Preparation of single-stranded DNA complementary to histone messenger RNA's

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Title:
Preparation of single-stranded DNA complementary to histone messenger RNA's a probe for histone gene expression
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xi, 124 leaves : ill. ; 28 cm.
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English
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Thrall, Cary Lee, 1949-
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DNA   ( lcsh )
RNA   ( lcsh )
Histones   ( lcsh )
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bibliography   ( marcgt )
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non-fiction   ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 116-122).
Statement of Responsibility:
by Cary Lee Thrall.
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Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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Full Text
PREPARATION OF SINGLE-STRANDED DNA COMPLEMENTARY TO HISTONE MESSENGER RNA'S: A PROBE FOR HISTONE GENE EXPRESSION
By
CARY LEE THRALL
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA 1975


CONTENTS
Page
LIST OF TABLES .............. ..... Y
LIST OF FIGURES.................. vi
KEY TO ABBREVIATIONS................ viii
ABSTRACT...................... IX
CHAPTER 1
INTRODUCTION ................... 1
CHAPTER 2
MATERIALS AND METHODS ........ ...... 12
I. Cell Culture................ 12
II. Cell Synchronization............ 12
A. Single Thymidine Block ..... .... 12
B. Double Thymidine Block ........ 12
C. Selective Detachment .......... 13
D. Thymidine Block Synchrony Monitoring 13 III. Isolation of Histone Messenger PNA's .... 14
IV. Characterization of Histone Messenger RNA's 20
A. Size Distribution SDS polyacrylamide
gel electrophoresis.......... 20
B. Base Composition............ 21
C. Cell-Free Translation ......... 22
D. Poly A Affinity Binding........ 25
V. Synthesis of Complementary DNA....... 26
A. Isolation of Rous Sarcoma Virus .... 26
B. Assay for Reverse Transcriptase Activity 27
C. Polyadenylation of Histone Messenger
RNA's................. 28
ii


Page
D. Preparation of the Complementary DNA 29 VI. Characterization of the Complementary DNA 31 A. Size Distribution-Alkaline Sucrose Gradi-
ent Centrifugation ........... 31
B. RNA-DNA Hybridization......... 3.1
VII. Utilization of the cDNA as a Probe..... 32
A. Chromatin Isolation .......... 32
B. Chromatin Dissociation ......... 33
C. Chromatin Reconstitution ........ 35
D. In vitro Chromatin Transcription 36
CHAPTER 3
RESULTS AND DISCUSSION ............... 38
I. Isolation of Histone Messenger RNA's 38
A. Cell Synchrony............. 38
B. RNA Fractionation........... 39
II. Characterization of Histone Messenger RNA 47
A. Size Distribution......... 47
B. Base Composition............ 51
C. Cell-Free Translation ......... 55
D. Poly A Affinity Binding........ 63
III. Synthesis of Complementary DNA....... 71
A. Assay for Reverse Transcriptase 73
B. Polyadenylation of Histone Messenger
RNA's................. 77
C. Reverse Transcriptase Assays of Polyadenylated Histone mRNA Templates 79
D. Preparation of the Complementary DNA 81
iii


Page
IV. Characterization of the Complementary DNA 84
A. Specific Activity ............ 84
B. Size Distribution............86
C. Single-Strandedness ........... 89
D. Hybridization Properties ........ 91
V. Utilization of the Complementary DNA as a
Probe....................103
A. Polysomal RNA's.............103
B. Transcripts of Native Chromatin .....105
C. Reconstituted Chromatin Transcripts .10 8
CHAPTER 4
SUMMARY AND CONCLUSION...............Ill
REFERENCES.....................116
BIOGRAPHICAL SKETCH.................123
iv


LIST OF TABLES
Page
1. Base Composition of 18S RNA High Voltage
Paper Electrophoretic Fractionation ....... 53
2. Base Composition of 7-12S RNA High Voltage
Paper Electrophoretic Fractionation ....... 53
3. Base Composition of 18S RNA Thin Layer Electrophoretic Analysis ...... ..... 53
4. Base Composition of 7-12S RNA Thin Layer Electrophoretic Analysis ......... ... 53
5. Binding of 7-12S RNA to Nitrocellulose Filters 65
6. Oligo (dT)-Cellulose Affinity Chromatography of
Poly A.....................65
7. Oligo (dT)-Cellulose Chromatography of 7-12S RNA 65
8. Utilization of 7-12S RNA as a Template for Reverse Transcriptase ............. 76
9. Utilization of Polyadenylated 7-12S RNA as a Template for Reverse Transcriptase ..... 80
10. Cr0t-jy2 Values for Selected Nucleic Acid
Hybridization Reactions ............ 96


LIST OF FIGURES
Page
la. Flow diagram for the isolation of polysomal
RNA...................... 18
lb. Flow diagram for the fractionation of
polysomal RNA and isolation of the RNA containing the histone mRNA's..........19
2. Incorporation of ^^C-thymidine into HeLa cells released from a single thymidine block .... 40
3. Optical density profile of polysomal RNA fractionated by zonal linear sucrose gradient centrifugation ................ 41
4. Optical density profile of 4-18S polysomal RNA fractionated in a 5-30% linear sucrose gradient
in a Beckman SW 27 rotor........... 43
5. Optical density profile of polysomes fractionated on sucrose gradients ....... 45
6. SDS Polyacrylamide gel electrophoretic absorbance profile of total polysomal RNA
before sucrose gradient fractionation ..... 48
7. SDS Polyacrylamide gel electrophoretic absorbance profiles of zonal sucrose gradient fractionated polysomal RNA's.........49
8. SDS Polyacrylamide gel electrophoretic absorbance profiles of 4-18S RNA1s fractionated
on sucrose gradients ............. 50
9. Acetic acid urea Polyacrylamide gel electrophoretic absorbance profile of stained wheat germ extract proteins, and endogenous radioactivity incorporation profiles ...... 58
10. Acetic acid urea Polyacrylamide gel electrophoretic fractionation profile of in vitro products from 7-12S RNA in the cell-free translation system derived from wheat germ 61
11. Separation profile of -%-cDNA product by Sephadex G-50 exclusion chromatography ..... 85
12. Alkaline sucrose gradient radioactivity profile
of histone ^H-cDNA preparation ......... 88
v i


Page
13. Melting curves for poly A(-) 7-12S RNA-cDNA hybrid .............. ...... 93
14. Rate of poly A(-) 7-12S RNA-cDNA hybridization
as a function of temperature.........95
15. Reannealing kinetics of histone cDNA to the
poly A(-) 7-12 S histone mRNA preparation 98
16. Hybridization analysis with histone cDNA of in vivo cell cycle stage-specific polysomal RNA's....................104
17. Hybridization analysis with histone cDNA of
in vitro native chromatin transcripts .107
IS. Hybridization analysis with histone cDNA of transcripts from chromatin reconstituted with or S phase nonhistone proteins .110
vii


KEY TO ABBREVIATIONS
AMD ..... actinomycin D
BSA ..... bovine serum albumin
CPK ..... creatine Phosphokinase
CrQt ..... concentration ribonucleotides x time
DEPC ..... diethyl pyrocarbonate
DTT ..... dithiothreitol
EDTA ..... ethylene diamine tetracetic acid
HEPES ..... N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid
POPOP ..... l,4-di[2-(5 phenyloxazoyl)] benzene
PPO .....2,5 diphenyloxazole
rAdt polyadenylic acid oligo thymidylic acid
REB .....RNA extraction buffer (100 mM Na CI,
10 mM Na acetate, 1 mM EDTA, pH 5.4)
REBS.....RNA extraction buffer + 1% SDS
REBs.....RNA extraction buffer + 0.1% SDS
RSB ..... Reticulocyte standard buffer (10 mM KCl,
10 mM Tris, pH 7.4, 1.5 mM Mg Cl2)
RSV .....Rous sarcoma virus
SDS ..... sodium dodecyl sulfate
SSC .....saline sodium citrate (150 mM Na CI, 15 mM
Na citrate, pH 7.0)
TCA ..... trichloroacetic acid
TEMED ..... N,N,N',N' tetramethylethylenediamine
TLC ..... thin layer chromatography
Tm ..... melting temperature
viii


Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
PREPARATION OF SINGLE-STRANDED DNA COMPLEMENTARY TO HISTONE MESSENGER RNA'S: A PROBE FOR HISTONE GENE EXPRESSION
By
Cary Lee Thrall December, 1975
Chairman: Gary S. Stein
Major Department: Biochemistry
Histone mRNA's have been found specifically on polysomes during the DNA synthetic period (S-phase) of the cell cycle, suggesting that histone gene transcription may only occur at this time. -^H-DNA complementary to isolated histone mRNA's was synthesized for the purpose of studying the regulation of histone gene expression by sensitive hybridization analysis techniques based on nucleic acid sequence recognition.
The histone mRNA's were isolated by size fractionation on sucrose gradients of polysomal RNA from S-phase HeLa cells; and the isolated RNA was analyzed for the reported properties of histone mRNA's including size by SDS pclyacrylamide gel electrophoresis, base composition by high voltage paper or thin layer electrophoresis, poly A content by affinity binding to nitrocellulose filters or oligo (dT)-cellulose, and trans-lational activity in a cell-free system derived from wheat germ. The isolated RNA was also tested for reverse trans-
ix


criptase template activity, and then polyadenylated with an ATP: polynucleotidylexotransferase enzyme isolated from maize seedlings to provide a sufficient template for oligo dT primed reverse transcription. -^H-dCMP plus 3H-dGMP labelled single-stranded complementary DNA (cDNA) was synthesized with the RNA-dependent DNA polymerase derived from Rous sarcoma virus; and the cDNA was characterized for size by alkaline sucrose gradient sedimentation, for single-strandedness by nuclease digestion, for sequence complexity representation by hybridization of the cDNA to the histone mRNA preparation with a CrQt analysis, and for complementarity specificity by hybridization to other defined RNA fractions. The cDNA was utilized as a probe in the quantitation by hybridization of histone specific sequences from in vivo cell cycle stage specific polysomal RNA's, as well as from iri vitro native or reconstituted chromatin transcripts.
The isolated histone mRNA preparation had a size distribution of 7-12S containing four distinct peaks, and a slightly GC rich base composition, contained less than 10% poly A, and was shown to stimulate histone polypeptide synthesis in vitro. The sequential addition of 20-30 AMP's per RNA molecule was determined to provide a sufficient template for oligo dT primed preparative synthesis of complementary DNA. The cDNA product had a size distribution of 200-400 nucleotides, and was at least 95% single-stranded. The RNA sequence complexity which the cDNA represented would


accommodate the five histone mRNA's. In hybridizing the cDNA to the other, primarily ribosomal, S-phase RNA fractions or to total polysomal RNA of cytosine arabinoside treated cells, no complementarity was found to a complexity 100 times that for histone sequences. The application of the cDNA probe also demonstrated the presence of histone sequences specifically among S-phase polysomes as well as among native S-phase chromatin transcripts. Furthermore, chromatin reconstituted with S-phase specific nonhistone proteins yielded transcripts containing an identical level of histone sequences, therefore, providing evidence for the key role of nonhistone chromatin associated proteins in the regulation of histone gene expression.
Although no single method has been established to determine absolute specificity, the properties of the complementary DNA indicated that the cDNA represented RNA's with characteristics consistent for histone specific sequences. The ^h-cDNA was demonstrated to be a high resolution probe which was utilized and can further be utilized to study histone specific RNA metabolism and the molecular mechanism for its regulation in intact cells as well as in vitro model systems.


CHAPTER 1 INTRODUCTION
During development of multicellular organisms proliferation and differentiation of cells to form tissues with specific functions are determined by controlled expression of the cell genome (1). Gene expression is mediated at several levels within the cell. It is initiated with the transcription of genes into RNA's from DNA by the RNA polymerase enzymes. The RNA's are generally processed and finally are translated at the ribosome in the synthesis of cellular proteins. Regulation can occur at each of these phases of gene expression, and the RNA represents the central biomolecule involved. To fully understand the complex yet ordered processes of cell proliferation and differentiation, a study of various factors which affect gene regulation is necessary.
A foundation has been established in the procaryotic system for understanding the regulatory mechanisms of gene expression. The structure of the basic genetic unit, the operon, and the interaction of specific regulatory proteins with this unit have been determined (2). Similar but more complex models for gene structure and its regulation in the eucaryotic system have been proposed vy Britten and Davidson {3}, Paul (4, 5), and Georgiev (6). These models introduce multiple regulatory gene sequences that interact with or code


2
for polypeptides which act as general or specific regulators of transcription. In support of this type of model is the general work on chromatin-associated proteins which has suggested that these proteins are important in controlling transcriptions both quantitatively by template activity analysis (7,8), and qualitatively by competitive hybridization analysis of tissue specific RNA transcripts (9,10). Although there appear to be regulatory proteins among the chromosomal proteins, more precise information of the structure of the genome and specific protein interactions is required to confirm the proposed models. More understanding about eucaryotic RNA polymerases, RNA processing and translation is also required in extending the model to all phases of gene expression.
Although such general information has been gained about the regulation which occurs at the various levels of gene expression, to learn about more specific regulatory mechanisms it has become essential to monitor a specific gene or set of specific genes controlled under defined cellular conditions. This means that a gene system must be carefully selected for study and implies the ability to detect the specific RNA product.
Several genes and their expression under certain well defined cellular conditions in eucaryotic systems are currently being studied. Identification and isolation of the gene products, messenger RNA1s, have been facilitated because these messenger RNA's code for a main protein product of a


final differentiated cell type. Examples of a few such proteins and the cells involved are: Globin of red blood cells (11,12); crystallin of lens epithelial cells (13,14); myosin of muscle cells (15); collagen of cartilage and tendon fibroblasts (16,17; keratin of hair, feather, and nail follicular cells (18); silk fibroin of Bombyx mori silk gland cells (19); avidin (20) and ovalbumin (21,22) of oviduct epithelial cells; and immunoglobulin (23,24) of plasmacytoma and myeloma cells. Systems of this type may provide information about the determination and the maintenance of the final differentiated cell stage, and about the modulation of the defined function for that tissue.
Elucidating the control of cell proliferation, a fundamental aspect of development, requires understanding the regulation of specific genes during the cell life cycle. The major events of the cell cycle are DNA replication and mitosis. Only a few defined proteins have been studied in relation to cell cycle events. One class of proteins and their mRNA's, which have been identified specifically in association with DNA synthesis, is the histones (25-28). Histones are nuclear proteins which are found bound to DNA, are proposed to serve as general repressors of the cell genome, and are synthesized when DNA replication takes place (29). The expression of histone genes offers a unique opportunity to study a process which occurs in the proliferative stage of all tissue cells and does not represent a final differentiation event. A study of the regulatory mechanism


4
for histone gene expression will also provide insight into the factors influencing organismic growth and development.
Histone mRNA's were discovered as a distinct size class of RNA, which appeared at the same time as histone protein synthesis occurred (25-27,30) and was found later to be the RNA responsible for the synthesis of histone polypeptides by cell free translation (26,27,31-34). These mRNA's have a size range of 7-12S and contain the five molecules coding for the five histone proteins (35,36). The mRNA's were found on small polysomes of cells replicating their DNA (25-28) and in unfertilized eggs of sea urchin (37,38) and Xenopus (39). Their appearance on polysomes of proliferating cells has been determined to be dependent upon DNA synthesis (27). In the absence of DNA synthesis, no detectable RNA species were found in the 7-12S size class in these early studies (25-28,40). Additional unique properties have been determined for this RNA size class representing the histone messages. They have a base composition of 54% G + C (28), placing them in a category between other mRNA's which are generally A + U rich and ribosomal RNA's which are more G + C rich, and work is presently proceeding on the base sequence for the histcne mRNA's (35). They lack the poly-adenylate sequence at their 3'OH ends (28) common to most mRNA's, rapidly associate with polysomes after initiation of DNA synthesis, and possess a relatively short polysomal half-life on the order of a few hours (40-44). The reiteration frequency of the histone genes is within the class of moderately repeated sequences. For sea urchin, each histone


gene was determined to be 400- to 1200-fold reiterated (36, 45); and recently for HeLa cells, each gene was found to be less reiterated at 10-20 copies per haploid genome (46). More recently, a gene frequency of 80 copies was estimated with human placental DNA (47).
The circumstantial evidence discussed above has suggested that histone mRNA. as well as histone protein synthesis, occurs during the DNA synthetic period (S-phase) of the cell cycle (25-28). Therefore, histone gene expression represents a promising system for studying specific gene control, in particular, during the cell cycle. The distinct size and compositional qualities of the histone mRNA's make its isolation possible. The histone mRNA system differs from other mRNA systems currently being investigated since it is not a result of a final differentiated state, but is related to the cell cycle. The regulatory mechanism for this set of genes may, therefore, also be different, requiring a proliferating cell system for its study.
The HeLa S3 cell system was chosen for several reasons, including rapid and continuous proliferation of these cells, ease in growth and synchronization, and its availability in this laboratory. This cell line was originally derived from human cervical carcinoma tissue by Gey (48) and cloned and characterized by Puck (49). The S2 clone was first propagated by Puck and is now grown in suspension culture having a 19-hour cell cycle separated into the four phases as follows: 1 hr mitosis, 5 hr Gp 9 hr S, and 4 hr G2 (50). Because
1


6
HeLa cells are hypotetraploid, they have a relatively large nucleus providing an enrichment in chromosomal material and conceivably regulatory molecules, which is an important advantage for studying specific gene expression at the level of the genome. Another reason for choosing this cell system was that it had already been used for the study of histone mRNA metabolism providing essential background information.
The monitoring of gene expression implies the ability to detect the specific RNA product. For mRNA's, translational activity can be used, either in vivo, or by isolation of polysomes or mRNA followed by translation in vitro. This procedure is indirect; dependent upon protein product analysis; and, if isolated RNA is used, requires several yg of material, especially if the RNA is not pure. The presence of messenger RNA can also be detected by the immunoreactivity of the polysomes synthesizing a specific antigenic polypeptide (51,52). This again is indirect, requires antibody preparation, and only detects RNA being translated. To detect directly a given RNA, not just messenger RNA, hybridization analysis using complementary sequences to the defined RNA can be employed. A specific complementary probe could be either fractionated from the cellular DNA, or synthesized as RNA or DNA. In either case, obtaining complementary sequences requires the isolation of the specific RNA to be studied. The probe must also be highly labelled for sensitive hybridization analysis. To obtain the complementary sequences from cellular DNA, preparative


hybridization with the RNA, and separation of the hybrids from the remaining DNA would be involved. This would require large amounts of DNA for reasonable yields, and would present purification problems. Cellular DNA could also not be radioactively labelled to an adequately high level. Alternatively, a complementary probe could be synthesized in vitro, and labelled to a defined extent. RNA copies could be produced by using an enzyme such as QB replicase which does not require a primer for initiation (53); however, purification of the synthesized RNA probe from its template is not currently possible. Hybridization analysis is, therefore, more difficult to interpret since the template RNA is always present and RNA-RNA hybrids are less stable. On the other hand, a complementary DNA (cDNA) probe, synthesized by the RNA-dependent DNA polymerase (Reverse Transcriptase) of RNA tumor viruses, offers a specific RNA detection system which provides a more direct analysis, can by highly labelled, is more stable than RNA, can be purified away from its template, allows more straightforward interpretation of hybridization data, and has already been extensively developed (54-75). Complementary DNA's have been synthesized to rabbit globin mRNA (54,55), mouse globin mRNA (56), human globin mRNA (57), mouse kappa chain immunoglobulin mRNA (24,67,68), and ovalbumin mRNA (70,71). The cDNA's have been characterized for physical properties and copy specificity. Size of the cDNA has been determined by alkaline


8
sucrose gradient sedimentation (54,56,24,67,68,71) and on neutral sucrose gradients (55,57,70). Buoyant density measurements have been made on isolated cDNA products and on cDNA-mRNA hybrids to determine base composition (56,57), single-strandedness (54,56,57), and extent of copy (56). In one case the base composition was determined by nuclease digestion and electrophoresis of the nucleotides (67) The specificity of the complementary DNA was examined by hybridization back to the mRNA preparation or to other RNA's. In some cases, the specificity was dependent partially upon the established purity of the mRNA (54,55,56,71). In other cases, sequence complexity was estimated by comparative kinetic hybridization determination (24,67,70), and in most cases, specificity was confirmed by hybridization attempts to other RNA species, such as: rRNA's, tRNA's, E. coli RNA's, or RNA from other cell types (54,55,56,67,70,71). Additional evidence for specificity has been determined by correlating translational activity of the RNA with the level of cDNA hybridization to the RNA at various stages during purification of the RNA (70). Complementary DNA sequences are particularly necessary in the study of in vitro RNA transcripts, because such RNA's can be directly and specifically analyzed by base sequence with such a probe and do not have to be analyzed by an indirect functional assay such as translation, which may not specifically detect the proper sequences within a transcribed RNA mixture. For these reasons, synthetic cDNA was prepared and employed for the detection of histone specific RNA gene expression in this study.


The study to be described involves the preparation of a cDNA to histone mRNA's of HeLa cells, for the purpose of investigating the regulatory mechanism for the expression of these specific genes. The preparation of the cDNA probe involved, first, isolation and characterization of the histone mRNA preparation; then, synthesis and extensive characterization of the cDNA sequences. The messenger RNA isolation procedure was developed from a number of published procedures (28,32,76), and was based on sucrose gradient fractionation of polysomal RNA from synchronized S-phase HeLa cells. Several analyses were conducted to monitor the RNA preparation using the known general properties of histone-specific messenger RNA. The RNA was sized, was analyzed for base composition and poly A content, and was tested for cell-free translational activity.
Isolation and characterization of the histone mRNA's were based upon the previous reports from several laboratories on the identification of these RNA species (25-29,30,35-55). Criteria for the purity of the histone mRNA preparations, however, have not been established. None of the characterizations including size, base composition, poly A content, or cell-free translational ability can rule out the presence of other RNA species which may have similar properties or are not detectable by these methods. Some of the strongest evidence for the purity of a histone mRNA preparation has come from the sea urchin system where some of the individual messenger RNA's have been separated by polyacrylamide gel


10
electrophoresis (35). The RNA bands were arranged in the order expected for the size of the histone proteins and when the RNA's were translated, the products comigrated with the specific histone proteins. The most distinctly separated mRNA, that coding for F2 -^ histone' was also subjected to ribonuclease digestion and oligonucleotide fingerprinting. Minor electrophoretic RNA bands, slightly larger in molecular weight than F2al ^and, were also fingerprinted and yielded an identical pattern to the F2ai band. This suggested that the limited number of minor RNA species of sea urchin in the histone region are possibly histone specific sequences. Only with improved separation and translation and sequence analysis of all bands can the purity of such a mRNA preparation be more conclusively established.
The histone mRNA preparation purified by exclusion from poly A binding supports was utilized as template for the synthesis of single-stranded complementary DNA by the RNA-dependent DNA polymerase derived from Rous sarcoma virus. The reverse transcriptase enzyme reaction has been shown, in the case of virus infection (63), as well as with exogenous mRNA's (54-75), to require a double-stranded priming sequence at the 3' end of the RNA template to initiate complementary DNA synthesis. Such a priming system is provided by the virus for the replication of its genome. Conveniently, the 3'OH polyadenylate sequence on most mRNA's has provided the means of initiating complementary DNA sequence synthesis with the utilization of oligo dT as the second strand primer when annealed to the poly A sequence. Since histone mRNA's


11
do not contain poly A sequences at their 3'0H termini, it was necessary to enzymatically furnish the polynucleotide sequence. Current work on polyadenylating enzymes (ATP: polynucleotidylexotransferase) (77-80), is being carried out by Dr. R.J. Mans (Department of Biochemistry, University of Florida), and this system was applied to the histone mRNA preparation, providing a template for oligo dT primed reverse transcription. Adequately polyadenylated templates were selected and JH-labelled cDNA or known specific activity was preparatively synthesized, and isolated. The cDNA was characterized by size, single-strandedness, renaturation to histone mRNA which determined representative sequence complexity, and finally by hybridization to other RNA fractions to evaluate the possibility of contamination. Since it was necessary to add poly A to the histone mRNA preparation, all RNA's could havebeen polyadenylated and subsequently copied with reverse transcriptase. The hybridization analysis provided further evidence for the relative purity of the histone mRNA preparation.
The cDNA probe was further utilized in this laboratory to evaluate the level of histone gene expression during the HeLa cell cycle. Stage specific polysomal RNA's, and in vitro RNA transcripts from stage specific native chromatin were analyzed by hybridization for the relative amount of histone mRNA sequences. The additional technique of chromatin reconstitution was employed with subseauent hybridization of in vitro transcripts to study the differential cell cycle control of histone gene transcription by the nonhistone chromosomal proteins.


CHAPTER 2 MATERIALS AND METHODS
I. Cell Culture
HeLa S2 cells were maintained in exponential growth at a density of 2.0 x 105 to 6.0 x 105 cells/ml in Joklik-modified Eagle's Minimum Essential Medium (Grand Island Biological Co.-Powder), supplemented with 1 mM glutamine and 3.5% each of calf and fetal calf serum. Before the medium was supplemented, it was sterilized by Millipore filtration and stored at 4C. The cultures exchanged gases with the atmosphere in cotton plugged and magnetically stirred bottles in an environmental room at 37C. Cell density was determined by the packed cell volume technique, in which 10 ml of culture was placed in 30 ml tubes with a 0.1 ml bottom stem and centrifuged at 1300 x g in a PR-6 centrifuge (International Equipment Company) for three minutes. A packed cell volume of 0.01 ml represented 2.5 x 10^ cells/ml in culture.
II. Cell Synchronization
A. Single Thymidine Block (91)
The cell cultures were brought to a concentration of 2 mM thymidine and incubated for 12-16 hours.
B. Double Thymidine Block (81)
The cells were released from a single thymidine block by pelleting the cells at 1300 x g for five minutes in an
12


13
International Model UV centrifuge at 37C, resuspending the cells in warm supplemented growth medium minus thymidine, and incubating for nine hours. The cultures were brought again to a concentration of 2mM thymidine and incubated 12 hours.
C. Selective Detachment (82)
All procedures were carried out at 37C. Partial synchronization was achieved by a single thymidine block for 12 hours. The cells were released into warm supplemented Eagle's Minimal Essential medium or Eagle's Basal medium at a concentration of 5 x 10^ cells/ml, and 50 ml aliquots were transferred to 1 liter Blake bottles. After six hours, cells not adhering to the glass surface were removed by vigorous shaking and by decanting the medium. The cell monolayers were washed twice with non-supplemented medium; 50 ml supplemented medium were added; and 9-10 hours after plating began, mitotic cells were gently shaken into suspension and accumulated in 100 ml aliquots at 5 x 10^ cells/ ml. Additional mitotic cells were obtained by repeating the gentle shake procedure immediately.
D. Thymidine Block Synchrony Monitoring
Cell cycle synchrony by thymidine block was monitored by a radioactive thymidine pulse-label assay (83). Two ml of culture (1 x 10^ cells) were removed at timed intervals following release of the cells from the thymidine block; 0.2 yCi of ^C-thymidine (New England Nuclear) was introduced, and


14
the cells were labelled in suspension for 30 minutes at 37C. The labelling was terminated by addition of 10 volumes of ice-cold spinner salts solution, and the cells were pelleted by centrifugation. Thymidine incorporation was determined by adding cold 10% trichloroacetic acid (TCA) to the cell pellet, collecting the precipitate on a 25 mm HA 0.45y Millipore filter by filtration, washing the filter with cold 10% TCA, dissolving the filter in 1 ml ethylene glycol monoethyl ether (cellusolve), and counting to toluene-based scintillation fluid containing 26% (v/v) cellusolve, 4.23 g/1 of 2,5 diphenyloxazole (PPO), and 52 mg/1 of 1,4-di (2-(5 phenyloxazolyl)) benzene (POPOP) (Liquifluor New England Nuclear). This scintillation fluid will be referred to as cellusolve toluene.
III. Isolation of Histone Messenger RNA's
In the method finally developed for the isolation of histone mRNA's, 32 liters of cell culture were grown in eight six-liter bottles containing four liters each of culture at a density of 3-4 x 10^ cells per ml. The cells were synchronized into S phase by a single thymidine block and a release of three hours. Cell synchrony was monitored
by the *^C-thymidine pulse method. The cells were harvested
q
by centrifugation, with 4.5 liters of culture (1.5 x 10 cells) pelleted at a time and resuspended in 50 ml of ice-cold Earle's balanced salt solution in 50 ml capped plastic tubes. These suspensions were kept on ice until all cells were harvested. The cells were washed by centrifugation and


15
resuspension twice more with cold balanced salt solution and were prepared for hypotonic lysis by draining the final pellet well with aspiration. The cells were resuspended to 240 ml in 10 mM KC1, 10 mM Tris, 1.5 mM MgCl2, pH 7.4 (RSB)
(84) at 4 x 107 cells/ml. The RSB buffer was autoclaved and treated with diethylpyrocarbonate (DEPC) (0.004%) to eliminate ribonuclease activity (85,86). DEPC treatment involved adding the compound to the solution and stirring vigorously overnight. The cell suspension was stirred in a 250 ml chilled graduated cylinder for 15 minutes and monitored by light microscopy for cell swelling. The cells were homogenized in a sterile 40 ml Dounce homogenizer by 12 strokes with a tight-fitting (B) pestle at 4C. The lysate was placed in sterile 50 ml polycarbonate tubes on ice, and was centrifuged at 15,000 RPM for 15 minutes (27,000 x g) at 4C in an SS-34 rotor and Sorvall RC-2 centrifuge. The post-mitochondrial supernatant was transferred aseptically to 28 ml capped polycarbonate tubes and each tube was brought to a uniform volume with RSB and centrifuged at 40,000 RPM
(approximately 100,000 x g) for 90 minutes at 4C in a Beckman 60 Ti rotor. The supernatant was decanted; and the polysomal pellets were resuspended at room temperature in 40 ml 0.1 M NaCl, 10 mM Na acetate, 1 mM ethylenediamine tetracetic acid (EDTA) and 1% sodium dodecyl sulfate (SDS), pH 5.4 (REBS) by transferring them to a sterile 40 ml Dounce homogenizer and dispersing them into solution by gentle homogenization with a loose-fitting (A) pestle. The polysomal homogenate was transferred to a sterile 500 ml separatory


funnel. An equal volume of phenol, buffered with REB (-SDS), was added and the mixture was shaken for five minutes followed by the addition of one volume of chloroform:isoamyl alcohol (24:1 v/v). The mixture was shaken again for five minutes, and the milky white suspension was decanted into 30 ml Corex centrifuge tubes and centrifuged at 10,000 RPM for 10 minutes in a Sorvall SS-34 rotor. The aqueous layer was removed and set aside. A half volume of REBS was added to the organic layer and interface. The mixture was shaken, centrifuged, and the aqueous portion was pooled with the previous extract. The pooled aqueous fraction was extracted again with phenol: chloroform:isoamyl alcohol and finally with chloroform: isoamyl alcohol until there was no visible interface of protein (usually twice). Two volumes of ethanol were then added to the aqueous fraction and the polysomal RNA was precipitated at -20C for at least 12 hours.
In preparation for sucrose gradient fractionation, the RNA was quantitated. The RNA was pelleted by centrifugation at 17,000 x g for 10 minutes at 4C. The pellets were drained at 4C and aspirated to dryness. The RNA was resuspended in 40 ml of REB containing 0.1% SDS (REBs), and the RNA concentration was determined by an absorbance measurement at 260 nm. The relationship that a 1 mg/ml solution represents 20 &260 un^ts was employed (86) The RNA was then reprecipitated with two volumes of ethanol.
Fractionation of the polysomal RNA was first carried out by zonal gradient centrifugation. The zonal gradient system required centrifugation during gradient formation, running,


17
and elution. A Beckman L2-50 ultracentrifuge was used with a Beckman Al-14 zonal rotor and seal assembly. For loading and unloading the gradient, the Beckman Model 141 High Capaci Gradient pump was used. The gradient was linear and continuous from 5-30% sucrose in REBs buffer with a volume of 400 ml. The rotor also contained a cushion of 200 ml of 30% sucrose and a REBs buffer overlay of 50 ml. Half of the RNA was suspended in 20 ml of gradient buffer, brought to 2-3% sucrose, applied to the gradient, and run at 32,000 RPM for 16 hours at 22C. The gradient was pumped out through an ISCO UA-4 absorbance monitor, and fractionated with an ISCO Model 567 Fraction collector. A second gradient was run with the remaining half of the RNA. Four sets of pooled fractions 28S, 18S, 4-18S, and 4,5S were precipitated with two volumes of ethanol.
The ethanol precipitated 4-18S RNA was pelleted in four 50 ml polycarbonate tubes by centrifugation, drained, and dried under nitrogen. It was resuspended in 2 ml REBs and fractionated on four 5-30% linear sucrose REBs gradients formed on an ISCO Model 570 Gradient former in 38 ml nitrocellulose tubes and run in a Beckman L2-65B ultra-centrifuge with a Beckman SW 27 rotor at 26,000 RPM for 22 hours at 22C. The gradients were fractionated with an ISCO Model 640 Density Gradient Fractionator coupled to a UA-4 absorbance monitor. The 18S, 14S, 7-12S and 4,5S containing fractions were individually pooled and precipitated with ethanol. The 7-12S fraction contained the histone messenger RNA's. See Figure 1 for a flow diagram of the isolation procedure.


18
Pellet (nuclei and mitochondria)
Organic phases (discard)
HeLa cells 1. 2.
synchronization by a 2 mM thymidine block
lysis in 10 mM KC1, 10 mM Tris, 1.5 mM MgCl2, pH 7.4
Lysate
centrifuge at 27,000 x g, 15 min
Supernatant
centrifuge at 100,000 x g, 90 min
Pellet (polyribosomes)
Supernatant
2.
resuspend in 100 mM NaCl, 10 mM Na acetate, 1 mM EDTA, 1% SDS, pH 5.4
extract 2x with phenol: chloroform:isoamyl alcohol (24:24:1 v/v/v)
3. extract lx with chloroform: isoamyl alcohol (24:1 v/v)
Aqueous phase
add 2 volumes of ethanol and precipitate RNA at -20C
Polysomal RNA
Figure la. Flow diagram for the isolation of polysomal RNA.


19
Polysoma1 RNA
1. suspend in 100 mM NaCl, 10 mM Na acetate, 1 mM EDTA, 0.1% SDS, pH 5.4
2. fractionate on 5-30% sucrose gradient in a zonal rotor
3. precipitate 4-18S fraction with 2 volumes of ethanol at -20C
4-18S RNA
1. suspend in 100 mM NaCl, 10 mM Na acetate, 1 mM EDTA, 0.1% SDS, pH 5.4
2. fractionate on 5-30% sucrose gradients in an SW 27 rotor
3. precipitate 7-12S fraction with 2 volumes of ethanol at -20C
7-12S RNA
bind poly A by nitrocellulose filtration or oligo (dT)-cellulose chromatography in 0.5 M KC1 (Methods IVD)
Bound
(poly A containing RNA)
--^
Unbound poly A(-) 7-12S RNA (containing histone mRNA)
Figure lb. Flow diagram for the fractionation of polysomal RNA and isolation of the RNA containing the histone mRNA's.


20
IV. Characterization of Histone Messenger RNA's A. Size Distribution SDS polyacrylamide gel electrophoresis
The RNA gel procedure was based on that of Loening (87). The gels were prepared with 2.7% (w/v) acrylamide (Eastman) and 0.675% (w/v) N,N methylene bisacrylamide (Eastman) as the cross-linker in 40 mM Tris, 20 mM Na acetate, 1 mM EDTA, pH 7.2, and 10% glycerol. The gels were polymerized with 0.04% N,N,N',N-tetramethyl ethylene diamine (TEMED) (Eastman) and 0.06% ammonium persulfate. The gel mixture was prepared and immediately poured to 8 cm in 0.6 cm glass tubes, over-layed gently with 5 mm of H20, and allowed to polymerize. Complete polymerization required approximately 40 minutes. The running buffer was 40 mM Tris, 20 mM Na acetate, 1 mM EDTA, pH 7.2, 0.2% SDS. The gels were subjected to electrophoresis prior to sample application for at least 30 minutes at 4 mA/gel with the positive electrode at the bottom. The RNA sample was generally prepared in 10 mM Na acetate, 100 mM NaCl, 1 mM EDTA, 0.2% SDS, 15% sucrose buffer at pH 6.0, and at a concentration of approximately 1 mg/ml. The RNA sample (10 to 100 pg) was applied by micropipette and was subjected to electrophoresis for 2.5 hours at 5 mA/gel. Immediately following electrophoresis, the gels were removed from the tubes and were scanned at 260 nm in a linear transport device (6 cm/min. transport speed) attached to a Beckman Acta CII Spectrophotometer with recorder (5 in./min. chart speed) on a scale of 0-3 Absorbance units.


21
B. Base Composition
1. Paper electrophoresis
The method of paper electrophoresis for the separation of nucleoside monophosphates was based on standard procedures (88-90). The RNA was hydrolyzed in 0.3 M KOH at 37C for 18 hours. The solution was brought to pH 1 with HCIO^, and the KCIO^ precipitate was pelleted by centrifugation. The supernatant was adjusted to pH 4 with KOH, and the nucleotide concentration to approximately 2 mg/ml. This mixture was spotted on a 58 cm strip of Whatman #1 paper. A minimum of 50 yg of ribonucleotides was applied to the paper three inches from one end by multiple application with drying to maintain small concentrated spots, and picric acid was used as a front marker. The paper was wetted by spraying with electrophoresis buffer (1.25% pyridine-1.25% acetic acid, pH 4.8) and run at 2000 V on a Gilson Electrophorator for 2-2.5 hours with samples migrating toward the anode. Following electrophoresis, the paper was removed and air dried in a hood. The nucleotide spots were visualized under a short wave UV light, marked, and cut out. The nucleotides were eluted with 0.01 N HC1. The absorbance at 260 nm was measured on the eluate solutions using the following extinction coefficients (E^q) (acidic pH) : AMP, 14.3; CMP, 6.8; UMP, 10.0; GMP, 11.8 (91).
2. Thin layer electrophoresis
The RNA sample was hydrolyzed in the same manner as for paper electrophoresis and was applied 3 cm from the bottom


22
edge of a 20-centimeter square cellulose-coated glass TLC plate. Picric acid was used as a marker. The plate was wetted by spraying with electrophoresis buffer and placed in an apparatus having buffer compartments at opposite ends with paper wicks which contact the cellulose layer. Electrophoresis was at 400 V for various times ranging from 2.5 to 4.5 hours. The samples migrated toward the anode.
The plates were dried, spots visualized as previously described, and the samples were collected by scraping onto glassine paper and deposited into test tubes. The material was extracted with 1-2 ml of 0.01 N HC1 for several hours and centrifuged. The supernatant, free of particles, was then measured for absorbance at 260 nm. C. Cell-Free Translation 1. Wheat germ lysate
All solutions and glassware were sterilized by autoclaving and diethylpyrocarbonate treatment. The method was adopted from that of Roberts and Paterson (92) The extract preparation involved mixing 6 gm of Crown wheat germ with 6 gm of autoclaved dry sand and cold sterile extract buffer 20 mM N-2-hydroxyethylpiperazine-N1-2-ethane-sulfonic acid (HEPES) pH 7.6, 0.1 M KC1, 1 mM Mg acetate, 2 mM CaCl2, and 6 mM 2-mercaptoethanol in a sterile mortar and pestle at 4C. The mixture was ground to a relatively homogeneous consistency and was centrifuged in two 30 ml Corex tubes at 16,000 RPM (30,000 x g) for 10 minutes at 4C. Avoiding the fat layer on top, the supernatant was removed and applied to a 1.5 x 85 cm G-25 (coarse) column equilibrated with 20 mM HEPES pH 7.6, 0.12 M KCl, 5 mM Mg acetate, 6 mM 2-mercaptoethanol, and eluted


23
with this buffer at approximately 1.5 ml/minute. The excluded very turbid fractions were pooled, frozen at -76C in 200 yl aliquots, and served as the S-30 wheat germ extract for cell-free protein synthesis. An intense yellow pigment was separated completely from the extract material.
The reaction mixture of 0.1 ml was composed of the following :
HEPES, pH 7.6 22 mM
KC1 56 mM
Mg acetate 3.5 mM
dithiothreitol (DTT) 2 mM
amino acids, except those labelled 120 yM each
ATP 1.4 mM
GTP 0.3 mM
phosphocreatine 17 mM
creatine phosphokinase 400 yg/ml
wheat germ extract 30% (v/v)
3H-leu and other 3H-a.a. 100 yCi/ml (2-4 yM)
exogenous mRNA 100-250 yg/ml
Incubations were for 90 minutes at 30C. Amino acid incorporating activity was measured by spotting 5 yl of the reaction mixture on a Whatman 3 MM filter disc; which was then dipped for 10 minutes in cold 10% TCA, 100 yM labelled amino acids; boiled for 20 minutes in 5% TCA; washed two times with cold 5% TCA; dried with ethanol: ether (1:1), and ether; and counted in 100% toluene scintillation fluid containing 0.4% PPO (w/v), and 0.005% POPOP (w/v). Material was prepared for


24
polyacrylamide gel electrophoresis by adding 40 yl of 1.8 N acetic acid, 5 M urea, 30% sucrose to 40 yl of the reaction mixture, and the resultant solution was applied to acetic acid-urea polyacrylamide gels.
2. Acetic acid-urea polyacrylamide gel electrophoresis
The method was based on that of Panyim and Chalkley (93). The gels with dimensions of 9 x 0.6 cm consisted of 15% acrylamide, 0.1% bisacrylamide, 2.5 M urea, and 0.9 N acetic acid; were polymerized in glass tubes with 0.5% TEMED and 0.125% ammonium persulfate; and were overlayed with 1.2 M urea. When an interface appeared, they were transferred to an ice-water bath to continue polymerization for two hours. Heat generated upon polymerization otherwise caused bubbles in the gel. The gels were subjected to electrophoresis for at least four hours at 2 mA/gel with 0.9 N acetic acid running buffer and the positive electrode at the top. The samples were run for 4.5 hours at 2 mA/gel. The gels were removed from the tubes; stained overnight in 40% ethanol, 7% acetic acid, 0.1% analine blue-black (other names include: amido black (schwarze) 10B, naphthol blue-black, buffalo black NBR (Ci 20470)); destained electrically with a Canalco quick gel destainer in 10% ethanol, 7% acetic acid for 15 minutes; and scanned with a Beckman Acta CII spectrophotometer at a wavelength of 620 nm, a transport speed of 1.5 cm/min., and a chart speed of 2 in/min. The gels were prepared for radioactivity analysis by freezing on dry ice, and slicing in a multi-razorblade gel slicer; the slices were placed in scintillation vials with 0.2 ml of 30% H2^2' an<^ were digested overnight at 37C. Radioactivity


25
incorporation was determined by adding 5 ml of a toluene-based scintillation fluid containing 33% (v/v) Triton X-100 (Palmetto Chemicals), 4.42 gm/1 of PPO, 55 mg/1 of POPOP (Liquifluor New England Nuclear) and counting in a liquid scintillation counter. This scintillation fluid will be referred to as triton toluene. D. Poly A Affinity Binding
1. Nitrocellulose filtration
The method used was directly adapted from that of Lee, Mendecki, and Brawerman (94) Poly A binding required a high salt buffer of 0.5 M KCl, 10 mM Tris, pH 7.6, and 1 mM MgCl2. RNA suspended in this buffer at 10 pg/ml was filtered through a wetted 25 mm HA 0.45y nitrocellulose filter held in a steel filter holder with syringe adaptor at 4C. The filtrate and successive washes were collected. Material bound to the filter was eluted and isolated using a 0.5% SDS, 0.1 M Tris solution, pH 9.0. RNA concentrations were measured by absorbance spectroscopy at 260 nm.
2. Oligo @T)-cellulose chromatography
The technique was directly adapted from the work of Aviv and Leder (12). The same salt conditions were applied in this procedure as in nitrocellulose filtration. All solutions and glassware were autoclaved and treated with DEPC. The procedure was carried out at room temperature. A slurry of oligo (dT)-cellulose with a reported capacity of 250 nmoles (85 yg) poly A per ml (P-L Biochemicals) was made in 0.5 M KCl, 10 mM Tris, pH 7.5. A pasteur pipet serving as a column was packed by gravity flow. The column was washed extensively


26
with the KCl solution and the optical density at 260 nm was brought to 0.0 absorbance units. Upon application of sample in high salt buffer, the column was run at a flow rate of 3-6 ml/hr. Fractions of 0.5 ml were collected for absorbance monitoring. The column was eluted until the absorbance returned to 0.0. More rapid elution was then carried out with 10 mM Tris, pH 7.5, and the absorbance monitored. Peak fractions were pooled from both elutions and final absorbance quantitation was made.
V. Synthesis of Complementary DNA A. Isolation of Rous Sarcoma Virus
Virus particles were isolated from tissue culture fluid by precipitation with 50% ammonium sulfate. The precipitate was resuspended in 15% sucrose, 50 mM Tris, pH 7.4, 10 mM EDTA, and 0.05% 2-mercaptoethanol. This suspension was added to 1" x 3-1/2" nitrocellulose centrifuge tubes containing a 3 ml 60% sucrose cushion and 15 ml of 15% sucrose in the same buffer. The material was centrifuged at 22,500 RPM in a Beckman SW 27 rotor for one hour at 4C and the material on the cushion was collected by fractionation. This was applied to 5 ml, 20 to 60% linear sucrose gradients in the same buffer, and centrifuged at 36,000 RPM for three hours at 4C in a Beckman SW 39 rotor. These gradients were fractionated and the turbid fractions within the gradients were pooled and frozen at -76C in 0.5 ml aliquots. This material represented the isolated virus particle preparation.


27
B. Assay for Reverse Transcriptase Activity
The basis for the composition of the reaction mixture had been established (95) A viral lysate was used as the enzyme source (54). Lysis was accomplished with a virus pre-treatment solution (VPT) consisting of 180 mM DTT and 0.12% Triton X-100. This was combined in a 1 to 5 VPT to virus ratio at 0C for 10 minutes. The chief template for determining enzyme activity was poly(rA)oligo(dT) referred to as rAdT. This template was made available and was prepared by hybridizing poly A with oligo dT sequences yielding a single nucleotide template with several initiation sites (95).
The reaction mixture was approximately 0.1 ml, and was composed of the following:
Tris, pH 8.3 50 mM
KC1 20 mM
MgCl2 10 mM
DTT 15 mM
RSV lysate 15% (v/v)
and the following were used as variables:
dNTPs (not labelled) 0.8 mM each
3H-dNTP (New England Nuclear or Schwarz Mann)
either 3H-TTP 100 yCi/ml, 42 yM (2.2 Ci/m mole)
or 3H-dCTP and 100 yCi/ml, 45 yM (2.22 Ci/m mole)
3H-dGTP 100 yCi/ml, 56 yM (1.78 Ci/m mole)
rA'dt template 1-5 yg/ml
RNA template 5 yg/ml
oligo dT1Q (Calbi- 0.4 yg/ml chem)


28
actinomycin D (AMD)
(Sigma) 100 yg/ml
The amount of oligo dT^Q utilized resulted in a dT-^Q to mRNA (10^ daltons) molar ratio of 2.4:1. The above concentrations were within the range specified by a number of investigators (54,55,57).
The reaction was carried out at 37C for various lengths of time. Incorporation of radioactivity was measured by placing 50 yl aliquots of the reaction mixture on Whatman 3 MM filter discs. The filter discs were then soaked in cold 10% TCA, 2 mM Na pyrophosphate for 10 minutes, washed three times in 10% TCA, 2 mM Na pyrophosphate for five minutes at room temperature; dehydrated by one wash with ethanol: ether (1:1), and two washes with ether; dried in a warm oven; and counted in 100% toluene scintillation fluid.
C. Polyadenylation of Histone Messenger RNA's
The histone mRNA preparation was polyadenylated with the specific enzyme ATP:polynucleotidylexotransferase isolated from maize seedlings and characterized by Dr. R.J. Mans (77,78,70). The mRNA was provided either from nitrocellulose filtration in 0.5 M KC1, 10 mM Tris, pH 7.6, 1 mM EDTA from which it was chromatographed into 1 mM Tris, pH 7.6 on Sephadex G-25; or in H20 as untreated RNA. The reaction mixture contained 70 mM Tris, pH 8.8, 10 mM DTT, 1 mg/ml BSA, 1 mM MnCl2, 0.11 mM 8-14C ATP (3.2 Ci/mole), 50 ug/ml histone mRNA, and 50 pg/ml fraction V ATP:polynucleotidylexotransf erase (80). Incubation was at 30C for various lengths of time, and the reaction was terminated by addition of an


29
equal volume of chloroformzoctanol (5:1, v/v). The reaction mixture was thus deproteinized and the organic layer was re-extracted with 10 mM Tris, pH 8. The aqueous portions were pooled and chromatographed on a Sephadex G-50 column equilibrated with 5 mM Tris, pH 8.0, 0.6 mM DTT, 2 mM KC1, 10 yM EDTA, which was approximately 1/10 reverse transcriptase reaction buffer. The excluded volume contained the polyadenylated RNA. D. Preparation of the Complementary DNA
In the preparative synthesis of the cDNA, changes were made in the assay reaction mixture to achieve a higher efficiency of the enzyme and a higher specific activity of the cDNA. The reaction mixture, from which complementary DNA with the highest specific activity was prepared, was composed of the following in 0.5 or 1 ml:
Tris, pH 8.3 50 mM
KC1 20 mM
MgCl2 10 mM
dATP 80 mM
TTP 80 yM
DTT 15 yM
polyadenylated histone mRNA 10 yg/ml
AMD 100 yg/ml
oligo <3T10 1.6 yg/ml
RSV lysate 20% (v/v)
3H-dCTP 300 yCi/ml, 30 yM(9.8 Ci/mmole)
3H-dGTP 200 yCi/ml, 32 yM(6.2 Ci/mmole)


30
In the initial preparative (0.5 ml) attempt, 3H-dCTP and 3H-dGTP were utilized at 200 yCi/ml (6.67 Ci/mmole) and 200 yO/rol (3.84 Ci/mmole) respectively, and represented nucleotide concentrations of 30 yM dCTP and 52 yM dGTP. The optimum ratio of oligo dT^ to RNA template had been reported for globin mRNA as 2 oligo dT^0/mRNA molecule (55), and this was approximately the amount used under assay conditions with histone mRNA preparation. In the preparation of cDNA as described, a ratio of 5 dT^^/mRNA molecule (10^ daltons) was employed. With the indicated modifications of the reverse transcriptase assay reaction mixture, cDNA was synthesized during a two-hour incubation period at 37C. After incubation, 100 yg of carrier E. coli DNA (donated by Dr. R. McMacken, laboratory of Dr. R.P. Boyce, Department of Biochemistry, University of Florida), which was further sonicated and denatured, was added to the reaction mixture. SDS was then added to 1% and the mixture incubated for 10 minutes at 37C. The reaction was deproteinized by extraction with phenol and chloroform:isoamyl alcohol as described previously for RNA isolation. The aqueous extract was brought to 0.25 N NaOH and incubated for 18 hours at 37C to hydrolyze all RNA. The hydrolysate was neutralized with HC1, concentrated to 0.5 ml by evaporation, and chromatographed on Sephadex G-50 in 1 mM HEPES buffer, pH 7. The excluded fractions contained the complementary DNA separated from unincorporated nucleotides.


31
VI. Characterization of the Complementary DNA
A. Size Distribution-Alkaline Sucrose Gradient Centrifu-gation
Linear alkaline sucrose gradients from 5-20% sucrose in 0.9 M NaCl, 0.1 N NaOH, and 5 mM EDTA were formed in 5 ml nitrocellulose tubes with a double well, gravity gradient former. These gradients were centrifuged at 50,000 RPM for eight hours at 20C in an SW 50.1 Beckman rotor, and were fractionated into 0.2 ml fractions on an ISCO Density Gradient Fractionator into scintillation vials. Five ml of triton-toluene scintillation fluid was added, and the samples were counted for radioactivity.
B. RNA-DNA Hybridization
The method of hybridization involved incubation of 15 pi samples containing variable amounts of RNA and 0.04 ng 3H-cDNA in sealed glass capillary tubes for various lengths of time at various temperatures in 25 mM HEPES buffer pH 7.0, 0.5 M NaCl, and 1 mM EDTA (58) with or without 50% formamide. At the end of the incubation period the extent of hybridization was determined by S^ single-strand specific deoxyribonuclease digestion with enough enzyme to degrade at least 95% of the cDNA. The S^ enzyme was available in this laboratory as prepared by the method of Vogt (96). The samples were incubated with S^ nuclease for 20 minutes in 1.8 ml of 30 mM Na acetate, 0.3 M NaCl, 1 mM ZnS04, and 5% glycerol, pH 4.6. Acid-precipitable radioactivity was determined by adding 4 ml of 15% cold TCA and collecting the precipitate on 0.45p HA nitrocellulose filters, washing


32
the filters with 50 ml of 15% TCA, and counting in cellu-solve-toluene scintillation fluid.,
Melting temperature (TM) determinations on DNA-RNA hybrids were made in the indicated hybridization buffer. Hybrids were formed to a maximum level as monitored by parallel samples and were then maintained in an ice bath. Separate sample incubations were then made at increasing temperatures for 15 minutes to allow equilibrium to occur. The samples were again placed in ice water and immediately assayed for S^ nuclease-resistant, acid-precipitable radioactivity.
VII. Utilization of the cDNA as a Probe A. Chromatin Isolation
In this study the term chromatin will be defined operationally as the nucleoprotein complex isolated from cell nuclei by a given procedure. The technique utilized in this laboratory for the isolation of chromatin follows:
The chromatin isolation procedure (83,97) was carried out at 4C. Harvested cells were washed with Earle's balanced salt solution to remove serum proteins. The cells were lysed in 80 mM NaCl, 20 mM EDTA, and 1% Triton X-100 (Sigma), pH 7.2 by vortexing. This medium caused hypotonic and detergent lysis of cells with fragmentation of the plasma membrane, the endoplasmic reticulum, and the outer nuclear membrane (83,97). Inner nuclear membrane may also have been partially or totally delipidated leaving a proteinateous lamina layer as determined for other cell types (98). Nuclei were rinsed in the cell lysis medium three times,


33
and appeared free of cytoplasmic contamination by phase-contrast microscopy. The detergent was removed from the nuclei by two washes in 0.15 M NaCl, 10 mM Tris, pH 8.0. The nuclei were drained well and lysed in cold distilled water (approximately 1 ml/2.5 x 108 cells) by incubation for 15 minutes followed by gentle homogenization or vortexing. Two volumes of water were added and the chromatin was pelleted by centrifugation at 20,000 x g for 15 minutes. The gelatinous pellet could then be resuspended in 10 mM Tris, pH 8.3.
Chromatin of HeLa S^ cells isolated by the procedure described above is composed of histones, nonhistone proteins, DNA, and RNA in approximate ratios of 1:0.8:1:0.09(8,29,99). Such chromatin as initially isolated is referred to as native chromatin because it functions similarly to the interphase nuclear material histochemically defined as chromatin. Specifically chromatin has been shown to transcribe the same specific sequences, such as globin (58,59), SV40 (60,60), and histone (62), which are expressed in the intact cell. Repetitive sequence expression has also been demonstrated to be tissue specific from such chromatin preparations (9,10). B. Chromatin Dissociation 1. DNA isolation
DNA was isolated from log phase HeLa S^ cells. A modification of the Marmur procedure (100) was used with subsequent treatment with ribonuclease, pronase, and phenol


34
(101) Cells were washed with Earle's balanced salt solution, suspended in 1.5% SDS at 60C, and shaken for 20 minutes. After cooling, the suspension was brought to 1 M NaC104 and deproteinized by repeated extractions with equal volumes of chloroform:isoamyl alcohol (24:1 v/v) by mixing for 20 minutes, centrifuging, and discarding the organic layer and interface. When interfacial material was no longer present, the aqueous phase was poured through two volumes of cold ethanol. The mixture was gently swirled and the precipitated DNA was spooled onto a glass rod.
DNA was resuspended in 0.15 M NaCl, 15 mM Na citrate, pH '7.0 (SSC). Heat-treated bovine pancreatic ribonuclease-A (Sigma) was added at a concentration of 50 mg per ml and the mixture was incubated for 30 minutes at 37C. Self-digested pronase (Calbiochem) was added to 50 mg per ml and was incubated one hour at 37C. The mixture was then extracted with SSC buffered phenol and chloroform. Extraction was repeated until interfacial material was absent. The DNA was precipitated with ethanol, resuspended in 1/10 SSC, chilled, made 0.33 M Na acetate, 10 mM EDTA, precipitated with 0.54 volumes of cold isopropyl alcohol, and resuspended and stored in SSC at 4C. 2. Protein isolation
The proteins were isolated from chromatin preparations
(102) Generally, the chromatin was derived from cells in a given stage of the cell cycle. First, the chromatin-associated proteins were separated from the DNA by adding solid NaCl and urea to 3 M and 5 M respectively to the


35
chrcniatin preparation at 4C in 10 mM Tris, pH 8.3. The mixture was shaken or vortexed vigorously to dissolve the salts and dissociate the chromatin. Such conditions have been found to release the proteins from the DNA and prevent aggregation (102,103). This mixture was centrifuged at 50,000 RPM for 48 hours in a Beckman 60 Ti rotor at 4C. The DNA was pelleted and proteins were in the supernatant, which was then dialyzed against 5 M urea, 10 mM Tris, pH 8.3 to remove the NaCl. Histones and nonhistone proteins were fractionated by a QAE-Sephadex method (102). The protein was added to QAE-Sephadex (Pharmacia) (A-50) previously equilibrated with 5 M urea, 10 mM Tris, pH 8.3 (2 gm of Sephadex per 150 ml of protein solution). A slurry was made and the histones were collected by filtration. The slurry was washed with 3 M NaCl, 5 M urea, 10 mM Tris, pH 8.3 which removed the nonhistone proteins. The proteins were then dialyzed separately against 3 M NaCl, 5 M urea, 10 mM Tris pH 8.3. To prepare for reconstitution, isolated DNA was also so dialyzed. C. Chromatin Reconstitution
DNA and appropriate histone and nonhistone protein fractions were mixed in 3 M NaCl, 5 M urea, 10 mM Tris, pH 8.3 and the NaCl was removed stepwise by gradient dialysis (103). The components were combined in a 2:2:1 histone to nonhistone to DNA ratio. Dialysis was carried out at 4C for a minimum of three hours per step against 200 volumes of 5 M urea, 10 mM Tris, pH 8.3 and 2.5, 2.0, 1.5, 1.0,


36
0.8, 0.6, 0.4, 0.2 M NaCl buffers and finally against 5 M urea, 10 mM Tris, pH 8.3 overnight, followed by centrifu-gation at 30,000 x g for 30 minutes. D. In vitro Chromatin Transcription
RNA was synthesized from chromatin preparations utilizing the procaryotic E. coli RNA polymerase fraction V isolated by the method of Berg et al. (104). The standard reaction mixture contained (105): 40 nM Tris (pH 8.0); 4 mM MgCl2; 20 yM EDTA; 0.008% 2-mercaptoethanol; 0.4 mM each of ATp, CTP, UTP, GTP: 15 yg/ml DNA as chromatin; and 60 units/ml RNA polymerase (1 unit = 1 nmole of ATP incorporation per 10 min. assay on 50 yg of calf thymus DNA). Incubation was for 70 minutes at 37C in a Dounce homogenizer with a wide-clearance pestle, and the reaction mixture was periodically homogenized to maintain the dispersion of chromatin in solution. The RNA was isolated as follows: At the end of the incubation, the reaction mixture was brought to a concentration of 1% SDS, 100 mM Na CI, 10 mM Na acetate, 1 mM EDTA, pH 5.4, incubated for 15 minutes at 37C, and was extracted twice with equal volumes of phenol with subsequent addition of chloroform: isoamyl alcohol (24:1 v/v). Two more extractions were performed with equal volumes of chloroform: isoamyl alcohol (24:1 v/v) and the nucleic acids were precipitated with two volumes of ethanol at -20C. The precipitate was collected by centrifugation and resuspended in 10 mM Tris, 0.1 M NaCl, 5 mM MgCl2 (pH 7.4) containing 40 pg/ml deoxyribonuclease I (ribonuclease free Sigma) and incubated at 37C for one


37
hour. Following one extraction with phenol:chloroform: isoamyl alcohol and two with chloroform:isoamyl alcohol, the aqueous phase containing the transcrpts was chromatographed on Sephadex G-50 fine, eluted with 50 mM Tris, 100 mM NaCl, 1 mM EDTA (pH 7.2). The RNA was monitored by UV, was found to be excluded, and was precipitated with three volumes of ethanol and stored at -20C.


CHAPTER 3 RESULTS AND DISCUSSION
I. Isolation of Histone Messenger RNA's
The main goal of this investigation was the synthesis of single-stranded DNA sequences complementary to histone messenger RNA molecules. The first required step toward this objective was the isolation of sufficient quantities of histone mRNA to characterize and to utilize as template for reverse transcription. The histone mRNA preparation was isolated from synchronized S phase HeLa cells and was fractionated by its distinct size from total polysomal RNA (described in Methods III) The final isolation procedure was derived from a number of published methods by analyzing various parameters with respect to histone mRNA yield. A. Cell Synchrony
S phase HeLa S^ cells were obtained for histone mRNA isolation by treatment of exponentially growing cells with a high dose (2 mM) of thymidine (81), a reversible metabolic block, which inhibits DNA synthesis by reducing cytidine synthetase activity, thus interrupting precursor biosynthesis. The cells are blocked in S phase and at the Gjl~S boundary by this treatment, and one thymidine block results in an enrichment of S phase cells (28) with the remainder of the cells in G2 phase. This treatment does
38


39
not affect the viability of HeLa cells as determined by
plating efficiency and colony formation (106). Cell
synchrony was monitored by the pulse label assay as described
in Methods (IID) (Fig. 2) The results indicated that the
cells were synthesizing DNA during the period immediately
following release from the block; however, incorporation 14
of C-thymidine during the first few hours following release from thymidine block did not accurately reflect the extent of DNA synthesis, since re-equilibration of the thymidine pool was occurring at this time. The decline of ^^C-thymidine incorporation starting at five hours following release reflected the exit of cells from S phase into G2-These results verified the synchrony of the cells and supported the rationale for choosing to release the cells for three hours from the single thymidine block prior to harvesting S phase cells. B. RNA Fractionation
The RNA containing the histone mRNAfe were separated from polysomal RNA of S phase cells by sucrose gradient fractionation as described in Methods (III) In the preparation of polysomal RNA, 170 mg of RNA were obtained and were fractionated on two zonal gradients. The resulting optical density profile is shown in Figure 3. The indicated fractions (A, B, C, D) were pooled and precipitated with two volumes of ethanol at -20C. The 18S peak appeared at the bottom of the 5-30% gradient and the 28S RNA was contained in the bottom 1/3 of the 30% sucrose cushion. Parameters were chosen for maximum separation between 4S and 18S RNA's,


40
5r
01 23456789 10 Time after release (hours)
Figure 2. Incorporation of 14C-thymidine for 30 minutes into HeLa cells released from a single 2 mM thymidine block for the indicated times.


1.0i
Fraction
Figure 3. Optical density profile of polysomal RNA fractionated by zonal linear sucrose gradient centrifugation as described in Methods (III) Sedimentation values of 28S, 18S, 5S, and 4S were assigned to the major polyribosomal peaks in conjunction with the known sedimentation values of the principal RNA species associated with eucaryotic ribosomes.


Optimal resolution for this amount of material required two gradient runs, each with half the total RNA.
In the 4-18S fraction, 5-6 mg of RNA were recovered, and the RNA was further fractionated on 5-30% linear sucrose gradients in a Beckman SW 27 Rotor as described in Methods (III). An optical density profile of such a gradient is shown in Figure 4. The indicated fractions (A, B, C, D) were precipitated with two volumes of ethanol at -20C. The 7-12S RNA, fraction C, contained the histone mRNA's, and a yield of 5 00 yg was obtained.
During development of the method for the isolation of RNA containing histone mRNA's as described in Methods (III) several isolation parameters were tested. The first parameter investigated was the RNA extraction procedure. Cells were hypotonically lysed and polysomes were pelleted by centri-fugation from a post-mitochondrial supernatant. Two procedures were compared. One involved a pH 7.3 buffer containing napthalene disulfonic acid as a ribonuclease inhibitor, and buffered phenol at room temperature as the organic extraction agent (32) The second procedure utilized a Tris buffer, pH 7.4, to dissolve the pelleted polysomes, and the solution was then adjusted to pH 5.5 with Na acetate to reduce ribonuclease activity. In this case, a chloroform:isoamyl alcohol mixture served as the organic extraction agent (28). Only the latter procedure yielded a detectable amount of 7-12S RNA,but the recovery was less than 10% of that indicated by the originators of the method.


43
2.0 r
in
CM
<
1.0
0
Figure 4. Optical density profile of 4-18S polysomal RNA fractionated in a 5-30% linear sucrose gradient in a Beckman SW 27 rotor as described in Methods (III) Sedimentation values of 18S and 5S were assigned to the chief ribosomal RNA species in this fraction. S values for other RNA species were extrapolated assuming a linear relationship between S values and rate of sedimentation in linear sucrose gradients.


44
Another parameter investigated was the cell lysis procedure. Instead of hypotonic lysis, the detergent lysis method used to isolate nuclei for chromatin was applied. Both pelleted polysomes and crude cytoplasm were extracted by the two methods described in the previous paragraph. Even though SDS was added as soon as possible after lysis in an attempt to reduce ribonuclease activity, detergent cell lysis was found to be inadequate in each case. This was probably because non-ionic detergents such as Triton X-100 used in the nuclear isolation procedure disperse membranes, including those of lysosomes which in particular are known to contain a large amount of ribonuclease activity.
A last variation of the method for specific RNA isolation tested was polysomal fractionation on sucrose density gradients. This technique was used by others to show that the small polysomes are enriched in S phase cells and contain the histone mRNA's (25,27,28,30,40). Cells were lysed hypotonically and the ionic detergents Na deoxycholate and Brij-58 were added to final concentrations of 0.5% each to the post-mitochondrial supernatant to dissociate polysomes from membranes. This mixture was layered on 15 to 30% sucrose gradients which were centrifuged at 26,000 RPM for two hours (180,000 g x hrs.) in a SW 27 rotor, and fractionated. Ribosome subunits, monosomes, and a distribution of polysomal size classes were separated on these gradients and three regions were pooled; monosomes, small polysomes, and large polysomes (Figure 5). The three fractions were then


45
i
Figure 5. Optical density profile of polysomes from 1.5 x 108 S phase HeLa cells prepared and fractionated on sucrose gradients as described in Results (I B). Inserts (a,b) are from gradient preparations analyzing smaller amounts of material and reflect increased resolution.


46
precipitated with two volumes of ethanol and stored at -20C for several hours. While investigating fractionated polysomal preparations, an improved organic extraction procedure for nucleic acid isolation was applied involving a mixture of phenol and chloroform:isoamyl alcohol (107). Phenol was used because it is a more efficient protein denaturant than chloroform, but chloroform adds density to the organic layer and reduces aqueous partitioning in phenol. Isoamyl alcohol was used because it reduces foaming due to the SDS when the mixture is vigorously agitated. This method has been discussed recently (76) With the use of this organic extraction mixture and pH 5.4 Na acetate buffer, 80 yg of 7-12S material were isolated with the small polysome fraction on subsequent SW 27 5-30% sucrose gradients. This yield from the small polysome preparation was less than that previously reported (28). Hence, the procedure was not considered adequate.
It was difficult to assess all the multiple parameters of RNA isolation independently, but the combination of hypotonic lysis, polysome pelleting, low pH buffer, and the mixed organic extraction medium appeared to be the most efficient for isolation of the 7-12S RNA fraction. The polysomal fractionation procedure was eliminated because histone messenger RNA was of a distinct size and was readily isolated by sucrose gradient fractionation of total polysomal RNA. These preliminary studies formed the basis for the simpler and more direct procedure for isolation of 7-12S RNA1s described in Methods (III) .


47
II. Characterization of Histone Messenger RNA The 7-12S RNA isolated as described was monitored using the characteristics expected of histone mRNA as criteria. Determinations were made of size, nucleotide composition, translational ability in a cell-free system, and poly A content of the 7-12S RNA. Although these criteria are inadequate to quantitatively evaluate the purity of the histone mRNA preparation, gross deviation from expected values would have shed doubt upon the identity of the 7-12S RNA. Later hybridization experiments with the histone complementary DNA product were conducted to provide more conclusive information on its compositional nature. A. Size Distribution
SDS polyacrylamide gel electrophoretic RNA size determination was carried out at stages of the polysomal RNA fractionation (Figs. 6-8). The absorbance profiles of the polysomal RNA at three principal stages of the preparation showed a progressive enrichment and purification of the 7-12S RNA species. The resultant 7-12S RNA1s appeared to be composed of at least four distinct components. Predicated on the molecular weight of the individual histone proteins, it is reasonable to anticipate that the 7-12S RNA fractions may represent the mRNA's for the individual histones. The detection of distinct bands instead of a random distribution of material suggested that extensive degradation had not occurred. The 14S material, which was very pronounced in the 4-18S fraction, has been empirically


48
28S
h (?)
Figure 6. SDS polyacrylamide gel electrophoretic absorbance profile of A. total polysomal RNA before sucrose gradient fractionation, and B. blank gel. S values for electrophoretic mobility were assigned to the major ribosomal RNA species fractionated and identified in sucrose gradients. S values for other RNA species were extrapolated assuming a logarithmic relationship between S values and electrophoretic mobility in this gel system.


49
28S
O
CD C\J
Figure 7. SDS polyacrylaraide gel electrophoretic absorbance profiles of zonal sucrose gradient fractionated polysomal RNA's pooled as indicated in Figure 3. A. 28S, B. 18S, C- 4-18S, D. 4,5S. S values were assigned as in Figure 6.


Figure 8. SDS Polyacrylamide gel electrophoretic absorbance profiles of 4-18S RNA's fractionated on linear sucrose gradients in a Beckman SW 27 rotor and pooled as indicated in Figure 4. A. 18S, B. 14S, C. 7-12S, D. 4,5S. S values were assigned as in Figure 6.


51
identified to consist, at least in part, of a degradation product of 18S rRNA. This contention was based on the observation that prolonged storage of 18S rRNA results in accumulation of the 14S product and does not yield additional lower molecular weight RNA fractions. The 14S RNA species caused concern, but the final 7-12S RNA preparation contained none of this material. Furthermore, hybridization analysis with the histone cDNA to rRNA (to be discussed) also indicated the absence of rRNA breakdown products in the 7-12S RNA preparation. B. Base Composition
There has been only one report of the analytical base composition of the histone mRNA's (28): 31.4% guanosine, 26.2% adenosine, 22.4% cytidine, and 21.0% uridine. Neither the purity nor the specific characteristics of this RNA preparation were reported, but their analysis provided a basis for comparing similarly isolated RNA1s associated with polyribosomes during the S phase of the cell cycle which lack poly A. Additionally, a theoretical base composition for histone mRNA's has been predicted from the amino acid compositions of the histones (45). Thus, another useful criterion applied to the 7-12S RNA was its base composition. 1. Paper electrophoresis
Two chromatographic methods were in current use. One method involved ion exchange chromatography (10 8), and the other method involved high voltage paper electrophoresis (88). Because a quick semimicro method was desired, and paper


52
electrophoresis had already been employed for determining the nucleotide base composition of the histone mRNA's, this method was attempted. The separated nucleotides were eluted in concentrated form and quantitated by appropriate absorbance measurements, but the small amount of RNA utilized resulted in low precision. The use of PO^-labelled RNA's as commonly reported yields a more consistent and sensitive analysis (28,88-90,109). However, 32P04~labelling was not attempted since the 7-12S RNA's were preparatively isolated from 30-40 liters of HeLa cell culture and the cost of PO4 needed to fully label the RNA's during such a large scale synchronization was prohibitive. Highly labelled RNA's would also not be as stable as nonradioactive RNA's. Labelling the RNA's with 32PO^ was, therefore, impractical for the extended analysis and application carried out with the 7-12S RNA's. Tables 1 and 2 summarize the results of nucleotide base composition analysis of 18S and the 7-12S RNA's respectively. Nucleotides were fractionated by the paper electrophoresis procedure described in Methods (IV Bl).
The variability noted is a common problem in this technique. Elution and recovery of particle-free eluate in concentrated form from paper was not reproducible. Total recovery of estimated applied material was not obtained and recoveries were variable. A method with more efficient recovery was therefore sought.


53
Table 1
Base Composition of 18S RNA High Voltage Paper Electrophoretic Fractionation
Trials Base X% (a) Reported (109)
3 U 22 (3.7) 23
G 28 (1.8) 26
A 22 (2.4) 23
C 28 (1.4) 28
Table 2
Base Composition of 7-12S RNA1 s High Voltage
Paper Electrophoretic Fractionation
Trials Base X% (g) Reported (28)
3 U 18 (4.3) 21
G 21 (5.8) 31.4
A 29 (3.0) 26.2
C 32 (8.8) 22.4
Table 3
Base Composition of 18S RNA Thin Layer Electrophoretic Analysis
Trials Base X% (a) Reported
8 U 22 (1.3) 23
G 32 (4.5) 26
A 18 (2.2) 23
C 28 (4.2) 28
Table 4
Base Composition of 7-12S RNA's Thin Layer
Electrophoretic Analysis
Trials Base X% (a) Reported
3 U 25 (1.2) 21
G 24 (2.1) 31.4
A 20 (0.6) 26.2
C 31 (3.1) 22.4


54
2. Thin layer electrophoresis
Another method utilized for separation of nucleotides was high voltage thin layer electrophoresis. Standard cellulose thin layer glass plates were used as the inert matrix and the same buffer system as in paper electrophoresis was utilized. The method represented a more effective extraction system because the spots were concentrated and could be extracted in fine particulate form. The procedure is described in Methods (IV B2) and the nucleotide base compositions of 18S and 7-12S RNA's, determined by this technique, are presented in Tables 3 and 4.
Once again, wide variability was seen in composition as well as recovery. The same 18S and 7-12S RNA samples were utilized in paper electrophoresis and thin layer electrophoresis. In comparing the average figures for the two types of analysis, relatively consistent data were obtained for the 18S RNA fraction in terms of G and C being greater than A and U. This is the distinctive pattern for ribosomal RNA's (109). On the other hand, while the two techniques were less consistent for the 7-12S RNA's, the relative amounts of C and G compared to A and U were similar. Discrepancies with the reported values could not be accounted for; however, standard deviations for the one published nucleotide base composition analysis of histone mRNA's had not been reported. It should be noted that in all analyses the base composition of the 7-12S RNA's differed from that of ribosomal RNA.


55
C. Cell-Free Translation
Cell-free protein synthesizing systems provided the opportunity to examine the ability of RNA fractions to direct the synthesis of specific polypeptides. One characteristic of polypeptides synthesized in such systems is size. In some instances the product may also be detected specifically by antibody reactivity. In the case of histone mRNA's, effective antibodies were not easily obtainable due to the interspecies relatedness of histone polypeptides. Reports on antibodies to histones (110,111) have been considered equivocal, since the antigens were histone-nucleic acid complexes or histone-albumin complexes, and total specificity was not demonstrated. For these reasons and due to problems of nonspecific trapping and cross reaction, immunoprecipitation of histones synthesized in vitro or histone mRNA isolation by polysome precipitation were not attempted. It should also be noted that the use of histone antibodies for anlyzing in vitro translation products would preclude detecting proteins other than histones which might be templated by the 7-12S RNA's. However, the histone proteins were readily identified with respect to size and charge by acetic acid-urea polyacrylamide gel electrophoresis, and electrophoretic analysis allowed the determination of other synthetic activity. Preparations of histone mRNA from HeLa cells as well as from developing sea urchins had been examined utilizing the established systems of rabbit reticulocyte (31) as well as Krebs Ascites tumor cell extracts


56
(32,33). Thus, it was considered worthwhile and realistic to pursue in vitro cell-free translation as a fundamental part of the analysis of the 7-12S RNA preparation. Such analysis would indicate only whether histone mRNA sequences were contained in the isolated RNA, and would demonstrate the presence of other mRNA sequences if substantially different from those of histone. Of course, the possibility arises that RNA's other than those which template histones may be present in the 7-12S RNA fraction, but if nontranslatable in the cell-free system their presence would not be detected. Because the 7-12S RNA's were isolated by size and would be expected to produce polypeptides of a limited size class, fractionation of the translation products solely on the basis of molecular weight could not be used as a definitive criterion for the presence of histone mRNA in the 7-12S RNA preparation. Success of the method was dependent on producing an electrophoretic pattern characteristic of the histone proteins in a system whose resolution is based on charge as well as size. Despite the limitations, valuable information was obtained about the qualitative nature of the isolated RNA.
The cell-free system applied to the translation of the 7-12S histone mRNA preparation was derived from unprocessed wheat germ. The reticulocyte system was initially attempted but was not employed further due to high levels of endogenous globin synthesis. The Krebs II Ascites tumor cell extract was not pursued due to problems concerning the procurement


and maintenance of tumor-bearing animals and other inherent
problems concerning preincubation requirement (112,113) and
ribonuclease activity (114). The newly developed wheat germ
cell-free protein synthesizing system had been proven
superior in ease of preparation and in having lower endogenous
activity than the Krebs II Ascites system (114). A specific
brand of wheat germ was obtained commercially, and lysate was
prepared and was tested by the procedures described in
Methods (IV CI).
In characterizing endogenous translational activity,
incorporation of JH-labelled amino acids as a function of
time, as well as polyacrylamide gel electrophoretic analysis
was carried out under conditions anticipated to be appropriate
for histone synthesis with the three amino acids arg, lys,
3
and leu used as H-labelled precursors. There was some incorporating activity by the extract with no added mRNA under these conditions as demonstrated by TCA precipitable radioactivity and by gel electrophoresis where many discrete radioactivity peaks were seen (Fig. 9). Precipitable radioactivity was not due to aminoacylated tRNA's, since hot TCA deacylates these RNA species. To resolve this problem of high endogenous activity, preincubation of the wheat germ extract prior to addition of mRNA as well as sample dialysis was attempted. Dialysis of the reaction mixture after incubation and prior to electrophoresis was seen to remove over 90% of the radioactivity, yet similar peaks of incorporation on gels as without dialysis were observed. Preincubation also did not eliminate the observed synthesis. It was con-


Fraction
Figure 9. (-) Acetic acid-urea polyacrylamide gel electrophoretic absorbance profile
of stained wheat germ proteins plus standard calf thymus histones (Sigma) as indicated.
(---) Endogenous (no mRNA added) radioactivity profile of wheat term extract in the
presence of 3H-lys, and ^H-leu. (......) Endogenous radioactivity profile of wheat
germ extract in the presence of 3pj-leu only.


59
sidered that an excessive amount of H-labelled amino acid precursor was being used. By carrying out the reaction with just 3H-leucine or 3H-lysine present, a substantial decrease in activity was seen (Fig. 9), and further analysis with 3H-arg indicated that the arginine label was solely responsible for the incorporating activity. It was unknown what arginine compound(s) were being synthesized. Polyarginine seemed unlikely due to its complex genetic codes. Perhaps arginine was forming protein adducts of some sort in the reaction mixture, but this is only speculation. The 3H-arg utilized was 97% pure at the time of purchase as specified by the supplier (New England Nuclear) and was not further tested.
Conditions for minimal background with the wheat germ system were found to include the use of only 3H-leucine, or 3H-lysine, or both. Several hundredfold stimulation over background by several exogenous RNA's was purported to occur in this system (92,114), but this was not seen in early attempts with histone mRNA in the presence of 3H-arginine. Later experiments with crude globin mRNA and the 7-12S RNA demonstrated 40- and 10-fold stimulation, respectively. Although 15 yg of globin mRNA elicited fivefold higher stimulation than 20-25 yg of 7-12S RNA, others have reported less stimulation with histone mRNA than with globin mRNA in both the Ascites and reconstituted cell-free systems (30, 93,98,103,104,89-91). Figure 9 demonstrates typical patterns for the arginine label and leucine label endogenous backgrounds, and the optical density profile of stained protein bands with this system plus marker histones. Figure 10 shows the 3n-leucine incorporation pattern stimulated by two


Figure 10. Acetic acid urea polyacrylamide gel electrophoretic fractionation in the presence of standard histone markers (indicated by arrows) of 3H-leu labeled in vitro products from 25 yg 7-12S RNA of S phase HeLa cells in the cell-free translatlonal
system derived from wheat germ. (.....) Endogenous (no mRNA added) radioactivity
profile. () Trial 10a, with added 7-12SHistone mRNA preparation and no endogenous background subtracted. Trial 10b (insert), with added 7-12S RNA distinct from trial a where only the histone region was examined and 3H-leu endogenous background illustrated in Figure 9 was subtracted.




62
separate 7-12S histone mRNA preparations. The pattern observed in trial 10a demonstrated a definite stimulation in the region corresponding to histones F^, F2b' F2a2 as resolved on this particular gel, and it should be noted that the low endogenous activity toward the upper region of the gel (fractions 15-40) was actually diminished in the presence of the 7-12S RNA. In trial 10b, where only the histone region was examined and background was subtracted, resolution of stimulated activity revealed a pattern consistent with the presence of the five histone species. No other reproducible incorporation over background was found, and any activity within the histone region could not be distinguished from that of histone. The results are in agreement with those of others for the cell-free translation of histone mRNA preparations, where relatively low and &2al activities and low resolution of the more highly stimulated intermediate histones are often found (26,27, 31-33,44,115). The qualitative pattern characteristic of histone polypeptides obtained gave positive functional evidence for the isolation of the histone mRNA's within the 7-12S size class, but could not be used to specify the purity of the preparation. The possibility cannot be eliminated that other RNA's may be present in the preparation which are not translatable or are not of sufficient concentration to stimulate detectable incorporation. The observed stimulation of incorporated activity distinctly comigrating with standard histone, therefore, suggests that the histone mRNA's represent a major proportion of the 7-12S preparation.


63
D. Poly A Affinity Binding
Advantage of another reported property was taken with respect to the further analysis and purification of the 7-12S RNA for histone specific sequences. It has been indicated that this RNA species does not contain extensive poly A sequences at the 3'0H end (28). Utilizing methods which have been developed to specifically bind poly A sequences, the 7-12S RNA preparation was analyzed for poly A content. Poly A analysis techniques of nitrocellulose filter binding (94) and oligo (dT)-cellulose affinity chromatography (12) were employed. The effectiveness of poly A removal was determined by oligo dT dependent reverse transcriptase activity analysis (described in Methods VB) of unbound material compared to untreated 7-12S RNA. RNA1s not bound to these supports were reanalyzed by size and translational ability to determine if changes occurred in the histone messenger RNA content. Retention of poly A containing material by nitrocellulose filters or oligo (dTJ-cellulose would suggest the presence of RNA sequences other than those which code for histones in the 7-12S RNA preparation. The poly A affinity method further served as a means to eliminate such poly A containing RNA species from the 7-12S RNA1s thus functioning as a purification step in the isolation of histone mRNA's. 1. Nitrocellulose fi1tration
Nitrocellulose filtration, utilizing Millipore HA 0.45p nitrocellulose filters as described in methods (94)(IV Dl), was the first procedure employed for selective binding of


64
poly A containing RNA's in the 7-12S RNA preparation. As an initial evaluation of the filter binding technique and to determine the poly A binding capacity of the nitrocellulose filters, a pure poly A (Schwarze-Mann) sample was tested, along with RNA's which contain minimal poly A, HeLa 4S and 28S RNA's. These nonradioactive nucleic acid samples were applied in 100 yg/10 ml solutions. No binding of tRNA or rRNA to the nitrocellulose filters was observed. In contrast, poly A was bound completely and upon elution from the filters in the SDS buffer 100% of the poly A applied was recovered. Quantitation of nucleic acids was carried out spectrophotometrically. The capacity of nitrocellulose filters for binding poly A was, therefore, considered sufficient to completely retain any poly A containing material present in 100 yg of the 7-12S RNA preparation.
The nitrocellulose filter binding technique was then applied to the isolated 7-12S RNA's. A known amount of RNA was applied to the nitrocellulose filters and the filtrate fractions were quantitated by optical density. The results are summarized in Table 5. Precise measurements of 7-12S RNA recovery from nitrocellulose filters were not obtained. Erratic recoveries may in part be attributable to problems encountered with (a) potassium dodecyl sulfate precipitation in the eluants containing filter bound material or (b) the quantitation of low amounts of RNA. The recoveries of RNA's in the filtrate fractions indicated that 0 to perhaps 25% of the 7-12S RNA's contained sufficient poly A to bind to the nitrocellulose filter under these conditions. In trials 1 and 2, where lower amounts (46.5 yg and 60 ug) of RNA were


65
TABLE 5
Binding of 7-12S RNA's to Nitrocellulose Filters
Trial 1 2 3
Initial RNA 60_ pg 46.5 pg 100 pg
Filtrate 63 43 75
Bound 7.5 7 20
The procedure for nitrocellulose filtration is described in Methods IV Dl. Non-poly A containing RNA's are eluted with 0.5 M KC1, 1 mM MgCl9, 10 mM Tris, pH 7.6, and are quantitated in the filtrate.
TABLE 6
Oligo (dT)-Cellulose Affinity Chromatography of Poly A
Trial 12 3
Column size 0.2 ml 0.5 ml 0.5 ml
Sample volume 0.5 1.0 1.0
A260 units:
Initial 1.26 0.95 0.97
KC1 Salt Wash 1.27 0.611 0.72 @ 4 C #3 only
Tris Wash 0.118 0.334 0.16 @ RT
(pg bound) (6) (17) (8)
The procedure for chromatography on oligo (dT)-cellulose described in Methods IV D2. Non-poly-A containing RNA's and poly A are eluted in 0.5 M KC1, 10 mM Tris, pH 7.5 (KC1 Salt Wash). Bound poly A is eluted with 10 mM Tris (Tris Wash).
TABLE 7
Oligo (dT)-Cellulose Affinity Chromatography of 7-12S RNA
Trial 1 2a 2b
Initial RNA 82.0 pg 84.0 pg 56.4 pg
KC1 Salt Wash 79.6 72.0 44.0
Tris Wash 2.4 3.4 2.5
The procedure is described in Methods IV D2. Binding is explained in Table 6. Trial 1 was carried out with one isolated RNA sample, and trials 2a,b were carried out with another isolated 7-12S RNA sample .


applied, less than 10% of the RNA's were retained by the nitrocellulose filters. The higher extent of RNA binding observed when 100 yg of 7-12S RNA was applied (trial 3) was based on a single determination. The limited amount of poly A containing material in the 7-12S RNA preparation was further substantiated by the other affinity method to be discussed.
Further analyses of the 7-12S RNA's which were not retained by nitrocellulose filters in trials 1 and 2 were performed. Reverse transcription assays (discussed in Section III A) showed that the nitrocellulose filter purification of the 7-12S RNA preparation resulted in a sixfold reduction in oligo @T)-stimulated RNA-dependent DNA synthesis. Reverse transcriptase activity provided a stringent measurement of the effectiveness of this method. 2 Oligo (dT)-cellulose chromatograpy
The second method employed for selective binding of poly A containing RNA's in the 7-12S RNA preparation was chromatography on oligo dT attached to a cellulose support (12). The capacity of oligo (dT)-cellulose to bind pure poly A was determined prior to utilizing the immobilized oligo dT for selective removal of ply A containing RNA's from cellular RNA fractions. Trials were made with various size columns and with approximately 1.0 absorbance unit of poly A in different volumes. Table 6 summarizes the results. When 28S RNA (100 yg (2.0 A2gg) per ml) was chromatographed on oligo (dT)-cellulose, 100% of the material was recovered in the KC1 salt wash. The conclusion drawn from these results


6 7
was that oligo (do)-cellulose bound a proportion of poly A, but only up to 40% of the reported capacity (85 pg poly A/ml, P-L Biochemicals). The discrepancy between the quantitation of the oligo dT binding capacity and that of the supplier may be attributable to differences in the conditions employed for determination of poly A binding or variations in the poly A, or both. However, the observed binding of poly A by oligo (dT)-cellulose demonstrated that this method, which has more defined affinity properties than nitrocellulose filters, would serve as another device for analyzing poly A content of the 7-12S RNA preparation.
Analysis of the 7-12S RNA was carried out under sterile conditions at room temperature with a 0.5 ml packed column of oligo (dT)-cellulose. The following results were obtained from three trials with two different preparations of 7-12S RNA (Table 7). By this procedure it appeared that less than 5% of the 7-12S material was bound to oligo (dT)-cellulose. From the first trial it appeared that total recovery of RNA was possible; however, in trials 2a and 2b an apparent loss in recovery of RNA was observed. The yield of bound material was consistent in all trials; therefore, the error probably arose from combining and quantitating only peak fractions of KC1' eluted RNA in trials 2a and 2b. The binding of 7-12S RNA's to oligo (d1)-cellulose was less than the binding of these RNA's to nitrocellulose filters, suggesting that in this case oligo (3T)-cellulose chromatography was not as effective as nitrocellulose filtration for removal of


68
polyadenylated RNA's from the 7-12S RNA preparation. This contention is further supported by comparison of reverse transcriptase activities of 7-12S RNA's following nitrocellulose filtration and oligo (3T)-cellulose chromatography. In contrast to the sixfold reduction in oligo GD-stimulated reverse transcriptase activity observed in the 7-12S RNA's following nitrocellulose filtration, oligo GThcellulose chromatography resulted in only a twofold reduction in reverse transcriptase activity when assayed under identical conditions. The 7-12S RNA's were also analyzed for size and translational properties prior to and following removal of poly A containing material by oligo @T)-cellulose chromatography. Both analyses indicated that the 7-12S RNA's were similar to the original isolated material.
In summary, procedures for detection and removal of poly A containing RNA species were shown to be functionally operative to a limited extent with the isolated 7-12S RNA. The relative amount of 7-12S RNA's selectively removed by nitrocellulose filtration and oligo 0T)-cellulose chromatography were somewhat inconsistent. However, the small percentage of the 7-12S RNA's which bound to the nitrocellulose and oligo fiT)-cellulose supports was probably responsible for the slight stimulation of oligo dT dependent reverse transcriptase activity characteristic of 7-12S RNA prior to removal of poly A containing material. Since no major change was detected in size or translational properties of the 7-12S RNA's following oligo dTrcellulose chromatography, it appeared


69
that only a small quantitative purification was achieved, but that a poly A containing contaminant which would ultimately have yielded cDNA products may have been eliminated. Furthermore, the importance of this procedure for removal of poly A containing RNA has been recently demonstrated because RNA coding for tryptophan containing polypeptides was eliminated from a histone mRNA preparation by such a technique (116).
Not only was it useful to monitor the 7-12S RNA preparation for properties of histone mRNA, but additionally it was important to test for contaminating species in characterizing the preparation. Past translational (32,33) and more recent electrophoretic evidence (35,44) (described in the Introduction) has suggested thatthe major 7-12S RNA found in S phase cells is probably mRNA coding for histone polypeptides. Thus, there probably would not have been a sufficient contribution of other 7-12S RNA species to interfere with hybridization experiments due to the low individual concentration of other RNA's. Degradation products of larger mRNA's also would probably not have contributed in individual concentrations sufficient to interfere. Furthermore, if sequences of large mRNA's were present, a considerable amount of poly A containing fragments would have been detected by the nitrocellulose filter and oligo (dT>-cellulose affinity methods described. The chief source of possible contamination would probably have arisen from degradation products of large RNA's, principally ribosomal. Thus, it was necessary to demonstrate the absence


70
of rRNA sequences in the 7-12S RNA preparation. There has been evidence that rRNA is not an efficient template for reverse transcriptase (55,70,75). This is probably due to the extensive amount of secondary structure in these RNA's and may also be based upon their lack of poly A. Upon degradation and polyadenylation, however, ribosomal RNA fragments could be rendered effective templates for reverse transcriptase. Detecting rRNA sequences among the 7-12S RNA's would have been difficult. Ribosomal RNA's and histone mRNA's are G-C rich, 5 8 and 54% respectively; hence, unless there were extensive contamination of rRNA's, such contaminating species would be difficult to detect by nucleotide composition analysis. Recognition of contaminating sequence from larger RNA's could theoretically be accomplished by complex competition hybridization techniques, e.g., 18S and larger RNA's in excess could be used to compete with 7-12S RNA for hybridization to denatured cellular DNA. Highly labelled cellular RNA would have been required and a considerable amount of 7-12S RNA would have been used in such competition hybridization experiments. This type of hybridization analysis also requires consideration of gene frequencies making interpretation more difficult. A more straightforward assessment of the possible presence of ribosomal RNA sequences in the 7-12S RNA preparation can be made by testing the abilities of ribosomal RNA's to hybridize to a DNA probe complementary to the 7-12S RNA's. This latter approach was pursued rather than the competitive hybridization method and will be discussed in section IV D4.


71
Characterization of the 7-12S RNA's isolated from the polysomes of S phase HeLa cells by the methods described above substantiated the presence of histone mRNA's in this fraction. Size distribution, general base composition, and poly A content revealed characteristics which were consistent with the presence of histone mRNA as a major component of the isolated 7-12S RNA's. The translational properties further substantiated this conclusion. Although the results do not conclusively establish the absolute purity of the histone mRNA preparation, the evidence supports the presence of histone mRNA's as major components of the 7-12S RNA fraction. The 7-12S RNA fraction was utilized to pursue the primary goal of histone cDNA synthesis.
III. Synthesis of Complementary DNA Lysates of Rous sarcoma virus were utilized as the source of RNA-dependent DNA polymerase for the synthesis of histone cDNA. Initially, the utilization of viral endogenous RNA's as templates from DNA synthesis by the enzyme were compared with utilization of exogenous 7-12S HeLa cell RNA's, both before and after removing poly A containing material. Evaluation of endogenous cDNA synthesis by the viral lysate was important in determining the potential contribution of viral sequences to a histone cDNA probe. However, viral (RSV) DNA sequences would not interfere with experimental hybridizations, since viral RNA's would not be encountered in analyses of HeLa cell derived RNA's. Another property of the synthesis directed by the enzyme was its dpendance on dNTP's, which demonstrated that the incorporation of


labelled dNTP's represented DNA synthesis coded by a template. Dependence on all four dNTP1s for enzyme activity was established by an increased incorporation of a given labelled dNTP in the presence versus the absence of the other dNTP's. On a poly (rA)oligo (dT) template no dependence on dATP, dCTP or dGTP was expected, but on mRNA templates, the dependence on these dNTP's should have been found. Reverse transcriptase activity in the presence of actinomycin was monitored to evaluate the effect of the antibiotic on double-strand DNA synthesis, which has been reported to be inhibited by this drug (64). Dependence on exogenous oligo dT primer for increased activity was monitored to indicate that poly A containing RNA's were being transcribed by the RNA-dependent DNA polymerase. It was expected that the isolated histone mRNA and especially the poly A (-) preparations would have minimal template ability for transcription by RNA-dependent DNA polymerase, since a defined priming system was lacking. These assays for endogenous activity, dependence on dNTP's, the effect of AMD, and oligo dT dependence provided valuable information pertaining to the properties and requirements of the reverse transcriptase preparation. The 7-12S RNA's lack poly A at their 3'0H termini and as such are ineffective for oligo dT primed reverse transcription of histone cDNA. AMP residues were therefore enzymatically added to the 3'0H termini of the 7-12S RNA's and these polyadenylated molecules were utilzied as templates for the preparation of histone cDNA.


A. Assay for Reverse Transcriptase
1. Endogenous activity
Endogenous activity levels of the reverse transcriptase
preparation (endogenous activity being defined as synthesis
without exogenous RNA added) were consistently low, with
variations within the error limits of the filter background.
Filter background is defined as 3H-dNTP absorbed to the 3MM
filter in a complete reaction but without viral lysate or no
enzyme. This value was subtracted in all experiments for
the particular 3H-dNTP utilized. After subtracting a 500
3
cpm background per filter for H-TTP, the average endogenous
3
activity was 492 cpm H-TMP incorporated with the other
dNTP's present, and 223 cpm without the other dNTP's,
3
reflecting expected dNTP dependence. This level of H-TMP
incorporation was observed without added primer oligo cITtq.
With oligo dT-j_o present, the activity increased to 1230 cpm
3H -TMP which indicated that there may be poly A or A rich
regions in the RSV preparation. Actinomycin D, under
optimum conditions with oligo dT and in the presence of all
dNTP's did not appreciably alter H-TMP incorporation
indicating that perhaps little double-stranded DNA synthesis
occurred in the absence of the drug. Alternatively, the
3
similar levels of H-TMP incorporation observed in the presence and absence of actinomycin D may reflect the same amount of DNA synthesis under both conditions with shorter, partially double-stranded DNA synthesized in the absence of AMD and longer single-stranded DNA synthesized in the


presence of the drug. In characterizing endogenous activity 3 3
further, H-dCMP and H-dGMP incorporation was measured. In the presence of dT-^Q / incorporation of 3H-dCMP and 3H-dGMP was approximately twofold greater than that of TMP reflecting a mRNA-like base composition. Thus, it was found that endogenous activity was low, was dependent on dNTP's, was stimulated by oligo dT-^g, was unaffected by actinomycin D, and reflected a mRNA-like base composition by 3H-dCMP and ^H-dGMP incorporation.
Exogenous rAdT was used as a template to monitor enzyme activity. A dependence on dNTPs was not seen with this template. In fact, higher activity was detected without dNTP's which was probably due to a lack of competition for
nucleotides. With 0.5 yg of rAdT, an average of 30,000 cpm
3 ...
JH-TMP were incorporated, and with increasing amounts of
tempate additional synthesis was observed; therefore, under
these conditions, enzyme excess was realized. Incorporation
of 3H-dCMP and 3H-dGMP was found to be less than 1000 cpm,
which was within the limits for the background of this label
combination. The rAdT template has been used as a basis
for unit activity by others (57,70,75,117). Unfortunately
not enough information on the amount of rAdT or the
polyAroligo dT ratio is available in the literature to
establish specific activity units under these conditions.
2. Isolated and poly A (-) histone mRNA template activity
It was pertinent to quantitatively evaluate the
utilization of HeLa cell 7-12S RNA's as template by reverse


75
transcriptase. Because some oligo dT^Q dependent endogenous activity was found with the reverse transcriptase preparation, it was necessary to test the dT^Q dependent activity of the 7-12S histone mRNA preparation in a similar manner. As alluded to earlier, this led to a sensitive test for effectiveness of the nitrocellulose and oligo @T)-cellulose affinity supports for removal of poly A containing RNA sequences. Thus the histone mRNA after passage over nitrocellulose or oligo (dT>cellulose was also analyzed for dT^Q primed activity levels. Dependence on dNTP as well as effects of actinomycin D were monitored. Table 8 summarizes the data on the analytical assays of the 7-12S histone mRNA preparations. The isolated 7-12S RNA fraction showed a dependence on oligo dT-^Q for an increased reverse transcriptase activity, and in the absence of dT^Q, an endogenous level of activity was found. In general, the poly A(-) 7-12S RNA had a much lower activity than the untreated RNA, and it is apparent that nitrocellulose treatment was more effective than oligo fciTj-cellulose in removing the oligo dT primed activity. Dependence on dNTP's for activity was not seen in the limited cases examined; but this aspect was not vital to study, since it was necessary in practice to supply dNTP's to copy mRNA. The effect of actinomycin D was again inconclusive, as explained previously, but it was used in most experiments. These results indicated, as stated before, that oligo dT dependent reverse transcription was a sensitive method for detecting the presence of poly A containing species in the


76
TABLE 8
Utilization of 7-12S RNA as a Template for Reverse Transcriptase
Template (0.5ug/0.1 ml) hi Label No. trials dT10 NTP AMD Mean CPM 50 ul-60 min. Largest % deviation
Isolated
7-12S RNA T 5 + + + 3775 28
4 0 + + 385 121
1 + 0 + 5350
1 + + 0 5176 -
dC + dG 2 + + + 4103 57
1 0 + + 924 -
Poly A(-)7-12S
RNA by: nitro-
cellulose T 2 + + + 550 58
1 0 + + 250 -
1 + 0 + 1160 -
Oligo ST )
cellulose T 2 + + + 2048 23
2 0 + + 750 22
dC + dG 2 + + + 2896 29
1 0 + + 1265
Notes: 1. Assay procedure described in Methods (VB).
2. Poly A(-) RNA is the 7-12S RNA preparation excluded from nitrocellulose or oligo (dT/-cellulose.
3. Specific activities were 2.2 Ci/mmole for TTP, and 2.22 and 1.78 Ci/mmole for dCTP and dGTP respectively.
4. CPM is averaged from similar trials with background subtracted, and no subtraction for endogenous (no mRNA added).
5. Background CPM is defined as 3H-NTP absorbed to the filter from a reaction mixture without viral lysate (no enzyme).
6. Deviation is represented by a percent difference from the widest data value to the mean.
7. dT^Q, dNTP, and AMD were in concentrations reported in Methods (VB).
8. Zero NTP maintains labeled nucleotide at original specific activity, and omits unlabeled nucleotides.


7-12S RNA preparation, which were removed by the affinity methods described. Furthermore, translational ability and size distribution of the poly A(-) RNA indicated no observable change in histone mRNA content. Taken together this information provided an indication of the purification obtained by the poly A affinity methods in preparation for polyadenylation and finally for preparative reverse transcription.
B. Polyadenylation of Histone Messenger RNA's
The next requirement for the synthesis of cDNA by RNA-
dependent DNA polymerase was establishing a priming system
for this enzyme on histone mRNA's. A defined homopolynucleo-
tide sequence was added to the 3' end of 7-12S RNA's for
this purpose. The enzyme (ATP: polynucleotidylexotransferase)
and procedure utilized for sequential addition of AMP was
available in the laboratory of Dr. R.J. Mans {75-80). In
contrast to the fixed lengths of poly A on all other
eucaryotic mRNA's thus far isolated, it was possible to vary
the extent of histone mRNA polyadenylation and thus
optimize conditions for efficient reverse transcription.
The adenylation of 7-12S RNA's was carried out (as described
in Methods VC) in enzyme excess to promote initiation on a
14
maximum number of RNA molecules. C-ATP was present in the adenylation reaction mixture to allow an average incorporated chain length to be determined by calculation of a mean number of AMP residues for RNA molecule of a given size. The average molecular weight assigned to 7-12S RNA's for this


78
calculation was 10^ daltons. Poly A incorporation was determined by measuring acid precipitable radioactivity (l^C-AMP) (77). There was no direct means of estimating the distribution of poly A sizes on RNA molecules without resorting to nuclease digestion of the RNA and polyacryla-mide gel electrophoretic sizing of the remaining poly A sequences (28). It has been shown for this enzyme, however, on substrates such as tRNA, that poly A chains increase in size linearly with time, resulting in a narrow size distribution range (78).
The first preparation of polyadenylated 7-12S (AdI) had relatively long poly A sequences; a chain length of 40 AMP1s/molecule was determined by the radio-labelled AMP incorporation. The extent of l^C-AMP incorporation in this preparation allowed an analytical determination of the average poly A chain length to be made. By hydrolysis of a sample of the RNA, paper chromatography of the nucleotides, and determination of the AMP: Adenosine ratio, a poly A chain length of 92 AMP's/chain was calculated. The combination of these two analyses indicated that approximately 50% of the RNA molecules were polyadenylated. There has been no major template specificity shown for this enzyme; therefore, there was no reason to believe that any component of the 7-12S was preferentially polyadenylated.
In subsequent preparations of polyadenylated 7-12S RNA's smaller poly A tails were synthesized and only radioactivity incorporation was measured. The second mRNA preparation was adenylated three consecutive times with mean poly A chain


79
lengths determined to be 0.1, 3 and 14 AMP's/molecule (Ad II a, b, c respectively). The third 7-12S preparation had an estimated mean incorporation per molecule of 25 AMP's (Ad III).
C. Reverse Transcriptase Assays of Polyadenylated Histone mRNA Templates
Analytical measurements were made on reverse transcriptase activity of the adenylated 7-12S RNA preparations with respect to 3H-TMP versus 3H-dCMP + 3H-dGMP incorporation. The specific activities of the deoxynucleotides were the same as indicated in Table 8, approximately 2 Ci/mmole for each and assuming a 50% CMP + GMP base composition of the histone mRNA preparation, incorporation of two times as much dCMP and dGMP as TMP was expected if the mRNA sequences were primarily transcribed. However, with increasing poly A tails and a fixed amount of oligo dT primer, increased 3H-TMP incorporation was expected.
Results on oligo dT primed JH-dCMP and H-dGMP incorporation were thus employed to determine which templates were more efficient for cDNA synthesis. Table 9 summarizes the results of reverse transcriptase assays on the three polyadenylated 7-12S RNA preparations. It was not possible to determine the per cent contribution of endogenous viral cDNA synthesis in these trials, since background was somewhat variable and dT-^Q stimulated endogenous activity may not have been constant in the presence of exogenous templates. Without endogenous activity levels subtracted, however, the reverse transcriptase activity on Ad II was negligible. Thus, relatively short poly A sequences were assumed to be


TABLE 9
Utilization of Polyadenylated 7-12S RNA as a Template for Reverse Transcriptase
Template Mean CPM
(0.5 ug/0.1 ml)_ H Label_dT10_50 ul-60 min.
Ad I T + 31,600
(40 AMP's/RNA) 0 408
dC + dG + 2,500
0 429
Ad II a T + 1,061
(0.1 AMP/RNA) 0 2,858
dC + dG + 1,942
II b T + 4,106
(3 AMP's/RNA) 0 977
dC + dG + 414
0 1,338
II c T + 2,450
(14 AMP's/RNA) 0 950
dC + dG + 1,850
0 1,795
Ad III T + 8,597
(25 AMP's/RNA) 0 1,099
dC + dG + 7,759
0 1,074
See notes 1, 3, 4, 5, 7 for Table 8


81
insufficient for oligo dT primed transcription of mRNA molecules. AD I showed high 3H-TMP but low 3H-dCMP and *^H-dGMP incorporation, yet was totally dependent upon oligo dT^Q. Ad III was shown to provide the best template for appreciable 3H-dCMP and 3H-dGMP incorporation. Thus, it appeared that a moderate size poly A stretch accommodated cDNA synthesis well into the mRNA sequence under these conditions. This analysis provided an insight into the efficiency of templates for synthesis of cDNA on a preparative scale. The selection of adequately polyadenylated
histone messenger RNA preparations for reverse transcription
3
was based upon the appreciable incorporation of H-dCMP and 3H-dGMP.
D. Preparation of the Complementary DNA
Complementary DNA was preparatively synthesized under the conditions outlined in Methods (VD) A trial experiment was conducted initially to develop product isolation procedures. The cDNA had to be separated from the RNA template, viral proteins, and unincorporated label. Several procedures had been reported (54,55,57) in which: Alkaline hydrolysis, or ribonuclease, or both were used to digest the RNA template, SDS denaturation treatment and organic extraction were used to remove protein, and either ethanol precipitation or Sephadex G-50 exclusion chromatography, or both were commonly employed to separate product from unincorporated label. In the first trial, Ad I was used as template at 5 pg/0.5 ml with 3H-dCTP and 3H-dGTP at the


82
specific activity of 5.25 Ci/mmole, and the attempt was made to deproteinize and digest RNA first (as described in Methods VD) then separate product by ethanol precipitation. The total TCA precipitable material from the original reaction represented 163,000 cpm or 28 ng of mRNA-like product. The first ethanol precipitate following HC1 neutralization contained 1.8 x 10^ cpm, and a second precipitate contained 7.5 x 10^ cpm. This illustrated the inefficiency of ethanol precipitation for separation of cDNA from dNTP contamination. At this point, the material was chromatographed on a 4 ml Sephadex G-50 (fine) column. The excluded material was pooled and quantitated. The yield of polynucleotide was 48,000 cpm or 8 ng or only 29% of the originally precipitable material. This material was ethanol precipitated again, but only 1.8 ng were recovered, and an absorbance spectrum of the final solution failed to indicate clearly nucleotide character. It appeared that loss of small amounts of cDNA resulted from ethanol precipitation even with carrier DNA, and it was also possible that single-stranded DNA adhered to the glass surfaces and polyallomer even in the presence of carrier DNA.
A second preparation was also made with Ad I; this time with the higher specific activity of 3H-dCTP and 3H-dGTP indicated in Methods VD representing 9000 cpm/ng cDNA. The initial TCA precipitable material amounted to 231,000 cpm or 26 ng, which was very similar in yield to the previous preparation. After alkaline hydrolysis, there were 11 ng of acid precipitable DNA, indicating substantial loss in the


83
initial treatments. The loss could have occurred during organic extraction or hydrolysis at extreme pH. The material was then treated by the more efficient method of separation, that of Sephadex G-50 chromatography into 1 mM HEPES buffer which was the buffer used in hybridization. Such chromatography dilutes the substrate slightly, but the excluded material was concentrated by evaporation. The excluded polymer was quantitated after evaporation, and 8 ng of cDNA remained. Thus this separation technique yielded good recovery under complete separation conditions, and all radioactivity was determined to be acid precipitable. The loss in the initial steps of this procedure was overcome by substituting ethanol precipitation with Sephadex G-50 chromatography and by utilizing evaporation for concentration which taken together produced a very straightforward and quantitative procedure.
This technique was then applied to 10 yg of Ad III in a 1 ml reaction. The reaction produced 95 ng of product as expected from the assay of this template. After alkaline hydrolysis and neutralization, 72 ng were measured. Following the exclusion chromatography on a 21.5 ml Sephadex G-50 column in 1 mM HEPES at a 0.08 ml/min. flow rate, 56 ng were obtained in 4 ml; and 40 ng were finally recovered after evaporation and suspension in hybridization buffer. Only 20-25% loss was determined at any one step in this preparation. The initial loss during extraction and hydrolysis was less than that in the previous preparation,


8:4
and subsequent loss occurred from tailing on the Sephadex G-50 column as illustrated in Figure 11.
The preparative synthesis of DNA complementary to the 7-12S RNA's containing histone messages was accomplished as discussed. The RNA had been fractionated by poly A affinity binding methods, polyadenylated, and assayed for oligo dT-primed reverse transcriptase template ability with 3H-dCTP and 3H-dGTP. The templates Ad I and Ad III were found sufficient for incorporation of these nucleotides. Ad I yielded primarily synthesis of poly TMP and Ad III yielded an increased 3H-dCMP + 3H-dGMP incorporation pattern. Ad I was used to develop the cDNA isolation procedure; and its cDNA was characterized to a limited extent, and was utilized in preliminary hybridization experiments. The cDNA to Ad III was utilized in the final characterizations and hybridization applications to be discussed.
IV. Characterization of the Complementary DNA Several approaches were pursued to characterize the complementary DNA sequences prepared by reverse transcription (RSV) of polyadenylated histone mRNA templates. The parameters of specific activity, size distribution, single-strandedness, and specificity of hybridization provided information relative to the usefulness of the cDNA as a specific probe for examining histone gene expression. A. Specific Activity
One property of the cDNA molecules to Ad III already alluded to was their specific activity of 27,250 dpm per ng,


85
Fraction
Figure 11. Separation of JH-cDNA product from H-dCTP and 3H-dGTP precursors by Sephadex G-50 exclusion chroma-tpgraphy in 1 mM HEPES buffer. Fractions 1-40 which represent polynucleotide are 0.4 ml taken at a flow rate of 0.08 ml/min. Fractions 41-54 which represent nucleotide precursor are 1.2 ml taken at a flow rate of 2 ml/min. Radioactivity measurements were made on 10 yl aliquots.


86
based on a 25% contribution of each deoxynucleotide and the known specific activities of the labelled precursors. An empirically determined counting efficiency of 33% for 3H implied a specific activity of 8992 cpm/ng which was similar to that of others in this field. This property was used to quantitate the recovery of the cDNA through purification as described in the previous section. B. Size Distribution
The size of the cDNA reflected the extent to which his-tone mRNA sequences were represented. In addition, the size distribution was an important factor in determining the quantity of the cDNA which would hybridize at a fixed temperature under optimum hybridization conditions. Larger sequences (greater than approximately 50 bases) will hybridize uniformly at a higher fixed temperature than shorter sequences will hybridize. 1. Sephadex G-100 chromatography
During the analytical assay of Ad I, large incorporation of TMP was seen. Identical conditions were used to synthesize product with H-TTP from 2.5 yg of Ad I from which the cDNA was sized by Dr. R.J. Mans via Sephadex G-100 chromatography in 8 M urea. Calibration of this chromatography system with sized deoxynucleotide polymers had been previsouly achieved by Dr. Mans. The reverse transcription reaction mixture was brought to 0.3 N KOH to hydrolyze the RNA template prior to chromatography. The single-stranded


87
cDNA was found to be mostly excluded from this column,
indicating a size greater than 180 nucleotides.
2. Alkaline sucrose gradient sedimentation
The most utilized technique for establishing size of
cDNA has been alkaline sucrose gradients. This procedure
was applied; but, since markers of the appropriate size
were not available, analytical measurements of sedimentation
velocity were used in conjunction with tabular values and
equations of McEwen to determine the mean S2o,w value for
the DNA (118). McEwen has derived the equation S /u2dt =
20 ,w
/ G(Z'T'PP> dz, where G(z,T,p ) = n(2'T) Pp-p20,w Z~ZQ n20fw P -p(z,t)
and z represents sucrose concentration, and he has tabulated
values for the integrals. By using the physical parameters
of the gradient, determining the percent sucrose at which
peak sedimentation occurs, and using these figures to
extrapolate integral values, the S value was calculated.
20 ,w
For a complete discussion see McEwen's publication. Further application of the equation of Prunnel and Bernard!, w =
0.0388 MW0,434, for denatured (single-stranded) DNA with a particle density of 1.8 gm/ml (119) was used to determine molecular weight.
Linear alkaline sucrose gradients were prepared, run and fractionated as described in Methods (VI A). The results are illustrated in Figure 12. The main band at 5.88 S corresponds to a molecular weight of 105,000 daltons or 325 nucleotides, which is sufficient to account for the majority of the nucleotide sequences of each of the histone mRNA's.


88
Fraction
Figure 12. Alkaline sucrose gradient radioactivity profile of histone 3H-cDNA preparation.


89
A contribution by smaller DNA's was also detected in the size range of 100 nucleotides and somewhat greater. Recent data, of others, on the effect of nucleotide concentration on the extent of reverse transcription illustrated similar findings by polyacrylamide gel electrophoresis of cDNA product synthesized in the presence of 25 yM deoxynucleotides (65). Apparent full copies of globin mRNA and chorionic mRNA's were seen, as well as distinct species of about half the size, at that nucleotide concentration (this study utilized 30 yM deoxynucleotides as described in Methods V D). The results therefore indicated that the cDNA product was of appropriate size for further hybridization, characterization, and experimental application. C. Single-Strandedness
It had to be shown thatthe cDNA synthesized was single-stranded, and therefore appropriate for hybridization. Two methods for the analysis of single- and double-stranded-ness of nucleic acids are hydroxylapatite chromatography and single-strand specific nuclease digestion. The first method is based on preferential binding of single and double-stranded nucleic acids to a hydroxylapatite crystal matrix under different buffer and ionic strength conditions (120). As with many chromatographic techniques, quantitative elution could be a critical problem. Nuclease digestion is more consistent, since excess enzyme can be used and quantitative analysis can be made without chromatography. The obvious difference in these techniques