82.9 -------> M00101 CdxA 82.9 <------M00101 CdxA 82.9 <------M00223 STATx 82.7 ------------------------> M00138 Oct-1 81.8 <-----------------------M00138 Oct-1 81.1 ---M00160 SRY 81.1 ------------> M00131 HNF-3b 80.9 ------> M00100 CdxA 80.8 -------> M00100 CdxA 80.8 ------> M00100 CdxA 80.8 -------> M00100 CdxA 80.8 ------> M00100 CdxA 80.8 -----M00137 Oct-1 80.7 ---> M00101 CdxA 80.7 ------> M00101 CdxA 80.7 <-----M00101 CdxA 80.7 <----------M00080 Evi-1 80.7 <----------M00080 Evi-1 80.7 <----------M00080 Evi-1 80.7 <----------M00080 Evi-1 80.7 <----------M00082 Evi-1 80.7 <----------M00082 Evi-1 80.7 <----------M00082 Evi-1 80.7 <----------M00082 Evi-1 80.7 ------------> M00130 HFH-2 80.5 <---------------M00136 Oct-1 80.0 201 ACCAGCACTG TGGCTCAGCA GGTTAAGCCA CCATATGCGA AGATAGCATC entry score ----------> M00076 GATA-2 92.1 <----------M00037 NF-E2 90.1 ---------> M00077 GATA-3 86.9 ----------> M00075 GATA-1 86.5 ------> M00271 AML-1a 83.7 <-----M00271 AML-1a 83.4 ------------------> M00059 YY1 83.3 M00223 STATx 82.7 ---------> M00199 AP-1 82.6 <--M00141 Lyf-1 81.8 <-----M00076 GATA-2 81.4 --------------> M00127 GATA-1 80.7 <----M00087 Ik-2 80.3 <-----M00075 GATA-1 80.0 251 CCATAGGAGT GCTGGTTTGG GTCCCAGTAC ATGGGCCCAT GCCATCTACA entry score <--------------M00257 RREB-1 85.1 <---------M00075 GATA-1 84.1 <--M00100 CdxA 83.3 ----M00141 Lyf-1 81.8 <---------M00076 GATA-2 81.8 --------------M00059 YY1 81.7 --M00076 GATA-2 81.4 -----M00087 Ik-2 80.3 --------------> M00033 p300 80.2 --M00075 GATA-1 80.0 301 AAGGAGACCT GGAATTCCAG GCTTCTGGTT TCAGCCTGAC CCAGTCCTAG entry score -----------> M00173 AP-1 84.5 <--------M00199 AP-1 83.4 -----> M00271 AML-1a 83.4 --M00100 CdxA 83.3 <---------M00032 c-Ets82.4 --> M00059 YY1 81.7 <--------------M00133 Tst-1 81.2 ----------> M00053 c-Rel 81.0 <--------M00053 c-Rel 81.0

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130 <------M00101 CdxA 80.7 ------> M00101 CdxA 80.7 <---------M00108 NRF-2 80.7 ---------> M00199 AP-1 80.6 ----------------> M00222 Th1/E4 80.4 -----------------> M00222 Th1/E4 80.0 351 CTATTATGGC CATTTGGAGA GTGGACCAGA GGATAGACTA TTTCTCTCCT entry score ------> M00101 CdxA 90.7 ----------> M00076 GATA-2 90.5 <------M00241 Nkx-2. 88.2 ------------------> M00059 YY1 86.5 <-----M00101 CdxA 86.4 -------------> M00159 C/EBP 85.4 ----------> M00075 GATA-1 84.5 <----M00271 AML-1a 83.4 -------> M00101 CdxA 82.1 -------> M00100 CdxA 82.1 --------> M00083 MZF1 80.0 <--M00083 MZF1 80.0 401 CTCTCTCTCT CTCTCTCTGT AACTCTGACT TTCAAATAAA TAAATTATAA entry score ------> M00101 CdxA 98.6 <---M00101 CdxA 92.9 <------M00101 CdxA 90.0 <------M00101 CdxA 90.0 <------M00100 CdxA 89.7 <------M00100 CdxA 89.7 <-----------M00131 HNF-3b 89.6 --------------> M00267 XFD-1 88.3 <-----------M00130 HFH-2 88.2 ------M00216 TATA 87.4 ---------------M00099 S8 87.3 <---M00100 CdxA 87.2 -------> M00101 CdxA 87.1 ---------> M00096 Pbx-1 86.3 ------> M00148 SRY 84.5 -------> M00148 SRY 84.5 <-M00101 CdxA 84.3 ------> M00100 CdxA 83.3 <-M00100 CdxA 83.3 <-----------M00129 HFH-1 81.5 <-----M00101 CdxA 81.4 <-----M00161 Oct-1 81.1 <-----------M00130 HFH-2 80.9 ---------> M00096 Pbx-1 80.4 -----M00099 S8 80.4 <-M00137 Oct-1 80.4 -----M00252 TATA 80.1 ---M00083 MZF1 80.0 451 AAATTATAAA TATATTAGAT AATGTATAAT AGAATAGAAA AATAAATAAT entry score <------M00101 CdxA 100.0 ------> M00101 CdxA 98.6 -----M00101 CdxA 98.6 <------M00100 CdxA 96.2 -M00101 CdxA 92.9 <----------M00130 HFH-2 91.6 -------> M00101 CdxA 91.4 <------M00101 CdxA 91.4 <----------M00131 HNF-3b 90.2 <-----M00101 CdxA 90.0 <-----M00100 CdxA 89.7 <----------M00129 HFH-1 89.2 --> M00216 TATA 87.4 > M00099 S8 87.3 ---------> M00077 GATA-3 87.2 -M00100 CdxA 87.2 -------> M00101 CdxA 87.1 <------M00101 CdxA 87.1 ------> M00101 CdxA 87.1

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131 <-----------M00045 E4BP4 86.3 -------> M00101 CdxA 85.7 -------> M00101 CdxA 85.7 <------M00101 CdxA 85.7 -------> M00101 CdxA 85.7 -------M00137 Oct-1 85.5 ------------M00267 XFD-1 85.0 -------> M00148 SRY 84.5 -------M00042 Sox-5 84.3 -----M00096 Pbx-1 84.3 ---M00101 CdxA 84.3 < M00101 CdxA 84.3 -----------> M00203 GATA-X 83.9 ---M00100 CdxA 83.3 ------> M00100 CdxA 83.3 < M00100 CdxA 83.3 --------------> M00126 GATA-1 83.1 ----------> M00260 HLF 83.1 --M00101 CdxA 82.9 <-M00101 CdxA 82.9 --------> M00096 Pbx-1 82.4 -------------> M00128 GATA-1 82.1 --------------> M00116 C/EBPa 81.9 <-----M00101 CdxA 81.4 -----------> M00080 Evi-1 81.4 <----M00101 CdxA 81.4 ---------> M00096 Pbx-1 81.4 ----------> M00228 VBP 81.4 ------M00161 Oct-1 81.1 --------M00160 SRY 81.1 <---------------M00026 RSRFC4 80.9 <-M00100 CdxA 80.8 ------> M00101 CdxA 80.7 ----------> M00076 GATA-2 80.6 ---------> M00099 S8 80.4 ---------M00137 Oct-1 80.4 -------------> M00137 Oct-1 80.4 -----------> M00079 Evi-1 80.3 <--M00130 HFH-2 80.2 --------> M00252 TATA 80.1 ---M00099 S8 80.1 -----------> M00082 Evi-1 80.1 --------------> M00267 XFD-1 80.0 <-----M00101 CdxA 80.0 501 AAAAATTATT TCCTAAAGGC AGAGAGAGAT AGACAAAGAC AGATTTCCCA entry score > M00101 CdxA 98.6 M00130 HFH-2 91.6 M00131 HNF-3b 90.2 M00129 HFH-1 89.2 <-------M00087 Ik-2 88.6 ------> M00101 CdxA 88.6 <-----M00101 CdxA 88.6 <---M00075 GATA-1 88.6 ---------> M00077 GATA-3 86.6 <---M00076 GATA-2 86.2 <-----M00100 CdxA 85.9 ----> M00137 Oct-1 85.5 -> M00267 XFD-1 85.0 ----------> M00076 GATA-2 85.0 <--------M00141 Lyf-1 84.4 -> M00042 Sox-5 84.3 --> M00096 Pbx-1 84.3 -----M00101 CdxA 84.3 <--------M00109 C/EBPb 83.6 -----M00100 CdxA 83.3 ------------> M00131 HNF-3b 83.2 --------> M00227 v-Myb 82.9 ---> M00101 CdxA 82.9 ---M00101 CdxA 82.9 -------> M00148 SRY 82.7

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132 <------------M00074 c-Ets82.6 ----------> M00075 GATA-1 82.4 M00101 CdxA 81.4 <-----------M00087 Ik-2 81.1 --> M00160 SRY 81.1 ---M00100 CdxA 80.8 -----------> M00203 GATA-X 80.6 <--M00032 c-Ets80.4 -------M00130 HFH-2 80.2 ------------> M00099 S8 80.1 ------> M00148 SRY 80.0 551 TCCTGAGGTT TACTCCCCAA ATGGCCACAA CAGCTGAACC AGGAGCCTGA entry score -----> M00271 AML-1a 88.7 <-------M00083 MZF1 88.7 --M00087 Ik-2 88.6 ----M00075 GATA-1 88.6 <----------------M00059 YY1 86.5 ----M00076 GATA-2 86.2 <-------------M00033 p300 85.1 <----M00271 AML-1a 83.7 <-----------M00001 MyoD 83.7 ---M00109 C/EBPb 83.6 <--------M00141 Lyf-1 83.1 <--M00100 CdxA 82.1 <-----M00271 AML-1a 81.4 <------------M00159 C/EBP 80.8 <------M00100 CdxA 80.8 <-------M00141 Lyf-1 80.5 -----M00032 c-Ets80.4 601 AATTCTATTC AGGTCTCCCA CATGGAGTCC TTTAGAATAA ATAAACCTAT entry score ------> M00101 CdxA 92.9 <------M00101 CdxA 90.0 M00101 CdxA 87.1 M00130 HFH-2 87.0 --------> M00217 USF 86.4 ---------> M00096 Pbx-1 86.3 <-----------M00130 HFH-2 85.9 <-----M00101 CdxA 85.7 <-----------M00131 HNF-3b 85.5 -------> M00148 SRY 84.5 <-----------M00087 Ik-2 83.8 <--------M00227 v-Myb 83.4 ------------> M00055 N-Myc 83.0 M00101 CdxA 82.9 < M00101 CdxA 82.9 <-----M00271 AML-1a 82.7 M00129 HFH-1 82.3 <-------M00217 USF 82.1 --M00100 CdxA 82.1 -------> M00100 CdxA 82.1 <-----------M00130 HFH-2 80.9 <------M00100 CdxA 80.8 M00100 CdxA 80.8 -----M00131 HNF-3b 80.3 <----------------M00059 YY1 80.2 <--M00082 Evi-1 80.1 --------------> M00267 XFD-1 80.0 651 TTTATTTATT TGAAAGGCAG AGTTACAGAG TGAGAGTGAC AGAGATGGAG entry score ------------M00267 XFD-1 90.0 ------> M00101 CdxA 90.0 ------> M00100 CdxA 89.7 -----------> M00131 HNF-3b 87.9 -----------> M00173 AP-1 87.6 --------> M00077 GATA-3 87.2

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133 <-----M00101 CdxA 87.1 -----------> M00130 HFH-2 87.0 <-------M00096 Pbx-1 86.3 ---------> M00075 GATA-1 85.7 <-----M00148 SRY 84.5 <------M00148 SRY 84.5 -----> M00101 CdxA 82.9 -----M00101 CdxA 82.9 -----------> M00129 HFH-1 82.3 ---------> M00076 GATA-2 82.2 -----> M00100 CdxA 80.8 ----------> M00228 VBP 80.5 -----> M00131 HNF-3b 80.3 ------M00082 Evi-1 80.1 701 AGAGATTGCC CATCCTCAGG TCCACTCACC AAAAGCCTGT AACAACCAGG entry score <---------M00075 GATA-1 89.4 <---------M00076 GATA-2 89.3 --------------> M00116 C/EBPa 84.9 <------------M00084 MZF1 84.7 <-------------M00109 C/EBPb 84.3 <------------M00159 C/EBP 83.8 --M00008 Sp1 83.6 ---------> M00075 GATA-1 82.9 -------> M00148 SRY 82.7 <-----M00271 AML-1a 82.7 ---------> M00076 GATA-2 82.6 -----------> M00073 deltaE 82.6 -----------> M00072 CP2 81.2 --------> M00077 GATA-3 80.9 --------------> M00117 C/EBPb 80.0 751 GCTGGGCCAG GCCAAAGTCA GGAGCTGGAA TCTCAATCCA CGTTGCCCGC entry score ------> M00008 Sp1 83.6 <---------M00075 GATA-1 82.4 -----------> M00072 CP2 81.2 801 ATGGGTGTCA GGGATCGATG TACTTTAGTC AACACATATT ACCTCCCAGG entry score <--------M00106 CDP CR 99.5 <--------M00104 CDP CR 91.0 ----------> M00106 CDP CR 87.2 <-------M00141 Lyf-1 84.4 --------------> M00269 XFD-3 83.6 <------------M00159 C/EBP 82.3 <----M00271 AML-1a 81.7 <----------M00173 AP-1 81.4 <------M00148 SRY 80.9 ---------> M00199 AP-1 80.7 851 GTGCACATTA GTGGGAAACT GATGTAGAGA GTGGATCTGG GACCCAAACC entry score <------M00101 CdxA 86.4 -------> M00148 SRY 84.5 -------> M00101 CdxA 83.6 --------> M00083 MZF1 82.6 ------------> M00087 Ik-2 82.5 ----------> M00075 GATA-1 80.8 ------------> M00087 Ik-2 80.3 --------> M00083 MZF1 80.0 ----M00148 SRY 80.0 901 CAGGCAAACT GATAGGGGAC ACAGCCATCC CAAGCAGTGG CTTAACCACT entry score ---------> M00077 GATA-3 93.4 ----------> M00075 GATA-1 91.8 --------------> M00126 GATA-1 91.5 -------------> M00128 GATA-1 91.5 <---------M00076 GATA-2 90.9 ----------> M00076 GATA-2 90.1 <-----------M00087 Ik-2 89.5 -------> M00083 MZF1 87.8 <---------M00075 GATA-1 87.3 <----M00271 AML-1a 85.4

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134 -------> M00148 SRY 84.5 --------------> M00127 GATA-1 83.1 <--------M00141 Lyf-1 83.1 -----------> M00203 GATA-X 82.4 <----------M00159 C/EBP 80.8 -> M00148 SRY 80.0 951 GGGCCAAACA CCCACCCTTA TCTGAGCATA TTAACACAGC ATGTTGTACA entry score <------------M00128 GATA-1 95.1 <--------M00077 GATA-3 91.9 <-------------M00126 GATA-1 90.1 <----------M00203 GATA-X 89.2 -------> M00148 SRY 89.1 <------M00101 CdxA 87.9 -------> M00101 CdxA 87.9 <---------M00076 GATA-2 87.7 <-------------M00127 GATA-1 86.7 <------M00050 E2F 86.2 <---------M00075 GATA-1 82.4 <----M00271 AML-1a 81.7 -----> M00271 AML-1a 81.4 <--------------M00136 Oct-1 80.8 -M00159 C/EBP 80.8 <------------M00159 C/EBP 80.8 <---------------M00081 Evi-1 80.2 1001 CCTCCTATAC AGTCAACTTT TTAAAGGTCT GTCTTAGCCC CTTGCAGGCC entry score <---------M00216 TATA 91.7 M00073 deltaE 88.5 <------M00100 CdxA 84.6 <-M00002 E47 82.7 < M00001 MyoD 81.4 <-------M00141 Lyf-1 80.5 1051 TTCACCTGCC TCTGAAAACA CCACAGCTCA CGGTCCTGGA GGTCTCTAAC entry score <-----M00271 AML-1a 100.0 -------> M00148 SRY 89.1 ---------> M00073 deltaE 88.5 -------> M00217 USF 86.9 -----M00227 v-Myb 86.1 ------------M00002 E47 82.7 -----------M00001 MyoD 81.4 ---------> M00227 v-Myb 80.7 1101 AGCCCCAGGA AAGATCAACA CAGCAGATTA CCAGTGATTT TTGGAAAGCG entry score -------------> M00159 C/EBP 89.2 -------> M00100 CdxA 87.2 --> M00227 v-Myb 86.1 ----------> M00147 HSF2 84.6 --------------> M00109 C/EBPb 84.3 <------------M00137 Oct-1 83.6 M00101 CdxA 83.6 <------------------------M00250 Gfi-1 83.3 -------> M00101 CdxA 82.1 ---------> M00141 Lyf-1 81.8 ---------> M00141 Lyf-1 81.8 <-----M00271 AML-1a 81.7 ------------> M00087 Ik-2 81.1 ----------> M00076 GATA-2 81.0 ----------> M00075 GATA-1 80.8 ----------> M00075 GATA-1 80.4 1151 TGTTTCTCTC CTGCAGGATG TTTACATGCC AGATATCCTA TAAACGAAGA entry score ----------> M00076 GATA-2 89.7 <--------M00076 GATA-2 89.7 ------> M00148 SRY 88.2 <------M00101 CdxA 87.9 <------M00148 SRY 87.3 <------M00100 CdxA 87.2 ----------> M00216 TATA 86.8

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135 ----------> M00032 c-Ets86.3 -------------> M00074 c-Ets85.8 ----------> M00075 GATA-1 85.3 <---------------M00222 Th1/E4 84.3 <--M00101 CdxA 84.3 ----------> M00075 GATA-1 84.1 ----M00148 SRY 83.6 <-------------M00267 XFD-1 83.3 ----------> M00076 GATA-2 83.0 --------------> M00162 Oct-1 81.6 -------------M00252 TATA 80.9 <------M00101 CdxA 80.7 M00109 C/EBPb 80.5 ------------> M00129 HFH-1 80.0 1201 AATGAGGAAA CCATATAAAA GTGTGCTAGG CTGCAGCTTT GTTTTGCTTT entry score <------M00148 SRY 100.0 <-----M00101 CdxA 92.9 ----------> M00216 TATA 89.9 <-----M00100 CdxA 87.2 ---------------> M00252 TATA 86.9 <-----M00148 SRY 86.4 M00271 AML-1a 81.7 <-------------M00162 Oct-1 81.6 -------> M00240 Nkx-2. 81.4 -> M00252 TATA 80.9 -------------> M00109 C/EBPb 80.5 1251 TGGTTCTCAT TCTGACTCCA ACTCCAGGCA GTCCCCATTG GCAGAGAAAT entry score <------M00083 MZF1 93.9 ----M00148 SRY 85.5 <-------------M00033 p300 85.1 -M00062 IRF-1 83.6 ---> M00271 AML-1a 82.7 -----------> M00173 AP-1 82.5 <-----M00101 CdxA 82.1 <-----M00100 CdxA 82.1 <-----------M00087 Ik-2 82.0 <------------M00137 Oct-1 81.5 <-------M00199 AP-1 81.1 <---------M00054 NF-kap 81.0 <---------M00051 NF-kap 80.9 <---------M00053 c-Rel 80.2 -------> M00101 CdxA 80.0 1301 GCCGGCCTAG TCATAGCCTG GCACGCTGGG CAGAAGCAGG TGTGCAGGAA entry score ---------------> M00002 E47 89.4 ----------------> M00071 E47 86.2 <-------M00217 USF 85.5 ------M00032 c-Ets83.3 <----------M00073 deltaE 82.1 ---------M00025 Elk-1 81.7 --------------> M00122 USF 81.1 <-------------M00122 USF 81.1 ------M00108 NRF-2 80.7 1351 GGGCTCCAGG TGGGGATGTC CAGTGTGTGG GAGAGGAACC CAGGGAGAGG entry score --------> M00083 MZF1 98.3 <----------M00073 deltaE 90.3 ---------> M00141 Lyf-1 88.3 ----------> M00076 GATA-2 87.4 ----------> M00053 c-Rel 86.8 ----------> M00051 NF-kap 85.8 ----------> M00052 NF-kap 85.4 ----------> M00054 NF-kap 85.4

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136 ----------> M00075 GATA-1 85.3 <-------M00217 USF 85.0 -------> M00240 Nkx-2. 83.7 --> M00032 c-Ets83.3 ------> M00271 AML-1a 82.7 ---> M00025 Elk-1 81.7 ------------> M00001 MyoD 81.4 -----------M00033 p300 81.2 --> M00108 NRF-2 80.7 ------------> M00087 Ik-2 80.7 -------M00084 MZF1 80.6 -M00084 MZF1 80.6 --------> M00217 USF 80.6 <-----------M00055 N-Myc 80.6 ----M00033 p300 80.2 1401 GAGAGGGAGA GGGCAGGTGG GCAGTAGAGG CACCAGCCAG TCCGGATACG entry score --------M00076 GATA-2 93.3 --------M00075 GATA-1 91.8 ----------------> M00002 E47 90.4 <------M00217 USF 89.4 <----------M00073 deltaE 84.7 ---------> M00032 c-Ets84.3 -------M00077 GATA-3 82.8 ---------M00128 GATA-1 82.1 ---------------> M00122 USF 81.9 <--------------M00122 USF 81.9 <-----------M00055 N-Myc 81.6 ------------> M00001 MyoD 81.4 M00025 Elk-1 81.3 --> M00033 p300 81.2 ------------M00074 c-Ets81.0 -----------M00126 GATA-1 81.0 --------> M00083 MZF1 80.9 ----> M00084 MZF1 80.6 -----------> M00084 MZF1 80.6 ------M00203 GATA-X 80.6 --------> M00033 p300 80.2 1451 GAGCAGGAAG CAGAGCTTCC TCTTGTGCTC ATCACCTGGA TCCTCCTCCC entry score > M00076 GATA-2 93.3 > M00075 GATA-1 91.8 -----------> M00073 deltaE 90.8 ----------> M00032 c-Ets90.2 <---------M00075 GATA-1 88.6 <------------M00074 c-Ets84.6 <------M00217 USF 82.8 > M00077 GATA-3 82.8 -------> M00217 USF 82.6 --> M00128 GATA-1 82.1 ---------------> M00122 USF 81.9 <--------------M00122 USF 81.9 <-----M00083 MZF1 81.7 -----> M00271 AML-1a 81.7 -----------> M00220 SREBP81.6 <------------M00001 MyoD 81.4 -------------> M00025 Elk-1 81.3 > M00074 c-Ets81.0 <---------M00076 GATA-2 81.0 --> M00126 GATA-1 81.0 <---------M00076 GATA-2 80.6 ---> M00203 GATA-X 80.6 --M00131 HNF-3b 80.3 -------------> M00074 c-Ets80.2 1501 TGTATGTTTG GATCATCAGT CACACCCCAA GCCAGCTGGG GGACAGCTCC entry score <-----M00148 SRY 90.9 <----M00271 AML-1a 87.4 --------> M00083 MZF1 85.2 ---------> M00199 AP-1 82.5 M00083 MZF1 81.7

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137 -------------> M00159 C/EBP 81.5 -----------> M00072 CP2 81.2 --------> M00131 HNF-3b 80.3 <-------------M00033 p300 80.2 1551 AAGAGGGCTC CCCCAACTAA AGCCCCACCT CAACGGCCCT TTTTGCCTCT entry score ---------> M00227 v-Myb 91.4 -----------> M00073 deltaE 90.0 <-----M00271 AML-1a 88.7 <------------------M00004 c-Myb 87.1 <-------M00083 MZF1 85.2 <------M00083 MZF1 83.5 -------> M00100 CdxA 83.3 ------------> M00131 HNF-3b 82.1 <---M00127 GATA-1 81.9 <---------M00008 Sp1 80.8 <---------M00051 NF-kap 80.2 1601 TGTCCACCTT CCTTGGAGGG TGCGAAGGGA TAGAAAAGTA TCTGAATAAT entry score -----M00101 CdxA 98.6 <-M00101 CdxA 90.0 <-M00100 CdxA 89.7 <---------M00076 GATA-2 87.4 <-----M00101 CdxA 87.1 ----------> M00076 GATA-2 84.6 -----M00096 Pbx-1 84.3 ---------> M00077 GATA-3 83.8 -------M00137 Oct-1 83.6 <-------M00199 AP-1 83.5 -----------> M00073 deltaE 82.9 <---------M00075 GATA-1 82.4 --------M00127 GATA-1 81.9 -------M00042 Sox-5 81.7 <----M00101 CdxA 81.4 <----------M00072 CP2 81.2 ----------> M00075 GATA-1 80.4 M00101 CdxA 98.6 ----------> M00075 GATA-1 96.7 ---------> M00077 GATA-3 92.2 ----------> M00076 GATA-2 90.9 ---M00101 CdxA 90.0 ---M00100 CdxA 89.7 --------------> M00159 C/EBP 84.6 --> M00096 Pbx-1 84.3 ----> M00137 Oct-1 83.6 M00042 Sox-5 81.7 M00101 CdxA 81.4 <----M00011 Evi-1 80.4 -----------M00074 c-Ets80.2 ------> M00101 CdxA 80.0 1701 TGTTGCTGTA ACAGAAAGCT AGAAACTGGG TGATTTATAA AGAAGATGGT entry score ------> M00101 CdxA 100.0 ------> M00100 CdxA 96.2 <------M00100 CdxA 92.3 --------M00075 GATA-1 89.8 < M00100 CdxA 88.5 <------M00101 CdxA 87.9 --------M00076 GATA-2 87.0 <-----M00100 CdxA 85.9 < M00101 CdxA 84.3 ----------> M00075 GATA-1 83.7 ---------> M00227 v-Myb 82.9 <--------------M00252 TATA 82.7 ----M00148 SRY 82.7 <--------M00096 Pbx-1 82.4 ---------------> M00252 TATA 82.2

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138 <---------M00228 VBP 82.2 -------------> M00137 Oct-1 82.2 <------M00101 CdxA 82.1 <-----M00101 CdxA 81.4 -------> M00148 SRY 80.9 ---------> M00223 STATx 80.8 <---------M00216 TATA 80.6 --M00011 Evi-1 80.4 <------M00148 SRY 80.0 1751 TTAAATCGGC TCGTGGTCCT AGAGACTGGG AGATTTGAGG TTAAGGGGCA entry score M00075 GATA-1 89.8 ------> M00271 AML-1a 88.7 -----M00100 CdxA 88.5 M00076 GATA-2 87.0 <-M00025 Elk-1 87.0 <-M00074 c-Ets87.0 --------> M00041 CRE-BP 86.2 <--M00054 NF-kap 86.0 ----M00053 c-Rel 86.0 <---M00007 Elk-1 85.4 ---M00054 NF-kap 84.7 -----M00101 CdxA 84.3 <----M00033 p300 84.2 -----> M00271 AML-1a 84.1 <-------M00217 USF 83.5 ------> M00100 CdxA 83.3 ----M00054 NF-kap 83.2 ----------> M00075 GATA-1 82.9 ---------> M00141 Lyf-1 81.8 ----M00052 NF-kap 81.5 ------> M00240 Nkx-2. 81.4 M00077 GATA-3 80.3 <-----------M00055 N-Myc 80.2 <----------M00073 deltaE 80.0 1801 CTTCCCGTGC AGGTTTCTTG CCTGTGGGGA CTTTCTGCAG AGTCCCAAGG entry score -------> M00083 MZF1 95.7 -------M00032 c-Ets90.2 -------M00108 NRF-2 87.7 -----------M00025 Elk-1 87.0 ---------M00074 c-Ets87.0 -------> M00217 USF 86.3 ----------> M00054 NF-kap 86.3 -----M00054 NF-kap 86.0 ----> M00053 c-Rel 86.0 ------> M00100 CdxA 85.9 -----------M00007 Elk-1 85.4 ------------> M00208 NF-kap 85.1 -----> M00054 NF-kap 84.7 -------M00033 p300 84.2 ----------> M00052 NF-kap 83.4 ----> M00054 NF-kap 83.2 -----> M00271 AML-1a 82.7 ----------> M00053 c-Rel 82.6 <-----------M00208 NF-kap 82.5 <---------M00146 HSF1 82.2 <-----------M00087 Ik-2 82.0 ----------> M00053 c-Rel 81.8 ----> M00052 NF-kap 81.5 ------> M00101 CdxA 81.4 ----M00240 Nkx-2. 81.4 ------> M00208 NF-kap 80.7 -----------> M00037 NF-E2 80.2 1851 CAGTGCAGGG TAAGGGAAGG ATACATCCAA CAGAGTCAAG AGGACTTTTA entry score ----M00101 CdxA 92.9

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139 <----M00216 TATA 89.0 <-----------M00252 TATA 87.8 ----M00100 CdxA 87.2 -------> M00240 Nkx-2. 83.7 ---------> M00227 v-Myb 83.4 ----------> M00076 GATA-2 82.2 -----------> M00203 GATA-X 81.8 <----------M00173 AP-1 80.4 -------------> M00087 Ik-2 80.3 1901 TATTAGACCC GCTCTCTAGA TAATTCACTA AACCATTAAT CCAATAATGA entry score <-----M00101 CdxA 99.3 ------> M00101 CdxA 97.9 -> M00101 CdxA 92.9 ---------M00137 Oct-1 91.3 <----------------M00099 S8 90.0 ---M00216 TATA 89.0 <-----M00100 CdxA 88.5 -------> M00101 CdxA 87.9 --M00252 TATA 87.8 -> M00100 CdxA 87.2 <---------M00147 HSF2 87.2 ----M00101 CdxA 87.1 <------M00101 CdxA 86.4 -----------> M00203 GATA-X 86.4 <-----------M00131 HNF-3b 85.5 <-----M00101 CdxA 83.6 --------> M00241 Nkx-2. 82.4 <------M00241 Nkx-2. 82.4 <------M00240 Nkx-2. 81.4 ---------> M00096 Pbx-1 81.4 ----------> M00075 GATA-1 80.8 -------> M00100 CdxA 80.8 -----M00109 C/EBPb 80.5 <-----M00101 CdxA 80.0 1951 TGAAAGTCAG AGTCCTGAGA ACTCATCAAC TATTAAAGTC CCTCTCCATG entry score <-----M00101 CdxA 92.9 --> M00137 Oct-1 91.3 -> M00101 CdxA 87.1 ----------> M00216 TATA 85.9 <--------M00075 GATA-1 84.1 ------> M00101 CdxA 83.6 <----M00008 Sp1 82.2 ----------------> M00252 TATA 81.5 ------> M00100 CdxA 80.8 -------> M00109 C/EBPb 80.5 2001 CCTCTACAAT GGGGATTACA TTTCAATGTG AGAGCTGCAG ACATTTGGCC entry score --------> M00083 MZF1 93.9 <-------------M00162 Oct-1 91.8 <------------M00249 CHOP-C 89.8 ------------> M00087 Ik-2 86.4 ---M00008 Sp1 82.2 ----------------> M00133 Tst-1 81.2 ----------> M00042 Sox-5 81.0 ---------> M00076 GATA-2 80.6 2051 TAGGAGCTAA GATGCCAGTT CCATTACCCT GTCCCATATG GGATTGCCTG entry score ------------> M00087 Ik-2 89.5 ----------> M00076 GATA-2 87.4 ----------> M00053 c-Rel 84.3 -------------> M00086 Ik-1 83.9 ----------> M00052 NF-kap 83.4 ----------> M00075 GATA-1 82.9 <------------M00137 Oct-1 81.5 ----------> M00076 GATA-2 81.0 <---------M00008 Sp1 80.8 2101 GATTCAATTC TCATCTCCAG TTACTGATTC CTGCCAGTGC AGGCTCTGGG entry score -----M00141 Lyf-1 93.5

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140 <---------M00075 GATA-1 84.5 <---------M00003 v-Myb 82.8 ----------> M00075 GATA-1 81.6 -------> M00101 CdxA 81.4 -------> M00101 CdxA 81.4 <--------M00077 GATA-3 81.2 -------> M00101 CdxA 80.7 <---------M00032 c-Ets80.4 <----------M00203 GATA-X 80.2 2151 AGGCAGCAGG GACGGCTCAA GAAGTTGAGT CTCTGCCACC CAATAAGGGG entry score --> M00141 Lyf-1 93.5 -----M00083 MZF1 90.4 ------> M00101 CdxA 82.9 2201 GACCTGGATG GAGTTCCCAG CTCCCAGCCC CACCAACAGC CCAGACGTGG entry score -> M00083 MZF1 90.4 <---------M00008 Sp1 90.4 <-----------M00087 Ik-2 86.8 ----------> M00075 GATA-1 86.1 <------------------M00004 c-Myb 85.2 <-------------M00088 Ik-3 83.8 <-------M00083 MZF1 83.5 ----------> M00052 NF-kap 83.4 ----------> M00076 GATA-2 81.8 <------M00217 USF 81.5 M00032 c-Ets81.4 <-------------M00086 Ik-1 81.3 ----------> M00054 NF-kap 80.7 --------> M00227 v-Myb 80.2 2251 CAGGAATTCT AGGTATGAGC TAGTGAATGG GAGCTCTCGT TCTCTATCAT entry score -------> M00101 CdxA 92.9 <-------M00075 GATA-1 90.6 <-------M00076 GATA-2 89.7 <-------M00077 GATA-3 89.1 <----------M00128 GATA-1 83.6 ------------M00085 ZID 82.2 M00087 Ik-2 81.6 <-----M00101 CdxA 81.4 --------> M00032 c-Ets81.4 <--M00127 GATA-1 80.7 <--M00126 GATA-1 80.3 <----------M00203 GATA-X 80.0 2301 CTATCTATCT ATCTATCTAT CTATCTATCT ATCTACCTAC CTACCTACCT entry score <--M00077 GATA-3 91.6 M00075 GATA-1 90.6 M00076 GATA-2 89.7 <--M00076 GATA-2 89.3 <--M00075 GATA-1 89.0 <----M00128 GATA-1 86.0 <----M00126 GATA-1 85.9 --M00145 Brn-2 84.2 <---------M00075 GATA-1 84.1 <---------M00076 GATA-2 83.8 -M00128 GATA-1 83.6 > M00085 ZID 82.2 <-------------M00127 GATA-1 81.9 ------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <-------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <--------M00077 GATA-3 81.9 <------------M00128 GATA-1 81.2 <-----M00203 GATA-X 80.9 ---------M00127 GATA-1 80.7

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141 <------------M00128 GATA-1 80.5 <------------M00128 GATA-1 80.5 <-------------M00128 GATA-1 80.5 <------------M00128 GATA-1 80.5 <------------M00128 GATA-1 80.5 <------------M00128 GATA-1 80.5 ---------M00126 GATA-1 80.3 2351 ATCTCTAATA AACAATTTTA ACATGAGATT TGGTGGGGAC ATTCAAACCA entry score -------> M00083 MZF1 98.3 ----------> M00042 Sox-5 94.1 ----M00077 GATA-3 91.6 -------> M00148 SRY 90.0 -----M00076 GATA-2 89.3 <-----------M00130 HFH-2 89.3 <-----------M00129 HFH-1 89.2 -----M00075 GATA-1 89.0 ---------> M00077 GATA-3 86.2 ------M00128 GATA-1 86.0 -------M00126 GATA-1 85.9 <--------------M00162 Oct-1 85.7 ------------> M00160 SRY 85.4 -------------> M00145 Brn-2 84.2 <-----------M00131 HNF-3b 83.8 <--M00271 AML-1a 83.4 ------> M00101 CdxA 82.9 ------> M00271 AML-1a 82.7 ----------> M00075 GATA-1 82.0 ------M00042 Sox-5 81.7 --------------> M00267 XFD-1 81.7 <----------M00072 CP2 81.2 ------------> M00208 NF-kap 81.2 ---M00203 GATA-X 80.9 <------M00100 CdxA 80.8 -------> M00101 CdxA 80.7 ----------> M00076 GATA-2 80.6 ------> M00101 CdxA 80.0 <-------------M00267 XFD-1 80.0 2401 TAGCGGTCCC CCCAAGCAAT CTCTTTCCCT TAATTTCTTC CAGCACTTAC entry score <-------M00083 MZF1 92.2 --------> M00241 Nkx-2. 91.2 ---------> M00223 STATx 90.4 <-----M00240 Nkx-2. 88.4 <---------M00075 GATA-1 84.5 <------M00101 CdxA 84.3 ------> M00101 CdxA 84.3 <----------------M00099 S8 84.1 -M00271 AML-1a 83.4 <---------M00076 GATA-2 83.0 <-----------M00244 NGFI-C 82.8 <------M00101 CdxA 82.1 --> M00042 Sox-5 81.7 <------M00240 Nkx-2. 81.4 <---------M00032 c-Ets81.4 <--------M00077 GATA-3 80.9 <-----------M00246 Egr-2 80.8 <-----------M00243 Egr-1 80.7 <------M00148 SRY 80.0 2451 AGCCTGATAA TGACAACTGC ACACTGTATT ATTTCTTACC CTGCCTTATT entry score <------M00101 CdxA 98.6 -------------> M00137 Oct-1 93.5 ----------> M00075 GATA-1 89.8 -------------> M00128 GATA-1 89.4 --------------> M00127 GATA-1 89.2 -----------> M00203 GATA-X 87.5 ----------> M00076 GATA-2 87.0 <---------M00008 Sp1 86.3 -------> M00101 CdxA 85.7 ---------> M00077 GATA-3 85.3

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142 ------> M00101 CdxA 84.3 <-----M00101 CdxA 82.9 <---------M00042 Sox-5 81.7 -------> M00101 CdxA 81.4 <----M00199 AP-1 80.9 <---------------M00081 Evi-1 80.2 <------M00267 XFD-1 80.0 2501 CACTAGTGCT GCTTGGTTAA CTTGGCCTCT CCAACTCCAG CCCAACTTCC entry score <-----M00032 c-Ets88.2 <------M00074 c-Ets88.1 ---------------> M00209 NF-Y 84.7 --------------> M00159 C/EBP 83.1 <--------------M00033 p300 82.2 <------M00025 Elk-1 82.2 --M00199 AP-1 80.9 ---------------> M00117 C/EBPb 80.8 ---M00223 STATx 80.8 <----------M00033 p300 80.2 -----M00267 XFD-1 80.0 <------------M00159 C/EBP 80.0 2551 TGGAGGACAG AGAATAGCAT AGTTTTTGGT ACCATTTCTC CCCACCACCC entry score <-------M00083 MZF1 98.3 --M00032 c-Ets88.2 ----M00074 c-Ets88.1 <------M00148 SRY 84.5 <----M00271 AML-1a 84.1 <-M00083 MZF1 83.5 -----------> M00221 SREBP83.1 -----> M00271 AML-1a 82.7 -----M00025 Elk-1 82.2 ------> M00101 CdxA 82.1 ------> M00100 CdxA 82.1 ---M00257 RREB-1 81.0 <-----M00008 Sp1 80.8 ----> M00223 STATx 80.8 --M00033 p300 80.2 <------M00101 CdxA 80.0 -------------> M00159 C/EBP 80.0 2601 CCACCACCCC GACCCATTAA CCTCAAGTTG AGCTCCATGA GTTGATGAAA entry score <-----M00271 AML-1a 88.7 <------M00101 CdxA 87.1 <-------M00041 CRE-BP 86.2 ------> M00240 Nkx-2. 86.0 <---------M00008 Sp1 84.9 <----M00271 AML-1a 84.1 ----M00083 MZF1 83.5 -----------> M00073 deltaE 81.5 ---------> M00257 RREB-1 81.0 --M00008 Sp1 80.8 -----------M00109 C/EBPb 80.5 2651 ATGAACTAGG AGCAGTCACA CACATGATGA CAGGCTGGCC CAGATATGTA entry score ----------> M00075 GATA-1 88.6 ----------> M00076 GATA-2 88.1 --------M00203 GATA-X 85.7 --------> M00217 USF 84.8 <-----M00240 Nkx-2. 83.7 ------------M00128 GATA-1 83.0 --------------> M00122 USF 82.5 <-------------M00122 USF 82.5 ----------> M00075 GATA-1 82.4 --------M00045 E4BP4 82.4 <-------M00217 USF 82.1 <-----------M00123 c-Myc/ 80.9 <-----M00101 CdxA 80.7 <--M00101 CdxA 80.7 --------> M00077 GATA-3 80.6 <------------M00157 RORalp 80.5

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143 --> M00109 C/EBPb 80.5 2701 AAATACTTGC CCAAAAGAAC CACAGTAAAA GAGCCTGGCA TGAGTAGGTT entry score <-----M00271 AML-1a 100.0 M00148 SRY 86.4 -> M00203 GATA-X 85.7 > M00128 GATA-1 83.0 ------> M00101 CdxA 82.9 --------------> M00117 C/EBPb 82.4 --> M00045 E4BP4 82.4 --------------> M00116 C/EBPa 81.1 --M00101 CdxA 80.7 <--------M00141 Lyf-1 80.5 ----------> M00216 TATA 80.2 2751 TCTTTTGACA GACAGTAAAA TTCAGCCTCA AGCGGAAGCT GCTGCTAGAG entry score ----M00148 SRY 90.0 --------------> M00074 c-Ets87.0 <-----M00101 CdxA 86.4 ---------------> M00025 Elk-1 85.2 ---------> M00108 NRF-2 84.2 -------> M00240 Nkx-2. 83.7 <--------------M00133 Tst-1 83.3 ----------> M00147 HSF2 83.3 <------M00101 CdxA 82.9 ---------> M00032 c-Ets81.4 2801 TGTGTGGGCT GGGGAGGTGT CGCCAGTTGC AGCCTGGCTT CCTCTGTGGC entry score --------> M00083 MZF1 90.4 -----> M00271 AML-1a 83.7 -------M00159 C/EBP 83.1 -----> M00271 AML-1a 82.7 <------------M00074 c-Ets81.8 ------------> M00001 MyoD 81.4 ----------> M00008 Sp1 80.8 ---------> M00008 Sp1 80.8 ----------------> M00002 E47 80.8 2851 CAGTCTTAAG AGAGGCATTC AAAATAGGTG GAGTGCAAGG TTTGTTCCCT entry score ----------------> M00145 Brn-2 91.1 --------------> M00033 p300 84.2 ----> M00159 C/EBP 83.1 <-----M00148 SRY 82.7 <----------M00073 deltaE 81.7 -------> M00240 Nkx-2. 81.4 ----M00223 STATx 80.8 ------------> M00130 HFH-2 80.2 2901 GGAACAGGGT AGGGGATGTC CCCTGTGGCC AAATGAAGTT GAATCAGTGT entry score <---------M00054 NF-kap 90.7 ---------> M00199 AP-1 89.9 ----------> M00076 GATA-2 87.4 <-------M00083 MZF1 87.0 ----------> M00053 c-Rel 86.8 ----------> M00054 NF-kap 86.6 --------> M00083 MZF1 86.1 ----------> M00051 NF-kap 85.8 ----------> M00052 NF-kap 85.4 ----------> M00054 NF-kap 85.4 ----------> M00075 GATA-1 85.3 -----> M00271 AML-1a 83.7 <--------M00199 AP-1 83.5 <-----------M00208 NF-kap 83.0 ---> M00223 STATx 80.8 -----------------M00250 Gfi-1 80.5 -----------> M00173 AP-1 80.4 <---------M00053 c-Rel 80.2 <-----------------M00059 YY1 80.2

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144 2951 CTGAAAGGAA ACCCAGATTC TTCCCTCCAG GTGAGGACTA CTCCAGGTGC entry score <----------M00073 deltaE 93.6 <---------M00147 HSF2 91.0 ----------> M00075 GATA-1 86.1 -------> M00217 USF 85.0 <-------M00073 deltaE 84.9 ----------> M00147 HSF2 84.0 --------> M00083 MZF1 83.5 ------------M00071 E47 83.0 --------> M00217 USF 82.8 <---------M00053 c-Rel 82.6 <-------M00217 USF 82.6 -----------M00002 E47 81.7 ------> M00250 Gfi-1 80.5 ----------> M00076 GATA-2 80.2 -------> M00148 SRY 80.0 3001 TCCCATCACA GACAGCCAGC AGCCACTCAG GCACAACAGG ACATCCAGGC entry score <--------M00075 GATA-1 88.6 -M00073 deltaE 84.9 <--------------M00209 NF-Y 84.6 ----------> M00032 c-Ets83.3 <---------M00032 c-Ets83.3 ---> M00071 E47 83.0 <--------M00076 GATA-2 83.0 <-------M00077 GATA-3 82.8 <----M00271 AML-1a 82.1 -------------> M00074 c-Ets81.8 ---> M00002 E47 81.7 <----M00271 AML-1a 81.4 <---------M00076 GATA-2 80.2 <---------M00075 GATA-1 80.0 3051 GACTAGAGGC AGCAGGGGGC TGGCCCCACG TTCCCTCTTC AGTACAGGTC entry score -------------------> M00005 AP-4 85.0 --------> M00217 USF 83.5 ------------> M00055 N-Myc 82.7 ----------> M00008 Sp1 82.2 -----------M00156 RORalp 81.5 <------M00083 MZF1 80.9 <---------------M00236 Arnt 80.0 3101 AACGCTCCGG GACACCTGAA GACACCAGGG ACTGCCAAAG GCCATGGCAG entry score -----------> M00073 deltaE 87.7 ----------> M00108 NRF-2 84.2 <-----M00240 Nkx-2. 83.7 <---------------M00002 E47 83.7 -------> M00217 USF 83.3 ------------> M00087 Ik-2 81.6 -> M00156 RORalp 81.5 <------------M00001 MyoD 81.4 <------M00217 USF 80.6 <-------M00141 Lyf-1 80.5 ---------------> M00122 USF 80.3 <--------------M00122 USF 80.3 ---------> M00227 v-Myb 80.2 3151 CAGCAGCAGC AGCAGCTAAC ACTGATCCCT GGCAAAAGCT GGAAACTGTA entry score ----------> M00106 CDP CR 84.5 ----------> M00042 Sox-5 80.4 3201 CAAGGCCAGA GAACGTGACA ATAGAAACTC TTTTGTCCAG TTTGCTGGTA entry score <----------------M00236 Arnt 83.0 ----------> M00042 Sox-5 82.4 <-----M00101 CdxA 82.1 ------------> M00160 SRY 82.0 <------M00217 USF 81.1 ------------> M00123 c-Myc/ 80.9 ---------------> M00122 USF 80.7 <--------------M00122 USF 80.7

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145 3251 AGTCTACTTC TTCACACTTG ACACCAGCAG AGATAAGCAG CCCACTTCCC entry score <------M00240 Nkx-2. 100.0 -----------> M00203 GATA-X 89.5 -M00240 Nkx-2. 88.4 --------------> M00127 GATA-1 86.7 ---------> M00077 GATA-3 86.6 -------------> M00128 GATA-1 86.0 <----M00141 Lyf-1 85.7 ---M00055 N-Myc 84.5 <------M00087 Ik-2 84.2 -M00217 USF 83.0 ----------> M00076 GATA-2 82.6 --------------> M00126 GATA-1 82.4 --------> M00217 USF 81.9 <-----M00240 Nkx-2. 81.4 --------M00072 CP2 81.2 <-----------M00033 p300 81.2 --------------> M00122 USF 80.9 <-------------M00122 USF 80.9 <-------M00217 USF 80.6 ----------> M00075 GATA-1 80.0 3301 AAGTGTAGTT GTAATTCACC AATTTTTAAA ATTGCACCAT CACTTCTCCA entry score <---------M00075 GATA-1 91.4 ---------------> M00116 C/EBPa 90.0 <--------------M00109 C/EBPb 89.9 <--------------M00133 Tst-1 89.6 ----> M00240 Nkx-2. 88.4 <------M00101 CdxA 86.4 --M00141 Lyf-1 85.7 <--------M00077 GATA-3 85.6 ---------------> M00117 C/EBPb 84.8 -------> M00055 N-Myc 84.5 ------> M00101 CdxA 84.3 ---M00087 Ik-2 84.2 <---------M00076 GATA-2 84.2 <------------M00159 C/EBP 83.8 ------> M00100 CdxA 83.3 -----> M00217 USF 83.0 <------M00101 CdxA 82.9 <-------------M00116 C/EBPa 81.9 -----> M00271 AML-1a 81.4 <-----M00240 Nkx-2. 81.4 <---------M00216 TATA 81.3 -> M00072 CP2 81.2 -M00033 p300 81.2 <----------------M00099 S8 81.1 <---------M00042 Sox-5 81.0 ----------> M00216 TATA 80.4 3351 TGAGTCCCCT CCCTCCACAC CTGCCAACAC ACTACATCCA CCCCCACCTC entry score <-------M00083 MZF1 93.0 --------> M00217 USF 85.5 -----------> M00073 deltaE 84.6 <-----------M00001 MyoD 83.7 <-------M00083 MZF1 83.5 <------M00083 MZF1 83.5 <----M00271 AML-1a 83.1 <--------------M00002 E47 81.7 <-----M00271 AML-1a 81.7 <----M00271 AML-1a 81.4 --------M00073 deltaE 80.8 <--------M00141 Lyf-1 80.5 <-------M00083 MZF1 80.0 3401 CCTATATTCC ATCACTAAAG CTTGAATGGG GCTTCACTCT GGGCACTCTG entry score <---------M00075 GATA-1 83.3 <-------------M00109 C/EBPb 83.0 ----------> M00054 NF-kap 82.2 <-----M00240 Nkx-2. 81.4 -> M00073 deltaE 80.8

PAGE 158

146 --------> M00096 Pbx-1 80.4 --------------> M00117 C/EBPb 80.0 3451 GGCACTACTT CTCTGTGCCA CACCTGGCCC AAAAGTTGGA ACAAGGCTAG entry score -----------> M00073 deltaE 86.1 <-----------M00001 MyoD 86.0 < M00073 deltaE 85.6 <-----M00271 AML-1a 83.7 <-------M00217 USF 83.0 <-M00101 CdxA 82.1 <-M00100 CdxA 82.1 <--------------M00002 E47 81.7 --------> M00217 USF 81.1 3501 AAAGGTGGAA AGTGAGCAGC AGCCACCTGT ACAACGACTC TCACCAGAGG entry score <-----------M00001 MyoD 88.4 -------> M00217 USF 87.4 ---------M00073 deltaE 85.6 -----------> M00073 deltaE 85.5 <---M00147 HSF2 84.6 <----M00271 AML-1a 83.4 M00073 deltaE 83.1 ---M00101 CdxA 82.1 ---M00100 CdxA 82.1 -------> M00240 Nkx-2. 81.4 -------------> M00159 C/EBP 80.8 ---------------> M00122 USF 80.1 <--------------M00122 USF 80.1 3551 TTTCCAGTAA ACAGTGAACT GTCAGTTTCA TAAAATTGTG TATTTCGTTA entry score <------M00101 CdxA 92.1 <------M00100 CdxA 91.0 --------> M00223 STATx 89.4 <------M00101 CdxA 86.4 ------------> M00129 HFH-1 86.2 <-----------M00131 HNF-3b 86.1 ------------> M00130 HFH-2 85.1 ----M00147 HSF2 84.6 <-------M00223 STATx 84.6 <-----M00148 SRY 84.5 <-------------M00162 Oct-1 83.7 -------M00032 c-Ets83.3 <-----M00101 CdxA 82.9 <-------------M00109 C/EBPb 82.4 ------> M00101 CdxA 82.1 <-------M00260 HLF 81.6 ----------> M00228 VBP 81.4 ---M00228 VBP 81.4 <----------M00109 C/EBPb 81.1 <-------M00137 Oct-1 81.1 ----------> M00042 Sox-5 81.0 -------> M00101 CdxA 80.7 <---------M00042 Sox-5 80.4 ----------> M00216 TATA 80.2 -------> M00148 SRY 80.0 3601 CCTCTTTCTG CAAATCTTTC TTTGGAGAAA AGATACAAAG CAGAGCTCCT entry score <------M00148 SRY 90.0 -----------> M00082 Evi-1 88.1 --------------> M00159 C/EBP 86.9 -----------> M00079 Evi-1 86.4 ------> M00100 CdxA 85.9 -----------> M00080 Evi-1 84.6 ------------> M00130 HFH-2 84.4 <-----M00100 CdxA 83.3 M00260 HLF 81.6 ------> M00101 CdxA 81.4 -----> M00228 VBP 81.4 --M00109 C/EBPb 81.1 ---M00137 Oct-1 81.1

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147 -------> M00148 SRY 80.9 ------> M00148 SRY 80.9 -------------------> M00135 Oct-1 80.8 <--M00108 NRF-2 80.7 ---------> M00011 Evi-1 80.4 -------------> M00128 GATA-1 80.2 3651 TCCGATGACC CTGCTGCTTC AGTTTAGACT AGAATCTACT CTCCCCTCCA entry score <------M00083 MZF1 93.0 ---------> M00147 HSF2 87.2 <------M00148 SRY 84.5 ---------> M00076 GATA-2 81.8 ------> M00148 SRY 81.8 <----------M00084 MZF1 81.5 <--------M00147 HSF2 80.8 <------M00101 CdxA 80.7 -----M00108 NRF-2 80.7 <------------M00156 RORalp 80.6 ---------> M00146 HSF1 80.1 <-------M00083 MZF1 80.0 3701 ACTCTGAAGG ACCTGTGATG TGTGATCTCT GCACAAGCTG TCAATGCCAT entry score <----M00076 GATA-2 87.0 <----M00075 GATA-1 86.5 ----------> M00075 GATA-1 85.7 -----> M00271 AML-1a 82.1 -----> M00271 AML-1a 82.1 -M00084 MZF1 81.5 ----------> M00076 GATA-2 80.6 3751 CTTCTTGTCC CTTAAGAGTT AATGAACGGC CGGCGCTGCG GCTCACTAGG entry score -------> M00101 CdxA 87.1 ---M00076 GATA-2 87.0 ---M00075 GATA-1 86.5 ----------------> M00099 S8 82.1 --------> M00227 v-Myb 81.8 <------M00240 Nkx-2. 81.4 ------> M00240 Nkx-2. 81.4 -------------> M00137 Oct-1 80.7 3801 CTAATCCTCC GCCTTGCAGC GCCGGCACAC TGGGTTCTAG TCCTGGTCAG entry score -------------------> M00192 GR 81.8 ----------------> M00205 GR 81.5 3851 GGCACTGGAT TCTGTCCCGG TTGCCCCTCT TCCAGGCCAG CTCTCTGCTG entry score <------------M00084 MZF1 91.1 <-----M00240 Nkx-2. 83.7 -M00271 AML-1a 83.7 <---------M00108 NRF-2 82.5 ----------> M00075 GATA-1 82.4 -------> M00101 CdxA 80.0 <------M00083 MZF1 80.0 3901 TGGCCAGGGA GTGCAGTGGA GGATGGCCCA AGCACTTGGG AGACCAGGAT entry score ---M00203 GATA-X 94.5 ----------> M00076 GATA-2 91.3 ---------> M00141 Lyf-1 89.6 <-----M00240 Nkx-2. 88.4 ----------> M00075 GATA-1 87.8 -----M00076 GATA-2 87.7 -------M00127 GATA-1 86.7 --------------> M00033 p300 86.1 <------M00217 USF 85.0 ------------> M00087 Ik-2 84.6 -----M00075 GATA-1 84.5 ---> M00271 AML-1a 83.7 -------------> M00123 c-Myc/ 83.2 <------------M00055 N-Myc 80.6 ------M00128 GATA-1 80.2 3951 AAGTACCTGG CTCCTGCCAT CCTATCAGCA CGGTGCGCTG GCCGCAGCAC entry score

PAGE 160

148 ------> M00203 GATA-X 94.5 <---------M00075 GATA-1 93.1 <---------M00076 GATA-2 90.1 ---> M00076 GATA-2 87.7 <--------M00077 GATA-3 87.5 <---------M00076 GATA-2 87.0 <------------M00128 GATA-1 86.9 -----> M00127 GATA-1 86.7 ---> M00075 GATA-1 84.5 <----M00235 AhR/Ar 83.3 <--------------M00126 GATA-1 83.1 <--------------M00127 GATA-1 80.7 <---------M00075 GATA-1 80.4 -----> M00128 GATA-1 80.2 4001 GCCAGCCATG GCGGCCATTG GAGGGTGAAC CAACGGCAAA GGAAGACCTT entry score ---------> M00227 v-Myb 94.1 <-----M00147 HSF2 85.9 ----------> M00003 v-Myb 85.3 <-----M00146 HSF1 85.1 ---------M00235 AhR/Ar 83.3 -------------> M00074 c-Ets83.0 <--------M00227 v-Myb 82.9 -------> M00148 SRY 81.8 ------M00147 HSF2 80.8 ----------> M00108 NRF-2 80.7 4051 TCTCTCTGTC TCTCTCTCTC ACTGTCCTCT CTGCCTGTCA AAAAAAAAAA entry score --M00147 HSF2 85.9 --M00146 HSF1 85.1 -------> M00148 SRY 84.5 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5 -----M00148 SRY 84.5 ----M00148 SRY 84.5 ---M00148 SRY 84.5 --M00148 SRY 84.5 -M00148 SRY 84.5 M00148 SRY 84.5 <----------M00130 HFH-2 82.1 <--------M00130 HFH-2 82.1 <-------M00130 HFH-2 82.1 <------M00130 HFH-2 82.1 <-----M00130 HFH-2 82.1 <----M00130 HFH-2 82.1 <---M00130 HFH-2 82.1 <--M00130 HFH-2 82.1 <-M00130 HFH-2 82.1 M00147 HSF2 80.8 <-------M00083 MZF1 80.0 4101 AAAAAAAAAA AAAAAAAAGA GTTAATGAAG AAGTAACTCA CTGGCGTCAT entry score <-------M00113 CREB 92.6 ------> M00101 CdxA 87.1 <------M00039 CREB 87.1 -------> M00041 CRE-BP 86.2 <------M00041 CRE-BP 86.2 --------------> M00137 Oct-1 85.8 -------> M00039 CREB 85.6 > M00148 SRY 84.5 -> M00148 SRY 84.5 --> M00148 SRY 84.5 ---> M00148 SRY 84.5 ----> M00148 SRY 84.5 -----> M00148 SRY 84.5 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5

PAGE 161

149 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5 -------> M00148 SRY 84.5 -------> M00148 SRY 84.5 -------> M00148 SRY 84.5 -------> M00148 SRY 84.5 -------> M00148 SRY 84.5 -------> M00148 SRY 84.5 ------> M00148 SRY 84.5 ------> M00148 SRY 84.5 <----M00076 GATA-2 83.8 <---M00087 Ik-2 82.9 M00130 HFH-2 82.1 -M00130 HFH-2 82.1 --M00130 HFH-2 82.1 ---M00130 HFH-2 82.1 ----M00130 HFH-2 82.1 -----M00130 HFH-2 82.1 ------M00130 HFH-2 82.1 -------M00130 HFH-2 82.1 --------M00130 HFH-2 82.1 ---------M00130 HFH-2 82.1 -----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 82.1 <-----------M00130 HFH-2 81.7 ------------------> M00206 HNF-1 81.6 <---------M00228 VBP 81.4 <----M00075 GATA-1 81.2 <-----------M00130 HFH-2 80.9 -----------------> M00099 S8 80.4 -------------M00036 v-Jun 80.3 4151 CCCTGTCCCA TAGCCAGAGA ATCCCAGACT TAAATTCTGC TCTTTGGATT entry score --M00113 CREB 92.6 <------M00100 CdxA 92.3 ------> M00101 CdxA 92.1 --M00100 CdxA 89.7 <----------------M00222 Th1/E4 85.9 --------------> M00162 Oct-1 85.7 <-----------M00087 Ik-2 85.1 --M00101 CdxA 84.3 ---M00076 GATA-2 83.8 <------M00240 Nkx-2. 83.7 <------M00101 CdxA 83.6 ------M00087 Ik-2 82.9 ------M00133 Tst-1 81.2 <-----M00072 CP2 81.2 ---M00075 GATA-1 81.2 <---------M00076 GATA-2 80.6 --> M00036 v-Jun 80.3 4201 TAGGGTGTTT CTAATGGATT TTCTTTTTGT AACTTCAAGT ATTTATCTAT entry score <----------M00203 GATA-X 92.8 ------> M00100 CdxA 92.3 M00101 CdxA 92.1 <------M00148 SRY 90.0 ---> M00100 CdxA 89.7 <-----M00148 SRY 89.1 -------> M00240 Nkx-2. 88.4 ------> M00101 CdxA 87.9 <------M00101 CdxA 87.1 <-------M00041 CRE-BP 86.2 <-----------M00128 GATA-1 84.8 <---------M00228 VBP 84.7 ---> M00101 CdxA 84.3

PAGE 162

150 <-------------M00116 C/EBPa 84.2 ---------> M00227 v-Myb 83.4 ------> M00100 CdxA 83.3 <---------M00260 HLF 83.1 <------M00101 CdxA 82.9 --------M00099 S8 82.4 -------> M00101 CdxA 82.1 -M00100 CdxA 82.1 <-----------M00127 GATA-1 81.9 ------------> M00045 E4BP4 81.4 -------> M00133 Tst-1 81.2 ---M00072 CP2 81.2 <----------------M00099 S8 81.1 -------------> M00074 c-Ets81.0 <-------------M00267 XFD-1 80.0 <-M00101 CdxA 80.0 4251 TTAATGTATT TGAAAGACAA AGACACAGAG ATGTCTTCCC CCTTACTGGT entry score -----> M00101 CdxA 92.1 <-------M00083 MZF1 90.4 M00128 GATA-1 84.8 <------------M00084 MZF1 83.9 -------M00025 Elk-1 87.0 ------> M00148 SRY 82.7 -------> M00148 SRY 82.7 ------> M00099 S8 82.4 ----> M00100 CdxA 82.1 -M00127 GATA-1 81.9 <-----------M00087 Ik-2 81.6 ------> M00101 CdxA 81.4 ----------> M00075 GATA-1 81.2 ----------> M00076 GATA-2 81.0 ---M00101 CdxA 80.0 -------> M00148 SRY 80.0 4301 TCACTCTCCA AATGTCCCCA AAGCAGGGGC TGGGCCAAGA GGGAGCCAGG entry score ----------> M00008 Sp1 89.0 <-------M00083 MZF1 88.7 <------------M00159 C/EBP 85.4 M00087 Ik-2 89.5 <------M00217 USF 86.4 ----------> M00008 Sp1 84.9 ---------> M00141 Lyf-1 84.4 -----M00072 CP2 83.3 -------------> M00159 C/EBP 83.1 ---------> M00227 v-Myb 82.9 ------> M00271 AML-1a 82.7 -------> M00217 USF 82.1 -------M00106 CDP CR 81.7 <-----------M00123 c-Myc/ 81.4 <-----------M00087 Ik-2 81.1 <-----------M00055 N-Myc 80.9 -------------> M00086 Ik-1 80.8 ------------> M00087 Ik-2 80.7 ------------> M00055 N-Myc 80.2 4401 CCCAAATACT TGAGCAGTCC CTGCTGCCTC CCAGGGCATG TATTAGCAGG entry score ----M00032 c-Ets91.2 <------M00240 Nkx-2. 88.4 -------M00007 Elk-1 85.4 <--------M00141 Lyf-1 84.4 ------M00074 c-Ets83.4 ----> M00072 CP2 83.3 -------> M00101 CdxA 82.9 <-----M00100 CdxA 80.8 4451 AAGTCAGAAT TGGGAGGAGG GGCGGTGCTT GAATCCAGGT ACTCTGATAA entry score <------M00101 CdxA 92.1

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151 ----> M00032 c-Ets91.2 -----M00203 GATA-X 90.9 ---------> M00141 Lyf-1 89.6 --------M00128 GATA-1 89.4 -----> M00025 Elk-1 87.0 ----------> M00008 Sp1 86.3 -------> M00007 Elk-1 85.4 -----M00079 Evi-1 84.1 -----M00082 Evi-1 83.5 -----> M00074 c-Ets83.4 -M00008 Sp1 84.9 ----------> M00053 c-Rel 81.8 ------M00077 GATA-3 82.5 --------> M00241 Nkx-2. 82.4 -------M00076 GATA-2 81.8 ------------> M00087 Ik-2 81.6 ---------M00127 GATA-1 80.7 -------M00075 GATA-1 80.0 4501 GACAGGCAGA CGTCTCCAGC GCTGCACCAA ATGGTCACCC CAGATCCTGG entry score ----> M00203 GATA-X 90.9 ---> M00128 GATA-1 89.4 <-----M00271 AML-1a 87.4 <-------M00050 E2F 86.2 -----------> M00221 SREBP85.9 ----------> M00075 GATA-1 85.7 ----> M00079 Evi-1 84.1 <-----------M00113 CREB 83.6 ----> M00082 Evi-1 83.5 -----------> M00072 CP2 83.3 <-----M00271 AML-1a 82.7 <---------M00076 GATA-2 82.6 -> M00077 GATA-3 82.5 <---------M00075 GATA-1 82.4 ----------------> M00066 Tal-1a 82.3 ----------> M00076 GATA-2 82.2 -> M00076 GATA-2 81.8 ---> M00127 GATA-1 80.7 --------> M00039 CREB 80.7 <-------M00039 CREB 80.7 <----------------M00059 YY1 80.2 -> M00075 GATA-1 80.0 4551 CCTTGAGGAG CCAAGGGAAG GGGCGAAAAC TTGTGTGTGC AGTACTGTGG entry score <------M00050 E2F 86.2 ----M00271 AML-1a 82.7 <------M00101 CdxA 82.1 ------> M00101 CdxA 82.1 ----------------> M00024 E2F 81.4 --------> M00083 MZF1 80.0 4601 GTCGGGGTCT CCATCGATCC GGAGTCTGGG GGTGTCATCC TAAATTCTGC entry score ----------> M00106 CDP CR 99.1 ------> M00101 CdxA 92.1 <---------M00106 CDP CR 90.0 <------M00100 CdxA 89.7 <---------M00104 CDP CR 89.2 ----------> M00104 CDP CR 89.2 ----------------> M00145 Brn-2 86.1 -------> M00008 Sp1 84.9 <---------M00075 GATA-1 84.5 <------M00101 CdxA 84.3 <--------M00141 Lyf-1 83.1 <---------M00076 GATA-2 83.0 > M00271 AML-1a 82.7 <-----------M00087 Ik-2 82.5 <---------M00075 GATA-1 81.2 <---------M00076 GATA-2 80.6 4651 TAACAACTGG GCGCAGCACC ACGCAGCCCG GGACCCCAGA GCCCTCCACG entry score <----M00271 AML-1a 87.4

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152 <-----M00271 AML-1a 84.1 ------> M00148 SRY 82.7 <----M00083 MZF1 80.0 4701 CTCACTGACC CAGACCCTCC ATCCCCAACC CCTCTCTCCT CCAAAATGGC entry score <---------M00075 GATA-1 91.4 <------M00083 MZF1 91.3 -----------> M00173 AP-1 89.7 <---------M00076 GATA-2 88.9 <--------M00141 Lyf-1 88.3 <------------M00059 YY1 85.7 <--------M00199 AP-1 83.4 <--------M00077 GATA-3 82.5 <--------M00141 Lyf-1 80.5 <------------M00159 C/EBP 86.9 <-----M00075 GATA-1 84.1 <-------M00141 Lyf-1 80.5 <--------M00077 GATA-3 81.9 <--------------M00126 GATA-1 81.0 ---------> M00199 AP-1 80.6 <-----------M00087 Ik-2 80.3 --------------> M00257 RREB-1 80.2 -M00083 MZF1 80.0 4751 TTCAGAATTC CCAAATTCTG ATGTAGATGC TGCACAGGGT ATACCAGCGT entry score <-----------M00087 Ik-2 96.5 <-----M00101 CdxA 92.1 ------> M00101 CdxA 92.1 <------------M00086 Ik-1 90.7 --------------> M00162 Oct-1 87.8 <--------M00141 Lyf-1 87.0 ---M00059 YY1 85.7 ----------> M00075 GATA-1 84.9 <-------------M00109 C/EBPb 82.4 <------------M00088 Ik-3 82.1 ----------> M00075 GATA-1 81.2 <------M00100 CdxA 80.8 4801 CTCCTTAGAG TCTCTCGCGG CTGGGCCCAC AGTGGCGGTT AACCCAGATC entry score <-----M00076 GATA-2 84.6 ------M00075 GATA-1 84.1 <-----M00271 AML-1a 82.7 ----------> M00008 Sp1 82.2 ------M00076 GATA-2 80.6 <-----M00074 c-Ets80.2 4851 CGCTCCCCAA GCGACTTGAC CTTCACTCTG AGAGTGCAGC TGCTACTGGA entry score <-------M00083 MZF1 88.7 <------------M00156 RORalp 87.0 <-----M00240 Nkx-2. 86.0 --M00076 GATA-2 84.6 -------M00074 c-Ets84.6 M00249 CHOP-C 84.3 --> M00075 GATA-1 84.1 --M00075 GATA-1 84.1 -----M00032 c-Ets82.4 M00076 GATA-2 80.6 -----M00074 c-Ets80.2 4901 ACTGCAATTT CCTCTCCTCT GCTTACATAT CTGTATAAAC CCCTTTATGG entry score ------> M00100 CdxA 96.2 <-----M00101 CdxA 87.9 <-----M00100 CdxA 87.2 ------> M00101 CdxA 87.1 <------------M00074 c-Ets86.6 <------------M00128 GATA-1 85.4 M00074 c-Ets84.6 ------------> M00249 CHOP-C 84.3 <---------M00076 GATA-2 84.2

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153 ----------> M00052 NF-kap 84.1 <-----------M00045 E4BP4 82.8 <----------M00203 GATA-X 82.8 ---> M00032 c-Ets82.4 <---------M00075 GATA-1 81.6 -------------M00133 Tst-1 81.2 <------M00100 CdxA 92.3 <-------------M00162 Oct-1 83.7 --------> M00083 MZF1 88.7 ------------> M00113 CREB 80.3 ----------> M00053 c-Rel 81.0 -------> M00101 CdxA 80.7 <--------M00077 GATA-3 80.6 <-------M00216 TATA 80.5 4951 GTTAGCAAAT GAAATTTTAT AAAGACAAGT GTGTAGGGGT TCCCACGAAA entry score -------> M00101 CdxA 92.9 -------> M00240 Nkx-2. 90.7 <------M00101 CdxA 87.9 -------> M00100 CdxA 87.2 <------------M00086 Ik-1 86.5 <---------M00216 TATA 86.4 -----------M00159 C/EBP 85.4 <-----------M00087 Ik-2 84.6 <------------M00088 Ik-3 84.3 ----------------> M00252 TATA 83.5 ------> M00101 CdxA 82.9 ------> M00148 SRY 82.7 -------------> M00137 Oct-1 82.2 <-------M00217 USF 82.0 -------> M00148 SRY 80.9 <---------M00216 TATA 80.6 M00216 TATA 80.5 --------------> M00122 USF 80.5 <-------------M00122 USF 80.5 5001 GCTTGAACAG GGAGTGGGAG CACCCGGAGC GCGGAGCCTC AGCAGCCCCG entry score --------------> M00033 p300 97.0 ------------> M00087 Ik-2 84.6 <----------M00037 NF-E2 81.5 ----------> M00032 c-Ets80.4 M00075 GATA-1 85.3 <---------M00075 GATA-1 84.5 ----------> M00076 GATA-2 84.2 <---------M00076 GATA-2 83.8 -------> M00083 MZF1 83.5 -------------> M00074 c-Ets82.2 ---------> M00008 Sp1 82.2 ------------M00257 RREB-1 80.2 5101 TGCTGAGCAC AGAGGGCTAC TGCGGAGCTG AAGGCGTTGT TCCAAGCGCC entry score <---M00050 E2F 86.2 <-----M00271 AML-1a 81.7 <------------M00159 C/EBP 80.8 5151 AAGGATTTGG GACCCGGCCC GGAGACGCCC CACGCCGCTG TGTTCGGCTC entry score --M00050 E2F 86.2 <---------M00008 Sp1 84.9 ---------> M00141 Lyf-1 83.1 ------------> M00087 Ik-2 82.9 ------> M00271 AML-1a 81.7 <-------M00083 MZF1 80.9 -------> M00100 CdxA 80.8 --M00223 STATx 80.8 5201 CTGGAAGGAA TTGGGTCCCC AGCCCCGGAC TCTCCCTGCC TCTTGCCATA entry score <-------M00083 MZF1 90.4 <---------M00008 Sp1 87.7

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154 <---------M00008 Sp1 86.3 < M00008 Sp1 80.8 -----> M00223 STATx 80.8 <-------------M00033 p300 80.2 ----------> M00053 c-Rel 80.2 5251 GCCAGCCCGG TCCCGGACTG CGCATCCTCG GTTTCCCAGC CCCCTGGGGT entry score <---------M00008 Sp1 90.4 <------------M00087 Ik-2 89.9 -----> M00271 AML-1a 87.4 <---------M00076 GATA-2 85.4 <---------M00075 GATA-1 85.3 --------M00008 Sp1 80.8 5301 GTCTGCAGGC CGGGCTACTT GCACAGCAGC AGGTGCGTAG GCGGGGCGCG entry score ------------> M00246 Egr-2 96.0 ------------> M00245 Egr-3 93.1 <-----M00271 AML-1a 84.1 -------> M00217 USF 81.7 ------------> M00243 Egr-1 90.7 ------------> M00244 NGFI-C 90.7 ----------> M00008 Sp1 84.9 <----------M00073 deltaE 84.9 <-------M00217 USF 84.3 ---------------> M00002 E47 81.7 ------------> M00001 MyoD 81.4 -------M00008 Sp1 80.8 5351 CAGCATTTAA GGCGGACACC ACCTCCCCTG GGCAGCGGCT GGCGATCGGC entry score -------> M00100 CdxA 92.3 ---------> M00075 GATA-1 90.2 <--------M00075 GATA-1 87.3 <--------M00076 GATA-2 87.0 <-------M00241 Nkx-2. 85.3 ---------> M00076 GATA-2 85.0 <-----------M00001 MyoD 83.7 -------> M00101 CdxA 83.6 <-------M00083 MZF1 82.6 <-----M00101 CdxA 81.4 <-----M00240 Nkx-2. 81.4 -> M00008 Sp1 80.8 5401 TGCGGAGGTG CGCGCAGGGC CCGCGTGGCT GTGGGTACCT CCTTCGCCAG entry score ------> M00271 AML-1a 82.7 ------------> M00055 N-Myc 82.7 5451 CACCGTCGCC ACTACCAACG CCGCCACCGC GGGACCCTAC CCCGCATCGG entry score <-------M00075 GATA-1 86.9 <---------M00008 Sp1 86.3 -----M00104 CDP CR 84.9 -----M00106 CDP CR 84.0 <-------M00083 MZF1 83.5 <---------M00008 Sp1 82.2 <------M00106 CDP CR 82.2 <-------M00076 GATA-2 81.8 <---------M00008 Sp1 80.8 5501 TCGCCGCCGC CACCGCAGGT CCCACGACCC CTCCTGCCCT CCGGCGCCCC entry score <----M00271 AML-1a 92.0 M00075 GATA-1 86.9 ---> M00104 CDP CR 84.9 <-----M00002 E47 84.6 ---> M00106 CDP CR 84.0 -M00106 CDP CR 82.2 M00076 GATA-2 81.8 <---M00083 MZF1 81.7
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155 <----------M00073 deltaE 87.6 ---> M00083 MZF1 80.9 <-----------M00246 Egr-2 88.3 <---------------M00002 E47 83.7 <-------M00050 E2F 86.2 -------M00002 E47 84.6 --------> M00217 USF 83.0 --------------M00002 E47 82.7 --M00083 MZF1 81.7 ------------> M00001 MyoD 81.4 -M00033 p300 81.2 <-------M00217 USF 81.1 ---M00083 MZF1 80.9 -------M00008 Sp1 80.8 ------------M00033 p300 80.2 5601 AGGAAGTGAC GCGGTGCGGA CTGAAGAGAA GTGCGGGAAA GGGTGAAGGG entry score <------M00050 E2F 90.8 -----------> M00173 AP-1 84.5 ------------> M00087 Ik-2 82.9 > M00002 E47 82.7 -------> M00083 MZF1 82.6 ------> M00240 Nkx-2. 81.4 -------> M00240 Nkx-2. 81.4 ------------> M00033 p300 81.2 --------------> M00033 p300 81.2 <-----------M00155 ARP-1 80.8 -> M00033 p300 80.2 5651 CTCCGTCCGG GGGTCTTTAC TCTGCAACCC TGTTCCAGCC GCCGAGCACC entry score --M00217 USF 82.9 <---------M00032 c-Ets81.4 ---------> M00053 c-Rel 81.0 ---M00155 ARP-1 80.8 5701 CGTGTGTCAC TCGGGAACTG GCTGGGTAAA GAGGTCAATC CAGACACGCG entry score ---M00083 MZF1 90.4 -------------> M00156 RORalp 88.9 <-------M00055 N-Myc 86.9 -------------> M00137 Oct-1 86.9 <-----M00217 USF 84.2 -------------> M00157 RORalp 83.9 ----> M00217 USF 82.9 <-------M00039 CREB 81.7 <--------------M00222 Th1/E4 81.6 <----------M00173 AP-1 81.4 ----------> M00008 Sp1 80.8 <--------M00106 CDP CR 80.4 ------------> M00087 Ik-2 80.3 <---------M00075 GATA-1 80.0 5751 GGGAAGGAGT TCCAGGGGTC AGCTCCGCCC TCGCACCTGC GGGCTCGGAT entry score ---> M00083 MZF1 90.4 -----------> M00073 deltaE 87.1 --M00055 N-Myc 86.9 <-----------M00244 NGFI-C 86.8 <-----------M00245 Egr-3 86.6 <-----------M00243 Egr-1 86.2 < M00223 STATx 84.6 -------> M00217 USF 84.3 M00217 USF 84.2 -----M00076 GATA-2 83.8 --------------> M00033 p300 83.2 -----M00075 GATA-1 82.4 M00222 Th1/E4 81.6 <-------M00083 MZF1 80.9 5801 TCGGAGAAAA GTGCTAGACT GGAGCTACAC GTATGCGTAG CGGTCTGGAA entry score <---M00208 NF-kap 86.2 -------M00223 STATx 84.6 ---> M00076 GATA-2 83.8

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156 <--M00054 NF-kap 82.9 <--M00052 NF-kap 82.8 <-M00053 c-Rel 82.6 ---> M00075 GATA-1 82.4 ------M00054 NF-kap 80.7 <--------M00223 STATx 83.7 --------> M00217 USF 85.0 <--M00053 c-Rel 81.8 --------M00159 C/EBP 81.5 -------> M00240 Nkx-2. 81.4 --------M00074 c-Ets81.0 <-M00052 NF-kap 80.8 -----------> M00203 GATA-X 80.8 <-M00054 NF-kap 80.7 --------------M00222 Th1/E4 80.0 5851 AATGCCCCAG GCTCGGGTCT GAGGGGCCCA AGTCTATGCA CCGCTGGTGT entry score ------M00208 NF-kap 86.2 -----M00054 NF-kap 82.9 -----M00052 NF-kap 82.8 ------M00053 c-Rel 82.6 -----M00053 c-Rel 81.8 -----------M00002 E47 81.7 ---> M00159 C/EBP 81.5 ---> M00074 c-Ets81.0 ------M00052 NF-kap 80.8 -> M00222 Th1/E4 80.0 5901 GACCCCGCAG GGCAACCCCG CGGTTAACTT CTCTCCTGCC CACCCCTAGA entry score <---------M00051 NF-kap 85.2 <----------M00079 Evi-1 84.1 <--------M00053 c-Rel 82.6 <----------M00082 Evi-1 81.8 ---> M00002 E47 81.7 <----------M00080 Evi-1 81.4 <-------M00083 MZF1 80.9 <--------------M00127 GATA-1 80.7 5951 GGTGTCTTCC TGGGAAGACG ATGGCAGGCG GTGCCCACCG AGCCGACCGT entry score ----------> M00075 GATA-1 88.2 ----------> M00076 GATA-2 86.6 ------------> M00087 Ik-2 86.0 -------------> M00088 Ik-3 85.1 <---------M00032 c-Ets83.3 -------------> M00086 Ik-1 82.4 ---------> M00223 STATx 81.7 <------M00083 MZF1 80.9 <---------M00108 NRF-2 80.7 <------------M00074 c-Ets80.2 6001 GCAACAGGGG AAGAGAGGAA GGAGGGAGGT GGGAGGTGGC GCGCTCCCCA entry score <----M00083 MZF1 95.7 --------> M00083 MZF1 87.0 <--M00271 AML-1a 82.7 ----------> M00108 NRF-2 82.5 <----------M00073 deltaE 82.3 ---------> M00141 Lyf-1 81.8 -------> M00083 MZF1 80.0 6051 CAGCCCTTCC CCTCCTGGCC CGCGAGGGTG TCCGGTCCCA CTCAAGGCAG entry score -M00083 MZF1 95.7 <-------M00083 MZF1 93.0 <---------M00032 c-Ets85.3 -M00271 AML-1a 82.7 <------M00240 Nkx-2. 81.4 6101 CTGCGCAGAG CCTGTGCAGA AAACCCACCT GGGGCCGGTA TTGCACTCTG entry score -----------> M00073 deltaE 90.0 <------------M00249 CHOP-C 89.8 <------M00101 CdxA 86.4 ------------------> M00005 AP-4 81.7

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157 <---------M00053 c-Rel 81.0 <------M00100 CdxA 80.8 <-------M00217 USF 80.6 ------> M00148 SRY 80.0 <------M00101 CdxA 80.0 6151 CTTCTCTTTC AGAGAAAGCT GGAAATTTAC TCCGTGGAGC ACCATGCAGC entry score -------------> M00074 c-Ets84.2 <--------M00223 STATx 82.7 <------M00101 CdxA 82.1 -------> M00100 CdxA 80.8 <-----M00148 SRY 80.0 6201 TACAGATATC AAGAAGAAGG AGGGGCGAGA TGGCAAGAAA GACAATGACT entry score ----------> M00075 GATA-1 91.0 ----------> M00076 GATA-2 87.4 ------M00173 AP-1 84.5 <---------M00075 GATA-1 84.5 ----------> M00076 GATA-2 83.4 -------------> M00128 GATA-1 83.0 -------> M00148 SRY 82.7 <---------M00076 GATA-2 82.6 -------------> M00084 MZF1 82.3 ----------> M00008 Sp1 82.2 ---------> M00077 GATA-3 81.6 <-----M00101 CdxA 80.7 -------> M00083 MZF1 80.0 6251 TGGAACTCAA AAGGAATCAG CAGAAAGAGG AGCTTAAGAA AGAACTTGAT entry score <---------M00075 GATA-1 87.8 -------> M00148 SRY 86.4 <-----M00240 Nkx-2. 86.0 <-----M00100 CdxA 85.9 ---> M00173 AP-1 84.5 --------------> M00109 C/EBPb 84.3 <----------M00037 NF-E2 84.0 <---------M00076 GATA-2 82.6 <-----M00101 CdxA 81.4 Total 1576 high-scoring sites found. Max score: 100.0 point, Min score: 80.0 point

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160 24. Kim, JK, SN Summer, and T Berl. 1984. The cyclic AMP system in the inner medullary collecting duct of the potassium-depleted rat. Kidney Int 26:384-91. 25. Koeppen, BM, and BA Stanton. 2000. Renal physiology, 3rd ed. Mosby, St. Louis. 26. Kone, BC, and SC Higham. 1998. A novel N-terminal splice variant of the rat H+-K+-ATPase alpha2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia. J Biol Chem 273:2543-52. 27. Kraut, JA, KG Helander, HF Helander, ND Iroezi, EA Marcus, and G Sachs. 2001. Detection and localization of H+-K+-ATPase isoforms in human kidney. Am J Physiol Renal Physiol 281:F763-8. 28. Kuwahara, M, WJ Fu, and F Marumo. 1996. Functional activity of H-K-ATPase in individual cells of OMCD: localization and effect of K+ depletion. Am J Physiol 270:F116-22. 29. Lagrange, T, AN Kapanidis, H Tang, D Reinberg, and RH Ebright. 1998. New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes Dev 12:34-44. 30. Laroche-Joubert, N, and A Doucet. 1999. Collecting duct adaptation to potassium depletion. Semin Nephrol 19:390-8. 31. Lodish, HF, and JE Darnell. 1995. Molecular cell biology, 3rd ed. Scientific American Books, New York. 32. Lutsenko, S, and JH Kaplan. 1995. Organization of P-type ATPases: significance of structural diversity. Biochemistry 34:15607-13. 33. Marsy, S, JM Elalouf, and A Doucet. 1996. Quantitative RT-PCR analysis of mRNAs encoding a colonic putative H, KATPase alpha subunit along the rat nephron: effect of K+ depletion. Pflugers Arch 432:494-500. 34. Meneton, P, PJ Schultheis, J Greeb, ML Nieman, LH Liu, LL Clarke, JJ Duffy, T Doetschman, JN Lorenz, and GE Shull. 1998. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J Clin Invest 101:536-42. 35. Modyanov, NN, KE Petrukhin, VE Sverdlov, AV Grishin, MY Orlova, MB Kostina, OI Makaravich, NE Broude, GS Monastyrskaya, and ED Sverdlov. 1991. The Family of Na,K-ATPase Genes: ATP1AL1 gene is transcriptionally competent and probably encodes the related ion transport ATPase. FEBS 278:91-94.

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161 J Physiol Renal Physiol 272:F357-65. 36. Nakamura, S, H Amlal, JH Galla, and M Soleimani. 1998. Colonic H+-K+-ATPase is induced and mediates increased HCO3reabsorption in inner medullary collecting duct in potassium depletion. Kidney Int 54:1233-9. 37. Otto, TC. 2001. Molecular analysis of F1F0 adenosine triphosphate synthase and renal H+,K+-adenosine triphosphatases. Dissertation. University of Florida, Gainesville. 38. Ryan, MJ, G Johnson, J Kirk, SM Fuerstenberg, RA Zager, and B Torok-Storb. 1994. HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int 45:48-57. 39. Sambrook, J, EF Fritsch, and T Maniatis. 1989. Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 40. Sangan, P, VM Rajendran, AS Mann, M Kashgarian, and HJ Binder. 1997. Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am J Physiol 272:C685-96. 41. Silver, RB, H Choe, and G Frindt. 1998. Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct. Am J Physiol 275:F94-102. 42. Simmons, NL. 1990. A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp Physiol 75:309-19. 43. Stevens, MS, and RW Dunlay. 2000. Hyperkalemia in hospitalized patients. Int Urol Nephrol 32:177-80. 44. Stokes, DL, and NM Green. 2003. Structure and function of the calcium pump. Annu Rev Biophys Biomol Struct. 45. Sverdlov, VE, MB Kostina, and NN Modyanov. 1996. Genomic organization of the human ATP1AL1 gene encoding a ouabain-sensitive H,K-ATPase. Genomics 32:317-27. 46. Toyoshima, C, M Nakasako, H Nomura, and H Ogawa. 2000. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647-55. 47. Toyoshima, C, and H Nomura. 2002. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature 418:605-11. 48. Verlander, J, R Moudy, WG Campbell, BD Cain, and CS Wingo. 2001. Immunohistochemical localization of H-K-ATPase alpha 2c in rabbit kidney. Am

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162 49. Weiner, ID, and CS Wingo. 1998. Hyperkalemia: a potential silent killer. J Am Soc Nephrol 9:1535-43. 50. Weiner, ID, and CS Wingo. 1997. Hypokalemia--consequences, causes, and correction. J Am Soc Nephrol 8:1179-88. 51. Wingo, CS, and BD Cain. 1993. The renal H-K-ATPase: physiological significance and role in potassium homeostasis. Annu Rev Physiol 55:323-47. 52. Zhang, W, T Kuncewicz, SC Higham, and BC Kone. 2001. Structure, promoter analysis, and chromosomal localization of the murine H(+)/K(+)-ATPase alpha 2 subunit gene. J Am Soc Nephrol 12:2554-64. 53. Zies, DL, CS Wingo, and BD Cain. 2002. Molecular regulation of the HKalpha2 subunit of the H+,K(+)-ATPases. J Nephrol 15 Suppl 5:S54-60.

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BIOGRAPHICAL SKETCH Deborah Milon Zies was born on December 6, 1964, to Frank and Barbara Milon. She grew up in New York with her older sister Patricia and younger brother Frank. She graduated from Pelham Memorial High School in May of 1982 and moved to Florida where she attended Rollins College. Deborah graduated from Rollins College in May of 1986 with a major in biology and a minor in teacher certification. Upon graduation, she remained in Florida where she taught biology at Oviedo High School. In May of 1988, Deborah married Peter Zies and moved to New Orleans, Louisiana, where she began graduate school at Tulane University. There, she worked under the supervision of Dr. David Mullin and completed a masters thesis project entitled The Genetic Characterization of Insertion Element IS511from Caulobacter cresentus. Deborah graduated from Tulane University with a Master of Science degree in December of 1990. She then returned to Orlando, Florida, and worked as a laboratory technician at the United States Department of Agriculture Horticultural Research Laboratory. During her four year appointment, she worked with Dr. Stephen Garnsey on the development of molecular techniques for the detection of citrus diseases. It was also during this time that she gave birth to her daughter Lee Ann and became divorced from her husband. At the end of the appointment, Deborah decided to return to teaching. Over the next three years she taught biology and chemistry at Apopka High School, Seminole Community College and Valencia Community College, all in the Orlando area. While teaching, Deborah 163 came to realize that although teaching was rewarding, she missed research. She therefore

PAGE 176

164 decided to return to graduate school for a Doctor of Philosophy degree. With this degree she would be able to obtain a teaching position that had a research component. In the fall of 1998, Deborah entered the Interdisciplinary Program at the University of Florida. She has carried out the work described in this dissertation in the laboratory of Dr. Brian Cain. She received a predoctoral fellowship from the American Heart Association which supported her for two years. Additionally, she received an outstanding graduate student award from the Interdisciplinary Program and the Boyce Award for outstanding research from the Department of Biochemistry and Molecular Biology. Upon graduation, Deborah will begin a postdoctoral position at the Mayo Clinic in Jacksonville, Florida. She will be studying changes in gene expression associated with colon cancer. From there, Deborah hopes to obtain a teaching position at a small college and combine her love for teaching and research into one career.


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MOLECULAR CHARACTERIZATION OF THE RABBIT HKa2 GENE


By

DEBORAH MILON ZIES

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2003

































This dissertation is dedicated to the memory of James Byrne McCracken, Jr. (July 19,
1967 to January 13, 1999). Jim's all-too-short-of-a-life has been, and always will be, an
inspiration to continue on, even in the toughest of times.















ACKNOWLEDGEMENTS

The work described in this dissertation could not have been completed without the

support that I have received on both a professional and a personal level. I would

therefore like to acknowledge specific individuals who have been there for me over the

past five years.

Professionally, the most significant support came from my mentor, Dr. Brian

Cain. He taught me to be an excellent scientist, an outstanding writer, and a supportive

colleague and friend. I thank my committee members, Drs. Charles Wingo, Thomas

Yang, Peggy Wallace, Sarah Chesrown, and Bruce Stevens, for their suggestions and

support throughout this process. Their contributions are evident in both this dissertation

and the scientist I have become. I thank the past and present members of the Cain

laboratory. I owe a great deal to those people who were members of the lab when I

joined, Drs. Paul Sorgen, James Gardner and Tamara Caviston Otto. They all helped

make the transition into a new laboratory smooth and fun. In particular, Tammy

remained in the the lab long enough to become a true friend. To this day, she continues

to offer understanding and encouragement when times are tough. I owe even more to the

current members of the Cain laboratory, Michelle Gumz and Tammy Bohannon Grabar.

We three have been through so much on every level that there are no words to express

my gratitude. I would also like to thank past and present members of our neighbors, the

Yang laboratory. These people have made the work environment both stimulating and

enjoyable. They are Christine Mione, Sara Jato-Rodriguez, Chien Chen and Sue Lee









Kang. Specifically, I would like to thank Sue for all her help on completing the DNaseI

hypersensitivity assay and Christine for her unending patience and guidance while

teaching so many things about the computer. Finally, on a professional level, I thank the

rest of the Department of Biochemistry and Molecular Biology. The entire group has

made my experience as a graduate student one that I will never forget.

On a personal level, the first people I would like to thank have already been

mentioned. In addition to all they provided on a professional level, Michelle and Tammy

have also provided emotional support and friendship. The rest of my thanks goes to my

family. I thank my dad, Frank Milon III, for setting a great example for me by going to

law school, and becoming an attorney, later in his life. I thank my mom, Barbara Milon,

for being a mom, a cheerleader, a babysitter, a friend, and so much more. The rest of my

family, Patricia Milon, Frank Milon IV, Christina Milon, Rebecca Milon, and Joshua

Milon, I thank for all of their encouragement, help, and reminders of what is really most

important in life. And finally, because she is the most important, I would like to thank

my daughter, Lee Ann Zies, just for being her. As a single parent with a five-year-old

daughter, it may not have been possible to attend graduate school and make such a big

change in my own life. Lee Ann, however, has not only made it possible, she has made it

all worthwhile. She is a blessing beyond her years. She has been happy to go the lab,

understanding when we have had to be apart, considerate when I have had to study, and

loving in every difficult moment. I could not have asked for a better companion. She

makes everything worthwhile. I love her and thank her with all my heart.















TABLE OF CONTENTS

Page

A C K N O W L E D G E M E N T S ......... ................................................................................. iii

LIST O F TA B LE S ...... .. .. .. ........................................ .. .. .... .............. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT .............. ................. .......... .............. xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Physiological Significance.......................................... ................................. 2
M em brane Potential........... ...................................................... ............... .2
H y p ok alem ia ......................................................................... 3
H yperkalem ia ....................... ...................... .. .. .. ..................4
M am m alian K idney ........................ ................... ................... .............
Evidence for H+, K+ ATPase Expression in the Collecting Duct .............................8
P ro te in ................................................9
m R N A ................................................................................. 9
Regulation of the Renal H+, K+ -ATPases.....................................................10
P o ta ssiu m ..................................................................................... 1 1
S o d iu m ........................................................................................................... 1 2
Acid-Base ........................................................... ... .... ......... 14
Structure of the H+, K ATPase Complex............... ................................14
P-Type A TPases .................. ................................. ..... .. ........ .... 15
The H K A TPase Subunits ................................ ................... ...... ........ 15
High Resolution Model of Rabbit HKua2a......................................................17
T h e R eactio n C y cle ................................................................... ................ .. 19
Structure of the HKa2 Gene .................. ..................................... 21
Eukaryotic G ene O organization ........................................ ........................ 21
H K u 2 cD N A s ............. ................. .................. .......... .. ............ 23
H um an H K u 2 G ene.................................................. ............................... 27
M house H Kc 2 G ene ........................ .. ....................... .... .. ........... 28
S u m m ary ......... ....................................................................2 8






v









2 CLONING OF THE HKa2 GENE ........................................ ........................ 29

M materials and M methods ..................................................................... ....................30
Screening the Lamda Genomic Library ................................... .................30
WH K u 2.1 Sequence ......................... .. .................... .. ...... ........... 34
WH K u 2.5 Sequence ......................... .. .................... .. ...... ........... 35
WH K u 2.8 Sequence ............ .. .... ..... ....... .......................... .............. 38
PCR Amplification of the Missing Fragment................................. ............... 38
R e su lts ..............................................................................................3 9
Screening the X Genom ic Library ............................................ ............... 39
Determ nation of Overlapping Clones...................................... ............... 43
WH K u 2.1 Sequence ......................... .. .................... .. ...... ........... 43
XH K c 2.5 Sequence ......................... .. .................... .. ...... ........... 44
WH K u 2.8 Sequence ......................... .. .................... .. ...... ........... 45
Completion of the HKc2 Gene Sequence ................. .................................47
D discussion ........... ........... .............................................................. ..... 48

3 MAPPING THE TRANSCRIPTION START SITES FOR THE HKc2 GENE....... 51

M materials an d M eth od s .................................................................... .....................52
A analysis of k Clone H K u2.1 ........................................ .......................... 52
R N ase P protection A ssay ........................................................... .....................52
R e su lts ......................... ..................................................................5 7
A analysis of C lone H K 2.1 ........................................................... .............. 57
T ran scription Start Sites ........................................................... .....................57
D isc u ssio n ............... .................................. .................... ................ 5 9

4 REPORTER GENE ANALYSIS OF THE HKu2 GENE PROMOTER...................64

M materials an d M eth od s .................................................................... .....................6 5
Reporter Gene Constructs...................... ...... ............................. 66
T issue C culture C ells ..................... ...... .. .................................. ............. 72
Transfection and Reporter Gene Activity............................................... 73
Normalization of the Luciferase Data .................................................. 74
R results ............. ....... ... ..... ....... ........................ ............. ......... 74
HKa2a and HKa2c Reporter Gene Activity ............................... ................ 74
HKa2a Reporter Gene Activity .................................................................. 79
Putative R epressor M utations...................... ............................. ... ........... 81
Putative TATA Element Mutations.................... ............. ..............82
Effect of Cell Type on Reporter Gene Activity......... ........... .............. 83
D iscu ssio n ....................................... ................... ............................ 8 5

5 CELL DIFFERENTIATION AND HKa2 GENE EXPRESSION..........................92

M materials an d M eth od s .................................................................... .....................93
D election of H K u 2 m R N A s ...............................................................................93









Detection of HKa2 Protein by Immunocytochemistry ................. ................95
DN aseI Hypersensitivity .................................. .....................................96
R results ................. ...... ............................................................................ .....97
RT-PCR ............. ............................................... ....... ......... 97
N northern A naly sis.......... .......................................................... .. .... ...... 98
Im m unocytochem istry ............................................................. ............. .99
DN aseI Hypersensitivity ............................................................................100
D iscu ssio n ................................................................................... 10 2

6 CONCLUSIONS AND FUTURE DIRECTIONS ................................................107

C losing the H K c 2 G ene ............... .. ..... ........ .. ................. ..................... 107
Transcription Start Sites for HKa2a and HK1 2c................ .................................. 110
Reporter Gene Analysis of the Region 5' of the HKc2 Gene................................111
Cell Differentiation and HKc2 Gene Expression....................................................112
Future Directions ..................................... .................. 113
Sum m ary ............. ......... ................................................................ ......115

APPENDIX

A RABBIT HKc2 GENE SEQUENCE ................. ....... .......................... ...............118

Sequence Data from X Clone HKu2.1 and HKu2.5 Exons 1-11 ............................118
Sequence Data from Products Exons 12, 13 and 14..............................................123
Sequence Data from k Clone HKu2.8 Exons 15-23 .............................................124

B TFSEAR CH RE SU LTS........................................ ..... .................... ............... 127

LIST OF REFEREN CES .................................................................. ............... 158

BIOGRAPHICAL SKETCH .............. ............ ............... 163
















LIST OF TABLES

Table pge

1-1 cDNA's for mammalian HKu2 transcripts. ................... .... ..................25

2-1 Primers used for pDZ10 sequencing. ........................... ..................................... 36

2-2 Primers used for HK2.8 sequencing ......................................................... 38

2-3 Primers for genomic PCR and sequencing.................................... .......................39

2-4 Exon and intron sizes for the known HKu2 genes...............................................49
















LIST OF FIGURES


Figure page

1-1 M am m alian k idn ey ............................................................................. .......... .. .. 7

1-2 Schematic representation of the rabbit H+, K+ ATPase............... ..................16

1-3 High resolution model of the rabbit HKa2a subunit.......... ...................................18

1-4 Reaction Cycle of the Ca++-ATPase as a representative of P-type ATPases...........20

1-5 Com m on eukaryotic gene prom other elem ents ...........................................................23

1-6 Formation of the alternative transcripts from the rabbit and rat HKc2 genes ..........26

2-1 Location of Sequencing Primers on the Complete pDZ10 sequence .......................35

2-2 Southern analysis of PCR screen of the X genomic library...................................41

2-3 Plaque lift screen of the X genom ic library...................................... ............... 42

2-4 Southern analysis to determine overlapping clones ............................................. 44

2-5 Clone MHKc2.5 ......................... ........ .. .. ...... .. ............. 46

2-6 Partial sequence from X clone HKu2.8 ........................................... ............... 47

3-1 Construction of RNase protection probes for HKa2a (A) and HKa2c (B)..................54

3-2 CpG dinucleotide analysis of subclone pDZ10 .............. ................................58

3-3 Putative transcription factor binding sites determined by TFSearch .........................58

3-4 RNase protection assay for HKa2a (A) and HKa2c (B)...........................................60

4-1 Deletion constructs containing the HKaC2a and HKaC2c transcription start sites..........67

4-2 H K 2a deletion constructs ................................................ .............................. 70

4-3 Percent activity for reporter gene constructs that contain the HKua2a and HKua2c
transcription and translation start sites .................................................. ............... 76









4-4 Sequence analysis of plasmid pDZ 15......................... ......................77

4-5 Sequence analysis of plasmid pDZ31 .................................. ...................78

4-7 Alignment of possible repressor sequences from human ATP1AL1 and rabbit HKa2
genes ......... ........................................................................... . ............. 8 1

4-8 Percent luciferase activity in repressor mutation constructs ....................................82

4-9 Alignment of the rabbit, human, rat and mouse DNA sequence upstream of the
H K 2 transcription start sites ............................................................................ 83

4-10 Percent activity in reporter gene constructs with mutations in the CATTTAA
e le m e n t ........................................................................... 8 4

4-11 Reporter gene activity in various cell types................................... ............... 85

5-1 M icrographs of tissue culture cells....................................... .......................... 93

5-2 RT-PCR products indicating the presence or absence of HKU2 transcripts ..............98

5-3 Northern blot of RCCT28A total RNA ........................................... ............... 99

5-4 Micrographs of the immunostaining of RCCT28A cells............... .... ..............101

6-1. The HKc2 gene ............... ........... ................ ........... ......... 109

6-2 Model for HKc2 gene expression ........................................................... 117















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MOLECULAR CHARACTERIZATION OF THE RABBIT HKc2 GENE

By

Deborah Milon Zies

December 2003

Chair: Brian D. Cain
Major Department: Biochemistry and Molecular Biology

Blood potassium concentration is critical for normal cellular functions. Failure to

maintain blood potassium levels within a very narrow range can lead to hypertension,

cardiac arrest, respiratory failure and death. The kidney is the major organ responsible

for maintaining a constant potassium level despite variations in dietary intake. One group

of proteins the kidney employs to carry out this function is the H K+ ATPases. These

ion transporters use the energy of ATP to absorb potassium ions from the nephron lumen

in exchange for hydrogen ions. Evidence suggests that the levels and the activities of this

group of transporters are responsive to potassium, sodium and possibly hormones. The

rabbit HKa2 gene produces two mRNAs encoding the HKUa2a and HKUa2c isoforms of the

alpha subunit of the renal H K ATPase. In order to study the regulation of these

isoforms, it was first necessary to clone the gene. Three genomic clones (HKu2.1,

HKu2.5, and HKu2.8) were identified by screening a lambda genomic library. These

three clones span a majority of the HKc2 gene. Subcloning and high-throughput









sequencing of the clone HKu2.5 provided information regarding gene structure and its

similarities to the human, mouse, and rat homologs. RNase protection assays mapped

two distinct transcription start sites that correspond to HKUa2a and HKUa2c transcripts.

Sequence analysis of the 5' clone, HKu2.1, suggested that this clone contained the gene

promoter and 5' regulatory elements. Subsequently, a luciferase reporter gene approach

was used to perform a deletion analysis of the region upstream of the transcription start

sites. The promoter and two putative negative regulatory elements were identified.

Furthermore, sequence alignments and mutational analysis provide evidence for a

functional TATA-like element within the promoter region. Finally, RNA and protein

analyses suggested that RCCT28A tissue culture cells must be differentiated in order to

express the HKc2 gene. Taken together, these data constitute the genomic organization

of the rabbit HKc2 gene, the initial characterization of the promoter, the first evidence of

cell differentiation as a signal for HKc2 gene expression,and a system for future studies

of regulation of the HKc2 gene at the molecular level.














CHAPTER 1
INTRODUCTION

The maintenance of extracellular and intracellular potassium ion (K )

concentrations is critical for normal cell functioning. The difference between these two

concentrations, along with the concentrations of other ions, creates an electrochemical

gradient that is known as membrane potential. When the extracellular concentration

becomes too low, or too high, the membrane potential is altered and serious side effects

occur. In order to maintain a constant blood K+ level the major organ employed by the

body is the kidney. The H K+ ATPases comprise one class of proteins found in the

collecting duct of the kidney. These proteins use the energy of ATP to bring K+ from the

nephron filtrate into the cell in exchange for hydrogen ions (H). This function is thought

to play a key role in K+ conservation. In the rabbit kidney, three isoforms of the alpha

(a) subunit of the H K+ ATPase have been identified. They are HKac, the gastric

isoform, HKa2a, the colonic isoform, and HKa2c, a novel splice variant of HKa2a (53).

Evidence suggests that when blood K+ is low, there is little to no change in transcription

from the HKaci gene. On the other hand, transcription from the HKca2 gene appears

increase under the same conditions (3). The purpose of our study was to characterize the

rabbit HKUa2 gene and initiate studies on its regulation. The specific aims were (1) to

clone the HKca2 gene from rabbit, (2) to map the transcription start sites for HKa2a and

HKa2c, (3) to perform promoter deletion analysis of the region 5' of the transcription

start sites and (4) to determine the effect of cell differentiation of HKc2 gene expression.









This chapter will review background information on the physiological significance of the

H K+ ATPases, the evidence for the presence of the H K+ ATPases in the kidney,

the evidence for regulation of the HKc2 gene products under a variety of cellular

conditions, the structure and function of H+, K+ ATPases, and the organization of the

known HKc2 cDNAs and genes.

Physiological Significance

Healthy individuals maintain a blood K level between 3.5 and 5.5

milliequivalents per liter (mE/L) (1). If blood K+ concentration drops below 3.5mE/L

(hypokalemia) or if blood K concentration increases above 5.5mE/L (hyperkalemia),

serious side effects, and even death, can occur. This section discusses the importance of

K concentration and the problems that are associated with low and high blood potassium

levels.

Membrane Potential

In a typical cell, the concentration of K inside the cell is much greater

(approximately 150 mE/L) than the concentration in the extracellular space and in the

blood (approximately 5 mE/L). This concentration difference, along with that of other

ions, such as sodium and chloride, creates a slight charge to the plasma membrane known

as membrane potential. The charge in an animal cell typically ranges from -50 to -100

millivolts (4). This charge is important for a variety of cell functions including ion

transport and cell signaling. In excitable cells, muscle cells and neurons, the membrane

potential is absolutely required for stimulation. In a resting state, sodium ion

concentration inside the cell is low (15mE/L) while potassium ion concentration is high

(150mE/L). When the cell receives a signal, Na+ channels open and allow Na+ into the









cell. The influx ofNa depolarizes the membrane and the membrane potential becomes

temporarily positive (+50mV). The change in membrane potential triggers K+ channels

to open and allow K+ to exit the cell. The efflux of K+ causes the membrane to repolarize

and the membrane potential once again becomes negative. These local changes in

membrane potential are propogated across the entire cell and cause a muscle cell to

contract or a nerve cell to fire a signal. K+ concentration plays a key role in this process,

and an imbalance of K+ concentration leads to some of the side effects associated with

hypokalemia and hyperkalemia.

Hypokalemia

Most individuals are capable of maintaining proper K+ levels. In fact,

hypokalemia occurs in less than 1% of the healthy population (1). There are two main

groups of people, however, that are very susceptible to developing hypokalemia. There

are individuals that suffer primarily from other disease states and acquire hypokalemia as

a secondary effect, and there are individuals who acquire hypokalemia as a side effect of

taking medications. The most common occurrence of hypokalemia is among patients

receiving diuretics, as many as 50% of these patients develop low blood K+. The second

largest group of individuals that develop hypokalemia suffer from hyperaldosteronism

associated with heart failure and hepatic insufficiencies. A third significant group of

individuals that suffer from hypokalemia are those with renal diseases that effect

potassium uptake.

The most predominant effects of hypokalemia are hypertension, muscle

weakness, and metabolic alkalosis. Blood volume, and therefore blood pressure, is

associated with ion balance in the blood. It appears that low blood K+ levels lead to

increased sodium retention thereby upsetting the normal blood ion balance and leading to









hypertension. Even very mild hypokalemia, a serum potassium level of 3.4 mEq/L, can

lead to increased blood pressure and risk for cardiovascular disease (50). When blood K

is low, muscle cells can become hyperpolarized, or more negative. The hyperpolarized

cell is difficult to depolarize, interfering with the ability of the cell to contract. In cardiac

muscle this condition can lead to ventricular arrhythmias. In skeletal muscle,

hypokalemia frequently leads to muscle weakness, cramping, and in severe cases,

paralysis (1). Hypokalemia also has a profound effect of blood acid/base balance.

Alkalosis, high blood pH, results from two main kidney activities. The first is the

stimulation of proximal tubule bicarbonate absorption and proton excretion. The second

is the increased activity of the H K+ ATPases. These complexes exchange K+ for H

eventually leading to acidification of the urine and alkalosis in the blood. A failure to

correct metabolic alkalosis can lead to additional cardiac and skeletal muscle weakness as

well as liver and brain damage.

Currently, the treatment for hypokalemia consists of oral or intravenous

replacement of lost K+. Unfortunately compliance with potassium supplements is low

due to its disagreeable taste and/or the inconvenience of intravenous replacement (50).

Additionally, there are cases where potassium supplementation does not result in the

desired increase in blood potassium levels (Wingo, personal communication). Finally,

overaggressive potassium replacement therapy can lead to hyperkalemia and its severe

side effects (see below). An understanding of the regulation of the H K+ ATPases

may lead to better mechanisms for controlling blood K+ levels.

Hyperkalemia

Hyperkalemia can result from as a disruption of the normal intracellular and

extracellular K concentrations or from a disruption in the balance between K intake and









K excretion (49). The kidney usually has an extraordinary capacity for excreting excess

K Therefore, hyperkalemia usually only occurs when there is a combination of

increased K+ uptake or a redistribution of cellular K+ along with decreased renal

efficiency. The most common cause for excess intake of K is its presence in

supplements and salt substitutes. The most common causes for a redistribution of K+

include acidosis, anesthetics and hypertonicity. Interestingly, in the case of acidosis, one

of the contributing factors to increased blood K+ is the H+, K+ ATPase found in the

collecting duct of the kidney. The H K+ ATPase functions in combination with other

cellular proteins to remove H+ from the blood. As part of this process, however, the H+ is

exchanged for K+, causing an increase blood K+. Reduced renal efficiency can be caused

by a variety of factors including reduction in the number of nephrons due to renal failure,

medications, and hormone imbalance (43).

The major adverse effect of hyperkalemia is on the cellular membrane potential.

Increased extracellular K+ makes the resting membrane potential more positive than

normal. The result is a decrease in the ability of the cell to propogate a signal and an

increase in the rate at which the cell repolarizes. The most severe effect is in cardiac

muscle where there is a delay in conduction of the muscle contraction which can lead to

fatal heart arrhythmias. In skeletal and smooth muscle, increased weakness and fatigue

are common. Additionally, the weakened muscles in the respiratory tract can lead to

severe respiratory depression and respiratory failure.

Currently there are two approaches to treating hyperkalemia. Calcium

supplements can be given as a treatment for the myocardial effects of hyperkalemia. This

treatment is very rapid, and relieves the major concern associated with hyperkalemia, but









it is not a long term solution to increased blood K Long term treatment of hyperkalemia

can be attained by stimulation of cellular uptake of K and stimulation of renal excretion

of K+. An understanding of the regulation of the H+, K+ ATPase may lead to a

mechanism for its inhibition thereby decreasing the uptake of K in the nephron and

reducing blood K+.

Mammalian Kidney

The mammalian kidney is an important regulatory organ. By excreting or

retaining water and solutes, the kidney can maintain proper blood volume and

composition despite changes in diet and activity (25). Figure 1-1A is a diagram of the

mammalian kidney. The kidney is divided into three sections: the cortex, the medulla

and the pelvis. Running through all three regions are millions of microscopic tubules

called nephrons. The nephrons are the functional units of the kidney and are depicted in

Figure 1-1B. As blood capillaries enter the nephron they form the glomerulus and come

in contact with the nephron at the Bowman's capsule. Blood pressure promotes the free

passage of water, urea, ions and solutes from the blood into the lumen of the nephron

where they become part of the filtrate. Throughout the nephron there are a variety of

proteins that function to pass additional waste products into the filtrate and recover

needed materials from the filtrate. The collecting duct of the nephron is the primary

location for the reabsorption of K+ when blood K+ concentration is low. Figure 1-1C is a

diagram of the cells that make up the cortical collecting duct. The three types of cells are

principal cells, type A intercalated cells and type B intercalated cells (30). In all three

cases, the apical membrane faces the lumen of the nephron and contains a different

constellation of proteins than the basolateral membrane, which faces the blood. There is

evidence to suggest that all three cell types have the H+, K+ ATPase present on their









apical membrane. Principal cells make up approximately 65% of the cells of the cortical

collecting duct. They appear lighter in color under the microscope because they


Glomerulus


Collecting
/ Duct


Blood
Vessel

/ I


Medulla -

Cortex
Pelvis


B Type


A Type


(A) Kidney


(B) Nephron


(C) Cells of the
Collecting Duct


Figure 1-1. Mammalian kidney. A) Cross-section of the mammalian kidney B) The
nephron C) Cortical collecting duct cells.



contain fewer mitochondria than either intercalated cell type. They have a highly folded

basolateral membrane and a very smooth apical membrane. Type B intercalated cells are

the second most abundant cell type, comprising about 25% of the population of cells in

the cortical collecting duct. Their basolateral surface is highly folded, similar to the









principal cells. Their apical membrane, however, is more extensive, containing several

microplicae. Additionally, tubulovesicular structures can be seen scattered throughout

the cell. Type A intercalated cells are the least abundant, making up the last 10% of the

cell population. They have extensive microplicae on the apical surface and a numerous

tubulovesicular structures just below the apical surface. Their basolateral membrane, in

contrast to the principal cells and B type intercalated cells, is smooth. It has been

suggested that the three cell types are capable of inter-converting in response to stimuli.

In fact, there is a progressive change in cell type along the collecting duct that includes

cell types that are intermediate to the three main types.

Evidence for H+, K' ATPase Expression in the Collecting Duct.

The earliest evidence for the existence of Ht, K+ ATPase activity in the

collecting duct came from studies involving the perfusion of microdissected rabbit

collecting duct tissue (51). By setting up a perfused tubule system, Dr. Wingo was able

to measure ion flux under a variety of controlled conditions. In this way, it became clear

that the collecting duct contained an apical ATPase that had properties similar to the

previously identified gastric H K+ ATPase (HKal). Pharmacologically, this ATPase

was sensitive to the gastric H+, K+ ATPase inhibitor omeprazole and insensitive to the

Na K+ ATPase inhibitor ouabain. It differed from the HKalu H, K+ ATPase in that

it was insensitive to the compound Schering 28080. Furthermore, removal of luminal K

had a profound effect on the proton secretion by the collecting duct segment of the

kidney. This section discusses the molecular evidence that supports these early findings.









Protein

Immunohistochemistry has been used to localize the various isoforms of the H+,

K+ ATPase in human (27) and rabbit (48). Kraut et al. (27) used antibodies raised

against the human HKal the rat HKu2 and the human ATP1AL1 (HKc2) proteins to

probe human cortical and medullary collecting duct tissue. Using the HKai antibody,

they observed a darker staining in the intercalated cells and a lighter, but consistent

staining of the principal cells in both the cortical and medullary collecting ducts. With

the rat HKc2 antibody, this group observed no staining. This result is not surprising

because although the rat and human proteins (HKU2 and ATP1AL1 respectively) are

considered homologous proteins (see below), they share only about 87% amino acid

identity. It is therefore likely that the antibody against rat HKc2 was unable to recognize

the human ATP1AL1 protein. Finally, with the antibody to ATP1AL1, light staining of

the intercalated cells and occasional staining of the principal cells in both the cortex and

the medulla was observed. In rabbits, Verlander et al. (48) used an antibody raised

against the HKU2c isoform of the H+, K+ ATPase and observed intense staining of the

apical membrane of both A and B type intercalated cells and a lighter staining of apical

membrane of the principal cells in both the corical and medullay collecting ducts. These

two reports are consistent and support the localization of the H+, K+ ATPase activity to

all the cell types present in the collecting duct.

mRNA

In rat, Ahn et al. (1) used in situ hybridization to show that the mRNA for HKa2

was present in the connecting tubule and intercalated cells throughout the collecting duct.

These experiments were not designed to distinguish between the two alternative









transcripts found in rat, HKUa2a and HKUa2b. A contrasting study by Jassier et al. (22),

detected HKca, but not HKu2, in the cortex and the medulla. Although both authors

used in situ hybridization for the detection of HKu2, Ahn et al. suggest that the

digoxigenin method used in their experiments is more sensitive technique than the 35S

labeling method used by Jassier et al. In a third report, Marsy et al. (33) consistently

found HKc2 mRNA in the cortical collecting duct of rats using quantitative RT-PCR.

The use of a different method in this report supports the presence of HKc2 in the

collecting duct in rat. Furthermore, two independent groups (6, 12) were able to use 5'

rapid amplification of cDNA ends (RACE) to detect HKc2 mRNA in samples from the

cortical collecting duct of rabbit.

The cloning of cDNAs for the HKu2 subunits from human (17, 35), rat (9, 26)

and rabbit (6, 12) kidney provided the strongest evidence for their expression in the

kidney. Additionally, the cDNA's from rat and rabbit were the first indications that there

were splice variants of the HKc2 mRNA's. The characteristics of the cDNA's

identified to date are listed in Table 1 and are discussed below.

Regulation of the Renal H+, K+ -ATPases

The microperfusion assays performed on rabbit kidney nephrons that led to the

discovery of renal expression of the H K+ ATPases also provided the first evidence

suggesting that the activity of the enzyme was regulated under certain cellular conditions

(51). Regulation by ion concentration, acid-base balance, and hormones has since been

confirmed predominantly by in vivo studies with rat. In considering the earlier work as

well as the work discussed below, is important to note that the studies measuring ATPase

activity were not designed to distinguish between the activity of pumps containing HKai









and HKa2 subunits. Furthermore, the molecular mechanisms responsible for controlling

these observed changes in activity have not been studied.

Potassium

In the cortical collecting duct, there is some controversy in the literature regarding

the regulation of HKu2 by low K+. Several investigators (26, 33) have been able to show

that K+ depletion results in an increase in the HKu2 message in the cortical collecting

duct. In contrast, Sangan et al. (40) saw a decrease in the amount ofHKu2 mRNA with

K -depletion. The rat model in these experiments, however, was exposed to a low K+ diet

for a longer period of time than previous studies and the effects of chronic hypokalemia

may differ from those of acute hypokalemia. Furthermore, Ahn et al. (1) did not observe

a change in HKUa2a message in the cortical collecting duct. More studies must be carried

out in order to clarify the regulation of HKu2 in the cortical collecting duct.

HKc2 transcripts are also present in the outer medullary collecting duct (OMCD)

and appear to be subject to upregulation during K+ restriction. RT-PCR (33), Northern

analysis (26), and in situ hybridization (1) have all been used to demonstrate increased

HKc2 gene activity. H+, K+ ATPase activity measurements reported also indicated

increased activity in both rat and rabbit (19, 28, 36). Studies of the type A intercalated

cells (23, 1), most abundant in the medullary collecting duct, showed that HKc2 mRNA

was present and that low K+ resulted in an increase in the message.

A greater controversy centers on the presence of H K -ATPase activity and the

induction of the HKa2 gene in the inner medullary collecting duct (IMCD). HKa2

mRNA was not always detectable (23). Marsy et al. (33) reported the presence of the

mRNA by RT-PCR. but she did not observe an upregulation of the message with









potassium restriction. In contrast, Kone and coworkers were able to detect HKa2

message and show an increase in the message during K+ restriction by both in situ

hybridization and Northern analysis (1, 26). Moreover, the Northern blot experiments

were reproduced independently by Nakamura et al. (36). These investigators were also

able to show an increase H+,K+-ATPase activity. Taken together, these data suggest that

HKu2 ATPase is present in the IMCD and is likely responsive to K+ restriction.

The observation that HKu2 gene products are likely to play a role in K

conservation led to the creation of an HKc2 gene knockout mouse by Meneton et al.

(34). The mouse knockout had no observable defects when fed a K+ replete diet. On the

other hand, these animals developed severe hypokalemia when the animals were fed a K

free diet. Interestingly, the kidney of the knockout mouse was still able to reduce K+ loss

by 100-fold suggesting that other kidney proteins are capable of compensating for a loss

of the HKc2 protein. The knockout mouse maintained on a K+ free diet, however,

developed a more severe case of hypokalemia than the normal mouse on the same diet.

Additionally, the bulk of the in vivo data indicates that the HKc2 gene is upregulated

when K+ is restricted. It is therefore very likely that the colonic H+, K+-ATPase (HKu2)

is regulated by low K+ and plays a role in K+ conservation.

Sodium

A reduction in dietary sodium leads to several alterations including

hyperaldosteronism and increased activity of the Na+, K+-ATPase. The Na+, K+-ATPase

is present on the basolateral membranes of principal cells in the cortical collecting duct

and functions by bringing K+ into the cell in exchange for Na+. As a result of this action,

the principal cells must possess mechanisms to remove additional cellular potassium.









The most likely mechanism for the removal of K is the opening of K specific channels

and/or increased activity of KCl cotransporters present on the apical membrane. It has

been suggested that these conditions stimulate the intercalated cells of the collecting

tubule to reabsorb potassium by use of the H K+-ATPase. Silver et al. (41) identified

intercalated cells of the cortical collecting duct by BCECF fluorescence and measured

their ability to recover from acid load under sodium depleted conditions. Increases in H

and K+ exchange were observed that could be attributed to either HKal or HKa2

containing ATPases. Sangan et al. (40) used a cDNA probe and a polyclonal antibody

specific for HKc2 to detect mRNA and protein from rats fed a low sodium diet.

Northern and Western analyses of kidney cortex and kidney outer medulla revealed that

sodium may have a slight effect on mRNA levels in the kidney cortex but had no

apparent influence on the protein level. It is possible that the increase in H+, K+-ATPase

activity that was observes may be a result of post-translational modification of the pump.

The major hormone released during sodium restriction is aldosterone. It follows,

therefore, that if low sodium increases the activity of H+, K+-ATPase in the cortical

collecting duct, aldosterone could do the same. However, aldosterone levels apparently

do affect HKc2 H K -ATPase activity. Eiam-Ong et al. (11) used adrenalectomized

rats in which aldosterone was replaced at either physiological or pharmacological levels.

When H+, K+-ATPase activity was measured in microdissected tubules there was no

apparent difference between rats that had no aldosterone and those that had either

physiological or pharmacological doses of aldosterone. In a similar set of experiments

using adrenalectomized rats, Jaisser et al. (23) directly measured HKc2 mRNA. In situ

hybridization demonstrated that HKc2 mRNA levels were very low in normokalemic rats









and did not increase significantly with the addition of aldosterone or dexamethasone.

Interestingly, experiments by Silver et al. (41) showed that when rats on a normal diet

were injected with aldosterone in order to simulate levels found during sodium

restriction, H K+-ATPase activity was not increased suggesting that the low sodium

induction of H+, K+-ATPase activity was not mediated by aldosterone.

Acid-Base

One might expect that blood pH would have a profound effect on H+, K+-ATPase

activity in the kidney and indeed the evidence for an increase in IH, K ATPase activity

from alkalosis seems clear. In the rabbit cortical collecting duct, Northern analysis of

mRNA derived from rabbits subjected to metabolic alkalosis generated a greater than

four-fold increase in HKc2 mRNA (13). On the other hand, metabolic acidosis

decreased (13) or had no effect (10) on the level of HKu2 mRNA. Collecting duct

tubules taken from animals fed a K+-depleted diet have 50% less bicarbonate absorption

when compared with the tubules from animals fed a K+-replete diet. At the same time,

the K+- depleted animals have a net increase in K+ absorption suggesting that an

H+, K+ ATPase pump is upregulated under low K+ conditions (30). Komatsu and Garg

also reported that metabolic acidosis increases in H K+ ATPase activity and metabolic

alkalosis suppresses the same activity (14).

Structure of the H+, KI ATPase Complex

The H K+ ATPases are considered P-type ATPases because they form a

phosphorylated intermediate during the reaction cycle. Based on the information known

about the structure and function of other P-type ATPases, models for the structure and









function of the HKa2 ATPases have been made. This section describes the P-type

ATPases, their reaction cycle, and the models of the HKa2 H+, K+ ATPases.

P-Type ATPases

The H K+ ATPases belong to a family of proteins known as the P-type

ATPases (32). This family uses the energy of ATP hydrolysis to translocate ions against

their electrochemical gradient. They are distinguished from other families of ATPases by

forming a phosphorylated intermediate during the reaction cycle. This phosphorylation

occurs at a highly conserved aspartate within the amino acid sequence DKTG. All P-type

ATPases share a core structure with highly conserved domains known as the ATP

binding domain (N), the phosphorylation domain (P) and the transmembrane domain.

Outside of these core domains there are several regions that define subtypes of the

family. In the P2-type ATPases, those that translocate non-heavy cations, the non-

conserved domains appear to be responsible for activities such as the regulation of

ATPase activity, cation specific conformational changes in the protein and proper

insertion of the protein into the plasma membrane. One subclass of P2-type ATPases is

the X+, K -ATPases. This family, which includes the H K+ ATPases, contains

ATPases that are made up of more that one protein subunit (32).

The H KI ATPase Subunits

The H K+ ATPase complex is a heterotetramer of two alpha (a) and two beta

(P) subunits (8). One of each of the subunits is diagrammed in Figure 1-2. The a subunit

is approximately 115 Kilodaltons (Kd) in size and contains 10 transmembrane segments

responsible for forming the channel for the translocation of ions. The a subunit also

houses the ATP hydrolysis activity located in the large intracellular domain between







transmembrane segments four and five. There are four possible 3 subunits that may pair
with HKu2 gene products. They are the gastric H+, K+ ATPase 3 (HK3) subunit and
the Na,K+ ATPase 3 subunits P31, 32, and 33. It is unclear whether the HKu2 proteins
pair with a specific 3 subunit or if they can pair with any of the four. All of the 3
subunits, however, share several characteristics. They are approximately 30 Kd in size
and contain one transmembrane domain. They contain one large intracellular domain
that has a varying number of potential glycolyslation sites. The 3 subunit has no catalytic
activity, but appears to be important for proper positioning and insertion of the a subunit
into the plasma membrane (37).


ATP ADP + Pi

K+

Intracellular




Extracellular

K+ H+


Oa


Plasma
Membrane









lI,


Figure 1-2. Schematic representation of the rabbit H+, K+ ATPase. Arrows indicate
direction of ion transport.









High Resolution Model of Rabbit HKa2a

Recently, the high resolution structure of the El state of the Ca+-ATPase from

rabbit sarcoplasmic reticulum was determined (46). Although this enzyme shares only

29% amino acid identity and 47% amino acid similarity with the rabbit HKua2a subunit,

the two enzymes are related. It was therefore possible to use the atomic coordinates for

the Ca+-ATPase to create a high resolution model of the H K+ ATPase c2 subunit

(18) (Figure 1-3).

In the transmembrane portion of the protein, the ten transmembrane segments are

shown to form a channel for the translocation of ions. The Ca+-ATPase does not have a

3 subunit. It was therefore not possible to model the 3 subunit into the H+, K+ ATPase

structure. Biochemical and low resolution structural data for the Na K+-ATPase

suggested that the 3 subunit would be positioned near membrane spanning domains M7

and M10. In the model, there is a space behind M7 that could hold the HK 3 subunit

(18).

In the cytoplasmic portion of the protein, three clear domains are represented: the

nucleotide binding domain (N), the phosphorylation domain (P) and the actuator domain

(A). The N domain is responsible for binding and hydrolysis of ATP. In Figure 1-3,

lysine 517 is highlighted in green. This amino acid forms a portion of the ATP binding

pocket. The P domain contains aspartic acid 385, shown in red. This amino acid

becomes phosphorylated during the reaction cycle. These two amino acids are far apart

in this model because the model represents the El state of the enzyme. When ions bind to

the channel, the enzyme goes through a conformational change that brings these amino

acids closer together so that the phosphate of ATP can be transferred to the aspartate.









The A domain contains the conserved TEGS loop that appears to play a role in catalysis

of the ATP. The A domain interacts with the P domain in the E1 state and appears to

modulate the ability of the P domain to interact with the N domain.




















Intracellular


Plasma
membrane


Extracellular


Figure 1-3. High resolution model of the rabbit HKa,2a subunit. Courtesy of Michelle
Gumz (18). This model is a representation of the E1 state of the reaction
cycle. Each transmembrane domain is numbered. Highlighted in green is
lysine 517, part of the ATP binding pocket. Highlighted in red is aspartic acid
385, the amino acid that becomes phosphorylated during the reaction cycle.
The arrow indicates the direction of movement of the P domain during the
conformational change to the E2 state.









The Reaction Cycle

It has long been established that the P-type ATPases go through significant

conformational changes while translocating ions (44). Biochemical data suggested that

the enzymes exist in two main conformational states. The E1 state binds ions and

transfers phosphate from ATP to an amino acid residue within the enzyme. The

hydrolysis of ATP causes a conformational change in the enzyme to the E2 state. This

confirmation has low affinity for ions and releases them to the opposite side of the

membrane. The recent crystal structures that represent the El (46) and E2 (47) states of

the Ca++ ATPase taken together with the biochemical data and lower resolution structures

of several P-type ATPases suggest that the reaction cycle is much more complicated,

consisting of a series of stable intermediate steps (44). The reaction cycle described

below and depicted in Figure 1-4 is for the Ca+ ATPase, but is likely representative of

the P-type ATPases as a whole.

The starting conformation is the closed (E2H) state (Figure 1-4A). The Ca

binding sites were protonated during the previous reaction cycle and face the lumen of

the sarcoplasmic reticulum. Deprotonation of these sites is accompanied by a rotation in

the P domain and a reorientation of the Ca++ binding sites towards the cytoplasm of the

cell. The P domain and the A domain are now able to interact with each other. This is

the state is the open conformation (Ei) (Figure 1-4B). Once Ca+ is bound to the channel,

the N domain binds ATP (EiMgATPCa2) and the P domain loses its interactions with the

A domain (Figure 1-4C). The P domain is then ready to accept a phosphate from an ATP

that is hydrolyzed in the N domain (EiMgP(Ca2)ADP) (Figure 1-4D). The release of

ADP from the enzyme causes a major conformation shift that orients the Ca++ ions to the

lumen (E2MgPCa2) (Figure 1-4E).











510


B


ADP


+ Ca
Cal


H+


C





3


ATP
Ca'

6


2


Figure 1-4. Reaction Cycle of the Ca++-ATPase as a representative of P-type ATPases.
The reaction intermediates are (A) E2H (B) El (C) E1MgATPCa2 (D)
E1MgP(Ca2)ADP (E) E2MgP. Numbers represent transitions between states
as described in the text.


This conformation has a low affinity for Ca+ and it is released (E2MgP). The sequential
release of H20, Mg+, and inorganic phosphate returns the enzyme in the E2 state ready
for protonation and the beginning of another reaction cycle (Figure 1-4A).









Structure of the HKa2 Gene

The gene that encodes the rabbit HKca2 subunit was previously unknown. One of

the aims of our study was to clone the gene from a rabbit genomic library. The genes for

the human and mouse gene, however, have been cloned. This section will review general

characteristics of eukaryotic genes and the genomic organization for the human and

mouse HKc2 genes.

Eukaryotic Gene Organization

By definition, a gene is considered to be the entire nucleic acid sequence that is

necessary for the synthesis of a functional polypeptide or RNA (31). According to this

definition, the nucleotide sequence that codes for the protein or functional RNA is only a

portion of the entire gene. The rest of the gene consists of non-coding sequences that

play a role in creating the final product. This section describes common features of

eukaryotic genes such as intron/exon organization, core promoter elements and

transcriptional control elements.

The nucleotide sequence that codes for a protein is not found as a continuous

sequence. Instead, the coding regions (exons) are interrupted by non-coding regions

intronss). Once the entire region is transcribed, RNA splicing machinery recognizes

specific splice junction sequences, removes the introns and joins together the exons

creating a complete mRNA sequence. Importantly, this arrangement allows for

alternative splicing. It is possible for the RNA splicing machinery to join some exons

and remove others creating alternative RNA transcripts from the same gene. The

production of alternative transcripts may be regulated by different stimuli or in a tissue

specific manner.









The core promoter elements are those that are necessary for basal transcription

(7). They are located near the transcription start site. Common core promoter elements

are diagramed in Figure 1-5. They include the TATA box, the initiator element (Inr), the

downstream promoter element (DPE), the TFIIB recognition element (BRE), and the

CpG island. The TATA element, with the consensus sequence TATAAA, is usually

located about 25-30 bases upstream of the transcription start site. This element is the

binding site for the TATA binding protein (TBP). TBP and its associated factors (TAFs)

make up the general transcription factor TFIID. Once bound, TFIID is capable of

recruiting and/or interacting with other general transcription factors and RNA

polymerase. The entire complex is then capable of determining the specific transcription

start site and initiating basal level transcription. In the absence of a TATA box, an

initiator element may be present and act to initiate transcription. This element has a very

loose consensus sequence of PyPyA+1NT/APyPy and usually overlaps with the

transcription start site. It is thought that the consensus sequence is recognized by TBP

associated factors and directs TBP to bind upstream in a TATA box independent manner.

The rest of transcription initiation occurs similarly to TATA box containing promoters.

The downstream promoter element is found about 30 bases downstream of the

transcription start site. It has a consensus sequence of RGA/TCGTG. Similarly to the

initiator, the DPE is thought to bind a TBP associated factor and direct specific initiation

of transcription. It is known to work in conjuction with the initiator sequence at TATA-

less promoters, but may also function to stabilize core promoters with weak TATA

elements. The TFIIB recognition sequence, G/CG/CG/ACGCC, is located immediately

upstream of the TATA box (29). As its name implies, this sequence binds the general









transcription factor TFIIB. This element is present at many, but not all, eukaryotic

promoters. Finally, some promoters do not have TATA box or an initiator element.

Instead these promoters contain a region of high GC content upstream of the coding

region. This GC rich region, known as a CpG island, can form multiple binding sites for

SP family members. The stimulatory protein family can bind and direct the formation of

preinitiation complexes. This process, however, is often imprecise and allows for

multiple transcription start sites. In addition to basal level transcription, many eukaryotic

genes are activated and repressed by environmental stimuli. This change in regulation is

modulated by the binding of sequence-specific DNA-binding proteins that can interact

with the core promoter proteins and either activate or repress transcription.


General
Transcription RNA
Factors Polymerase
m m


RN
SIi


A Transcript


V CpG Island

Regulatory Elements
activatorss, repressors)


Figure 1-5. Common eukaryotic gene promoter elements. TATA represents the TATA
element. INR represents the initiator element. DPE represents the
downstream promoter element. Adapted from Lodish et al. (31).

HKa2 cDNAs

Table 1-1 lists all known HKc2 cDNAs along with the sizes of their 5'

untranslated regions (UTR), open reading frames and the 3'UTRs. The HKa2a cDNA









sequences are very similar. The first rat HKU,2a cDNA was obtained by screening a

colonic cDNA library (9). Subsequently, Kone and coworkers (26) reported a cDNA

with an identical open reading frame that they cloned from kidney. The 3' UTRs were

also identical, but Kone obtained a longer 5'UTR by using primer extension analysis. In

addition to the HKUa2a sequence, a second HKu2 cDNA termed HKa2b was found (see

below). Two groups independently cloned the HKU,2a cDNA from rabbit kidney and

performed 5' and 3' Rapid Amplification of cDNA Ends (RACE) to determine the length

of the HKU,2a cDNA (6, 12). RACE has the capacity to produce a full-length cDNA, but

the length of the PCR product is dependent upon the efficiency of the reverse

transcriptase reaction. Therefore, a full-length cDNA is not necessarily produced. For

the rabbit HK,2a cDNAs, Fejes-Toth et al. (12) acquired more 5' UTR sequence while

Campbell et al. (6) obtained the longer 3' UTR sequence. Our study used the RNase

protection assay to determine the true 5' end of the HKU,2a and HKcU2c transcripts. The

3'UTR obtained by Campbell et al. extended to the likely poly A signal, and was viewed

as complete. It is notable that the complete 3' UTR of the human ATP1AL1 transcript

was apparently much shorter than that of other mammalian species. Whether this effects

mRNA stability or has some other regulatory significance has not been studied.

cDNA clones representing splice variants of HKc2 gene transcripts have been

obtained for rat (HKc2b) (26) and rabbit (HKC2c) (6). HKo2b and HKI 2c cDNAs differ

from the HK,2a cDNA only at the extreme 5' end but the deduced protein products differ

significantly.










Table 1-1. cDNA's for mammalian HKa2 transcripts.
Organism Apparent Open Reading Probable Genebank Reference
Subunit 5'UTR Frame/Amino acids 3'UTR Accession
Number
Human

ATP1AL1 168 3117/1039 290 U02076 [Grishin, 1999 #8]

Rat

HKo2a 202 3108/1036 650 M90398 [Crowson, 1992 #1]

HKo2a 275 3108 /1036 650 U94912 [Kone, 1998 #66]

HKo2b 739 2787 / 929 348 U94913 [Kone, 1998 #66]

Rabbit

HKo2a 190 3101 /1034 875 AF106063 [Fejes-Toth, 1999 #5]

HKo2a 39 3101 /1034 939 AF023128 [Campbell, 1999 #3]

HKo2c 198 3285 / 1095 939 AF023129 [Campbell, 1999 #3]

Guinea Pig

HKo2a 145 3101 /1034 996 D21854 Genbank Database

Mouse

HKo2a 253 3108/1036 629 AF350499 [Zhang, 2001 #31]].



Figure 1-6 is a schematic representation of the likely mechanism for the formation of the

alternative transcripts. In both species, HKu-2a transcription initiates at exon 1 and the 5'

end of the mRNA is produced by splicing exon 1 to exon 2. The rat HKua2b and the rabbit

HKUa2c transcripts arise from transcription initiation within what had been designated as

intron 1 of the HKu2 gene. The rat HKUa2b cDNA had a peculiar organization. The 5'

UTR was longer than any of the other HKu2 5' UTRs (739bp) and is in fact much longer

than a typical mammalian 5'UTR. In addition, it contained eight short open reading

frames prior to the HKU2b translation start site that may offer a mechanism for










Genomic


ron
1


DNA
Exon Other
2 Exons



Rabbit


I Protein


HK~a
HKca2c


Figure 1-6. Formation of the alternative transcripts from the rabbit and rat HKc2 genes.



translational regulation (26). Northern analysis of total RNA from various rat tissues

shows that the HKUa2b transcript was present in vivo. The HKaU2b protein appeared to be

truncated by 108 amino acids, but the presence of the protein in vivo has not been

reported. In rabbit, the HKUa2c 5' UTR contains only two short open reading frames prior

to the translation start and is of similar length to other HKc2 transcripts. Unlike the

truncated HKa2b of rat, the HKUa2c protein contains a 61 amino acid extension on the N-

terminus ofHKa2a. Fejes-Toth et al. (12) failed to detect HKa2c in their 5' RACE

experiments. However, Campbell et al. (6) demonstrated by both Northern and Western

analyses that HKa2c mRNA and protein were present in rabbit kidney tissue as well as in

tissue culture cells derived from the rabbit cortical collecting duct (RCCT28A). The


Exon Int

4 1 +
Ooo,.


Rat









cloning of homologous cDNAs from other species will be necessary to determine if the

formation of splice variants is common among mammalian species or whether it is

unique to rat and rabbit.

Human HKa2 Gene

The human HKc2 gene was originally identified as the ATP1AL1 gene (45). It

was initially unclear as to whether or not this H K+ ATPase should be considered

homologous to the rat HKaU2a (8). The amino acid identity for these two proteins was

much lower (87%) than that of the HKaci proteins from human, rat and rabbit (97%). The

cloning of the rabbit HKUa2a cDNA (6) suggests that all three proteins are homologous as

it had approximately 87% amino acid identity with both rat HKUa2a and human ATP1AL1

sequences (8). The genomic structure of the mouse HKc2 gene (below), the rat gene

(NCBI database), and the rabbit HKc2 gene (this study) confirm that these genes are

homologous genes.

The complete genomic organization of the human ATP1AL1 gene was reported

by Sverdlov et al. in 1996 (45). The gene is approximately 32Kb in length and contains

23 exons and 22 introns. The sizes of the exons and introns are included in Table 2-1.

The transcription start site was mapped using S1 nuclease protection and primer

extension. The S1 nuclease protection assay produced 4 clustered bands from -185 to -

188. The primer extension produced a single band marking the transcription start site at -

187 with respect to the ATG start codon. Analysis of the region immediately 5' of the

transcription start site identified possible regulatory elements including a TATA box, SP

family binding sites, AP-2 binding sites and NF-kB. Additionally, the region from -484

to +369 met the criteria for a CpG island. Analysis of the 3' region of the gene identified









3 possible polyadenylation sites. The authors suggest that these sites may be used in a

tissue specific manner.

Mouse HKa2 Gene

Recently, the complete sequence and structure of the murine HKc2 gene was

reported (52). Similarly to the human gene, the murine gene spans 23.5Kb and contains

23 exons. The transcription start site was mapped using primer extension. It is located at

-253 with respect to the ATG start codon. These authors did not observe an alternative

transcription start site as seen in rat (26) and rabbit (6). Computer analysis of 7.2Kb of

sequence immediately 5' of the start site identified many possible transcription factor

binding sites including a TATA box, CEBP, NF-kB, cAMP and glucocorticoids. There

appears to be one poly A signal designating the 3' end of this gene.

Summary

Intracellular and extracellular K+ concentrations play a critical role in normal cell

functioning. The collecting duct segment of the nephron is the major location where K

ions are reabsorbed when blood K+ becomes too low. The HKu2 subtype of the

H K+ ATPase, located on the apical membrane of collecting duct cells, appears to play

a major role in K+ reabsorption since its activity and its expression appear to be increased

when blood K+ concentration is low. There is additional evidence that the expression of

the HKu2 gene products are also regulated by Na+ levels, acid/base balance, and

hormones. All of the studies on HKu2 gene expression, however, have been performed

in vivo. There is nothing known about the molecular mechanisms responsible for the

change in gene expression. The goal of our study was to characterize the rabbit HKa2

gene and initiate studies on the molecular regulation of the gene.














CHAPTER 2
CLONING OF THE HKa2 GENE

The first specific aim of this dissertation was to clone the rabbit HKc2 gene.

There were two important reasons for carrying out this goal. First, cloning the gene

would provide sequence information essential for the future experiments designed to

study the gene's promoter and regulatory elements. The cDNAs for HKC2a and HKac2c

have been previously identified (6) and shown to be products of the same gene (5).

However, there is currently nothing known about the genomic sequence that is 5' of the

cDNA ends. This dissertation, therefore, provides the first data regarding the upstream

sequence that contains the gene's promoter and its regulatory elements. The second

purpose for cloning the rabbit HKc2 gene was to determine its genomic organization.

There has been some controversy over whether or not the HKc2 proteins identified from

several species were in fact homologous proteins (8). The a subunit proteins from rabbit

(HKa2a), rat (HKU2a), mouse (HKu2), guinea pig (HKu2) and human (ATP1AL1) share

an amino acid identity of 87%. This percentage is much lower than the amino acid

identity that the same species share for the HKal subunit (97%). One method for

resolving this controversy is to compare the intron and exon sizes of the genes that

encode the proteins since genes that were derived from common ancestors should

maintain a consistent organization. The genomic organization of the human ATP1AL

(45) and mouse HKu2 (52) genes have been determined independently from this

dissertation. The genomic organization of the rat gene was determined from the rat









genome database at the National Center for Biotechnology Information (NCBI). The

cloning and sequencing of the rabbit gene is the subject of this chapter. The comparison

of the organization of the four genes supports the conclusion that these genes are

homologous and have been derived from a common ancestor.

The rabbit HKc2 gene was cloned from a bacteriophage X library of the rabbit

genome. Three clones were identified that span a majority of the rabbit HKc2 gene. The

polymerase chain reaction (PCR) was used on rabbit genomic DNA in order to amplify

the remaining portion of the gene. This chapter discusses the cloning, sequencing and

analysis of the bacteriophage X clones and the PCR products that contain the HKc2 gene.

Materials and Methods

Screening the Lamda Genomic Library

A bacteriophage X library containing 15Kilobase pair (Kbp) inserts of rabbit

genomic DNA was purchased from Clontech, Inc. (Catalog #TL1008j). Two approaches

were used to screen the library. The first method was a PCR-based screen (21) and the

second was a more conventional plaque lift/hybridization screen (Clontech, Inc).

The PCR approach used primers designed to the HKU2a cDNA to identify aliquots

of the X library that contained bacteriophage with inserts that corresponded to the HKa2

gene. The primers BC334 (5'-TATCTGTAGCTGCATGGTGCTCCAC-3') and BC386

(5'-ACCCGCGCGCTCCAGCGCGACAT-3') were used in the PCR reaction. These

primers correspond to base pairs 69-93 and base pairs 16-40 the HKu2a cDNA and are

known to amplify a 647 base pair fragment from rabbit genomic DNA (5). The amplified

fragment is larger than expected because it includes intron sequence that is not present in

the cDNA. This reaction was repeated as a positive control for the PCR approach to









screening the X library. Additionally, 2[tl of the PCR reaction was ligated into the TOPO

cloning vector (Invitrogen, Inc. Cat# K4574-J10). This construct was designated pDZ6

and is referred to as the 5' probe for all future experiments. To screen the library, 500[tl

of E.coli strain K802 (Clontech, Inc.) was infected with 1 X 106 plaque forming units

(pfu) of the X genomic library and incubated at 370C for 15 minutes. The infected

bacteria were brought to a volume of 10mL with LB broth (tryptone, yeast extract, NaC1)

supplemented with 10mM MgSO4. 100tl of infected bacteria were placed into each well

of a 96 well plate and amplified by growth at 370C for 5 hours. 25[tl from each row and

each column were pooled to form 16 samples representing the 96 wells. 10[tl of the

pooled samples were used in a PCR reaction (250pm each primer, 250[LM dNTP mix, 1X

PCR buffer, 5U Taq polymerase, 10tl of pooled template, dH20 to 401t). The PCR

conditions were 940C for 1 minute 30 seconds; 940C for 15 seconds + 720C for 2 minutes

times 5 cycles; 940C for 15 seconds + 700C for 2 minutes times 5 cycles; 940C for 15

seconds + 680C for 2 minutes times 25 cycles; 680C for 8 minutes. Southern analysis of

the PCR products was carried out as described in Maniatis et al. (39). The PCR reactions

were run on a 1% agarose gel and visualized with ethidium bromide stain and UV light.

The gel was soaked gel in denaturation solution (1.5M NaC1, 0.5M NaOH) for 30

minutes, neutralizing solution (1.5M NaC1, 1.OM Tris-Cl pH 8.0) for 30 minutes, and

equilibrated in 10X SSC (1.5MNaC1, .15M sodium citrate) for 30 minutes. The DNA

was transferred to a nylon membrane by capillary action overnight. The DNA was UV

crosslinked to the nylon membrane, placed in a hybridization tube and incubated with

5mL of buffer (.25M Na2HPO4 pH 7.2, ImM EDTA, 1% BSA, 7% SDS) at 65C for at

least 15 minutes. A radioactive probe was prepared by digesting pDZ6 with EcoRI and









running the digestion on a 1% agarose gel. The 647bp band was cut from the gel and

extracted using the Gel Extraction Kit from Qiagen, Inc.(Catolog #28706). 25ng of the

DNA fragment were used to create a 32P labeled probe. The method used was the Prime-

It RmT Random Primer Labeling Kit from Stratagene, Inc. (Catolog #300392). The

probe was added directly to the prehybridization solution and incubated with the nylon

membrane overnight at 650C. The next day, the membrane was washed three times with

300mL of wash buffer (20mM Na2HPO4 pH 7.2, 1% SDS, ImM EDTA) at 65C,

wrapped in saran wrap and exposed to autoradiograph film for an appropriate length of

time. Positive rows and columns were aligned to determine possible positive wells. The

PCR was repeated on samples from individual wells. The contents of positive wells were

used to create plaques on agar plates. Plaque lift hybridizations were performed to purify

positive plaques (see below).

The second method for screening the library was to perform a series of plaque lift

hybridizations. The protocol was modified from the procedure recommended by the

rabbit genomic library manufacturer, Clontech, Inc. The initial screens were performed

with approximately 20,000 pfu per 150mm agar plate. The X library was diluted 1/500

with dilution buffer (1M NaC1, 0.1M MgSO4, 1M Tris pH 7.5) and 100[tl was used to

infect 600[tl of E. coli strain K802 at 370C for 15 min. 7mL of warmed soft agar (LB

broth + 7g/L agar) was added to infection and poured over a 150mm LB agar plate. After

overnight incubation at 370C, plaque lifts were performed by placing circular nylon

membranes on top of each plate for 2 minutes, the filter was removed placed plaque side

up on top of Whatman paper #3 soaked in denaturing solution for (1.5M NaC1, .5N

NaOH) for 5 minutes, neutralizing solution (1.5M NaC1, 1M Tris pH 8.0) for 5 minutes









and 2X SSC (.3M NaC1, .03M Sodium Citrate) for 5 minutes. The filters were baked dry

at 800C for 1 hour. The filters were probed with either a 5' probe (pDZ6), a mid probe

(HKU2a cDNA bases 1264 1569) or a 3' probe (HKc2a cDNA bases 3265 4073) as

described for the Southern blot filters. Positive plaques were pulled from the agar plates

with a Pasteur pipette and diluted into ImL of dilution buffer. Serial dilutions of the

plaque were used to infect E.coli strain K802, mixed with 3mL of soft agar and poured

over 100mm agar plates. The plaque lift procedure was repeated. Isolated positive

plaques were picked and the procedure was repeated until all plaques on a plate were

positive and each plaque was considered pure.

Pure plaques that were isolated by both screening methods were grown in large

scale and DNA was isolated using the Qiagen Lambda Maxi Kit (Cat# 12562). A 5mL

overnight culture of E.coli strain K802 was pelleted and resuspended in 1.5mL of

bacteriophage dilution buffer. Approximately 1 X 107 bacteriophage were added to the

bacterial cells and incubated at 370C for 20 minutes. The infected bacteria were added to

250mL of LB supplemented with 10mM MgSO4 and 0.2% maltose. The culture was

allowed to grow at 370C until the bacteria lysed (approximately 4 hours). The bacterial

debris was pelleted while the bacteriophage that remained in the supernatant was used for

DNA isolation. 400tl of buffer L1 (300mM NaC1, 100mM Tris-Cl pH 7.5, 10mM

EDTA, .2mg BSA, .2mg/mL RNaseA) was added to the lysate and incubated at 370C.

This step digests away any bacterial RNA. In order to precipitate and pellet the

bacteriophage, 50mL of buffer L2 (30% polyethylene glycol, 3M NaC1) was added and

the mixture was incubated on ice for 60 minutes and centrifuged at 10,000 rpm for 10

minutes. The pellet was resuspended in 9mL of buffer L3 (100mM NaC1, 100mM Tris-









Cl pH 7.5, 25mM EDTA) and 9mL of buffer L4 (4% sodium dodecyl sulfate (SDS) and

the mixture was heated to 700C for 10 minutes. This step denatured the bacteriophage

proteins and released the bacteriophage DNA. After cooling on ice, 9mL of buffer L5

(3M potassium acetate) was added and mixture was centrifuged for 30 minutes at 15,000

rpm in order to pellet bacteriophage proteins. The supernatant that contained the

bacteriophage DNA was poured over a Qiagen column that was equilibrated with buffer

QBT (750mM NaC1, 50mM MOPS, 15% isopropanol, .15% triton X-100). The DNA

bound to the column. The column was washed with 60mL of buffer QC (1M NaC1,

50mM MOPS, 15% isopropanol) and then the DNA was eluted off the column with

15mL of buffer QF (1.25M NaC1, 50mM Tris-Cl pH 8.5, 15% isopropanol). 10.5mL of

isopropanol was added to the eluate and centrifuged at 15,000 rpm for 30 minutes to

precipitate and pellet the bacteriophage DNA. The DNA was washed with 70% ethanol

and resuspended in TE (10mM Tris pH 7.5, ImM EDTA). The DNA from each X clone

was digested and used in Southern analysis in order to identify overlapping clones that

span the entire HKc2 gene.

WHKa2.1 Sequence

Clone HKa2.1 was the 5' most clone. Southern analysis showed that the EcoRI

fragment that hybridized to the 5' probe was attached to the X SP6 arm and sequencing

with the SP6 promoter primer (5'-ATTTAGGTGACACTATAG-3') indicated that the

orientation of the insert was such that the rest of the clone contained sequence 5' of the

HKu2 gene. In order to obtain the sequence of the region immediately 5' of the cDNA, a

6.3Kbp XhoI fragment was subcloned into pBluescript (pBS, Stratagene, Inc.) creating

plasmid pDZ10. The sequence of the entire fragment was determined by walking along









the sequence in both 5' and 3' directions. Sequencing was carried out by the University

of Florida Interdisciplinary Center for Biomedical Research (ICBR) sequencing core

facility. Complete sequences from both directions were obtained by compiling sequences

from individual primers. The two complete sequences were compared and any base pair

mismatches were resolved by additional sequencing through the region. The location of

each primer on the complete pDZ 10 sequence and the sequence of primers can be found

in Figure 2-1 and Table 2-1 respectively.







I I I I I 1 I I*





Figure 2-1. Location of Sequencing Primers on the Complete pDZ 10 sequence. Black
represents the 6.3Kbp Xhol fragment subcloned from WHKa2.1. Orange
represents the ends of pBS (Stratagene, Inc.). Blue represents primers used
for sequencing in the 5' to 3' direction. Red represents primers used for
sequencing in the 3' to 5' direction. The star indicates the location of the 5'
probe.



%HKa2.5 Sequence

Clone HKa2.5 was the middle clone. It hybridized to both the 5' and the mid

probes (HKU2a cDNA base pairs 16-93 and 1264-1569 respectively). Since this clone

was being used to determine the intron/exon boundaries within the rabbit HKU2 gene, it

was necessary to obtain the sequence of the entire clone. The k DNA arms were









removed by digestion of the clone with Xhol. The digestion was run on a 1% agarose gel

and visualized with ethidium bromide. The genomic DNA insert was cut from the gel

Table 2-1. Primers used for pDZ10 sequencing.
Name Primer Sequence 5' to 3" Location
T7 CCCTATAGTGAGTCGTATTA k arm
DZ12 CAATCCACGTTGCCCGCATGGG 784-805
DZ14 CCAGTCCGGATACGGAGCAGG 1437-1457
DZ15 CCCCACCAACAGCCCAGACG 2228-2247
DZ18 CCTCCAGGTGAGGACTACTCC 2974-2994
DZ25 CTCTCCCCTCCAACTCTGAAGG 3689-3710
DZ20 GAACGGCCGGCGCTGCGG 3774-3791
DZ26 GTGTCCCATGTGGGAAGCCAGG 4378-4399
DZ27 CTTGGGGGCTCCGGATCCTGG 5064-5083
SP6 ATTTAGGTGACACTATAG arm
DZ4 CGCATGTCGCGCTGGAGG 5587-5570
DZ5 CTGCACTCTCAGAGTGAAGG 4889-4870
DZ6 GGCTATGGGACAGGGATGACG 4165-4145
DZ16 GGCACAGAGAAGTAGTGCCC 3469-3450
DZ19 GAAACCTACTCATGCCAGGCTC 2752-2731
DZ21 GATGAGTTCTCAGGACTCTGAC 1977-1956
DZ23 GCTGCAGCCTAGCACAC 1237-1221
DZ24 GGGGAGTAAACCTCAGGATGGG 568-547


Blue represents primers used for sequencing in the 5' to 3' direction.
primers used for sequencing in the 3' to 5' direction.


Red represents


and extracted from the agarose using the QIAquick Gel Extraction Kit (Qiagen, Inc. Cat #

28706). This procedure was repeated until 25[tg of insert DNA was obtained. The insert

fragment was then sheared and shotgun subcloned into the TOPO cloning vector

according to Invitrogen, Inc. (Cat.# K7000-01). 25[tg of DNA was added to 7501tl

shearing buffer (TE, 20% glycerol) and placed in a nebulizer attached to a compressed air

pump. The DNA was sheared twice at 10psi for 90 seconds. The sheared DNA was

precipitated (80[tl 3M NaOAc, 4[tl glycogen, 700[tl 100% isopropanol) on dry ice for 15

minutes, pelleted by centrifugation at 12,000 rpm for 15 minutes, washed with 80%

ethanol and resuspended in 200[tl of sterile dH2O. In order to repair the sheared ends for









cloning, 2[tg of DNA was added to a blunt-end repair reaction (20[tl DNA, 5[tl 10X

blunting buffer, 1 tl BSA, 51tl dNTP mix, 2[tl T4 DNA polymerase, 2\tl Klenow DNA

polymerase) and incubated at room temperature for 30 minutes. The enzymes were

deactivated by heating the reaction mix to 750C for 20 minutes. Dephosphorylation of

the repaired ends was carried out by adding 35[tl sterile dH20, 10l 10X

dephosphorylation buffer, and 5[tl calf intestine alkaline phosphatase (CIP) to the blunt

end repair reaction and incubating the reaction at 370C for 60 minutes. The reaction was

phenol/chloroform extracted, precipitated and resuspended in 20[tl of sterile dH20.

Shotgun cloning of the k DNA was carried out with 3 concentrations of DNA (60ng,

20ng, 5ng), 1 Cl salt solution, and 1 C1 pCR4-blunt TOPO vector (Invitrogen, Inc.). The

ligations were incubated at room temperature for 5 minutes and then transformed into

chemically competent E. coli strain DH5a. The resulting bacterial colonies were

screened to identify 48 plasmids containing inserts of approximately 1500bp. Each

colony was grown overnight in 3mL of LB broth and then miniprep DNA was isolated

using the QIAprep Spin Miniprep Kit from Qiagen, Inc. (Catolog # 27106). The DNA

was digested with EcoRI, run on a 1% agarose gel and visualized with ethidium bromide

stain. When 48 bacterial colonies that contained plasmids of the appropriate size were

identified, 200[tl of an overnight culture of each colony was placed in an ELISA plate

and taken to ICBR for high throughput sequencing. Approximately 250bp of sequence

was obtained from each end of the plasmid inserts. These sequences were assembled into

ten contiguous fragments (contigs) by ICBR. The high-throughput sequencing core used

the Helix Finch program distributed by Giospiza, Inc. in orger to assemble the sequences.

The order of the contigs was determined by alignment of the contigs with the HKOa2a









cDNA and by determination of plasmids that contained sequence in two contigs. The

gaps between the fragments were closed by additional sequence from the plasmids that

spanned the gaps as well as sequence from the original X clone HKu2.5. The primers

used for the additional sequencing are listed in Table 2-2.

Table 2-2. Primers used for HKu2.8 sequencing
Name Sequence 5' to 3' Location
DZ83 CCCCGCTCTAAAGAAGGCCG 2418
DZ94 GGGCTTTCGGCCGACCTCACTG 2873
TC4 CCTGGAATGGACAGGCT 2983
DZ93 GCCTTCTGCCTCCAGGGC 3181
DZ96 GCCCCCGTTTTGACTCCC 3815
DZ95 GAGCGGGGGTGTCATTCACTCCG 2190
MG45 CGTCCATTCCTGTCCATAGCTATCTTCCAAGTCGTTCAGGTG 2897
MG49 CATCGTATACCCAGATCAGGATGGCATGGGGTACGGCCAC 3188
Blue represents primers used for sequencing in the 5' to 3' direction. Red represents
primers used for sequencing in the 3' to 5' direction. Location indicates the position of
the primer on the HKa2a cDNA.


%HKa2.8 Sequence

Clone HKa2.8 was the 3' clone. It hybridized to the 3' probe (HK.2a cDNA base

pairs 3265-4073). The approximate intron/exon boundaries were determined by

alignment of the cDNA from rabbit HKa2a to the exon sizes of human ATP1AL1 gene.

Primers were then designed near the expected end of each exon (Table 2-3) and were

used for partial sequencing of k clone HKa2.8. The sequencing was carried out by the

ICBR sequencing core.

PCR Amplification of the Missing Fragment

In order to obtain the exon boundaries for the portion of the HKa2 gene that was

missing from the three X clones, PCR primers were designed to the approximate ends of

the exons. The sequences of the primers, their orientations, and their location along the









HKa2a cDNA are listed in Table 2-3. Proofstart DNA polymerase (Qiagen, Inc. Cat#

202203) and RCCT28A genomic DNA were used for the PCR reaction. The PCR

reaction mix consisted of 1X Proofstart PCR buffer containing 15mM MgSO4, 300[tM

each dNTP, 1IlM each primer, 2.5U ProofStart DNA polymerase, 0.5tlg DNA template,

and dH20 up to 50[tl. The PCR cycle conditions were 95C for 5 minutes times one

cycle and 94C for 1 minute, 60C for 1 minute, 72C for 2 minutes times 40 cycles. The

PCR products were run on a 1% agarose gel and visualized with ethidium bromide. The

most intense band was cut from the gel and extracted using the QIAquick Gel Extraction

Kit (Qiagen, Inc. Cat# 28706) as described by the manufacturer. In order to obtain the

sequence of the exon boundary, 50ng of DNA was sent to ICBR sequencing core along

with the primers used to create the PCR product.

Table 2-3. Primers for genomic PCR and sequencing
Name Sequence 5' to 3' Location
DZ98 CCTTGGGTGCGGGGGGACAG Intron 11
DZ99 GGGCGGCCTGGGCGAGC 1852
TC80 GGCTCTCTTATCAATGATTCATCC 1979
DZ81 GCCATTGCCAAGAGTGTAGGG 2093
BC231 GCTTGTCATTGGGATCTTCC 1702
DZ100 CTGAGTCAAATGAGTAGGTCTCTGG 1913
DZ101 CCCTACACTCTTGGCAATGGC 2093
DZ97 CTGGGGAAACTTTGCCCTCC Intronl4
Blue represents primers used for sequencing in the 5' to 3' direction. Red represents
primers used for sequencing in the 3' to 5' direction. Location indicates the position of
the primer on the HKa2a cDNA.


Results

Screening the X Genomic Library

A majority of the rabbit HKa2 gene was cloned by screening a rabbit genomic

library using PCR and plaque lift hybridization methods. These two techniques identified

nine X clones. Two of the clones were identified by the PCR method (HKa2.2, HKa2.4)









and seven of the clones were identified by traditional plaque lift hybridization (HKc2.1,

HKu2.3, HKu2.5, HKu2.6, HKu2.7, and HKc2.8).

Figure 2-2 is an example of the PCR screen that identified X clone HKu2.2.

Figure 2-2A shows a Southern analysis of the initial screen. Samples were pooled across

rows and down columns and PCR was performed on 25[tl of the pooled sample. The

PCR products were run on a 1% agarose gel and Southern analysis was performed with

the 5' probe. In this example, rows F and H and columns 6, 7, 8 and 11 contain positive

clones. Figure 2-2B is the Southern blot that was performed on PCR products from the

screen of individual wells in row F. Well F7 was identified as a well containing a

positive X clone. The sample in well F7 was diluted and used in plaque lift experiment to

purify the positive clone that was designated HKu2.2. Additional PCR screening with

the 5' probe identified X clone HKu2.4. PCR amplification of positive fragments of

DNA quickly caused cross-sample contamination problem that was difficult to overcome.

This method was therefore abandoned and the remainder of the X clones were isolated

using standard plaque lift techniques.

Figure 2-3 is example of the plaque lift and purification procedure that used the 5'

probe to identify clone HKu2.1. In the first screen (Figure 2-3A) approximately 20,000

plaques were plated on a 150mm dish. After transfer to nylon and hybridization to the 5'

probe, only one positive plaque was identified. An agar plug was taken from that region

of the plate, diluted, and used to create a new plate with approximately 200 plaques.

About one half of the plaques on this plate hybridized to the 5' probe (Figure 2-3B). An

agar plug of an isolated plaque was taken from that plate and used to create a new plate

with a similar number of plaques. All of the plaques on the new plate hybridized to the











C D E FG H1

a. 6#


*^ <0 N 00

ue e ; u


5 6 7 8 9 10 11


(A) First screen of PCR
product pools


(B) Second screen of PCR
products from individual wells


Figure 2-2. Southern analysis of PCR screen of the X genomic library. A) First screen
1kb represents the 1kb ladder, GD represents genomic DNA, A-H represent
samples pooled across the rows of an ELISA plate, 1-12 represent samples
pooled down the columns of the same ELISA plate. Samples 2 and 10 were
cut of before DNA transfer. B) Second screen. Individual wells from positive
pools were screened for positive clones.



probe (Figure 2-3C). Any plaque from this plate was considered pure and could be used

for a X DNA maxiprep. This plaque lift method was used to identify seven clones; two

hybridized to the 5' probe (HKu2.1, HKu2.3), three hybridized to the mid probe

(HKu2.5, HKu2.6, 17-1), and two hybridized to the 3' probe (HKu2.7, HKu2.8).

















A.









SB..


e *






,.*,.-. o,






Figure 2-3. Plaque lift screen of the k genomic library. A) First screen. 20,000 plaques
per plate, one hybridizes to the 5' probe. B) Second screen. Plug from first
screen is diluted to 200 plaques per plate, /2 the plaques hybridize to the 5'
probe. C) Third screen. Individual plaque picked from second screen diluted
to 200 plaques per plate, all plaques hybridize to the 5' probe









Determination of Overlapping Clones

In order to determine which clones overlapped and spanned the HKu2 gene,

Southern analysis was performed on digested DNA from each clone. Figure 2-4 is an

example of a Southern analysis showing that HKu2.1 and HKu2.5 overlap in the region

of the 5' probe. HKu2.1, HKu2.5 and HKu2.6 DNA were digested with XAho and

HindlII individually and in combination. The digests were run on a 1% agarose gel and

visualized with ethidium bromide (Figure 2-4A). The DNA was transferred to nylon

membrane and hybridized to the 5' probe. The membrane was washed and exposed to

autoradiograph film (Figure 2-4B). In the lanes representing HKu2.1 and HKu2.5 a

single band appears that hybridizes to the 5' probe while in the lanes representing

HKu2.6 no band appears. These data clearly show that HKu2.1 that was isolated using

the 5' probe and HKu2.5 that was isolated using the mid probe overlap in the region of

the 5' probe. HKu2.6, that was isolated using the mid probe, does not extend to the 5'

probe. A similar analysis was carried out for the remainder of the X clones and with all

three probes. It was determined that three clones, HKu2.1, HKu2.5 and HKu2.8

spanned a majority of the HKu2 gene, but a gap existed in between HKu2.5 and

HKu2.8. Genomic PCR was carried out in order to obtain the missing portion of the

gene (see below).

kHKa2.1 Sequence

Clone HKa2.1 hybridized to the 5' probe and contains approximately 14Kbp of

sequence upstream of the HKa2 gene. Appendix A contains all of the known sequence

from the rabbit HKa2 gene. The 6300bp Xhol fragment subcloned from X HKa2.1 is

represented in base pairs 1 6298. This sequence was used to determine potential







44


promoter and regulatory elements. The complete analysis of this sequence is discussed in

Chapter three of this dissertation.


- = .2
0 -=
>< *~0
IIT
(c0(3(0
c o


(A) Ethidium bromide stained
Agarose gel


3 0 -~ = 03 O~ 3 0 -3










db W















(B) Autoradiograph of gel probed
With the 5' probe


Figure 2-4. Southern analysis to determine overlapping clones. (A) Each X clone was
digested as indicated and run on a 1% agarose gel. The DNA was visualized
by staining the gel with ethidium bromide. (B) The DNA was transferred to
nylon membrane and probed with the 5' probe (B).




XHKa2.5 Sequence

Clone HKu2.5 hybridized to both the 5' and the mid probes suggesting that it

contained many of the 5' exons for the HKc2 gene. ICBR high through-put sequencing

of the ends of 48 plasmids that were subcloned from HKu2.5 yielded 144 sequences.









The M13 forward primer (5'-GTAAAACGACGGCCAG-3') sequencing reaction was

performed twice and the M13 reverse primer (5'-ACAGGAAACAGCTATGAC-3')

sequencing reaction was performed once. The average read-length for the reactions was

307 bases. ICBR used a computer alignment algorithm to assemble the sequences into 10

contiguous fragments labeled 1-10 based upon size. Using the HKu.2a cDNA and

subclones in which sequence from opposite ends mapped into different contigs, nine of

the ten sequences were placed in order. Figure 2-5 depicts the nine sequences and the

subclones that spanned the gaps. In order to obtain the remainder of the HKa2.5

sequence, the gaps between the contigs were filled with additional sequence from the

indicated plasmids as well as with sequence directly from the HKu2.5 clone. Table 2-3

lists the DNA template and the primers that were used to complete the sequence.

Appendix A contains the known sequence of the rabbit HKc2 gene. The sequence

determined from HKu2.5 overlaps with the sequence from pDZ10 and is represented in

base pairs 4616 to 19766.

XHKa2.8 Sequence

Clone HKu2.8 hybridized to the 3' probe. Partial sequences were determined for

this clone. The purpose was to determine the precise intron/exon boundaries of the

remainder of the gene as well as the 3' end of the gene. The partial sequence revealed

that HKu2.8 contained sequence from exons 15-23. The sizes of the exons and introns

identified are listed in Table 2-5. A portion of the sequence of exon 25 is shown in

Figure 2-7. The red bases represent the 3' end of the HKa2a cDNA cloned by Fejes-Toth

et al. (12). Just upstream of the cDNA end there is a poly A signal sequence (blue) and

just downstream there is a T-rich region of DNA. It seems likely that this poly A signal












* Li


f7LZUI-~


r687U
I


El-
'i



I t


I


fC
< -





e-1
ol -


"-0-
ibs



- c


0o

coi
T3-e
c t



o
Sat

0 0




0T
0 5
ct 0

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" 0 0







00
F^ u &
U ;
F4


I





I

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167U-
88ZU-


17










represents one true end to the HKc2 mRNAs. Campbell et al. (6), however, cloned a

cDNA of HKa2a and HKU,2c that was slightly longer at the 3' end (green). Just

downstream of this cDNA end there are two possible poly A signals and T-rich region of

DNA. Therefore it seems likely that one of these two poly A signals, or both represent

alternative ends to the HKU2a and HKUa2c mRNAs. The partial sequence obtained from

HKa2.8 is located in Appendix A.



301 AGGTTTTTTT TTTTAAATAA AAGATGTTTT TAAGTAAAAT GTTTTATGAA

351 ACAAAATCTA ATTGTGATGT TTTACTTAAT TCAAGTTTTT CCAGAGGCAG

401 GCACGGAAAA TAC AAAA ATAAAATAAA ATAAGATTCT GGGTTTTTTT

451 TCTTTTTTGC TCCTTCTGGT CATTTTCTTT ACACACAGAG TGTCTGGAAA

501 TACAGGCTTT TCCTCGTGAG TGCTTCCCGC ACCTGTGCCC CCTCCCCCCC


Figure 2-6. Partial sequence from X clone HKu2.8. Blue represents 3 possible poly A
signals. Red represents the last three bases of the Fejes-Toth cDNA for
HKa2a. Green represents the last three bases of the Campbell cDNA for both
HKa2a and HKa2c.



Completion of the HKa2 Gene Sequence

The complete sequencing of k clone HKa2.5 and the partial sequence of k clone

HKa2.8 revealed that three HKa2 exons were not contained in either clone. Therefore,

genomic PCR was performed on RCCT28A cell DNA in order to amplify four DNA

fragments that contained the missing exons. Primer set DZ98/BC231 amplified a band of

approximately 4000 base pairs. This fragment contained a portion of intron 11 and the 5'

boundary of exon 12. Primer set DZ99/DZ 100 amplified a band of approximately 1500

base pairs. This fragment contained the 3' boundary of exon 12, intron 12 and the 5'









boundary of exon 13. Primer set DZ80/DZ101 amplified a band of approximately 700

base pairs. This fragment contained the 3' boundary of exon 13, intron 13 and the 5'

boundary of exon 14. Primer set DZ81/DZ95 amplified a band of approximately 4000

base pairs. This fragment contained the 3' boundary of exon 14 and a portion on intron

15. The sequences obtained from these PCR products are located in Appendix A.

Discussion

The screening of the X library generated three X clones that contained 87% of the

HKc2 exons and 65% of the HKc2 gene. Most importantly, clone HKu2.1 hybridized to

the 5' probe and contains approximately 14kbp of sequence upstream of the gene.

Obtaining this clone was a necessary first step in the study of the regulation of the HKa2

gene, which is the subject of the remaining chapters of this dissertation. Additionally, the

genomic organization of the rabbit HKc2 gene was determined using the complete

sequence of HKc2.5, the partial sequence of HKc2.8 and the genomic PCR fragments

that spanned the gap between the two X clones. HKu2.5 contained 15150bp of gene

sequence including the HKc2 exons 1-11, HKc2.8 contained exons 15 23, and the

PCR fragments contained exons 12, 13, and 14. The entire gene spanned approximately

30Kbp of genomic DNA. It is notable that intron 11 and intron 14 are approximately

4200bp each. The mid probe hybridizes to exon nine and the 3' probe hybridized to the

3' UTR. The size of the intervening DNA (18Kbp) is the likely reason why a clone

containing these three exons was not obtained when the bacteriophage X library was

screened.

Table 2-4 compares the sizes of the exons and introns of the rabbit HKc2 with

those of the rat HKc2 gene (ICBR database), the mouse HKc2 gene (52), and the human









ATP1AL1 gene (45). The exon sizes for the four genes are absolutely identical except

for the three 5' exons one, two and four. It is not surprising that the 5' end of the gene is

Table 2-4. Exon and intron sizes for the known HKc2 genes.
Exon Rabbita Ratb Mouse" Humand Intron Rabbita Ratb Mouse" Humand
1 208 287 262 195 1 567 677 658 (700)
2 141 153 150 159 2 2702 2133 2286 (2300)
3 60 60 60 60 3 3061 2511 2321 (2900)
4 204 201 201 204 4 1049 746 762 738
5 114 114 114 114 5 1032 742 726 937
6 135 135 135 135 6 113 124 129 123
7 118 118 118 118 7 173 233 227 258
8 269 269 269 269 8 854 939 970 1187
9 199 199 199 200 9 141 144 134 157
10 110 110 110 110 10 2282 1324 1269 (1700)
11 135 135 135 135 11 (4200) 4231 2010 (4200)
12 135 193 193 193 12 1600 1484 1471 (1600)
13 176 176 176 176 13 600 834 639 (900)
14 137 137 137 137 14 4200 1599 1597 (4200)
15 151 151 151 151 15 365 1419 557
16 169 169 169 16 88 90 87
17 155 155 155 17 (700) 1425 1311 (1900)
18 124 124 124 124 18 172 184 195 193
19 146 145 146 146 19 445 594 590 (600)
20 134 134 134 134 20 167 174 168 195
21 103 102 102 102 21 (1155) 434 388 431
22 92 92 92 92 22 87 137 161 83
23 658 658 658 905 23 -
Sources: a this dissertation, b NCBI database, c (52), d (45).Notes: () indicates introns
with sizes determined by estimating the size of restriction fragments. indicates sized
that could not be determined due to incomplete database sequence.


the most variable since it is in this region where the rabbit and rat genes can undergo

alternative splicing to create HKUa2c and HKUa2b while the mouse and human gene

apparently do not. The controversy over whether or not these genes are homologous was

partially resolved by a distance analysis of the HKu and NaKc subunit proteins (8). The

analysis showed that the three HKc2 proteins were more closely related to each other

than to any of the other X+,K+ ATPase a subunits suggesting that they are homologous.






50


The exon/intron sizes, compared in Table 2-4, support the existing evidence and confirm

that these four genes are homologous and were derived from a common ancestor.














CHAPTER 3
MAPPING THE TRANSCRIPTION START SITES FOR THE HKa2 GENE

The second specific aim of this dissertation was to map the transcription start sites

for the two alternative mRNAs produced by the HKc2 gene. The determination of the

transcription start sites was an important step in characterizing the HKc2 gene for two

reasons. The main goal of our study was to initiate an investigation of the regulation of

the rabbit HKc2 gene. The core promoter and regulatory elements directing transcription

from the gene are likely to be found just 5' of the transcription start sites. Additionally,

there was some controversy over the existence of HKa2c. Identification of the

transcription start site for HKa2c would resolve this controversy.

The first experimental goal was to identify potential promoter and regulatory

elements 5' of the transcription start sites. The 5' ends of the cDNAs for HKa2a and

HKa2c were previously identified by studies that used 5' Rapid Amplification of cDNA

Ends (RACE) (5). This method, however, is not likely to determine the true transcription

start site. 5'RACE uses a reverse transcription reaction to extend a primer annealed near

the 5' end of the mRNA to the true end of the mRNA. The reverse transcriptase reaction

often terminates before reaching the absolute end of the RNA and the cloned cDNA will

therefore end 3' of the true transcription start site. In fact, the HKa2a 5' RACE

performed by two independent groups produced two different 5' ends. Campbell et al.

(6) obtained a 5'UTR of 39 bp, while Fejes-toth et al. (12) obtained a 5'UTR of 190 bp.

Furthermore, Campbell et al. was able to obtain a 5' cDNA end corresponding to the









splice variant HKca2c while Fejes-toth et al. did not. The RNase protection assay used in

this dissertation more accurately determines the transcription start sites because it does

not rely on primer extension. This chapter describes the construction of RNase protection

probes using the analysis of the sequence from clone HKu2.1 and the use of the probes in

mapping the transcription start sites the for HKc2 gene.

The second experimental goal in mapping the transcription start sites for the

HKu2 gene was to confirm the existence of the HKca2c transcript. Our laboratory

previously showed that the HKUa2c transcript and protein were present in both rabbit

kidney and colon (6). A second laboratory, however, was unable to detect the HKu.2c

transcript while using similar detection techniques (12). This chapter describes the

successful mapping of the transcription start sites for both the rabbit HKa,2a and HKac2c

transcripts.

Materials and Methods

Analysis of Clone HKa2.1

Clone HKa2.1 was identified by hybridization to the 5' probe (see Chapter 2). A

6.3Kbp Xhol fragment from HKa2.1 was subcloned into pBS (creating pDZ 10) and

completely sequenced. The sequence was analyzed for elements commonly found at

eukaryotic promoters.

RNase Protection Assay

The RNase protection assay was used to map the transcription start sites for

HKa2a and HKa2c. This assay was performed in three steps. First, an antisense

radioactive RNA probe was created from a fragment of genomic DNA likely to contain

the transcription start sites as well as sequence 5' of the start sites. Second, the









radioactive probe was annealed to the specific mRNA thereby protecting it from RNase

digestion. And third, the protected fragment was run on an acrylamide gel along with a

sequencing ladder of known size. Once the size of the protected fragment was

determined, the transcription start site could be mapped on the genomic DNA sequence.

In order to create the RNA probe, a 1.1Kbp HincII fragment likely to contain both

transcription start sites was cloned into the pGEM vector (Promega, Inc) creating plasmid

pDZ 12. The pGEM vector contained the SP6 polymerase promoter that was used in an in

vitro transcription reaction to create a radioactive RNA probe. Preliminary experiments

showed that one probe could not be used to map both start sites. Therefore, pDZ12 was

modified to create one plasmid with the region likely to contain the start site for HKua2a

(pDZ44) and a second plasmid with the region likely to contain the start site for HKa2c

(pDZ43). Figure 3-1 depicts the construction of these two plasmids, the expected sizes of

the full-length probes, and the predicted size of the protected fragment based on the 5'

end of the cDNAs. Plasmid pDZ44 was created by digesting pDZ12 with SacII and SphI,

filling in the vector ends with Klenow DNA polymerase, and religating the vector. A

450bp fragment containing the HKUa2c start site was removed from the vector creating a

shorter HKa2a RNA probe (Figure 3-1A). The full-length probe was 235 base pairs and

the protected fragment was expected to be approximately 87 base pairs. Plasmid pDZ43

was created by digesting pDZ12 with XmnI and HincII. The resulting 168bp fragment

contained the HKU2c transcription start site and had blunt ends. The fragment was cloned

directly into the HincII site of pGEM vector (Figure 3-1B). This plasmid produced a 182

base pair full-length probe and an expected 105 base pair protected fragment.







54





A.



7 "s I' 6 S
T7 SP6
I I l I ", P6







Full length probe 235bp

Protected fragment
estimated 87bp
SN m \N








T7 | T ISP6







-* --

Full length probe 182bp

Protected fragment
Estimated 105bp








Figure 3-1. Construction of RNase protection probes for HKU2a (A) and HKU2c (B).
Orange represents the cloning region of pGEM vector (Promega, Inc.) Black
B.





























represents 1.2Kbp fragment of rabbit genomic DNA subcloned into the pGEM
vector. Brown represents pertinent restriction sites. Blue represents the 5'
ends of the HKa2a and HKa2, cDNAs. Purple represents the binding sites for
,-~ ~ -f VS -S C -h0





























T7 and SP6 polymerase. Red represents the expected sizes of the in vitro
transcription products. Green represents the sized of the protected fragments
estimated based on the end of the cDNAs.
estimated based on the end of the cDNAs.









Each plasmid was used in the MAXIscript in vitro transcription kit (Ambion, Inc.

Cat. # 1308) in order to create radioactive RNA probes. 1tg of the plasmid DNA was

added to the in vitro transcription reaction (2[tl 10X transcription buffer, 1 tl 10mM each

ATP, CTP and GTP, 2.5tl 10mCi/ml [32-aP] UTP, 2l1 SP6 polymerase, and dH20 up to

20[tl). The reaction was incubated at 370C for 10 minutes. ltl of DNaseI was added and

the reaction was incubated at 370C for an additional 15 minutes. After incubation, the

entire reaction was loaded on a 5% acrylamide gel and run at 300 volts for 30 minutes.

The probe fragment was visualized by wrapping the gel in saran wrap and laying down a

piece of Polaroid type 57 high-speed film. When developed, a white band appeared on

the film at the position of the probe. The film was aligned with the gel, the band was

excised, and gel fragment was pressed through a 1mm syringe containing 250 pl of

elution buffer (.5M NH4Acetate, ImM EDTA, 0.2% SDS). The probe was eluted from

the gel fragments by incubation of the mixture at 370C for one hour. The specific activity

of the probe was measured on using a Beckman LS3801 scintillation counter. An aliquot

of probe containing a specific activity of 8 x 104cpm was used in the ribonuclease

protection assay.

The RPAIII kit from Ambion, Inc. (Cat # 1414) was used for the ribonuclease

protection assay. The probe was co-precipitated with 10[tg of rabbit colon total RNA by

bringing the volume of the probe and RNA to 1001tl, adding 10[tl NH40Ac and 250[tl

100% ethanol, incubating at -200C for 15 minutes and centrifuging at 15,000 rpm for 15

minutes. The pellet was air dried, resuspended in hybridization solution (Ambion, Inc.),

heated to 950C for 5 minutes and incubated at 420C overnight. During this time, the

probe annealed to its specific mRNA. The next day 1.5[tl ofRNaseA/RNase T1 cocktail









was diluted 1:100 in RNase Digestion buffer (Ambion, Inc.), added to the hybridization

reaction and incubated at 370C for one hour. During this incubation, all the single

stranded nucleic acids were degraded and only the double stranded protected fragment

remained intact. After the incubation, the protected fragment was precipitated by adding

225[tl of RNase inactivation/precipitation buffer (Ambion, Inc.), incubating the tube at

-200C for 15 minutes and centrifuging the tube at 15,000 rpm for 15 minutes. The pellet

was air dried and resuspended in 5[tl of gel loading buffer (95% formamide, .025%

xylene cyanol and bromophenol blue, 18mM EDTA, .025% SDS).

In order to visualize the protected RNA fragment, the each sample was loaded

onto a 6% polyacrylamide sequencing gel along with a sequencing reaction of a known

size. The sequencing reaction was carried out using Sequenase 7-deaza-dGTP DNA

Sequencing Kit (USB, Cat # 70990) with the control M13 single stranded DNA provided

with the kit. The M13 single stranded template (1.0[tg) was annealed to the -40 primer

(0.5pM) by mixing with 2[tl of sequenase reaction buffer (200mM Tris HC1 pH 7.5, 2mM

DTT, 0. 1mM EDTA, 50% glycerol) and dH20 up to 10tl and then heating to 650C for

two minutes. After cooling to room temperature, the labeling reaction (annealed DNA,

0.1M DTT, 21tl labeling mix (1.5mM 7-deaza-dGTP, 1.5[LM dCTP, 1.5[LM dTTP), 0.5l1

[ac-32P]dATP, 2[tl Sequenase polymerase (1U/[tl)) was incubated at room temperature for

five minutes. The reaction was terminated by adding 3.5[tl of the labeling mixture to

each of four pre-warmed termination tubes. All termination tubes contained 80mM of

each 7-deaza-dGTP, dCTP, dATP, dTTP, and 50mM NaC1. Additionally, each tube

contained 80[tM of either ddGTP, ddATP, ddTTP, or ddCTP. The termination reaction

was incubated at 370C for five minutes. The termination reaction was stopped with 4[tl









of stop solution (95% dien, 20mM EDTA, 0.05% xylene cyanol). The sequencing

reactions were run on a 5% polyacrylamide gel along with the protected fragments from

the RNase protection assay. The gel was run at 65 volts for approximately five hours,

dried for two hours and exposed to autoradiograph film overnight at -800C.

Results

Analysis of Clone HKa2.1

In order to determine the region most likely to contain the HKc2 gene promoter,

the sequence of the 6.3Kbp Xhol fragment was analyzed for characteristics common to

eukaryotic promoters. First, it was determined that the 3' end of the sequence contains a

CpG island. Figure 3-2 is a graph that shows the number of CpG dinucleotides found in

50 base pair windows of the sequence. Most of the sequence contained very few CpG

dinucleotides, but there was a clear peak in the number of CpGs at the 3' end. Next, the

computer program TFSearch was used to determine if any possible transcription factor

binding sites were present along the sequence. The results showed a wide variety of

possible binding sites. Appendix B contains the entire search results. Figure 3-3 is a

cartoon depicting the possible transcription factor binding sites that seemed most rational

based on previously known data about the regulation of the HKc2 gene (see discussion).

These include a TATA-like element, five SP family member binding sites, a downstream

promoter element, a cyclic-AMP response element (CRE) and a steroid response element

(SRE).

Transcription Start Sites

The transcription start sites for HK,2a and HKa2c were determined using the

RNase protection assay. In each case two protected fragments were observed. Figure 3-4





























111 II


I I 1111


o o0 00 00 00 0
10 1 0 0 10 0 0
o C m 10o 0 Cm 10o 0 Cm
- N N N N C) Cm


111111II


n ~frww~ri w


00 00 00 00 0 00
10 0 0 0 0 0 1 0 1 0 1
10 0 m 1 ~ 0 C 10~


The sequence of the 6.3kbp insert of pDZ10



Figure 3-2. CpG dinucleotide analysis of subclone pDZ10.


SRE CRE


- -


.-I


SP1SP1 SP1 SP1/TATA


Figure 3-3. Putative transcription factor binding sites determined by TFSearch. The 5'
ends of the HKUa2a and HKU2c cDNAs are indicated. Blue represents a CAAT
box. Pink represents a sequence with weak homology to the TATA box.
Green represents possible binding sites for SP family members. Red
represents a cyclic AMP response element. Orange represents a sterol
response element. Slashes indicate a break in the sequence of approximately
500bp.


0 0 0 0
10 0 10 0
CO) 10 03


0
CN

I


CAAT









is an example of a polyacrylamide sequencing gel in which the protected fragments for

HKa2a and HKU2c were run next to a sequence of known size (M 13 single stranded

DNA). By comparison to the known sequence, it was determined that the two protected

fragments for HKUa2a were 94 and 95bp. These fragments correspond to the bases of

genomic DNA indicated in Figure 3-4. They are 10 and 11 bases upstream of the cDNA

end obtained by Fejes-toth et al. (12), making the 5'UTR for HKU2a 200 and 201 base

pairs. Similarly the HKU2c protected fragments were 116 and 118bp and correspond to

the bases of genomic DNA indicated in Figure 3-4. They are five and seven bases

upstream of the cDNA end obtained by Campbell et al. (6), making the 5' UTR for

HKU2c 203 and 205 base pairs. For the remainder of this dissertation, the first

transcription start site for HKU2a was designated as +1 and all other positions are

designated relative to that transcription start site. The HKu2c transcription start sites were

therefore designated +382 and +384.

Discussion

Mapping the transcription start site was an important step in the characterization

of the HKc2 gene promoter. The RNase protection assay was used to map the

transcription starts sites for HKa2a and HKa2c. The results of this assay yielded several

interesting observations. First and foremost, the protected fragments observed with the

HKa2c probe confirmed the existence of the transcript in rabbit colon. Second, each

probe yielded two protected fragments. Third, the sequence upstream of the two start

sites contained a variety of possible core promoter elements. And finally, further

upstream, the sequence revealed several possible cis acting regulatory elements.










G A T C


2a start site:
CAGCATTTAA GGCGGACACC


B. A


'. ,.


HKX2a

ACCTCCCCTG GGCA GGCT GGCGATCGGC TGCGGAGGTG


T C RCR


mm


HKc2c
2c start site:
GGTCAATCCA GACACGCGGG GAAGGAGTTC CAGIGG CCAG CTCCGCCCTC GCACCTGCGG


Figure 3-4. RNase protection assay for HKUa2a (A) and HKa,2 (B). GATC represents
those nucleotides for the M13 control sequence. RCR represents the protected
fragment from the RNase protection assay performed with rabbit colon RNA.
A portion of the genomic sequence 5' of the HKa2 gene is shown below each
figure. Arrows indicate the position of the transcription start sites. Pink
represents putative core promoter elements and blue represents the 5' end of
the respective cDNAs.


A.


RCR


--W









The RNase protection assay was used to map two transcription start sites for

HKa2c. Previously, Fejes-Toth et al. (12) questioned the existence of the alternative

transcript identified by Campbell et al. (6). Fejes-Toth states that their 5' RACE

experiments generated one amplicon corresponding to the 5' end of HK.2a. They go on

to state that the convergence of their data from that of Campbell may be due to the fact

that the 5' RACE of Campbell was carried out in tissue culture cells instead of rabbit

tissue and furthermore that Campbell et al. was unable to detect HKu.2c mRNA in the

renal cortex. Although these statements are true, Fejes-Toth failed to recognize that

HKU2c mRNA was detected in rabbit colon, and HKca2c protein was detected in both

rabbit renal cortex and colon. These facts alone substantiate that HKu2c was not an

artifact of working with tissue culture cells. The RNase protection assay performed in

this dissertation, however, confirmed the existence of the HKc.2c transcript at least in

rabbit colon.

There are several explanations for the fact that each RNase protection probe

yielded two protected fragments in very close proximity for both HKa2a and HKa2c. One

possibility is that RNA polymerase had difficulty lying down in an exact position and

starting transcription at a precise site because the GC content of a region is high. The

genomic sequence surrounding the HKUa2a and HKca2c start sites are 67% and 68% GC

respectively (Figure 3-4). Additionally, the putative TATA box upstream of the HKua2a

start site (see below) had very weak homology to the consensus TATA box. It is

therefore likely that the putatitive core promoter elements surrounding the TATA box

play a role in transcription initiation and my not precisely position RNA polymerase. A

second explanation for the two protected fragments comes from the RNase protection









technique itself. It is possible to observe a fragment slighty longer than the true protected

fragment because the RNases used in the assay (A and T1) are endonucleases and may

leave bases on the end of a protected fragment. It is also possible to get a protected

fragment shorter that the true fragment because the end of the RNA-RNA duplex may

occasionally separate. It might be possible to distinguish between these possibilities with

a primer extension assay. This assay, however, also has inherent problems with

distinguishing one correct start site as it relies on reverse transcription similarly to the 5'

RACE (see Chapter 3 Introduction).

The putative core promoter elements found upstream of the HKca2a transcription

start site were a weak TATA box at -31, four SP family binding sites at -47, -102, 154,

and -170, a downstream promoter element (DPE) at +17, a TFIIB responsive element at -

42, and a CpG island that extends from -49 to +504. Additionally, directly upstream of

the HKUa2c transcription start site a single CAAT box at +351 was observed (31 bases

upstream). The fact that this is the only promoter-like element immediately upstream of

the HKUa2c transcription start site suggests that the HKc2 gene has one core promoter that

is able to direct transcription from the two alternative starts. The CAAT box may be

important in directing the initiation of transcription form HKUa2c. The weak TATA

element (CATTTAA) may be serving as a binding site for the general transcription factor

TFIID. Additionally, the other core promoter elements found surrounding this element

may serve to stabilize the preinitiation complex at the weak element. The function of the

TATA-like element is further investigated in Chapter 4 of this dissertation.

Further upstream of the transcription start sites, a possible cyclic AMP response

element (CRE) at -187 and a possible sterol response element (SRE) at -852 were









identified. There is evidence in vivo that cyclic AMP is increased in hypokalemic rats

(24). The CRE could provide a mechanism for upregulating the HKc2 gene.

Additionally, there is evidence from our laboratory that aldosterone may upregulate the

HKu2 gene (5). The SRE may provide a binding site for aldosterone and its hormone

receptor.

In summary, the transcription start sites for HKUa2a and HKUa2c were mapped using

the RNase protection assay and rabbit colon total RNA. HKUa2c was confirmed as a

transcript in rabbit colon. Upstream of the transcription start site several putative

transcription factor binding sites were observed. This work is the first analysis of the

rabbit HKc2 gene 5' of the transcription start site. The sequence contains many putative

transcription factor binding sites. Additionally, determination of the sequence allowed

for the design of future experiments regarding the regulation of the HKc2 gene.














CHAPTER 4
REPORTER GENE ANALYSIS OF THE HKa2 GENE PROMOTER

The third specific aim of this dissertation was to analyze the HKc2 gene promoter

using a reporter gene system. At the time that this study was proposed, there was in vivo

evidence that expression of HKc2 gene products was regulated by a variety of cellular

conditions including ion concentration, acid-base balance and hormones. There was,

however, nothing known about the mechanisms by which the expression was altered.

cDNAs for rabbit, rat, guinea pig and human had been identified, but only the human

ATP1AL1 gene was known. There have been no studies undertaken to determine

promoter elements for the human gene. Recently, the mouse gene was identified and a

reporter gene analysis of its 5' flanking region was carried out in mouse inner medullary

collecting duct cells (mIMCD3) (52). In their reporter gene experiments, Zhang et al.

found that thier longest deletion construct had significant luciferase activity and the

deletion of bases -177 -7265 had little to no effect on activity. The authors suggest that

the core promoter elements as well as positive regulatory elements are located between

bases +235 and -177. Although putative regulatory elements were identified in a

database search, there were no attempts made to determine the functionality of any

specific core promoter or regulatory elements. Additionally, Zhang et al. tested their

promoter constructs in outer medullary collecting ducts cells (mOMCD1) and medullary

thick ascending limb cells (ST-1). All of the deletion constructs had significant activity

in the second collecting duct cell type (OMCD) but little to no activity in the ST-1 cells.

The results suggested that either positive regulatory elements are absent in ST-1 cells or









that negative regulatory elements, including a closed chromatin structure, are present in

ST-1 cells.

The experiments described in this chapter used the luciferase reporter gene assay

to analyze rabbit HKc2 promoter constructs in a rabbit cortical collecting duct cell line

(RCCT28A). It was determined that X clone HKu2.1 contained the HKc2 gene promoter

(see Chapter 2). Portions of the 6.3Kbp Xhol fragment from HKU2.1 were cloned in

front of the luciferase reporter gene in the pGL3 basic vector (Promega, Inc.). The

constructs were transfected into RCCT28 cells and reporter gene activity was measured.

Our goals were to provide the first data regarding the regulation of the rabbit HKc2 gene,

to identify possible regulatory elements, and to test the functionality of those elements by

mutating specific bases within the identified elements. In this way, important regulatory

regions would be identified for future studies.

Materials and Methods

The Promega dual luciferase reporter gene assay (Promega, Inc. Catalog # E1960)

was chosen for the promoter analysis. Each promoter construct was cloned in front of the

firefly luciferase reporter gene in the pGL3 reporter gene plasmid (Promega, Inc. Catalog

# E1751). The plasmids were then transfected into RCCT28A tissue culture cells using

the Superfect transfection reagent (Qiagen, Inc. Catalog #301305). The cells were

simultaneously transfected with the pRL control plasmid which contained the Renilla

luciferase reporter gene driven by the thymidine kinase promoter (Promega, Inc. Catalog

# E2241). After 24 hours the cells were lysed and both the firefly luciferase activity and

the Renilla luciferase activity were measured using a Berthold Sirius Luminometer.

These data were normalized using the Renilla luciferase activity and represented as a









percentage of the highest normalized activity observed. The results identified fragments

of DNA 5' of the HKc2 transcription start sites that may play a role in the regulation of

HKc2 gene transcription.

Reporter Gene Constructs

Four sets of reporter gene plasmids were constructed. The first two sets were

promoter deletion plasmids and the second two sets were mutation plasmids. The first set

of deletion constructs contained both the HK,2a and the HKU2c transcription start sites

cloned into the pGL3 reporter vector (Figure 4-1). These constructs had little to no

luciferase activity. Therefore, a second set of deletion plasmids that contained only the

HKa2a start site were created (Figure 4-2). These constructs had varying amounts of

activity as expected in a promoter deletion experiment. Based on the data obtained from

the deletion analysis, two sets of mutation constructs were created. The first set tested

the functionality of two potential repressor elements (Figure 4-3) and the second set

tested the functionality of a potential core promoter element (Figure 4-4).

The plasmid pDZ10, which contained the 6.3Kbp XhoI fragment of clone

HKa2.1, was used to create the first set of deletion constructs (Figure 4-1). A 5259bp

Stul/Xhol fragment was cloned into the pGL3 vector (pDZ15). This sequence extended

from -4339 to +930 and contained the transcription start sites (+1 and +382) and the

translation start sites (+200 and +585) for both HK,2a and HKa2c. In order to create a

plasmid that contained upstream DNA, but did not produce the HK.2a protein, the

Quikchange Mutagenesis Kit (Stratagene, Inc. Cat# 200-518-5) was used to mutate the

ATG start codon at +200. The primers created for the mutation were DZ24

(5'CTCCAGCGCGACACGTGCCAGGTGTGTGAGG3') and DZ25 (5'CCTCA









CACACCTGGCACGTGTCGCGCTGGAG3'). This plasmid was designated pDZ28.

Deletion plasmids were made from pDZ15 and pDZ28 by removing a 3463bp Nhel/AatII

fragment, filling in the ends using Klenow DNA polymerase, and religating the vector

fragment using T4 DNA ligase. These plasmids were designated pDZ29 and pDZ30

respectively. The construction of pDZ29 and pDZ30 inadvertently placed a potential

stop codon in the 5' UTR. In order to create a construct that removed the stop codon, two

Pac sites were inserted into pDZ28 and pDZ29 by Quikchange with primer sets

DZ41/42 (5'CAGAGAAAGCTGTTAATTAACTCCGTGGAGCAC CATGCAGC3',

5'GCTGCATGGTGCTCCACGGAGTTAATTAACAGCTTTCTCTG3') and DZ43/44




ATG -l-- pDZ15
GTG c pDZ28


0 P P
I, ATG Lul pDZ29

GTG I- Lc pDZ30



'ATG F-LU pDZ31

I GTGc u pDZ32


Figure 4-1. Deletion constructs containing the HKUa2a and HKUa2c transcription start sites.
Lines represent the HKc2 gene 5' DNA. Base pair numbers indicate the
position of the restriction enzyme recognition site with respect to the HKua2a
transcription start site. ATG represent the HKUa2a translation start site. GTG
represents the mutation created to abolish translation from the HKua2a
translation start site. P represents the Pac sites inserted by Quikchange. Luc
represents the cDNA for the luciferase reporter gene.









(5'CAGCTTGGCATTCCGGTACTTTAATTAAAGCCACCATGGAAGACGCC3',

5'GGCGTCTTCCATGGTGGCTTTAATTAAAGTACCGGAATGCCAAGCTG3').

The 85bp fragment was removed by digestion with Pac, and the vectors were religated

creating plasmids pDZ30 and pDZ31 (Figure 4-1).

The second set of deletion constructs contained only the transcription start site for

HKa,2a (Figure 4-2). Plasmid pDZ10 was used as a starting plasmid for these constructs

as well. A 5459bp XhoI/SacII fragment was cloned into the pGL3 vector (pDZ11). This

sequence extended from -5367 to +93. A series of plasmids were then constructed by

digestion ofpDZl 1 with Nhel for the 5'end and a second enzyme for the 3' end. The

overhangs on the vector ends were filled in using Klenow DNA polymerse and the vector

was religated using T4 DNA ligase. These plasmids digested with the indicated enzymes

were designated pDZ 16 (Stul), pDZ20 (Ndel), pDZ22 (Spel), pDZ21 (MscI), pDZ19

(AatII), pDZ18 (EcoRI), and pDZ23 (SmaI). There were no convenient restriction

enzymes recognition sequences that could be used to make deletions intermediate to

plasmids pDZ21 and pDZ22. Therefore, two plasmids of an intermediate size were

created by introducing Mlul sites into pDZ22 by Quikchange (Stratagene, Inc.). Primer

set DZ31/32 (5'GGGTAGGGGATGTCACGCGTGGCCAAATGAAGTTG3',

5'CAACTTCATTTGGCCACGCGTGACATCCCCTACCC3') introduced an Mlul site

at position -2464. Plasmid pDZ26 was then created by digestion with Mlul and Nhel,

filling in of the overhangs and religating the remaining vector fragment. Primer set

DZ33/34 (5'CTTCTCTGTGCCACGCGTGGCCCAAAAGTTGG3', 5'CCAACTT

TTGG GCCACGCGTGGCACAGAGAAG3') created an Mlul site at position -1916.









Digestion and religation of this vector fragment created pDZ27. Furthermore, a plasmid

intermediate to pDZ21 and pDZ19 was created by inserting an Mlul site at position

-1241. In this case, the Mlul site was introduced as part of a primer set (DZ48

5'ACGGCTCCCTGTCCCATAGCCAGAGAATCCC3') used for PCR. The PCR

reaction containslO0tM of primers DZ48 and DZ5 (5'CTGCACTCTCAGAGTGA

AGG3'), 10ng pDZ21, 501tl of Qiagen PCR Master Mix (Taq DNA polymerase, Qiagen

PCR Buffer with 3mM MgC12, 400[LM each dNTP) and dH20 up to a volume of 100[tl.

The PCR conditions were one cycle of 95C for 5 minutes, 30 cycles of 95 for 30

seconds, 680C for 30 seconds, 72C for 30 seconds, and one cycle of 72C for 5 minutes.

The PCR reaction was run on a 1% agarose gel and visualized with ethidium bromide.

The reaction produced a single 700bp band which was gel extracted (Qiagen, Inc.) and

cloned into the TOPO cloning vector (Invitrogen, Inc.). In order to create the deletion

construct, the TOPO clone was digested with EcoRI and MluI and the overhangs were

filled in. The blunt ended fragment was then cloned into the Smal site of pDZ23 in order

to create plasmid pDZ36. The shortest construct (pDZ25) was created by performing

Quikchange on plasmid pDZ18. Primer set DZ35A/35B (5'CGCGCAGCATTTAACGC

GTACAC CACCTCCCC3', 5'GGGGAGGTGGTGTACGCGTTAAATGCTGCGCG3')

inserted an MluI site at -26. Digestion of the plasmid with MluI and NheI, filling in the

overhangs and religation of the vector fragment completed pDZ25. One final construct

was made to ensure that the size of the deletion plasmid was not having and effect on

reporter gene activity. A 4.2Kbp HindIII fragment of non-specific DNA (from E. coli

F1Fo plasmid pAES9) was ligated into the HindIII site of pDZ18 located at -26. This






70

fragment made the construct pDZ49 approximately the same size as the largest construct,

pDZ 11. The sizes of all of these deletion constructs are indicated in Figure 4-2.




| pDZ l

F-[Lu-uE pDZ16

[-L--u-E pDZ20

[fLuc pDZ22

I I-Luc pDZ26

'- |I fLuc pDZ21
ILl pDZ27

SpDZ21

I' E Lu pDZ36

pDZ19

F- tfHL pDZ18



S -tI u pDZ25
Non-specific DNA
-Luc pDZ49


Figure 4-2. HKa2a deletion constructs. Lines represent the HKa2 gene 5' DNA inserted
into the pGL3 reporter gene plasmid. Base pair numbers indicate the position
of the restriction enzyme recognition site with respect to the FKau2a
transcription start site. Luc represents the cDNA for the luciferase reporter
gene.









The third set of luciferase reporter gene constructs were created to test the

functionality of two putative repressor elements identified by the deletion analysis (see

results). Three mutations were made using Quikchange. Primer set DZ55/56

(5'GCAGCACCACGCAGCCCGGGACCATTAATTAAAACGCTCACTGACCCAG

ACCCTCC3', 5'GGAGGGTCTGGGTCAGTGAGCGTTTTTAATTAATGGTCCC

GGCTGCGTGGTGCTGC3') mutated the element located at -700. Primer set DZ59/60

(5'GCCCTCCACGCTCACTGACCATTTAAATACATCCCCACCCC TCTCTCC3',

5'GGAGAGAGGGGTGGGGATGTATTTAAATGGTCAGTGAGCG TGGAGGGC3')

mutated the element at -680. The two putative elements were close together, so a third

primer set, DZ63/64 (5'GGACCATTAATTAAAAACGCTCACTGACCATTTAAA

TACATCCCCACCCCTCCTCTCC3', 5'GGAGAGAGGGGTGGGGATGTATTTAAA

TGGTCAGTGAGCG TTTTTAATTAATGGTCC3') was used to mutate both elements.

The fourth set of luciferase reporter gene constructs were created to test the

functionality of the TATA-like element found at -31 (see results). The second smallest

deletion construct that contained the element (pDZ18) and the smallest deletion construct

that did not contain the element (pDZ25) were used as positive controls for this set of

experiments. Three mutation constructs were made using Quikchange with three primer

sets. Primer set DZ53/54 (5'GCGGGGCGCGCAGCGGCCGCGGCGGACACCACC3',

5'GGTGGTGTCCGCCGCGGCCGCTGCGCGCCCCGC3') created a GC box in place

of the TATA element (pDZ34). Primer set DZ86/87 (5'GCGGGGCGCGCAGCGAT

CGAGGCGGACACCACC3', 5'GGTGGTGTCCGCCTCGATCGCTGCGCG

CCCCGC3') created a random sequence in place of the TATA element (pDZ48). And

finally, primer set DZ57/58 (5'GCGGGGCGCGCATATAAAAGGCGGACACCACC3',









5'GGTGGTGTCCGCC TTTTATATGCGCGCCCCGC3') created a consensus TATA

sequence at the same location (pDZ39).

Tissue Culture Cells

RCCT28A cells were chosen for the majority of the reporter gene analyses.

These cells were isolated from rabbit cortical collecting duct and transformed with the

SV40 virus by Arend et al. (2). They were characterized by their ability to bind several

antibodies and by their response to specific hormones. It was determined that RCCT28A

cells maintain characteristics most similar to the intercalated cells of the cortical

collecting duct. Furthermore, Campbell et al. (6) showed that these cells express the

HKu2 mRNA's and proteins. RCCT28A cells were therefore known to contain factors

required for expression of the reporter gene driven by the HKc2 gene promoter.

In addition to RCCT28A cells, three other cell types were used for one of the

reporter gene assays. The activity of several of the deletion constructs were tested in

HIG-82 cells, HEK293 cells and HK2 cells. HIG-82 cells were established by

spontaneous transformation of fibroblasts from rabbit soft tissue lining the knee joint

(15). They are not likely to express the HKc2 gene products. HEK293 cells are human

embryonic kidney cells transformed from sheared human adenovirus type 5 (42). They

display general characteristics of renal tubular cells, but it is not possible to relate their

characteristics to a specific renal segment. Grishin et al. (16) used these cells for the

functional expression of cloned ATP1AL1 cDNAs. Antibodies raised against a portion

of the ATP1AL1 protien did not react with untransfected HEK293 cells suggesting that

these cells do not express the HKc2 gene product. HK2 cells are human adult proximal

tubule epithelial cells immortalized by transduction with human papiloma virus (38). It









has been shown that rabbit proximal tubule cells do not express the HKac2 gene products

(12). It is therefore unlikely that HK2 cells express the ATP1AL1 gene product.

Transfection and Reporter Gene Activity

RCCT28A cells were grown in a 24 well plate format with each well containing

ImlL of media (Dulbecco's Modified Eagle Medium F12 (DMEM-F 12), 10% Fetal

Bovine Serum (FBS)) until they reached approximately 70% confluency (usually over

one night). For each transfection, 250pmoles of the deletion construct to be tested, non-

specific DNA to 1 tg and 0.2[tg of pRL control plasmid were mixed with 140[tl DMEM

and 40[tl of Superfect reagent (Qiagen, Inc.) and incubated at room temperature for 10

minutes to allow for complex formation between the transfection reagent and the DNA.

During this incubation, the RCCT28A cells were washed two times in PBS. The

completed solution was brought up to 400[tl with DMEM-F12 plus FBS and 200[tl was

pipetted on top of each of two duplicate wells. The transfection proceeded at 370C for 2

hours. The transfection reagents were washed off the cells with PBS and ImL of fresh

media was added to each well. After 24 hours at 370C, the media was removed and the

cells were lysed by adding 100[tl of lysis buffer (Promega, Inc.). Each well was scraped

with a 20[tl pipette tip to facilitate lysis. Ten microliters of the lysed cells were added to

100[tl of firefly luciferase substrate and the raw firefly luciferase activity was measured

for 10 seconds. One hundred microliters of quenching buffer plus Renilla luciferase

substrate was added and the raw Renilla luciferase activity was measured for 10 seconds.

Each construct was transfected into RCCT28A cells at least three times and in duplicate

each time in order to obtain statistically relevant data. Each round of transfections

included the plasmid that initially had the most activity (pDZ18) and the plasmid with the









least activity (pGL3 empty vector). All the constructs in one transfection were

normalized to the Renilla control data (see below). The pDZ18 activity was set to 100%

and data for the other constructs were calculated as a value relative to 100%.

Normalization of the Luciferase Data

Table 4-1 is an example of the calculations required for normalization of the

luciferase data. For each set oftransfections, the raw firefly reading and the raw Renilla

reading are taken directly from the luminometer (Column one and Column two

respectively). The first Renilla reading was divided into each of the subsequent Renilla

readings to create a normalizing factor for each sample (Column three). Each firefly

reading was multiplied by its normalizing factor to obtain the normalized firefly reading

(Column four). The average of the two readings for the plasmid that initially gave the

highest activity reading (pDZ 18) was divided into the normalized firefly reading for each

sample and multiplied by 100 to obtain a percentage of the highest activity (Column

five). The background activity observed for the empty vector (pGL3) was subtracted out

(Column six) and the activity for the plasmid with the highest activity (pDZ 18) was reset

to 100% (Column seven). Each plasmid was transfected at least three time and in

duplicate. The average percent relative activity for each plasmid was graphed and error

bars were added to indicate plus and minus the standard of error.

Results

HKa2a and HKa2c Reporter Gene Activity

Figure 4-3 is a graph of relative luciferase activity for the constructs that contain

both HKa2a and HKa2c transcription and translation start sites. All constructs were

transfected into RCCT28A cells in duplicate and at least three times. These data were

normalized using the Renilla luciferase internal control. Plasmid pDZ18 contains only









Table 4-1. Example of the normalization of raw luciferase data
Raw Raw Normalizing Normalized % Subtract Reset
Vector firefly Renilla factor fireflyb activity pGL3 to
100%
pGL3 616 13166 1.00 616 7.66 -0.45 -.050
623 11912 1.11 689 8.56 0.45 0.50

pDZll 4540 34459 0.38 1735 21.56 13.45 14.66
3887 26942 0.49 1899 23.61 15.50 16.90

pDZ18 12923 20904 0.63 8139 101.16 93.05 101.42
10513 17404 0.76 7953 98.84 90.73 98.90
a) Normalizing factor equals first renilla reading/each raw renilla reading
b) Normalized firefly equals raw firefly normalizing factor
c) % activity equal average for(normalized firefly/pDZ 18)*100


the HKa,2a transcription start site and contained the most firefly activity of all the

constructs created. This reading was set to 100% activity and the relative activity for the

remaining constructs was calculated. The original construct containing the HKUa2a and

HKUa2c transcription and translation start sites (pDZ15) had no activity. Since the ATG

start codons for HKUa2a and HKUa2c were part of this construct, it seemed possible that the

HKc2 amino acids added to the N-terminus of the luciferase protein could affect

luciferase activity. Therefore, Quikchange was used to create a plasmid in which the

ATG start codon for HKUa2a was mutated (pDZ28). This plasmid also had no luciferase

activity. At this point, reporter gene activity data from the second set of constructs, those

that only contained the HKUa2a transcription start site, had shown that shorter plasmids

contained more activity (see below). Therefore, plasmids pDZ15 and pDZ28 were

digested with AatlI and Nhel, the ends were filled in, and the vectors were religated.

These two plasmids, pDZ30 and pDZ31 still had no activity. A portion of the sequence

from plasmid pDZ30 is shown in Figure 4-4A. Analysis of the two alternative mRNA









transcripts that would be produced by this sequence (Figures 4-4B and C) revealed a

potential stop codon that was introduced by the ligation of the HKu2 gene fragment into

the pGL3 vector (shown in red). This stop codon would terminate translation from both


pDZ18

pDZ15 '

pDZ28

pDZ29

pDZ30

pDZ31

pDZ32

-20


I

I



I


0 20 40 60 80

Percent of Relative Luciferase Activity


Figure 4-3. Percent activity for reporter gene constructs that contain the HKa2a and
HKa2c transcription and translation start sites. Error bars represent the
standard of error.



the HKa2a translation start and the HKa2c translation start before reaching the ATG start

codon for the luciferase gene. A new set of constructs were created to fix this problem

and to remove some of the HKu2 amino acids that were added to the N-terminus of the

luciferase protein. Pac sites were introduced into pDZ29 and pDZ30 using Quikchange

(Figure 4-1). Subsequent digestion with Pac and religation of the vector resulted in

plasmids pDZ31 and pDZ32. The sequence for pDZ31 and the mRNA transcripts

produced by this plasmid are shown in Figure 4-5. Although the potential stop codon

was successfully removed, plasmid pDZ31 had no luciferase activity suggesting that the


I,


















GCGGGGCGCG
TGCGGAGGTG
ACTACCAACG
CCCACGACCC
ACATGCGCCA
GTGAAGGGCT
TGTGTCACTC
CAGGGGTCAG
AGCTACACGT
TCTATGCACC
CCCCTAGAGG
AACAGGGGAA
TCCTGGCCCG
TACCACCTGG
CGTGGAGCAC
CAATGACTTG
TCGAG


CAGCATTTAA
CGCGCAGGGC
CCGCCACCGC
CTCCTGCCCT
GGTGTGTGAG
CCGTCCGGGG
GGGAACTGGC
CTCCGCCCTC
ATGCGTAGCG
GCTGGTGTGA
TGTCTTCCTG
GAGAGGAAGG
CGAGGGTGTC
GGCCGGTATT
CATGCAGCTA
GAACTCAAAA


GGCGGACACC
CCGCGTGGCT
GGGACCCTAC
CCGCGCCCCC
GAAGTGACGC
GTCTTTACTC
TGGGTAAAGA
GCACCTGCGG
GTCTGGAAAA
CCCCGCAGGG
GGAAGACGAT
AGGGAGGTGG
CGGTCCCACT
GCACTCTGCT
CAGATATCAA
GGAATCAGCA


ACCTCCCCTG
GTGGGTACCT
CCCGCATCGG
TGCCCGCCGA
GGTGCGGACT
TGCAACCCTG
GGTCAATCCA
GCTCGGATTC
TGCCCCAGGC
CAACCCCGCG
GGCAGGCGGT
GAGGTGGCGC
CAAGGCAGCT
TCTCTTTCAG
GAAGAAGGAG
GAAAGAGGAG


GGCAGCGGCT
CCTTCGCCAG
TCGCCGCCGC
CCCGCGGCGC
GGAGAGAAGT
TTCCAGCCGC
GACACGCGGG
GGAGAAAAGT
TCGGGTCTGA
GTTAACTTCT
GCCCACCGAG
GCTCCCCACA
GCGCAGAGCC
AGAAAGCTGG
GGGCGAGATG
CTTAAGAAAG
GTTGGTAA


GGCGATCGGC
CACCGTCGCC
CACCGCAGGT
CTCCAGCGCG
GCGGGAAAGG
CGAGCACCCG
GAAGGAGTTC
GCTAGACTGG
GGGGCCCAAG
CTCCTGCCCA
CCGACCGTGC
GCCCTTCCCC
TGTGCAGAAA
AAATTTACTC
GCAAGAAAGA
AACTTGATCC
ATG


ATG CGC CAG AGA AAG CTG GAA ATT TAC TCC GTG GAG CAC CAT GCA GCT ACA GAT


ATC AAG AAG
AGG AAT CAG
TAA


ATG
GGA
TGT
GGC
GAG
GAC
CTT


GGC
GGG
GTC
TAT
CAT
GAC
CCT


AAG GAG GGG CGA GAT GGC AAG AAA GAC AAT GAC
CAG AAA GAG GAG CTT AAG AAA GAA CTT GAT CCT


GGT GCC
AGG TGG
CCA CTC
TGC ACT
GCA GCT
TTG GAA
CGA G
ATG


CAC
CGC
AAG
CTG
ACA
CTC


CGA
GCT
GCA
CTT
GAT
AAA


GCC
CCC
GCT
CTC
ATC
AGG


GAC
CAC
GCG
TTT
AAG
AAT


GCA
CCT
AGC
AGA
AAG
CAG


GGG
CCT
TGC
CTG
GGG
GAG


GAA
CCT
AGA
GAA
CGA
GAG


TTG GAA CTC AAA
CGA G
ATG


GAG
GGC
AAT
ATT
GAT
CTT


AGG
CCG
ACC
TAC
GGC
AAG


AAG
CGA
ACC
TCC
AAG
AAA


Figure 4-4. Sequence analysis of plasmid pDZ15. (A) Sequence of the plasmid. Black
indicates HKu2 sequence while green indicates pGL3 sequence. Dark purple
represents the transcription start sites and light purple the translation start site
for HKUc2a. Dark pink represents the transcription start site and dark pink the
translation start site for HKa2c. Blue represents the translation start site for
the luciferase gene. Orange indicates the two sets of bases mutated to Pad
sites for future experiments (see text). (B) mRNA transcribed by initiation
from the HKUa2a start site. Red indicates stop codon introduced before the
luciferase translation start site. (C) mRNA transcribed by initiation from the
HKa2c construct.














GCGGGGCGCG
TGCGGAGGTG
ACTACCAACG
CCCACGACCC
ACATGCGCCA
GTGAAGGGCT
TGTGTCACTC
CAGGGGTCAG
AGCTACACGT
TCTATGCACC
CCCCTAGAGG
AACAGGGGAA
TCCTGGCCCG
TACCACCTGG
ATG


CAGCATTTAA
CGCGCAGGGC
CCGCCACCGC
CTCCTGCCCT
GGTGTGTGAG
CCGTCCGGGG
GGGAACTGGC
CTCCGCCCTC
ATGCGTAGCG
GCTGGTGTGA
TGTCTTCCTG
GAGAGGAAGG
CGAGGGTGTC
GGCCGGTATT


GGCGGACACC
CCGCGTGGCT
GGGACCCTAC
CCGCGCCCCC
GAAGTGACGC
GTCTTTACTC
TGGGTAAAGA
GCACCTGCGG
GTCTGGAAAA
CCCCGCAGGG
GGAAGACGAT
AGGGAGGTGG
CGGTCCCACT
GCACTCTGCT


ACCTCCCCTG
GTGGGTACCT
CCCGCATCGG
TGCCCGCCGA
GGTGCGGACT
TGCAACCCTG
GGTCAATCCA
GCTCGGATTC
TGCCCCAGGC
CAACCCCGCG
GGCAGGCGGT
GAGGTGGCGC
CAAGGCAGCT
TCTCTTTCAG


GGCAGCGGCT
CCTTCGCCAG
TCGCCGCCGC
CCCGCGGCGC
GGAGAGAAGT
TTCCAGCCGC
GACACGCGGG
GGAGAAAAGT
TCGGGTCTGA
GTTAACTTCT
GCCCACCGAG
GCTCCCCACA
GCGCAGAGCC
AGAAAGCTGT


GGCGATCGGC
CACCGTCGCC
CACCGCAGGT
CTCCAGCGCG
GCGGGAAAGG
CGAGCACCCG
GAAGGAGTTC
GCTAGACTGG
GGGGCCCAAG
CTCCTGCCCA
CCGACCGTGC
GCCCTTCCCC
TGTGCAGAAA
TAATTAA


ATG CGC CAG AGA AAG CTG


GCA GGC GGT GCC CAC CGA
GGT GGG AGG TGG CGC GCT
CCG GTC CCA CTC AAG GCA
CGG TAT TGC ACT CTG CTT


GCC GAC
CCC CAC
GCT GCG
CTC TTT


CGT GCA ACA GGG GAA GAG AGG AAG GAG
AGC CCT TCC CCT CCT GGC CCG CGA GGG
CAG AGC CTG TGC AGA AAT ACC ACC TGG
CAG AGA AAG CTG


Figure 4-5. Sequence analysis of plasmid pDZ31. (A) Sequence of the plasmid. Black
indicates HKc2 sequence while green indicates pGL3 sequence. Dark purple
represents the transcription start sites and light purple the translation start site
for HKUa2a. Dark pink represents the transcription start site and dark pink the
translation start site for HKa2c. Blue represents the translation start site for
the luciferase gene. Orange indicates the remaining Pad site after digestion
and religation. (B) mRNA transcribed by initiation from the HKua2a start site.
(C) mRNA transcribed by initiation from the HKa2c2 construct.





HKcu2 amino acids added to the N-terminus of the luciferase protein were inactivating the


enzyme. Plasmid pDZ32, which has a mutation at the HKUa2a ATG translation start was


the first plasmid in this series to have luciferase activity. The amount of activity,


however, was about 50% of that seen for the similar sized plasmid that contains only the


ATG


ATG
GGA
TGT
GGC
ATG









HKUa2a transcription start site (pDZ 19). At this point in the study, it became clear that the

second set of deletion constructs, those that contain only the HK(a2a transcription start site

had significant reporter gene activity and would produce the desired deletion data (see

below). Therefore, attempts to restore luciferase activity to these constructs were

terminated.

HKOC2a Reporter Gene Activity

Figure 4-6 is a graph of the relative reporter gene activity for the all of the

plasmids in the second set of deletion constructs. These plasmids contain the HKu.2a

transcription start site and varying amounts of 5' DNA. These constructs were

transfected into RCCT28A cells and after 24 hours, the luciferase activity was measured.

Once again the normalized activity from plasmid pDZ18 was set to 100% and the percent

activity for the remaining constructs was calculated. The clear result was that

progressively shorter promoter fragments contained progressively more luciferase

activity. The one exception was that the shortest plasmid (pDZ25) contained activity

similar to background (pGL3). It is notable that the luciferase activity increased

gradually as the plasmid length decreased. In order to eliminate the possibility that the

size of the plasmid was affecting luciferase activity, plasmid pDZ49 was created. This

plasmid contained a random fragment of DNA placed in front of the pDZ 18 fragment

resulting in a plasmid the length of pDZ 11. The luciferase activity of this construct was

similar to pDZ18, not pDZ 11, suggesting that plasmid size does not affect reporter gene

activity. A one way ANOVA analysis was performed on the deletion data. The red stars

in Figure 4-6 indicate plasmids with a statistically significant difference in the level of


































o 0






rJ)



C)









CC

(0 0
1-


- D O N 0M ( N- CD 0) (0 M (0
CM CN CN CN C o C x- CN CN
N N N N N N N N N N N
o o o o o o o o o o o o
a- a- a- a- a a a a- a- a a a









luciferase activity when compared to the preceding plasmid. The differences between

pDZ19 and pDZ18, and pDZ23 and pDZ25 were the most significant and therefore their

sequences were analyzed for possible regulatory elements.

Putative Repressor Mutations

The difference in luciferase activity between plasmids pDZ19 and pDZ 18 was

examined. The sequence difference between these two plasmids came from a 245bp

deletion that extended from bases -631 to -876. A transcription factor binding site

database (TFSEARCH) analysis did not reveal any known repressor binding sites in this

region. The human ATPAL1 gene was the only HKu2 gene that also had known

sequence in this 5' region. An alignment of the rabbit sequence and the human sequence

in this region did show a short sequence that was well conserved between the two species

(Figure 4-7). Quikchange mutagenesis was used to randomize the sequence at -680



-713 CCAGAGCCCT--CCA] Human
-680 CCAGA-CCCT--CCA Rabbit
-700 ACAGA--CCTCTCCAJ



Figure 4-7. Alignment of possible repressor sequences from human ATP1AL1 and rabbit
HKc2 genes.



(pDZ38), the sequence at -700 (pDZ40), and both sequences together (pDZ41). These

constructs were transfected into RCCT28A cells along with pDZ18 and pDZ19. After 24

hours the luciferase activity was measured. The activity for pDZ 18 was set to 100% and

the relative activity for the rest of the plasmids was calculated. Figure 4-8 is a graph of

these data. Although there was a small increase in activity over that of pDZ 19, it did not









appear as though the conserved sequence that was analyzed has a major effect on HKa2

repression.






LX

X X


0 20 40 60 80 100 120

Percent of Relative Luciferase Activity


Figure 4-8. Percent luciferase activity in repressor mutation constructs. Red X indicates
the position of the putative repressor mutation.



Putative TATA Element Mutations

The dramatic drop in luciferase activity between plasmids pDZ23 and pDZ25

suggested that the core promoter for the HKa2 gene was deleted. An alignment of the

269 bases deleted in pDZ25 with the sequences known for the human ATP1AL1 gene,

the mouse HKu2 gene, and the rat HKu2 gene revealed a great deal of homology as

indicated by the stars at the bottom of Figure 4-9. In particular, the CATTTAA (red

lettering) element near the rabbit transcription start site was completely conserved in

human, rat and mouse. In order to test if this element was functioning as a TATA

element, pDZ18 was mutated in several ways. These data are represented graphically in

Figure 4-10. The mutation constructs that destroyed the element (pDZ34 and pDZ49)

show a drop, although not a complete loss, of reporter gene activity. It therefore appears







83


as though the element is necessary for full activity, but the surrounding bases are capable

of initiating an intermediate level of transcription despite the mutation to the TATA box.

The mutation that created a consensus TATA box (pDZ39) had activity that was not

significantly different from the wild type element, again suggesting that the native

element was functioning well as a consensus TATA box.



rabbit ATCCTCGGTTTCCCAGCCCCCTGGGGTGTCTGCAG-GCCGGGCTACTTGCACAGCAGCAG
human GCCGGCGGGTTTCCTACCCTCCGAGGCGTCCGCTG-GCC--------TGCGCCCTGGCGG
rat GCACTCGGGTTTCCTATCC-CAGGGGCGTCCCTGGTGCAGTG--AACTGTGTCGCCCCAG
mouse GTCCTCGGGTTTCCTACCC-CAGGGGCGTCCCCAGTGCAGTG--AGCTGCGTCGCCCCAG
*** ** ** ** ** *** ** ** *

rabbit GTGCGTAGGCGGGGCGCGCAGCATTTAAGGCGGACACCACCTCCCCTGGGCA GGCTGG
human GGACGTGGGCGGGGCGGGCGGCATTTAAGGCTGGTGCCACCTC CCGGTGCAGCGGCTGG
rat GTACGTGGGCGGAGCTCGCAGCATTTAAGGCGCGCTCCACCTCCCAGGG CAGTGGCTGG
mouse GTACGTGGGCGGAGCGCGCGGCATTTAAGGCGCGCTCCACCTCCCCAGGACAGCGGCTGG
*** ***** ** ** *********** ********* *** ******

rabbit CGATCGGCTGCGGAGGTGCGCGCAGGGCCCGCGTGGCT
human CGATCGGCCGCGGAGGTGCGTGCAGGGCCCGCGCCGCC
rat TGATCGGCCGCGGAG-TGCTTG-TGCTTCCGG-TTGGC
mouse CGGTCGGC GCGGAGGTGCGTGCTGGGTCCGGGTTGGT
***** ****** *** *** *


Figure 4-9. Alignment of the rabbit, human, rat and mouse DNA sequence upstream of
the HKc2 transcription start sites. Stars represent completely conserved
bases. Red indicates the completely conserved TATA-like element. Dark
purple represents the transcription start sites for rabbit HKa2a determined in
Chapter 2, human ATP1AL1 (45), rat HKU2a (26) and mouse HKc2 (52).


Effect of Cell Type on Reporter Gene Activity

In order to test the effect of cell type on reporter gene activity, three deletion

constructs were transfected into four cell types. Plasmids pDZ18, pDZ19 and pDZl 1

were transfected into HEK293, HK2, RCCT28A and HIG-83 cells. Within each cell line,

the normalized activity for pDZ 18 was set at 100% and the other two constructs are
















X Luc

X



0 20 40 60 80 100 120

Percent of Relative Luciferase Activity


Figure 4-10. Percent activity in reporter gene constructs with mutations in the
CATTTAA element. Red X represents mutations that randomize the
CATTTAA element. Green triangle represents a mutation that converts the
CATTTAA element into a consensus TATA element.



represented relative to 100%. Figure 4-11 is a graph representing the results of the

transfections. In the three adult cell lines (RCCT28A, HIG-82, HK2), the expression

pattern for the three constructs was similar. The longest construct was repressed and the

shorter constructs had increasing amounts of luciferase activity. The embryonic cell line

(HEK293), however, had a different pattern of expression. The most dramatic difference

was with the longest construct. Plasmid pDZ 11 was repressed in all three adult cell lines,

but was not repressed in this embryonic cell line. The activity was most similar to

pDZ 19 suggesting that the embryonic cell line did not contain some or all of the factors

required for HKac2 gene repression.












pGL3

pDZ 11

pDZ19

pDZ18

0 20 40 60 80 100

Percent of Relative Luciferase Activity


Figure 4-11. Reporter gene activity in various cell types. Plasmid constructs are as
indicated. Light pink represents HEK293 cells. Dark pink represents HK2
Cells. Light orange represents HIG-82 cells. Dark orange represents
RCCT28A cells.



Discussion

The use of the luciferase reporter gene assay to evaluate the region of DNA 5' of

the HKc2 gene provided the first data regarding the regulation of the rabbit HKc2 gene.

Four sets of reporter gene constructs and four tissue culture cell types were used in this

assay and each provided important information.

The first set of constructs contained the transcription and translation start sites for

HKa2a and HKc2c cloned in front of the luciferase reporter gene. These constructs had

little to no reporter gene activity. The removal of the stop codon introduced during

plasmid construction, and the mutation of the ATG start codon for HKa2a resulted in one

construct with significant luciferase activity (pDZ32). This activity, however, was still

low when compared to the construct with same deletion (pDZ 19) in the series that









contained only the HKUa2a start site (60% for pDZ19 vs. 30% for pDZ32). This result was

difficult to interpret. The mutation of the HKUa2a ATG should have removed the amino

acids that were added to the N-terminus of the luciferase protein. The luciferase activity,

however, was not restored to the expected level suggesting that there were negative

regulatory elements that effect the HKUa2a transcription in the downstream region that was

eliminated when the second set of constructs were made (+93 to +920). Plasmid pDZ31

removed the stop codon that would terminate translation from the HKu.2c transcript. This

construct, however, had no luciferase activity. There are several possible explanations

for the lack of activity. First, the 72 amino acids added to the N-terminus of the

luciferase protein could cause a loss of function (Figure 4-5C). Second, the splicing

machinery may not recognize the reporter gene construct and fail to produce the

alternative HKa2c transcript. And finally, the HKc2c transcript may be regulated, and

without the proper signal, it may completely repressed. As it would be difficult to

distinguish between these possibilities, and more importantly, the second set of deletion

constructs were producing results, this line of investigation was therefore abandoned.

The second set of reporter gene constructs provided the most information

regarding the regulation of the HKu2 gene. The 3' end of these were shorter, ending at

+92, and contained only the transcription start site for HKa2a. Interestingly, the longest

construct (pDZ 11) contained the least reporter gene activity and progressively shorter

constructs showed a gradual increase in reporter gene activity. These results suggested

that under the conditions of the luciferase assay, the HKu2 gene promoter was repressed

and significant reporter gene activity was seen only after deleting the DNA responsible

for binding repressor elements. The increase in luciferase activity, however, appeared as









a gradual increase rather than as distinct jumps in activity that would be expected when

repressor elements are deleted. It seemed possible, therefore, that the decrease in size of

the constructs, rather than the removal of repressor elements, caused the change in

luciferase activity. Plasmid pDZ49, however, eliminated this possibility. This construct

was made by taking the plasmid with the most activity, pDZ 18, and inserting a fragment

of E. coli DNA into a HindII site upstream of the putative promoter element. The result

was a plasmid the size of pDZ 11, but devoid of any additional eukaryotic transcription

factors. Plasmid pDZ49 has luciferase activity similar to pDZ18, not pDZ 11, meaning

that the size of the plasmid was not having an effect on luciferase activity. Although the

changes in luciferase activity were smaller than one might expect, the one way ANOVA

analysis of these data did reveal several deletions that caused a statistically significant

change in reporter gene activity. There are clear increases in luciferase activity between

constructs pDZ20 and pDZ22, pDZ21 and pDZ36, and pDZ19 and pDZ18. Additionally,

there is a dramatic decrease in luciferase activity between plasmids pDZ23 and pDZ25

(Figure 4-6). These changes in activity provided the basis for the construction of the

mutation plasmids that are discussed below. The fact that the HKc2 gene was repressed

in these assays was a surprise because RCCT28A cells have been shown to express the

HKu2 mRNA's and proteins (6). Additionally, Zhang et al. (52) performed a promoter

deletion analysis of the mouse HKc2 gene and did not observe repression with their

longest constructs. The discrepancy between the results of our study and the study of

Zhang et al. may be explained by the differences in the cell types. The RCCT28A cells

were derived from the cortical collecting duct while the cells used by Zhang et al. were

derived from medullary collecting duct cells. To date, it has been unclear whether or not









the cortical collecting duct normally expresses the HKc2 gene products (see

introduction) whereas the medullary collecting duct has been consistently shown to

express the HKc2 gene products. The reporter gene assay performed in our study

suggested that there may be certain cellular conditions necessary for HKc2 gene

expression in the cortical collecting duct. This may, in part, explain the discrepancies

present in the literature. One likely explanation for the repression observed in the

deletion analysis is the condition under which the RCCT28A cells were grown for the

assay. The transfection of the RCCT28A cells necessitated that they were grown to 70%

confluency. At this level of confluency the RCCT28A cells are undifferentiated. It was

hypothesized that the HKc2 gene was repressed in this state and then when the cells

reach 100% confluency and differentiate, they would begin to express the HKc2 gene

products. This hypothesis resulted in the experiments that are discussed in Chapter 5 of

this dissertation.

The third set of reporter gene constructs were designed to test the functionality of

putative repressor elements identified in the DNA fragment between plasmids pDZ19 and

pDZ18. The increase in luciferase activity between these two plasmids was the largest

observed (40%) and seemed a good point to begin a search for novel repressor elements.

A transcription factor database search did not identify any known binding sites for

transcription factors. An alignment of the rabbit DNA sequence in this region with that

of the human ATP1AL1 gene, however, did identify one sequence in human that was

well conserved to two sequences in rabbit (Figure 4-7). Unfortunately, random mutation

of these sequences both individually and combined did not have a significant effect on

luciferase activity (Figure 4-8) suggesting that these sequences are not directly involved