Isolation and characterization of a variant human H2B histone gene expressing alternative mRNAs regulated differentially...

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Title:
Isolation and characterization of a variant human H2B histone gene expressing alternative mRNAs regulated differentially during the cell cycle and differentiation
Physical Description:
xvii, 196 leaves : ill. ; 29 cm.
Language:
English
Creator:
Collart, David George, 1961-
Publication Date:

Subjects

Subjects / Keywords:
Gene Expression Regulation   ( mesh )
Histones -- genetics   ( mesh )
RNA, Messenger -- genetics   ( mesh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 178-195).
Statement of Responsibility:
by David George Collart.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 25487850
ocm25487850
System ID:
AA00011193:00001

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ISOLATION AND CHARACTERIZATION
OF A VARIANT HUMAN H2B HISTONE GENE
EXPRESSING ALTERNATIVE mRNAs REGULATED DIFFERENTIALLY
DURING THE CELL CYCLE AND DIFFERENTIATION







By

DAVID GEORGE COLLART


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

UNIVERSITY OF FLORIDA
1991


































Dedicated to Mary,

the mother of God.














ACKNOWLEDGEMENTS


I would like to thank Drs. Gary and Janet Stein for

their support and guidance during my graduate studies.

Their dedication as scientists has provided us with a

laboratory which has always been well equipped and supplied,

allowing us to carry out the experiments necessary for the

completion of our individual projects. In addition, I would

like to thank Gary and Janet for all their support on a

personal level during all the trials which life has offered

throughout my journey from a single, and sometimes

foolhardy, new graduate student to a married, and hopefully

more mature, scientist. I would also like to thank Drs.

Harry Nick, Philip Laipis, Harry Ostrer, Carl Feldherr, Lee

Weber and Eileen Hickey for their helpful advice as members

of my graduate committee. I am also grateful for the

support and help of Dr. Richard Boyce. In addition, I thank

Anna Ramsey-Ewing, Dr. Paul Romain, Suzie Pilapil, Dr. Jane

Lian, Shirwin Pockwinse, Dr. Rita Bortell, Dr. Carlo Croce,

Dr. Kay Huebner and Dr. Linda A. Cannizzaro for their

collaborative efforts on various aspects of this research.

I would like to acknowledge Christine Dunshee for her

photography.


iii








I would like to thank all of the members of the Steins'

lab for their companionship over the years. I thank Linda

Green, Farhad Marashi and Mark Plumb for their friendship

and helpful discussions during the early years of my

graduate studies. I thank Andr6 van Wijnen for his

friendship and helpful suggestions on writing manuscripts

and Anna Ramsey-Ewing for her friendship and scientific

expertise in tissue culture and transcriptions. I would

also like to thank Tim Morris for his friendship and

discussions on everything in the heavens and on earth, and

Gerry Zambetti for his friendship and for being the

godfather of little Timothy Gerard. In addition, I thank

all my roommates during my pre-marriage days in particular

Charlie for his Scottish outlook on life and money, and Andy

for all of his friendship and support during my years as a

graduate student.

Finally, I would like to thank my family. My parents

and brothers and sisters have always supported me and have

taught me so much about caring. I thank my wife Kathy,

whose prayers and constant love have always been a source of

joy and strength for me. I thank our son Timothy, whose

smiles, giggles and squiggles have taught me what happiness

is and for his suggested addition to this dissertation, "aah

ahh ook." Lastly, I give thanks to God for his constant

love, forgiveness and blessings.














TABLE OF CONTENTS


PAGE
ACKNOWLEDGEMENTS..........................................iii

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

KEY TO ABBREVIATIONS .....................................xii

ABSTRACT ................................................. xv

CHAPTERS

1) INTRODUCTION
General Background.................................... 1
Histone Proteins...................................... 2
Structure and Organization of Human Histone Genes... 4
Coupling of DNA Synthesis and Histone Gene
Regulation......................... .......... 4
Transcriptional and Post-Transcriptional Regulation
of Human Histone Genes............................ 5
H2B Histone Gene Expression......................... 7
Overview of Project................................... 7

2) MATERIALS AND METHODS
Materials.................................. ......... 9
Materials and Biochemical Reagents............... 9
Antibiotics, Growth Factors, Inhibitors and Media 10
Enzymes and Kits.................................. 11
Nucleic Acids and Nucleotides ................... 12
Propagation and Maintenance of Bacterial Strains.... 13
Growth of Bacterial Strains....................... 13
Storage of Bacterial Strains ..................... 13
Propagation and Maintenance of I Bacteriophage...... 14
Growth and Titration of I Bacteriophage.......... 14
Preparation of plating bacteria............... 14
Obtaining a titer of I bacteriophage.......... 15
Small scale liquid cultures of I bacteriophage 15
Large scale liquid cultures of I bacteriophage 16
Isolation and Purification of I Bacteriophage.... 17
Storage of I Bacteriophage Stocks................ 18
Recombinant Phage DNAs... .............. .............. 19
Isolation and Purification of Bacteriophage DNA..... 19
Large Scale I Bacteriophage DNA Isolation........ 19
Small Scale I Bacteriophage DNA Isolation........ 20








Single-Stranded M13 Bacteriophage Template
Isolation..................................... 21
Recombinant Plasmid DNAs............................. 23
Isolation and Purification of Plasmid DNA........... 23
Rapid, Small Scale Plasmid DNA Isolation......... 23
Large Scale Plasmid DNA Isolation ................ 24
Isolation and Purification of Eukaryotic Genomic DNA 26
Isolation and Purification of Mammalian RNA......... 27
Selection of Poly A* RNA ........................... 29
Spectrophotometric Quantitation of DNA and RNA...... 30
Preparation of Radiolabeled DNA .................... 30
Random Oligonucleotide Primed Labeling........... 30
Labeling the 3' Termini of DNA................... 31
Labeling the 5' Termini of DNA................... 32
Analysis of Recombinant DNA Clones .................. 34
Mapping by Single and Multiple Restriction
Endonuclease Digestions...................... 34
Indirect End-Labeled Mapping...................... 34
Southern Blot Analysis........................... 36
Recovery of DNA Fractionated Electrophoretically.... 37
Analysis of Mammalian RNA............................ 38
Agarose-Formaldehyde Denaturing Gel
Electrophoresis............................... 38
Northern Analysis................................ 38
S1 Nuclease Protection Analysis.................. 39
In Vitro Nuclear Run-on Transcription Analysis... 41
Library Screening.................................... 43
Lambda gtll cDNA Library Screening............... 43
Lambda EMBL4 Genomic DNA Library Screening....... 49
Cloning and Construct Preparation................... 51
Subcloning into Plasmid Vectors................. 51
Construct Preparation........................... 51
Subcloning into Bacteriophage M13 Vectors........ 59
Transfection of DNA into Bacterial Cells............ 59
Preparation of Competent E. coli cells for
Transfection ................................. 59
Transfection of Recombinant Plasmid DNA.......... 60
Transfection of Recombinant Bacteriophage M13 DNA 60
DNA Sequencing..................................... 61
Sanger Dideoxy-Mediated Chain Termination Method. 61
Chromosomal in situ Hybridization.................... 61
Mammalian Cell Culture............................... 62
HeLa Cell Culture and Synchronization............ 62
HL60 Cell Culture and Differentiation............ 63
3T3L1 Cell Culture.............................. 63
Transfection and Transient Expression of
Recombinant DNA in 3T3L1 Cells................ 63
Selection of Stable Polyclonal 3T3L1 Cell Lines.. 64
Induction and Differentiation of 3T3L1 Cells..... 65
Cell Culture for Chromosome Localization......... 65
THP-1 Cell culture................................ 66









3) ISOLATION AND CHARACTERIZATION OF A cDNA FROM A HUMAN
HISTONE H2B GENE WHICH IS RECIPROCALLY EXPRESSED IN
RELATION TO REPLICATION-DEPENDENT H2B HISTONE GENES
DURING HL60 CELL DIFFERENTIATION
Introduction...... .................................. 67
Results...................................... ...... 68
Isolation of an H2B cDNA Clone From a 1gtll
Poly A+ cDNA Library.......................... 68
Restriction Endonuclease Mapping of Positive
Clones Isolated From the Agtll Poly A+ cDNA
Library Screening............................. 71
Sequencing Strategy for the Positive Clones
Isolated From the Agtll Poly A+ cDNA Library
Screening.................................... 71
Sequencing of a Poly A+ H2B cDNA................ 78
Comparison of the AHHC289 Predicted Amino Acid
Sequence With Other H2B Amino Acid Sequences. 83
Comparison of 3' Non-translated Sequences From
1HHC289 and Other Histone cDNAs and Genes.... 86
HHC289 mRNA is Expressed Throughout the HeLa
Cell Cycle and During Inhibition of DNA
Synthesis.................................... 86
Transcription of the HHC289 Histone Gene During
the HeLa Cell Cycle........................... 93
HHC289 mRNA is Expressed in a Reciprocal
Relationship With Replication-Dependent H2B
mRNAs During the Onset of HL60 Cell
Differentiation.............................. 93
Discussion......................................... 100
Structural Analysis of the IHHC289 H2B cDNA..... 100
Cell Cycle Regulation of the HHC289 H2B Histone
Gene........................................ 103
Regulation of HHC289 mRNA During Differentiation
in HL60 cells.............................. 105

4) ALTERNATIVE 3' END PROCESSING IS INVOLVED IN
MODULATING EXPRESSION OF A HUMAN HISTONE H2B.1 VARIANT
GENE, LOCATED ON CHROMOSOME 1, IN RESPONSE TO CHANGES IN
THE PROLIFERATIVE STATE OF THE CELL
Introduction ..................................... 109
Results............................................ 110
Isolation of an H2B Genomic Clone From a XEMBL4
Human Adult Lymphocyte DNA Library.......... 110
Restriction Endonuclease Mapping of Positive
Clones Isolated From the AEMBL4 Genomic DNA
Library Screening .......................... 111
Cloning and Nucleotide Sequence of the H2B-GL105
Gene........................................ 118
Genomic Organization of 1HHG5E................. 126
Chromosomal Localization of the H2B-GL105 Gene.. 131
H2B-GL105 mRNA Levels in Proliferating THP-1
Cells....................................... 141


vii









H2B-GL105 mRNA Levels in Synchronized HeLa Cells 144
Mapping of the H2B-GL105 5' mRNA Start Site..... 145
Mapping of the H2B-GL105 mRNA 3' Termini........ 150
Discussion........................................ 155
Structure and Organization of the H2B-GL105 Gene 155
Transcriptional regulation of the H2B-GL105 Gene 156
Post-Transcriptional Regulation of the H2B-GL105
Gene.............................. ......... 157

5) SUMMARY AND FUTURE CONSIDERATIONS..................... 160

APPENDICES

A) REAGENTS FOR PROPAGATION AND MAINTENANCE OF BACTERIA
AND BACTERIOPHAGE....................................... 169

B) REAGENTS FOR NUCLEIC ACID ISOLATION AND PURIFICATION. 175

C) BACTERIAL STRAINS USED .............................. 177

REFERENCES........................................ .......... 178

BIOGRAPHICAL SKETCH...................................... 196


viii














LIST OF FIGURES


Figure Page

2-1 Schematic diagram of histone gene probes used for
the Agtll poly A+ cDNA library screening........... 46

2-2 Outline of the screening scheme for the Agtll
poly A+ cDNA library............................... 48

2-3 Outline of the cloning scheme for the construction
of the H2B construct pGL105SV...................... 54

2-4 Outline of the cloning scheme for the construction
of the H2B 3' deletion construct pGL110SV.......... 56

2-5 Outline of the cloning scheme for the construction
of the H2B hairpin motif minus construct pGL109SV.. 58

3-1 Plaque dot hybridization analysis of putative
positive clones from the Agtll poly A+ cDNA
library screening................................. 70

3-2 Restriction endonuclease Southern mapping of
IHHC185 ............................................ 73

3-3 Restriction endonuclease maps of positive clones
isolated from the Agtll poly A+ cDNA library
screening................................... .. .. 75

3-4 Sequencing strategy on the positive clones isolated
from the Xgtll poly A+ cDNA library screening...... 77

3-5 Sequencing strategy for the human H2B histone cDNA
AHHC289................. .. .... ................ 80

3-6 (A) Nucleotide and deduced amino acid sequence of
the human H2B histone cDNA XHHC289. (B) Restriction
map of the cDNA AHHC289............................. 82

3-7 (A) Comparison of the IHHC289 histone H2B amino
acid sequence with the sequences of other H2Bs.
(B) DNA sequence comparison of the 3' untranslated
region of selected histone sequences............... 85









3-8 IHHC289 mRNA levels during the HeLa cell cycle..... 88

3-9 H2B mRNA levels during the HeLa cell cycle......... 90

3-10 Transcription of the IHHC289 histone gene during
the HeLa cell cycle................................. 95

3-11 IHHC289 mRNA levels during proliferation and
differentiation of HL60 cells ..................... 97

3-12 H2B mRNA levels during proliferation and
differentiation of HL60 cells..................... 99

3-13 Densitometric analysis of the 500 nt replication-
dependent H2B mRNA and the 2300 nt IHHC289 mRNA
levels during differentiation of HL60 cells........ 102

4-1 Third round screening of a XEMBL4 human adult
lymphocyte DNA library with a IHHC289 3' non-
translated trailer probe............................ 113

4-2 Indirect end-labeled mapping of the putative
genomic H2B clone 1HHG6F ........................... 115

4-3 Restriction map of putative genomic H2B clones..... 117

4-4 Southern blot analysis of the genomic H2B subclone
pGL201R ............................................ 120

4-5 Sequencing strategy for the human histone gene
H2B-GL105 .......................................... 122

4-6 Nucleotide and deduced amino acid sequences of the
human H2B and H2A histone genes from the genomic
clone AHHG5E....................................... 124

4-7 Restriction data for the genomic clone AHHG5E...... 128

4-8 Southern blot analysis of the genomic clone AHHG5E
with probes for histones Hl, H2A, H2B, H3 and H4... 130

4-9 Southern blot analysis of HeLa genomic DNA with a
H2B-GL105 probe..................................... 133

4-10 Southern blot analysis of DNAs from mouse-human
somatic cell hybrids probed with a 32P-labeled
DNA fragment from the human histone gene H2B-GL105. 135

4-11 Presence of the human histone gene H2B-GL105 in a
panel of 21 rodent-human hybrids.................... 137








4-12 Regional localization of the human histone gene
H2B-GL105 on human chromosome 1.................... 140

4-13 Northern blot analysis of H2B-GL105 mRNA levels in
proliferating THP-1 cells........................... 143

4-14 Northern blot analysis of H2B-GL105 mRNA levels in
synchronized HeLa cells ............................ 147

4-15 Sl nuclease mapping of the H2B-GL105 5' mRNA start
site.............................................. 149

4-16 Sl nuclease mapping of the H2A-GL101 5' mRNA start
site ............................................. 152

4-17 Sl nuclease protection analysis of the 3' termini
of the H2B-GL105 mRNA............................... 154

5-1 Sl analysis of the 3' termini of H2B mRNA expressed
in 3T3L1 cell lines................................. 165















KEY TO ABBREVIATIONS


APS:

ATP:

bp:

BSA:

CIP:

oC:

DMEM:

dATP:

dCTP:

dGTP:

DNase I:

DNA:

DEPC:

dpm:

DTT:

ddH20:

EDTA:

g:

g:

GAPDH:

HBSS:

IAA:


Ammonium Persulfate

Adenosine-5'-triphosphate

Base pair

Bovine serum albumin

Calf intestinal alkaline phosphatase

Degree centigrade

Dulbecco's minimal essential medium

2'-Deoxyadenosine 5'-triphosphate

2'-Deoxycytidine 5'-triphosphate

2'-Deoxyguanosine 5'-triphosphate

Deoxyribonuclease I

Deoxyribonucleic acid

Diethyl pyrocarbonate

Disintegrations per minute

Dithiothreitol

Double-distilled water

Ethylenediaminetetraacetic acid

Gram

Gravitational force

Glyceraldehyde-3-phosphate-dehydrogenase

Hank's balanced salt solution

Isoamyl alcohol

xii









IPTG:

1:

Ag:

Am:

mg:

ml:

mm:

mM:

M:

MOPS:

ng:

nt:

OD:

PBS:

pfu:

Poly (A):

PEG:

PVP:

PVS:

RF:

RNase A:

RNA:

rpm:

SDS:

TCA:

TEMED:


Isopropyl-p-D-thiogalactopyranoside

Liter

Microgram

Micrometer

Milligram

Milliliter

Millimeter

Millimolar

Molar

Morpholinepropane-sulfonic acid

Nanogram

Nucleotide

Optical density

Phosphate buffered saline

Plaque forming unit

Polyadenosine

Polyethylene glycol

Polyvinyl-pyrrolidone

Polyvinylsulfonic acid

Replicative form

Ribonuclease A

Ribonucleic acid

Revolutions per minute

Sodium dodecyl sulfate

Trichloro-acetic acid

N,N,N',N'-tetramethylethylenediamine


xiii









tH2B: Testis specific H2B

TPA: 12-0-tetradecanoyl phorbol-13-acetate

TTP: Thymidine 5'-triphosphate

U: Unit of enzyme

X-gal: 5-bromo-4-chloro-3-indolyl-p-D-galactoside


xiv














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


ISOLATION AND CHARACTERIZATION
OF A VARIANT HUMAN H2B HISTONE GENE
EXPRESSING ALTERNATIVE mRNAs REGULATED DIFFERENTIALLY
DURING THE CELL CYCLE AND DIFFERENTIATION

By

David George Collart

May 1991


Chairman: Gary Stein
Major Department: Biochemistry and Molecular Biology


We have isolated and characterized a cDNA (AHHC289)

corresponding to a variant human histone H2B gene. The H2B

protein coding region of IHHC289 is flanked at the 3' end by

a 1798 nt non-translated trailer that contains a region of

hyphenated dyad symmetry and a poly(A) tail. Nuclear run-on

transcription analysis revealed a two fold increase in

transcription of the HHC289 gene during S phase. Northern

blot analysis indicated that the levels of the 2300 nt

HHC289 mRNA species were constant during the HeLa cell

cycle. Northern blot analysis also revealed that the levels

of the 2300 nt HHC289 H2B species increased 10-fold during

HL60 cell differentiation whereas the levels of replication-








dependent H2B mRNAs decreased to less than 1% of those in

proliferating cells.

Using a probe from the IHHC289 cDNA that detects only a

single copy region of the human genome we isolated a variant

human H2B histone gene (GL105) which expresses alternative

mRNAs regulated differentially during the HeLa cell cycle.

This H2B gene encodes both a 500 nt replication-dependent

mRNA and the 2300 nt HHC289 constitutively expressed mRNA.

The 3' end of the cell cycle regulated mRNA terminates

immediately following the region of hyphenated dyad symmetry

typical of most histone mRNAs, whereas the constitutively

expressed HHC289 mRNA has a 1798 nt non-translated trailer

that contains the same region of hyphenated dyad symmetry

but is polyadenylated. The cap site for both H2B-GL105

mRNAs is the same and is located 42 nt upstream of the

protein coding region. The H2B-GL105 histone gene was

localized to chromosome region 1q21-1q23 by chromosomal in

situ hybridization of a H2B-GL105 specific probe and by

analysis of rodent-human somatic cell hybrids. The

H2B-GL105 gene is paired with a functional H2A histone gene

and this gene pair is separated by a bidirectionally

transcribed intergenic promoter region containing consensus

TATA and CCAAT boxes and an OTF-1 element. These results

demonstrate that replication-dependent and constitutively

expressed histone mRNAs can be encoded by the same gene and

indicate that alternative 3' end processing is a major level


xvi








of regulation by which cells can modulate the synthesis of

variant histone proteins during the cell cycle and at the

onset of differentiation.


xvii














CHAPTER 1

INTRODUCTION



General Background

All cells of higher animals and plants carry genetic

information in chromosomes in the form of long linear

molecules called DNA. Within these DNA molecules are

regions containing units of genetic information, called

genes, whose expression results in the production of

proteins or RNA molecules necessary for cellular function.

In the human being more than 1 meter of DNA is contained

within the cell nucleus. DNA must be packaged efficiently

within the cell in such a manner that it can be replicated

when the cell divides, and transcribed at most stages

throughout the cell cycle. This packaging process is

carried out primarily by a group of proteins, approximately

equal in mass to the DNA itself, called histone proteins.

When DNA is associated with histone proteins it is called

chromatin.

When a cell divides, the DNA within a cell is

replicated and the cell must therefore also double the

amount of its histone protein. Because the cell requires a

large quantity of histone protein to package its DNA, it










must carefully regulate the production of these proteins

according to the cellular needs. The cell must allow for

production of large amounts of histone protein during

periods of rapid growth and division but when DNA

replication ceases, such as outside of S phase or at the

onset of differentiation, the cell must downregulate histone

production to a low level but sufficient to provide for

replacement of histone proteins and for reorganization of

chromatin. Therefore, the study of histone gene expression

provides an excellent opportunity to understand the changes

in gene expression during the transition from a

proliferative to a nondividing state.



Histone Proteins

Histones are a complex family of highly conserved basic

proteins responsible for packaging chromosomal DNA into

nucleosomes (85,86). There are five major classes of

histone proteins (H1, H2A, H2B, H3, and H4) (85,86). Histone

proteins exhibit two levels of diversity: (i) evolutionary

diversity between species and (ii) subtype diversity in a

class (HI, H2A, H2B, H3, or H4) within a species (164). The

subtypes within a species are often referred to as variants

(187,188,195). Replication-dependent histone variants

represent the majority of the histone proteins synthesized

in proliferating cells, and it is well established that

their synthesis is functionally and temporally coupled to










DNA replication (1,133,164). Replacement variants are

expressed in nonproliferating cells and are often expressed

in proliferating cells, although normally at a lower level

than their replication-dependent counterparts (188,195).

The third type of variant within a species is the tissue-

specific variant (17,40,83,84,172). Tissue-specific

variants are generally not synthesized in a replication-

dependent manner (40,83,84).

Histone H2B proteins have been studied in a variety of

species--including chicken, mouse, rat and human--with

respect to their subtype diversity (17,88 and reviewed in

164,195). Although the replication-dependent and tissue-

specific H2B proteins are easily detected in most species,

the replacement histone H2B proteins are not always observed

in humans, possibly due to the lack of sensitivity of the

assay (164). In mouse the H2B.2 variant is the replication

dependent subtype and is abundant in rapidly dividing cells

(195). The H2B.1 variant is the constitutively expressed

subtype and is present in small amounts in rapidly growing

tissues but increases sharply as proliferation slows,

especially after the postnatal growth phase (195). The H2B

subtype specific amino acids of the H2B.1 mouse protein

(Ser14, Ala21, Asp25, Lys27, Gly6, Asp6, Gly75, Glu76) differ

from those of the H2B.2 protein only at Ser7 (195),

however, the functional significance of this change is

unclear. In general, replacement histone variants are less










hydrophobic than replication-dependent or tissue specific

variants.



Structure and Organization of Human Histone Genes

Human histone genes are a family of moderately

reiterated sequences (75,91,154,164) and are arranged in

clusters; they show no evidence of a simple tandem repeat

organization (75,154,164,194) as do the histone genes of sea

urchin species (65,164,186) and Drosophila (102,164). In

humans, these clusters are localized on chromosomes 1, 6,

and 12 (62,164,171) and are associated with a series of

repetitive DNA sequences (36). Cell cycle dependent histone

genes do not contain introns (91,164), and they encode mRNAs

which are not polyadenylated and have short 5' leader and 3'

trailer sequences (77,164). Although replication-dependent

histone mRNAs are structurally simple, they do have a

characteristic 3' stem-loop motif (164). In contrast,

constitutively expressed histone genes are structurally more

complex. They may contain introns (20,43,182,183,185), and

they encode mRNAs which are polyadenylated and frequently

contain long 5' leader or 3' trailer sequences (16,20,28,45,

46,71-73,78,96,100,110,116,143,144,175,182,183).



Coupling of DNA Synthesis and Histone Gene Regulation

Replication-dependent histone genes are coordinately

expressed during the cell cycle and their expression is








5

coupled with DNA synthesis (3,74,126,130,131). The

abundance of their mRNAs is regulated at both the

transcriptional and post-transcriptional levels

(3,74,130,131,176 and reviewed in 113,150,151,163,164). In

comparison, the mRNAs for replacement histones

(10,19,25,45,71,89,152,155) as well as tissue-specific

histone mRNAs (27,34,64,83,84,93) are not always regulated

in a replication-dependent manner.



Transcriptional and Post-Transcriptional
Regulation of Human Histone Genes

Transcriptional regulation of human histone genes

involves a 2 to 5 fold enhancement in the rate of

transcription within the first 2 hours of DNA synthesis

followed by a return to basal level by mid to late S phase

(4,74,113,131,151,163). However, steady state levels of

histone mRNA are elevated 15-to 20-fold during S phase of

the cell cycle and parallel the rates of DNA synthesis

(3,74,130,131,176). Although the rate of histone gene

transcription peaks within the first 2 hours of DNA

synthesis, histone mRNA levels do not reach a maximum until

4-5 hours into S phase. Taken together these observations

suggest post-transcriptional mechanisms play a role in the

regulation of histone mRNA levels.

Post-transcriptional regulation of histone genes

involves a rapid and selective destabilization of histone

mRNAs toward the end of S phase or in response to inhibition








6
of DNA synthesis (3,76,117,127,128,130,142). However, there

is little or no effect on histone gene transcription by

inhibition of DNA synthesis (4,130,155). This

destabilization of histone mRNA upon inhibition of DNA

synthesis is dependent upon protein synthesis (3,22,76). In

contrast, the mRNA levels of replication-independent histone

genes are not regulated in the same cell cycle dependent

manner as their replication-dependent counterparts and

remain relatively constant upon inhibition of DNA synthesis

(10,19,25,35,45,71,89,152).

The 3' end of cell cycle dependent mRNAs contains a

region of hyphenated dyad symmetry (77) which is essential,

although not sufficient, for coupling histone mRNA stability

with DNA replication (2,101,106,125,159). Graves et al.

(61) observed that moving the 3' stem-loop motif affects

coupling of histone mRNA stability with DNA replication.

Destabilization of histone mRNA initiates at the 3' terminus

(140,142), and is continued rapidly in a 3' to 5' direction

by a ribosome complex-associated endonuclease (141). In

addition, subcellular localization may play a role in

histone mRNA stability (192,193).

The 3' end of most eukaryotic mRNAs is formed by an

endonucleolytic cleavage reaction followed by

polyadenylation of the 3' terminus (12,53,122,129). This

cleavage reaction requires two sequences, a highly conserved

AAUAAA sequence and a downstream GU rich sequence, which










flank the cleavage site (12,53,122,129,179). Most histone

mRNAs are not polyadenylated and their mRNA 3' end formation

requires two elements: the stem-loop motif and a purine rich

sequence 3' to the stem-loop motif (9,12,159,178). An

endonucleolytic cleavage reaction occurs 3' to the stem-loop

motif (12,55,95,118,167) and is dependent upon the

interaction of the U7 snRNA with the purine rich sequence

(12,39,118-120,156,167).



H2B Histone Gene Expression

To date, H2B histone gene organization and expression

have been studied most thoroughly in the chicken; five

replication-dependent, one partially replication-dependent,

one uncharacterized and one testis-specific chicken H2B

genes have been described (27,59,60,83,84). Human H2B

histone gene expression has not been reported in such

detail. Although several human histone H2B replication-

dependent genes and one pseudogene have been described

(74,112,131,134,154,164,194), no human replacement or

tissue-specific H2B genes have been reported.



Overview of Project

The overall aim of this work was to isolate and

characterize a variant human histone gene and study its

regulation in response to changes in the proliferative state

of the cell. When we began these studies, information in










the literature on nonhuman variant histone genes indicated

the genes for human variant histone proteins would be

structurally more complex than their replication-dependent

counterparts (20,43,71,185). This indication was supported

by the observations of Borun et al. (16) that a fraction of

human histone mRNAs contained short tracts of poly(A). In

addition, because constitutively expressed histone proteins

are synthesized throughout the cell cycle (164,188,195),

their mRNAs must be stable in the absence of DNA synthesis.

Presumably, structural features of constitutively expressed

histone mRNAs could render them resistant to the mechanisms

which rapidly and selectively destabilize replication-

dependent histone mRNAs toward the end of S phase or in

response to inhibition of DNA synthesis (3,76,117,142).

Alternatively, the absence of structural elements endogenous

to replication-dependent histone mRNAs could have the same

result. Therefore, we have focused our attention on the

cloning of a variant human H2B histone gene, and

characterization of its structural features and their effect

on expression in response to changes in the growth state of

the cell.














CHAPTER 2

MATERIALS AND METHODS



Materials

Materials and Biochemical Reagents

The majority of chemicals, reagents and solvents were

purchased from either Fisher Scientific, Springfield, NJ or

Sigma Chemical Co., St. Louis, MO., unless otherwise

indicated. Formamide and X-gal were purchased from Bethesda

Research Laboratories (BRL), Gaithersburg, MD. Bio-Gel

A-1.5m, A-5m, and A-15m (100-200 mesh) agarose beads for gel

filtration, Dowex 50W-X8 cation exchange resin (200-400

mesh) hydrogen form, ultrapure electrophoresis grade agarose

and Zeta-probe nylon membranes were purchased from Bio Rad,

Richmond, CA. Acrylamide (crystallized), APS, ultrapure

bis-acrylamide (N,N'-methylene-bis-acrylamide), DTT

(Cleland's reagent), glycogen (molecular biology grade), and

IPTG were purchased from Boehringer Mannheim Biochemicals

(BMB), Indianapolis, IN. Cronex x-ray film was manufactured

by Dupont, Wilmington, DE. Dimethyl sulfate,

dichlorodimethyl silane, hydrazine, PEG 8000, PVS, XAR-5

x-ray film and X-Omat duplicating film were manufactured by

Eastman Kodak Co., Rochester, NY. SPECTRA/POR








10

molecularporous membrane tubing, cellusolve (ethylene glycol

monoethyl ether), dimethyl sulfoxide, ethidium bromide,

2-piperidine, TCA and toluene were purchased from Fisher

Scientific, Springfield, NJ. Cesium chloride (special

biochemical grade 99.9%) was purchased from Gallard-

Schlesinger Industries Inc., Carle Place, NY. OPTI-FLUOR

scintillation cocktail was purchased from Packard Instrument

Co., Inc., Downers Grove, IL. Ficoll 400 was purchased from

Pharmacia Inc., Piscataway, NJ. Type 55 and type 57 instant

sheet film were purchased from Polaroid, Cambridge, MA.

Concentrated liquid scintillator (Liquifluor) was purchased

from Research Products International, Elk Grove Village, IL.

Elutip-d mini-columns, 82 mm and 132 mm nitrocellulose

circles (pore: 0.45 gm) and nitrocellulose sheets were

purchased from Schleicher and Schuell, Inc., Keene, NH. BSA

(fraction V), DEPC, N,N-dimethyl formamide, PVP and TEMED

were purchased from Sigma Chemical Co..



Antibiotics, Growth Factors, Inhibitors and Media

Antibiotics and media were prepared as described in

appendix A. Fetal bovine, calf bovine and horse sera were

obtained from Gibco Laboratories, Grand Island, NY or Flow

Laboratories, McLean, VA. Maltose was purchased from Becton

Dickinson and Co., Cockeysville, MD. Bacto-agar, bacto-

tryptone, bacto-yeast extract, and vitamin-free casamino

acids were purchased from Difco Laboratories, Detroit, MI.










Dextrose was purchased from Fisher Scientific, Springfield,

NJ. Geneticin (G418 sulfate) was purchased from Gibco

Laboratories. Chloromycetin sodium succinate

(chloramphenicol sodium succinate) was purchased from Parke-

Davis, Morris Planes, NJ. TGF-P1 was purchased from R&D

Systems, Inc., Minneapolis, MN. Ampicillin (D[-]-a

aminobenzylpenicillin) sodium salt, dexamethasone,

hydroxyurea, insulin (from bovine pancreas), 3-isobutyl-

1-methyl-xanthine, NZ amine (casein enzymatic hydrolysate),

penicillin-G (Benzyl penicillin) potassium salt,

streptomycin sulfate (streptomycin sesquisulfate),

tetracycline, TPA and trypsin (from porcine pancreas)

prepared in lX HBSS without calcium or magnesium were

purchased from Sigma Chemical Co.



Enzymes and Kits

Restriction endonucleases were purchased primarily from

BRL, BMB, and New England Bio Labs (NEB), Beverly, MA.

Enzyme reactions were carried out using the buffer and

incubation conditions described by the manufacturers unless

otherwise indicated. E. coli DNA polymerase I large

fragment (Klenow enzyme) was purchased from BRL.

Proteinase K, CIP, nuclease Sl1 and T4 DNA ligase were

purchased from BMB. Trypsin (0.25% in buffered saline) was

purchased from Hazleton Biologics, Inc., Lenexa, KS. The

EXOMETH DNA sequencing kit with reverse transcriptase and










the PRIME-IT random primer kit were purchased from

Stratagene, La Jolla, CA. DNase I (DN-EP), DNase I (DN-25)

and RNase A (Type III-A) were obtained from Sigma Chemical

Co.. The Sequenase 2.0 DNA sequencing kit and T4

polynucleotide kinase (cloned) were from United Stated

Biochemical Corp. (USB), Cleveland, OH. Lysozyme was

purchased from Worthington Biochemicals, Freehold, NJ.



Nucleic Acids and Nucleotides

[methyl-3H]Thymidine (20.0 Ci/mmole), [a-32P]dATP

(-3,000 Ci/mmole), [a-32P]dCTP (-3,000 Ci/mmole), [y-32P)ATP

(-3,000 Ci/mmole), [a-32P]UTP (-3,000 Ci/mmole) and

[a-35S]dATP (>1,000 Ci/mmole) radionucleotides were

purchased from Amersham Corp., Arlington Heights, IL, or NEN

Research Products, Wilmington, DE. Oligonucleotides were

synthesized in the recombinant DNA core facility of the

Department of Cell Biology at the University of

Massachusetts Medical Center using an Applied Biosystems

(AB) 380A DNA synthesizer purchased from AB, Foster City,

California. ATP (from equine muscle), dATP (sodium salt),

dCTP (sodium salt), dGTP (sodium salt), TTP (sodium salt),

oligo dT-cellulose and salmon testes DNA (type III) were

obtained from Sigma Chemical Co. RNA molecular weight

markers were purchased from BMB and from BRL. Ml3mpl8 RF

DNA and M13mpl9 RF DNA were obtained from BMB.










Propagation and Maintenance of Bacterial Strains

Growth of Bacterial Strains

Escherichia coli (E. coli) bacteria (see appendix C)

were grown at 370C in YTN medium with vigorous shaking or on

plates containing YTN bottom agar (see appendix A) unless

otherwise indicated. If necessary, ampicillin was added to

the media to a final concentration of 50 Ag/ml or

tetracycline to 15 Ag/ml immediately prior to inoculation of

liquid cultures or pouring of YTN agar plates. Liquid

cultures of bacteria were usually grown such that the volume

of the airspace above the culture was at least 3 times that

of the medium, unless otherwise indicated.



Storage of Bacterial Strains

Bacterial colonies were stored for short periods of

time on the surface of bottom agar plates at 4C. These

plates were wrapped in parafilm to prevent moisture loss and

stored inverted.

For long-term preservation of bacterial strains

bacteria were stored in medium containing 50% glycerol at

-70C. To prepare bacterial stocks, 5 ml of YTN was

inoculated with a single colony of bacteria and grown

anaerobically for 18 hours. A 0.5 ml aliquot of the

anaerobic culture was then placed into a vial containing

0.5 ml of sterile glycerol, the contents were mixed and the

vial stored at -700C.










Propagation and Maintenance of A Bacteriophage

Growth and Titration of I Bacteriophage

Lambda Charon 4A (13) and AEMBL4 (54) recombinant phage

were grown in the LE392 strain of E. coli (15,44,121) and

Igtll recombinant phage in the Y1088 strain of E. coli

(191). Lambda bacteriophage were grown either on NZCYM

plates as described by Maniatis et al. (108), in small scale

liquid cultures as described by Leder et al. (98) or in

large scale liquid cultures (13,109).



Preparation of plating bacteria (108). A single

bacterial colony (or 1 loop of bacteria from a glycerol

stock) was used to inoculate 50 ml of NZCYM medium,

supplemented with 500 pl of 20% maltose (see appendix A),

and grown for 10-15 hours at 370C with vigorous shaking.

Maltose induces the maltose operon, containing the gene

coding for the I receptor which is essential for efficient

absorption of I bacteriophage to bacteria. The cells were

then centrifuged at 3000 rpm in an IEC rotor (2000 X g) for

5 minutes at 40C. The supernatant was discarded and the

cell pellet resuspended in 20 ml (0.4 X the volume of the

original culture) sterile 10 mM MgSO4. These bacteria were

stored at 40C and used for up to 3 weeks; however, normally

the cells were used within 1-2 days of their preparation.










Obtaining a titer of I bacteriophage. Titration of

bacteriophage was carried out essentially as developed by

Felix d'Hdrelle in 1920 (165) and described by Maniatis

et al. (108). Serial dilutions of bacteriophage were

prepared in SM and 100 pl aliquots of each dilution to be

assayed were placed into 13-mm x 100-mm culture tubes. A

100 pA aliquot of plating bacteria was placed into each

tube, and the tube was vortexed gently and incubated at 370C

for 15-20 minutes to allow the bacteriophage to absorb.

Upon completion of the incubation period, 4 ml aliquots of

NZCYM top agar (450C) were added to each tube, and the

contents were vortexed gently and then poured onto NZCYM

bottom agar plates (see appendix A for preparation of NZCYM

top agar and NZCYM bottom agar plates). Upon solidification

of the top agar the plates were inverted and incubated at

370C for 12-18 hours to allow plaque formation.



Small scale liquid cultures of I bacteriophage (98).

Approximately 1/3-1/2 of a resuspended bacteriophage plaque

or 3 X 106 bacteriophage from a stock was mixed with 100 Al

of a fresh bacterial overnight culture in a sterile, 50-ml

polypropylene tube and incubated for 20 minutes at 370C.

Following absorption of the bacteriophage, 4 ml of NZCYM

medium was added and the tube incubated at 37C with shaking

for 6-12 hours until lysis occurred. After lysis, 40 Al of

CHC13 was added and the incubation continued for 15 minutes.










Upon completion of the incubation period, the culture was

centrifuged at 5800 rpm in a Beckman JA20 rotor (4000 X g)

for 10 minutes at 40C. Next, the supernatant was recovered,

12 Ml of CHC13 added and the stock stored at 40C. The

titers were normally in the range of 108-1010/ml.



Large scale liquid cultures of I bacteriophage

(13,109). A single bacterial colony (or 1 loop of bacteria

from a glycerol stock) was used to inoculate 100 ml of NZCYM

medium, which was grown for 10-15 hours at 37C with

vigorous shaking. The OD600 of the culture was measured and

the cell concentration calculated, assuming that

1 OD60=8 X 108 cells/ml (108). An aliquot containing 1010

cells was removed and centrifuged at 3000 rpm in an IEC

rotor (2000 X g) for 5 minutes at 22C. After

centrifugation the supernatant was discarded and the cells

were resuspended in 3 ml SM. Approximately 5 X 108

bacteriophage were added to the cell suspension and the tube

was incubated at 37C for 20 minutes with intermittent

shaking. Upon completion of the incubation period, the

contents of the tube were added to 500 ml of prewarmed NZCYM

and incubated at 370C with vigorous shaking for 7-12 hours

until lysis occurred. After lysis, 10 ml of CHC13 was added

and the incubation continued for 30 minutes. Upon

completion of the incubation, the I bacteriophage were

purified as described by Yamamoto et al. (189).










Isolation and Purification of I Bacteriophage (189)

After treatment of the large scale preparation of

I bacteriophage with CHCl3, the culture was chilled to room

temperature, pancreatic DNase I and RNase A were added to a

final concentration of 1 gg/ml each and the culture was

incubated for 30 minutes at room temperature. Upon

completion of the incubation, solid NaCl was added to a

final concentration of 1 M (29.2 g), dissolved by swirling

and the culture incubated for 1 hour on wet ice. The

culture was then poured into a 500 ml centrifuge bottle, the

CHC13 was carefully left behind, and the culture centrifuged

at 9500 rpm in a Beckman JA10 rotor (11,000 X g) for

10 minutes at 4C. The supernatant was poured into a clean

flask; solid PEG 8000 from Eastman Kodak (chemical source is

important) was added to a final concentration of 10% w/v

(50 g), dissolved by slow stirring, and incubated for at

least 1 hour on wet ice (often this precipitation step was

carried out at 4C overnight). After the incubation, the

precipitated bacteriophage particles were recovered by

centrifugation at 9500 rpm in a Beckman JA10 rotor

(11,000 X g) for 10 minutes at 4C. The supernatant was

poured off and the bottles were drained in a tilted position

for 5 minutes. The bacteriophage pellet was gently

resuspended in 8 ml of SM, using a Pasteur pipette and a

rubber bulb to dislodge the bacteriophage particles from the

wall of the centrifuge bottle. The bacteriophage suspension










was extracted with 8 ml CHC13/IAA (24:1, v/v) and

centrifuged at 2500 rpm in an IEC rotor (1400 X g) for

15 minutes at 4C. The aqueous phase was recovered and the

above extraction repeated until no PEG interface remained.

Upon completion of the CHCl3 extractions 0.75 g of solid

CsCl was added for every ml of solution and mixed gently.

The bacteriophage suspension was then centrifuged at 38,000

rpm in a Beckman 50Ti or 70.1Ti rotor (135,000 X g) for

24 hours at 4C. The band of bacteriophage particles was

collected using an 18 gauge needle and stored at 40C.



Storage of I Bacteriophage Stocks

Bacteriophage were stored in one of three ways: (i)

Pasteur pipette plugs of bacteriophage plaques were placed

into 1 ml aliquots of SM (initial titers of approximately

106-107/ml) and stored at 4C for 2-4 years, (ii)

supernatants obtained from small scale liquid cultures of

bacteriophage (initial titers of approximately 108-l01/ml)

were stored at 4C for 3-5 years, and (iii) suspensions of

bacteriophage in CsCl, obtained from large scale liquid

cultures of bacteriophage purified over CsCIl gradients

(initial titers of approximately l10"-10/ml), were stored

at 4C for over 5 years. Stocks of bacteriophage generally

experienced a drop in titer of one order of magnitude during

the first month of storage and an equivalent drop during

each year of further storage.










Recombinant Phage DNAs

The A Charon 4A recombinant bacteriophage used in this

study were previously isolated (24,154,196) from a I Charon

4A genomic DNA library. Also used in this study were Agtll

recombinant bacteriophage isolated (see chapter 3) from a

Agtll human liver cDNA library and AEMBL4 recombinant

bacteriophage which were isolated (see chapter 4) from a

AEMBL4 adult human lymphocyte genomic DNA library.



Isolation and Purification of Bacteriophage DNA

Large Scale I Bacteriophage DNA Isolation (109)

DNA was isolated from purified I bacteriophage

as described by Maniatis et al. (109). The purified

bacteriophage preparation was dialyzed twice at room

temperature for 1 hour against a 1000 X volume of 10 mM

NaCl, 50 mM Tris*Cl (pH 8.0), 10 mM MgCl2, to remove cesium

chloride. The bacteriophage suspension was transferred to a

glass, 50 ml extraction tube and EDTA added to a final

concentration of 20 mM. Proteinase K was added to a final

concentration of 50 Ag/ml, SDS to a final concentration of

0.5% (w/v) and the solution was mixed gently and incubated

at 650C for 1 hour. The solution was extracted with an

equal volume of equilibrated phenol (see appendix B) and

centrifuged at 2500 rpm in an IEC rotor (1400 X g) for

10 minutes at room temperature. Next, the solution was

extracted with an equal volume of a 1:1 mixture (v/v) of










equilibrated phenol and CHC13/IAA (24:1, v/v) and

centrifuged at 2500 rpm in an IEC rotor (1400 X g) for

10 minutes at room temperature. The aqueous phase was

recovered and extracted once with an equal volume of

CHCly/IAA (24:1,v/v). Upon completion of the extractions

the aqueous phase was transferred to a dialysis sac and

dialyzed sequentially for 6 hours, at 4C, against 3 1000 X

volumes of 10 mM Tris*Cl, 1 mM EDTA (pH 8.0). The purified

DNA was quantitated and stored at either 4C or -20C.



Small Scale I Bacteriophage DNA Isolation (149)

Small scale I bacteriophage DNA preparations were

carried out as described by Schweizer (149). A high titer

lysate of I bacteriophage, 0.5 ml, was transferred to a

1.5 ml microcentrifuge tube containing 10 Al 10% SDS (w/v).

Subsequently, 1 Al DEPC was added and the tube mixed by

inversion. Next, 100 gl of 1 M TrisoCl (pH 8.0) and 25 pl

0.5 M EDTA were added, the tube mixed again by inversion and

incubated for 5 minutes at 70C. After the incubation,

80 Al 3 M sodium acetate (pH 5.2) was added and the

incubation continued for 30 minutes on wet ice. The

solution was then centrifuged at 12,000 X g for 15 minutes

at 4C. Following centrifugation, the supernatant was

decanted to a fresh microcentrifuge tube. The tube was

filled with 95% ethanol (-20C), mixed by inversion and

centrifuged at 12,000 X g for 5 minutes at room temperature.










The supernatant was discarded and the pellet washed with

1.0 ml 70% ethanol at room temperature. The pellet was

dried briefly under vacuum and resuspended in 200 tl TES

buffer (20 mM Tris*Cl (pH 7.5), 10 mM NaCI, 0.1 mM EDTA).

The DNA was applied to a 2 ml column of Bio-Gel A-0.5m

agarose beads and eluted with TES buffer. Fractions were

collected and analyzed on a 1% agarose plate containing

5 gg/ml ethidium bromide. Fractions containing

A bacteriophage DNA were pooled and precipitated with 0.3 M

NaCl and 2.5 volumes of 95% ethanol (-200C), mixed by

inversion and centrifuged at 12,000 X g for 5 minutes at

room temperature. The supernatant was discarded and the

pellet washed with 1.0 ml 70% ethanol at room temperature.

The pellet was dried briefly under vacuum, resuspended in

50 il 10 mM Tris*Cl (pH 8.0), 1 mM EDTA containing DNase-

free RNase (50 Ag/ml) and incubated for 30 minutes at 370C.

Following incubation the DNA was stored at -200C.



Single-Stranded M13 Bacteriophage Template Isolation

Single-stranded M13 Bacteriophage templates for

sequencing cloned inserts were prepared as described by

Sanger et al. (146) and Messing (115). An exponentially

growing culture of host cells, XL1-Blue (21) (see

appendix C), was diluted 1:100 in 2 ml of YTN medium,

infected with a purified M13 plaque and incubated for 6-8

hours at 37C with vigorous shaking. Following the










incubation, 1.5 ml of the culture was transferred to a

microcentrifuge tube and spun in an Eppendorf centrifuge at

12,000 X g for 10 minutes at 4C. The supernatant (1.2 ml)

was transferred to a microcentrifuge tube containing 300 pl

of 20% PEG, 2.5 M NaCl and incubated at room temperature for

15 minutes. The remaining supernatant was placed into a

clean microcentrifuge tube and stored at -200C as a phage

stock. After the incubation, the phage suspension was

centrifuged at 12,000 X g for 5 minutes at room temperature

and the supernatant was carefully removed and discarded.

The phage pellet was resuspended in 150 pl TES buffer (20 mM

Tris*Cl (pH 7.5), 10 mM NaCl, 0.1 mM EDTA) by vortexing for

2 seconds. Equilibrated phenol (50 pl) was added to the

virus suspension, vortexed for two seconds, incubated at

room temperature for 5 minutes, vortexed for an additional

2 seconds and centrifuged at 12,000 X g for 4 minutes at

room temperature. Single-stranded template DNA was

precipitated from 130 pl of aqueous phase with 5 pl 3 M

sodium acetate, 1 pl glycogen (20 mg/ml) and 405 Al 95%

ethanol on crushed dry ice for 15 minutes and pelleted by

centrifugation at 12,000 X g for 30 minutes at 4C. The

supernatant was decanted and the pellet washed with 100 pl

70% ethanol at room temperature. The DNA pellet was dried

under vacuum, resuspended in 80 gl TES buffer and stored at

-200C.










Recombinant Plasmid DNAs

DNA fragments containing HI, H2A, H2B, H3 and H4 genes

from recombinant I Charon 4A phage (24,154,196) were

subcloned into pBR322 or pUC8 ((131,154,196) and unpublished

data). The plasmid RGAPDH-13 (52) was generously provided

by Dr. Ph. Jeanteur and the plasmid G32m (38) by Dr. Kenneth

Soprano after preparation from a 550 bp Pst I fragment of

human #2-microglobulin kindly provided by Dr. K. Itakura,

Harvard University.



Isolation and Purification of Plasmid DNA

Bacteria containing plasmids were grown in YTN medium

containing 50 Ag/ml ampicillin and amplification was induced

in the presence of 200 Ag/ml chloramphenicol. Plasmid DNA

was isolated and purified using the following methods.


Rapid, Small Scale Plasmid DNA Isolation (80)

Small scale plasmid isolation was carried out using the

rapid boiling method of Holmes and Quigley (80). An

overnight culture (1.5 ml) of plasmid-containing bacteria

was transferred to a microcentrifuge tube and centrifuged in

an Eppendorf centrifuge at 12,000 X g for 2 minutes at room

temperature. The supernatant was decanted and the pellet

vortexed for 2 seconds and resuspended in 150 li STET buffer

(8% sucrose, 5% triton X-100, 50 mM EDTA, 50 mM Tris*Cl

(pH 8.0)). After resuspension, 38 pl of freshly prepared










lysozyme (10 mg/ml) was added to the cell suspension, the

mixture boiled for 60 seconds and centrifuged at 12,000 X g

for 10 minutes at room temperature. The pellet was removed

from the microcentrifuge tube using a toothpick and the DNA

precipitated from the supernatant with 188 Al isopropanol at

room temperature for 10 minutes. Following precipitation,

the DNA was pelleted by centrifugation at 12,000 X g for

5 minutes at room temperature, the supernatant decanted and

the pellet washed with 100 pA 70% ethanol at room

temperature. The DNA pellet was dried under vacuum,

resuspended in 40 l 10 mM Tris*Cl, 1 mM EDTA (pH 8.0)

containing DNase-free RNase (50 Ag/ml) and incubated for

30 minutes at 37C. Following incubation the DNA was stored

at -200C.



Large Scale Plasmid DNA Isolation

Large scale isolation of plasmid DNA was carried out

using the alkaline lysis method described in Birnboim and

Doly (11) and modified by D. Ish-Horowicz (87). A 500 ml

culture of plasmid-containing bacteria was centrifuged at

5000 rpm in a Beckman JA10 rotor (4400 X g) for 15 minutes

at 4C. The supernatant was decanted and the cell pellet

resuspended in 10 ml alkaline lysis solution #1 (see

appendix B) containing 50 mg lysozyme and incubated for

5 minutes at room temperature. After the incubation, 20 ml

of alkaline lysis solution #2 (see appendix B) was added,










the solution was mixed gently by inverting until no clumps

remained and further incubated for 15 minutes at 40C

followed by the addition of 15 ml ice cold 5M potassium

acetate (pH 4.8) and incubation at 4C for an additional

15 minutes. Upon completion of the incubation the solution

was transferred to a 50 ml polycarbonate centrifuge tube and

centrifuged at 16,000 rpm in a Beckman JA20 rotor

(31,000 X g) for 20 minutes at 4C. The supernatant was

transferred to a 150 ml corex centrifuge bottle containing

28 ml isopropanol, incubated at room temperature for

15 minutes and the DNA collected by centrifugation at

6000 rpm in a Beckman JA7.5 rotor (6700 X g) for 30 minutes

at 200C. After the centrifugation, the supernatant was

decanted and the pellet washed with 10 ml 70% ethanol at

room temperature. The DNA pellet was dried under vacuum and

resuspended in 8 ml 10 mM Tris*Cl, 1 mM EDTA (pH 8.0).

The DNA collected after isopropanol precipitation was

purified by CsCl buoyant density gradient centrifugation as

described by Clewell and Helinski (33). Solid CsCl, 8 g,

was added to the resuspended DNA (8 ml) and the solution was

mixed gently until all of the salt was dissolved. A

10 mg/ml stock of ethidium bromide, 0.9 ml for every ml of

DNA solution, was added and the final solution transferred

to Beckman quick seal ultracentrifuge tubes. The plasmid

DNA was banded by centrifugation at 53,000 rpm in a Beckman

70.1Ti ultracentrifuge rotor (270,000 X g) for 24 hours at










20C. The band of supercoiled plasmid DNA was collected

using an 18 gauge needle and the DNA passed over a cation

exchange column of Dowex 50W-X8 followed by chromatography

through a 30 X 1.5 cm BioGel A-15m column, developed with

10 mM Tris*Cl, 1 mM EDTA (pH 8.0). Fractions containing

plasmid DNA (Vo) were pooled and ethanol precipitated. The

purified DNA was quantitated and stored at either 4C or

-20C. Alternatively, after collection of the supercoiled

plasmid DNA band, the DNA solution was transferred to a

dialysis sac and dialyzed sequentially for 6 hours, at 40C,

against 3 1000 X volumes of 10 mM Tris*Cl, 1 mM EDTA

(pH 8.0). The purified DNA was then quantitated and stored

at either 4C or -200C.



Isolation and Purification of Eukaryotic Genomic DNA

Eukaryotic genomic DNA was isolated and purified

essentially as described by Gross-Bellard et al. (66) and

modified by Blin and Stafford (14). Approximately 2 X 107

eukaryotic cells were collected by centrifugation at

2500 rpm in an IEC rotor (1400 X g) for 5 minutes at 40C.

The supernatant was decanted and the cells gently

resuspended in cold PBS and recollected by centrifugation at

2500 rpm in an IEC rotor (1400 X g) for 5 minutes at 40C.

After centrifugation, the supernatant was decanted and the

pellet was vortexed gently, resuspended in 10 ml DNA lysis

solution (0.1 X SSC (20 X SSC: 3 M NaCl, 0.3 M sodium










citrate; pH 7.4), 0.5% SDS (w/v), 200 gg/ml proteinase K)

and incubated for 24 hours at room temperature with very

gently shaking. Upon completion of the incubation the

solution was extracted with an equal volume of equilibrated

phenol and centrifuged at 2500 rpm in an IEC rotor (1400 X

g) for 10 minutes at room temperature. Next, the solution

was extracted with an equal volume of CHC1/IAA (24:1, v/v)

and an equal volume of equilibrated phenol and centrifuged

at 2500 rpm in an IEC rotor (1400 X g) for 10 minutes at

room temperature. This extraction was repeated until a

clear interphase was visible. The final extraction was with

an equal volume of CHC1/IAA (24:1, v/v) followed by

centrifugation at 2500 rpm in an IEC rotor (1400 X g) for

10 minutes at room temperature. Upon completion of the

extractions the aqueous phase was split into two fractions

of equal volume and transferred to 30 ml corex centrifuge

tubes. The DNA was precipitated with 0.5 ml 3 M sodium

acetate and 15 ml 95% ethanol overnight at 4C. The

precipitated genomic DNA was hooked out with a bent glass

rod, resuspended in 10 mM Tris*Cl, 1 mM EDTA (pH 8.0) and

stored at 4C. In addition, cellular DNAs used for

chromosome localization were isolated as described (81,82).


Isolation and Purification of Mammalian RNA (131)

Isolation of total cellular mammalian RNA was carried

out as previously described (131). Cells growing in








28

suspension culture were harvested by centrifugation at 2500

rpm in an IEC rotor (1400 X g) for 10 minutes at 4C and

washed twice with cold 1 X spinner salt solution (GIBCO).

The cells were lysed in a solution (1.3 X 07 cells/ml lysis

solution) containing 1.6 mM Tris*Cl (pH 7.4), 0.8 mM EDTA,

5.0 gg/ml PVS, 2.0% SDS (w/v) and 500 jg/ml proteinase K for

15-30 minutes at room temperature. Following the

incubation, NaCl was added to 0.25 M and the aqueous phase

was extracted once with an equal volume of a 1:1 mixture

(v/v) of equilibrated phenol and CHC13/IAA (24:1, v/v) and

twice with an equal volume of CHCly/IAA (24:1, v/v). Upon

completion of the extractions the total nucleic acid was

precipitated using 24 Al 5M sodium acetate (pH 5.5) and

three volumes of 95% ethanol overnight at -20C. The total

nucleic acid was collected by centrifugation at 6000 rpm in

a Beckman JA7.5 rotor (6700 X g) for 30 minutes at 200C.

After the centrifugation, the supernatant was decanted and

the pellet washed with 10 ml 70% ethanol at room

temperature. The pellet was dried under vacuum, resuspended

in DNase I digestion buffer (20 mM Tris*Cl (pH 8.0), 10 mM

CaCl2) and incubated at 370C for 20-40 minutes with

0.1 mg/ml DNase I (electrophoretically pure) which had been

pretreated with proteinase K for 2 hours (174). Following

the incubation, NaCl was added to 0.25 M, SDS to 2.0% (w/v)

and extractions were carried out as described above. Upon

completion of the extractions the RNA was precipitated using











a final concentration of 25 mM sodium acetate and three

volumes of 95% ethanol overnight at -200C. The total RNA

was collected by centrifugation at 6000 rpm in a Beckman

JA7.5 rotor (6700 X g) for 30 minutes at 200C. Following

the centrifugation, the supernatant was decanted and the

pellet washed with 10 ml 70% ethanol at room temperature.

The pellet was dried under vacuum, resuspended in ddH20,

quantitated and stored at -200C.



Selection of Poly A+ RNA

Poly A+ RNA was selected using a single pass over oligo

dT-cellulose as described by Maniatis et al. (108). A

single oligo dT-cellulose selection greatly enriches for

poly A+ RNA but does not totally remove all of the poly A'

RNA. Total cellular HeLa RNA in loading buffer (10 mM

Tris-HCl (pH 7.6), 500 mM LiCI, 1.0 mM EDTA, 0.1% SDS (w/v))

was heated to 65C for 5 minutes and applied to prewashed

oligo dT-cellulose columns. The columns had been prewashed

with i) three column volumes ddH20, ii) three column volumes

0.1 N NaOH, 5 mM EDTA and iii) three column volumes loading

buffer. After addition of the sample to the column the

flow-through was collected, heated to 65C for 5 minutes,

cooled and reapplied to the column. The column was then

washed with 5 to 10 column volumes of loading buffer,

followed by 4 column volumes of loading buffer containing

100 mM NaCl. The poly A+ RNA was eluted with 2 to 3 column










volumes of elution buffer (10 mM Tris*Cl (pH 7.5), 1.0 mM

EDTA, 0.05% SDS (w/v)) and precipitated with 300 mM sodium

acetate (pH 5.2) and 2.2 volumes 95% ethanol overnight at

-20C. RNA was collected by centrifugation at 10,000 rpm in

a Beckman JA20 rotor (12,000 X g) for 30 minutes at 200C.

Following the centrifugation, the supernatant was decanted

and the pellet washed with 10 ml 70% ethanol at room

temperature. The pellet was dried under vacuum, resuspended

in ddH20, quantitated and stored at -200C.



Spectrophotometric Quantitation of DNA and RNA

Nucleic acids, both DNA and RNA, were routinely

quantitated by OD at 260 nm. An OD at 260 nm (OD26) of 1

corresponds to approximately 50 Ag/ml for double-stranded

DNA, 40 jg/ml for single-stranded DNA and RNA, and 20 Ag/ml

for oligonucleotides (108). The purity of the nucleic acid

was often assessed by calculating the 260 nm:280 nm optical

density ratio (OD260: D280). Pure preparations of DNA and RNA

have an OD20:OD280 of 1.8 and 2.0, respectively.



Preparation of Radiolabeled DNA

Random Oliqonucleotide Primed Labeling

A random oligonucleotide priming technique (47,48,169),

simplified by Roberts and Wilson (139), employing T7 DNA

polymerase (57,168), was carried out using a random primer

kit. The volume of a sample, containing 25 ng of DNA










template linearizedd plasmid DNA or an isolated DNA

fragment) to be labeled, was adjusted to 23 Al with ddH2O.

The solution was then heated in a boiling H20 bath for

5 minutes and centrifuged briefly to collect the

condensation. After heat denaturation, 10 Al of 5 X primer

buffer (supplied by the manufacturer), 5 pl labeled

nucleotide (-50 Mci) ([a-32P]dCTP (-3,000 Ci/mmole)) and

1 Al diluted T7 DNA polymerase (2 units/Al in enzyme

dilution buffer supplied by the manufacturer) were added to

the template DNA and the mixture was incubated at 37C for

2-10 minutes. Upon completion of the incubation period, the

reaction was quenched by the addition of 2 gl of stop

mixture. The volume of the reaction mixture was adjusted to

1.2 ml with Elutip-d low salt buffer (200 mM NaCl, 20 mM

Tris*Cl (pH 7.4), 1 mM EDTA) and the labeled DNA purified

using a Schleicher & Schuell Elutip-d column as described by

the manufacturer. The labeled DNA was stored at 4C until

use.



Labeling the 3' Termini of DNA (124)

DNA hybridization probes labeled at the 3' termini were

prepared as described by O'Farrell (124). The volume of the

solution containing the DNA fragment to be labeled was

adjusted to 12 Ml with ddH20. BSA, 2.5 Ml of a 1 mg/ml

stock, was added to the DNA solution along with 2.5 Al of

Boehringer Mannheim restriction endonuclease 10 X digestion










buffer L and 2.5 Al T4 DNA polymerase (1 unit/Al) and the

solution incubated for 2-5 minutes at 37C. Immediately

following the incubation, 1 pl of each of the following was

added: 2 mM dGTP, 2 mM dATP, 2 mM TTP and 2 Al (-20 Mci) of

[a-32P]dCTP (-3,000 Ci/mmole), and incubation was continued

for 1 minute at 37C. Upon completion of the incubation,

0.5 Al of 2 mM dCTP was added and the incubation continued

for 10 minutes at 37C. The reaction was stopped by heating

the solution for 5 minutes at 70C followed by the addition

of 1 pl of 250 mM EDTA. The volume of the quenched reaction

mixture was brought to 1.2 ml with Elutip-d low salt buffer

and the labeled DNA purified using a Schleicher & Schuell

Elutip-d column as described by the manufacturer. The

labeled DNA was stored at 4C until use.


Labeling the 5' Termini of DNA

DNAs to be labeled at the 5' termini and used as

hybridization probes were treated with CIP as described by

Chaconas and van de Sande (26) and modified by Maniatis

et al. (108), followed by 5' end labeling with T4

polynucleotide kinase and [y-32p]ATP (-3,000 Ci/mmole) as

described by Maxam and Gilbert (114) and modified by

Maniatis et al. (108). The volume of the solution

containing the DNA fragment to be labeled was adjusted to

44 Ml with ddH20O. CIP 10 X buffer (500 mM Tris.Cl (pH 9.0),

10 mM MgCl2, 1 mM ZnCI2, 10 mM spermidine), 5 pl, and 1 Ml










of CIP (0.2 units/Ml for DNA fragments with protruding

5' termini or 1 unit/Ml for DNA fragments with blunt or

recessed 5' termini) were added to the DNA solution and

incubated for 15 minutes at 37C followed by incubation for

15 minutes at 56C. Upon completion of the 560C incubation

a second aliquot of CIP was added to the reaction mixture

and the successive incubations were repeated. The reaction

was quenched by the addition of 40 Al ddH20, 10 il 10 X STE

buffer (100 mM Tris*Cl (pH 8.0), 1 M NaCI, 10 mM EDTA) and

5 Al 10% SDS (w/v), and the CIP was heat inactivated for

30 minutes at 68C. Upon completion of the incubation the

reaction mixture was extracted twice with an equal volume of

equilibrated phenol and twice with an equal volume of

CHCl3/IAA (24:1, v/v). Following the addition of 6.3 pl 5 M

NaCl, 1 pl glycogen (20 mg/ml) and 330 Al 95% ethanol, the

DNA was precipitated on crushed dry ice for 15 minutes and

pelleted by centrifugation at 12,000 X g for 30 minutes at

4C. After centrifugation, the supernatant was decanted and

the pellet washed with 100 Al 70% ethanol at room

temperature. The pellet of DNA was dried under vacuum,

resuspended in 44 pl 10 mM Tris*Cl, 1 mM EDTA (pH 8.0) and

stored at -200C until the 5' end labeling reaction was

carried out.

Spermidine, 1 pl of a 100 mM stock, and 5 pl 10 X

kinase buffer (660 mM Tris*Cl (pH 9.5), 100 mM MgCl2, 100 mM

2-mercapto-ethanol) were added to the solution of CIP








34

treated DNA and incubated for 5 minutes at 70C. After the

incubation, the solution was snap frozen in crushed dry ice.

The frozen solution was quickly thawed, added to 10 1l

(-100 Aci) of [y-32P]ATP (-3,000 Ci/mmole) and incubated for

30 minutes at 37C. The reaction was stopped by the

addition of 1 Al 100 mM EDTA and 1.2 ml Elutip-d low salt

buffer, and the labeled DNA was purified using a Schleicher

& Schuell Elutip-d column as described by the manufacturer.

The labeled DNA was stored at 4C until use.



Analysis of Recombinant DNA Clones

Mapping by Single and Multiple Restriction Endonuclease
Digestions

Single and double digests were carried out on plasmid

and A bacteriophage DNAs using restriction endonucleases and

analyzed electrophoretically on horizontal agarose gels

(108).



Indirect End-Labeled Mapping

Restriction mapping of recombinant A phage by indirect

end-labelling was carried out as described by Rackwitz

et al. (137) using hybridization conditions defined by

Little and Cross (103). Oligonucleotides complementary to

the cos sequence of X phage were 5' end labeled using

T4 polynucleotide kinase. Oligonucleotides (33 ng) in 1 pl

were combined with 1 pl 10 X kinase buffer, 2.5 Al (-25 Mci)

of [y-32P]ATP (-3,000 Ci/mmole), 0.4 Al 100 mM spermidine,










3.8 4l ddH20O and 1.5 pl of 10 unit/4l T4 polynucleotide

kinase and the reaction mixture was incubated for 30 minutes

at 37C. The reaction was stopped by adding 0.4 Al 250 mM

EDTA and 100 pA 10 mM Tris*Cl, 1 mM EDTA (pH 8.0) and

heating for 10 minutes at 70C. The reaction mixture was

extracted with an equal volume of a 1:1 mixture (v/v) of

equilibrated phenol and CHCl3/IAA (24:1, v/v). Following

the addition of 6.7 pl 5 M NaCI, 1 .l glycogen (20 mg/ml)

and 350 pl 95% ethanol, the oligonucleotides were

precipitated on crushed dry ice for 15 minutes and pelleted

by centrifugation at 12,000 X g for 30 minutes at 40C.

After centrifugation, the supernatant was decanted and the

pellet washed with 100 4l 70% ethanol at room temperature.

The pellet was dried under vacuum, resuspended in 100 pl 10

mM Tris Cl, 1 mM EDTA (pH 8.0) and stored at 4C until the

hybridization was carried out.

To 10 Al of partially restricted A phage DNA

(0.5-1.0 Ag) was added 1 4l of a solution containing: 2% SDS

(w/v), 50 mM EDTA, 1.0 sg/4l proteinase k and 0.026 ng of

5' end labeled oligonucleotides, and the mixture was

incubated for 10 minutes at 75C. Immediately following the

incubation, the mixture was transferred to 45C and

hybridized for 60 minutes. Following the hybridization, the

samples were placed on wet ice and 1 4l TPE tracking dye

(36 mM Tris*Cl (pH 7.7), 30 mM NaH2PO4 (monobasic), 1 mM

EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol,








36

10% glycerol) was added. The samples were then loaded onto

a 0.5% agarose TPE (36 mM Tris*Cl (pH 7.7), 30 mM NaH2PO4

(monobasic), 1 mM EDTA) gel and electrophoresed for 24-28

hours at 40 watts with buffer recirculation. Following

electrophoresis, the gel was dried under vacuum for 1 hour

at 800C. Autoradiography was performed using pre-flashed

Kodak XAR-5 X-ray film and a Cronex Lightning Plus screen at

-70C. I would like to acknowledge Dr. Paul L. Romain and

Suzie Pilapil for their work on the mapping analysis of the

AHHG5E genomic clone.



Southern Blot Analysis

Restriction endonuclease digested DNAs were separated

by electrophoresis through agarose gels and transferred to

Zeta-Probe blotting membrane as described by Reed and Mann

(138). The agarose gel was soaked in 25 mM HC1 for

10 minutes at room temperature, rinsed briefly in ddH20 and

the DNA was transferred with 0.4 N NaOH overnight at room

temperature. Following transfer, the membrane was baked in

a vacuum oven at 80*C for 1 hour, prewashed in 0.5% SDS

(w/v), 0.1 X SSC for one hour at 65*C and then prehybridized

and hybridized using a modification of the method described

by Wahl et al. (180). Prehybridization was carried out for

two hours in 50 mM sodium phosphate (pH 6.5), 0.45% SDS

(w/v), 47% formamide, 9% dextran sulfate and 500 Ag/ml

double stranded E. coli DNA. After prehybridization,








37

32P-labeled probe was added to the mix to a concentration of

1 X 106 dpm/ml and hybridized for 6-18 hours at 48C. The

hybridized membrane was then washed three times as follows:

1) in 100 ml 100 mM potassium phosphate (pH 7.4) for

30 minutes at room temperature; 2) in 100 ml 100 mM

potassium phosphate (pH 7.4) for 30 minutes at 60C; 3) in

100 ml 1 X SSC and 0.2% SDS (w/v) for 30 minutes at 600C.

Autoradiography was performed using pre-flashed Kodak XAR-5

X-ray film and a Cronex Lightning Plus screen at -70*C.

32P-labeled probes used for Southern analysis of the genomic

H2B clone IHHG5E were as follows: HI, 1445 nt Pst I/Eco RI

fragment from pFNC16 (131); H2A, 980 nt Sst I fragment from

pFF435 (112); H2B, 340 nt Eco RI/Xho I fragment from AHHC289

(Figure 3-6); H3, 2100 nt Eco RI fragment from pST519 (111);

H4, 1740 nt Hind III/Eco RI fragment from pFO108A (131).



Recovery of DNA Fractionated Electrophoretically

An agarose slice containing the DNA fragment of

interest was cut into very fine pieces, using a clean

scalpel, and the resulting agarose puree was then placed

into the pocket formed by folding a large piece of parafilm

into quarters. The puree was frozen on crushed dry ice

followed by thawing while applying pressure with the thumb

and forefinger. The resulting liquid which was forced out

of the puree was drawn off with a pipette and placed into a

clean tube. This process, including the freezing step, was








38
repeated until no additional liquid was squeezed out of the

puree. The DNA fragment was purified using a Schleicher &

Schuell Elutip-d column as described by the manufacturer and

was stored at -200C.



Analysis of Mammalian RNA

Aqarose-Formaldehyde Denaturing Gel Electrophoresis

RNA was size fractionated in a 1.5% agarose, 1 X MOPS

(20 mM MOPS (pH 7.0), 5 mM sodium acetate, 1 mM EDTA), 6%

formaldehyde gel using a 1 X MOPS, 3.7% (v/v) formaldehyde

running buffer, a modification of the method used by Lehrach

et al. (99).



Northern Analysis

Northern blot analysis was carried out as follows: the

RNA was size fractionated as described above and transferred

as described by Thomas (170) to Zeta-Probe blotting membrane

using 20 X SSC as the transfer buffer. Following transfer,

the membrane was baked in a vacuum oven at 80C for 1 hour,

prewashed in 0.5% SDS (w/v), 0.1 X SSC for one hour at 65*C

and then prehybridized and hybridized using a modification

of the method described by Wahl et al. (180).

Prehybridization was carried out for two hours in 50 mM

sodium phosphate (pH 6.5), 0.45% SDS (w/v), 47% formamide,

9% dextran sulfate and 500 Ag/ml double stranded E. coli

DNA. After prehybridization, 32P-labeled probe was added to








39
the mix to a concentration of 1 X 106 dpm/ml and hybridized

for 6-18 hours (the temperature of the hybridization was

dependent upon probe size and percent GC content and ranged

from 380C-480C). The hybridized Zeta-Probe membrane was

then washed five times as follows: 1) 10 minutes at room

temperature with 5 X SSC, 1 X Denhardt's; 2) 30 minutes at

60*C with 5 X SSC, 1 X Denhardt's; 3) 30 minutes at 60C

with 2 X SSC, 0.1% SDS (w/v); 4) 30 minutes at 60C with

1 X SSC, 0.1% SDS (w/v); 5) 30 minutes at 600C with

0.1 X SSC, 0.1% SDS (w/v). Autoradiography was performed

using pre-flashed Kodak XAR-5 X-ray film and a Cronex

Lightning Plus screen at -70*C. The developed X-ray films

were analyzed by laser densitometry using a LKB 2400 GelScan

XL densitometer. P-particle analysis of hybridized

membranes was carried out using a Petagen (Waltham, MA)

Betascope 603 blot analyzer.


Sl Nuclease Protection Analysis

Sl analysis was carried out according to Berk and Sharp

(6) as modified by Haegeman et al. (67). DNA fragments used

as probes (preparation described above) were treated with

CIP as described by Chaconas and van de Sande (26) and

modified by Maniatis et al. (108) followed by 5' end

labeling with T4 polynucleotide kinase and [Y-32P]ATP

(-3,000 Ci/mmole) as described by Maxam and Gilbert (114)

and modified by Maniatis et al. (108). Alternatively, DNA










fragments were 3' end labeled with T4 DNA polymerase and

[a-32P]dCTP (-3,000 Ci/mmole) as described by O'Farrell

(124). The RNA to be analyzed was co-precipitated with

labeled DNA probe using 0.3 M NaCI, 1.0 Al glycogen

(20 mg/ml) and 2.5 volumes of 95% ethanol for 15 minutes on

crushed dry ice and pelleted by centrifugation at 12,000 X g

for 30 minutes at 4C. After centrifugation, the

supernatant was decanted and the pellet washed with 100 Al

70% ethanol at room temperature. The pellet was dried under

vacuum, resuspended in 5 pl 5 X Sl hybridization buffer

(5 X: 2 M NaCI, 200 mM Pipes (pH 6.4), 5 mM EDTA) and 20 Ml

deionized, recrystallized formamide. Hybridization

mixtures, 25 pl, were heated at 900C for 10 minutes and then

annealed at 55C for at least three hrs. Annealing

reactions were quenched with eight volumes, 200 Al, Sl

nuclease buffer (25 mM NaCI, 30 mM sodium acetate (pH 4.6),

1.0 mM ZnSO4) followed by incubation at 37C for 30 minutes

with 900 units of Sl1 nuclease in the presence of 20 Mg

salmon sperm DNA. After Sl digestion the samples were

extracted with an equal volume of a 1:1 mixture of

equilibrated phenol and CHC13/IAA (24:1, v/v) and ethanol

precipitated using 0.3 M NaCl, 1.0 pl glycogen (20 mg/ml)

and 2.5 volumes of 95% ethanol overnight at -20C. The

protected fragment was pelleted by centrifugation at

12,000 X g for 30 minutes at 4C. Following centrifugation,

the supernatant was decanted and the pellets were washed










with 100 Al 70% ethanol at room temperature. The pellets

were dried under vacuum, resuspended in 6 pl loading buffer

(95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05%

xylene cyanol) and the samples were heat denatured and

loaded onto a 6% polyacrylamide sequencing gel containing

7.8 M urea (108). After electrophoresis the gel was soaked

in 10% acetic acid, 12% methanol for 15-30 minutes to remove

urea, dried under vacuum at 80*C, and placed against

preflashed Kodak XAR-5 X-ray film and a Cronex Lightning

Plus screen at -700C.



In Vitro Nuclear Run-on Transcription Analysis

The in vitro nuclear run-on transcription assays were

performed using a modification (4) of the method used by

Flint et al. (51). Cells were harvested by centrifugation

and the cell pellets washed twice in cold isotonic buffer

(125 mM potassium phosphate, 30 mM Tris-HCl (pH 7.9), 5 mM

MgCl2, 10 mM f-mercaptoethanol). Cells were disrupted by

homogenization (6-12 strokes) with a Dounce homogenizer,

Wheaton type A pestle. After >90% of the cells had been

lysed, nuclei were pelleted by centrifugation at 2000 rpm

for 10 minutes in an IEC centrifuge at 40C and resuspended

in nuclei storage buffer (50 mM Tris-HC1 (pH 8.3), 5 mM

MgC12, 0.1 mM EDTA) containing 40% glycerol. Nuclei were

aliquoted and used fresh in the in vitro transcription

reactions. Reactions typically contained 107 nuclei,








42
100 jCi a-32P-UTP (3000 Ci/mmole), 1 mM ATP, 0.25 mM GTP and

0.25 mM CTP in a final volume of 130 Ml and were incubated

for 30 minutes with intermittent shaking at 30*C.

Radiolabeled RNAs were isolated by treatment of nuclei with

DNase I (100 Ag/ml) in the presence of 0.6 M NaCl, 50 mM

Tris-HCl (pH 7.5), 20 mM MgCl2 for 15 minutes at room

temperature. The mixture was then incubated with

proteinase K (200 Ag/ml) for 30-60 minutes at 37*C in the

presence of 150 mM NaCl, 12.5 mM EDTA, 100 mM Tris-HC1

(pH 7.5) and 20 mM MgC12. Sodium acetate (pH 5.5) was added

to 200 mM and nucleic acids were extracted several times by

the hot phenol method (32,148). To the aqueous solution of
32P-labeled RNAs, 150 pg of yeast RNA and 2.5 volumes of 95%

ethanol were added. Precipitation was overnight at -20C.

Radiolabeled transcripts were resuspended in 1 ml of 10 mM

Tris-HCl (pH 8.0), 1 mM EDTA and an aliquot of each sample

was precipitated with 150 Ag yeast RNA and cold 10% TCA.

TCA-precipitable counts were determined by liquid

scintillation spectrometry.

Following preparation of Southern blots (157) of

electrophoretically-separated restriction endonuclease-

digested plasmid DNAs or slot blots (prepared using a slot

blot apparatus with conditions as described by Schleicher

and Schuell Inc.) of linearized plasmid DNAs, DNA excess

hybridizations were carried out. Southern or slot blots on

nitrocellulose were prehybridized in 1 M NaCl, 20 mM










Tris-HC1 (pH 7.4), 2 mM EDTA, 0.1% SDS (w/v), 5 X

Denhardt's, 25 Ag/ml denatured E. coli DNA and 12.5 mM

sodium pyrophosphate at 650C for at least 6 hours.

Hybridizations were conducted at 65*C for 72 hours in

1 M NaCI, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.1% SDS

(w/v), 2.5 X Denhardt's, 25 Jg/ml E. coli DNA with

32P-labeled transcripts at 5 X 105-1 X 106 TCA-precipitable

dpm/ml of hybridization solution. Blots were washed at 65C

for 15 minutes in fresh prehybridization solution, 1 hour in

2 X SSC, 0.1% SDS (w/v), overnight in 2 X SSC, 0.1% SDS

(w/v) and 1 hour in 0.2 X SSC, 0.1% SDS (w/v).

Autoradiography was performed on air dried filters with pre-

flashed XAR-5 or Cronex film and Cronex Lightning Plus

screens at -70*C. The developed X-ray films were analyzed

by laser densitometry using a LKB 2400 GelScan XL

densitometer. I would like to acknowledge Anna Ramsey-Ewing

for her work on the transcriptional analysis of the

H2B-GL105 gene.



Library Screening

Lambda qtll cDNA Library Screening

A Agtll human liver cDNA library, made from total

cellular poly A+ RNA and generously provided by Dr. S. L. C.

Woo (Baylor College of Medicine, Department of Cell

Biology), was screened with the 32P-labeled human histone

probes indicated in Figure 2-1. The library screening was










carried out as indicated in Figure 2-2 according to the

procedure described by Benton and Davis (5) with the

following modifications in the hybridization conditions.

The filters were prehybridized at 600C for two hours in a

solution containing 5 X Denhardt's (100 X: 2% ficoll, 2%

polyvinyl-pyrrolidone), 5 X SSPE (20 X: 3.6 M sodium

chloride, 200 mM sodium phosphate, 20 mM EDTA; pH 7.4), 0.2%

SDS (w/v), 0.1% w/v BSA and 500 pg/ml double stranded

E. coli DNA. Hybridization was carried out at 60*C for 24

hours in a solution containing 0.5 X Denhardt's, 5 X SSPE,

0.2% SDS (w/v), 0.01% w/v BSA, 500 pg/ml E. coli DNA and

2 X 106 dpm/ml heat-denatured probe DNA fragment.

Hybridized filters were then washed at 24*C for 30 minutes

in 100 mM potassium phosphate (pH 7.4) followed by two

30 minute washes at 60*C in 2 X SSPE, 0.3% SDS (w/v).

Autoradiography was performed using Kodak XAR-5 X-ray film

and a Cronex Lightning Plus screen at -700C.

Plaque dot analysis was carried out according to the

procedure described by Powell et al. (132). A 100 A.

aliquot of plating bacteria was placed into a tube and 4 ml

of NZCYM top agar was added to the tube, vortexed gently and

poured onto a NZCYM bottom agar plate. Upon solidification
















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of the top agar, 0.5 Al aliquots of I bacteriophage

dilutions were spotted onto the plate and allowed to dry.

The plate was inverted and incubated at 370C for 5-12 hours

to allow plaque formation. Up to 3 nitrocellulose filter

lifts were prepared from the plate and hybridized to various

probes as described in the previous section.



Lambda EMBL4 Genomic DNA Library Screening

An adult human lymphocyte genomic library, containing

inserts of between 15 and 20 Kb, was prepared by inserting

Sau3A I partially digested adult human lymphocyte genomic

DNA into the Bam HI site of XEMBL4 and was generously

provided by Dr. Paul Dobner (University of Massachusetts

Medical Center Department of Molecular Genetics and

Microbiology). A complete equivalent of this AEMBL4 human

genomic DNA library (8 X 105 phage) was grown on 14 150 mm

diameter petri dishes and screened with a 32P-labeled H2B 3'

non-translated probe (900 nt Sac I fragment isolated from

IHHC289) according to the procedure described by Benton and

Davis (5). LE392 (121) plating bacteria, 0.3 ml, were mixed

with 50,000 pfu of recombinant phage and incubated for

20 minutes at 37C. Next, 6.5 ml melted top agarose (460C)

was added and the mixture poured onto a 150 mm NZCYM bottom

agar plate. The top agarose was allowed to solidify at room

temperature and then incubated 10-12 hours (Or until the

plaques reach a diameter of approximately 1.5 mm) at 370C.










Following overnight incubation the plates were chilled for

60 minutes at 4C. For filter lifting, plates were removed

from the cold box and nitrocellulose filters carefully laid

on top of the agarose for 2-3 minutes. The orientation of

the filters was marked in three asymmetric positions using a

nitrocellulose marking pen. The filters were then removed

and soaked in denaturing solution (1.5 M NaCl, 0.5 N NaOH)

for 30-60 seconds and transferred into neutralizing solution

(1.5 M NaCl, 0.5 M Tris*Cl (pH 8.0)) for 5 minutes.

Subsequently the filters were rinsed in 2 X SSPE, blotted

dry on Whatman 3 MM paper and baked for 2 hours at 800C in a

vacuum oven. Hybridization of these filters was carried out

essentially as described by Lawn et al. (97) with the

following modifications in the hybridization conditions.

The filters were prehybridized at 600C for two hours in a

solution containing 5 X Denhardt's, 5 X SSPE, 0.2% SDS

(w/v), 0.1% w/v BSA and 500 pg/ml double stranded E. coli

DNA. Hybridization was carried out at 60*C for 24 hours in

a solution containing 0.5 X Denhardt's, 5 X SSPE, 0.2% SDS

(w/v), 0.01% BSA, 500 gg/ml E. coli DNA and 2 X 106 dpm/ml

heat-denatured probe DNA fragment. Hybridized filters were

then washed (10 ml wash solution/filter) at 24C for 0.5

hour in 100 mM potassium phosphate (pH 7.4) followed by two

0.5 hour washes at 60C in 2 X SSPE, 0.3% SDS (w/v).

Autoradiography was performed using Kodak XAR-5 X-ray film

and a Cronex Lightning Plus screen at -70*C. Following










hybridization, the positive plaques (or plaque areas) were

picked and placed into 1 ml of SM containing a drop of

chloroform. These positive plaques were screened a second

time by hybridization. Single, well isolated, positive

plaques were picked and screened a third time.



Cloning and Construct Preparation

Subcloninq into Plasmid Vectors

DNA fragments were isolated from agarose gels, and

ligations into the appropriate vectors were carried out

using T4 DNA ligase as described by the manufacturer. The

ligation products were used to transfect competent E. coli

DH5a (8) (see appendix B) bacteria. The 6100 nt Pst I

fragment, 4100 nt Eco RI fragment and 6100 nt Eco RI

fragment from IHHG5E (Figure 4-7) were isolated and cloned

into pUC19 and the resulting subclones termed pGL105, pGLl01

and pGL102, respectively.



Construct Preparation

Construct I (pGL105SV) is the control construct

containing the entire IHHG5E H2B gene, promoter and 2000 nt

of 3' untranslated region (Figure 2-3). The 6200 nt Pst I

fragment from IHHG5E was isolated and ligated, using T4 DNA

ligase, into the Pst I site of pUC19 to obtain the

intermediate (pGL105). Construct I was obtained by

isolating the Eco RI/Eco RI SV40 enhancer element from










pSVE108A (153), blunt-ending its termini with the Klenow

fragment of DNA polymerase I and lighting it into the Ban HI

site of pGL105 which had previously been blunt-ended with

the Klenow fragment.

Construct II (pGLI10SV) is similar to construct I but

has the 3' Eco RI/Pst I fragment, containing the poly(A)

addition sequences, deleted (Figure 2-4). The 4100 nt Eco

RI fragment from AHHG5E was isolated, blunt-ended using the

Klenow fragment and ligated using T4 DNA ligase into the

Hinc II site of pUC8SV40E, which contains the SV40 enhancer

element from pSVE108A (153), resulting in pGL110SV.

Construct III (pGL109SV) is similar to construct I but

has the 170 nt Xho I fragment, containing the histone 3'

hairpin motif, deleted (Figure 2-5). Preparation of

construct III was carried out as follows. The intermediate

pGL106 was prepared by isolating the 4200 nt Hind III

fragment from pGL105 and lighting it, using T4 DNA ligase,

into the Hind III site of pUC19. Next, the intermediate

pGL107 was prepared by digesting pGL106 with Xho I to remove

the 170 nt Xho I fragment containing the hairpin motif

region and recircularizing the remaining fragment using T4

DNA ligase. The final construct was obtained by isolating

the 4200 nt Hind III fragment from pGL107 and lighting it

into the 4300 nt Hind III fragment from pGL105SV to give

pGL109SV.













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Figure 2-5. Outline of the cloning scheme for the
construction of the H2B hairpin motif minus construct
pGL109SV

Construct III (pGL109SV) is similar to construct I but
has the 170 nt Xho I fragment, containing the histone 3'
hairpin motif, deleted (Figure 2-5). Preparation of
construct III was carried out as follows. The intermediate
pGL106 was prepared by isolating the 4200 nt Hind III
fragment from pGL105 and lighting it, using T4 DNA ligase,
into the Hind III site of pUC19. Next, the intermediate
pGL107 was prepared by digesting pGL106 with Xho I to remove
the 170 nt Xho I fragment containing the hairpin motif
region and recircularizing the remaining fragment using T4
DNA ligase. The final construct was obtained by isolating
the 4200 nt Hind III fragment from pGL107 and lighting it
into the 4300 nt Hind III fragment from pGL105SV to give
pGL109SV. Restriction endonuclease sites are as follows: B,
Bam HI; E, Eco RI; H, Hind III; P, Pst I; S, Sac I; S',
SDh I; X, Xho I; a letter containing a semicircle hat
indicates a restriction endonuclease site which has been
destroyed due to a blunt-ending reaction. Plasmid GL105SV
is described in Figure 2-3.















E@ ZBPS' X XH E

pGL 105SV


2Kb


2Kb


H


2Kb
PH


EPH



)
i' I


E E PH

pGL106





S2Kb
E-





2Kb 2Kb
PH X PHPH


SH PH




S2Kb
E
E' PS'X XH X PH

SpGL1 O9SV )


f


H
\f


E










Subcloning into Bacteriophage M13 Vectors

DNA fragments to be sequenced were isolated from

agarose gels and ligated into M13mpl8 and/or mpl9

replicative form (123,190) using T4 DNA ligase as described

by the manufacturer. The ligation products were used to

transfect competent XL1-Blue (21) (see appendix C) bacteria.


Transfection of DNA into Bacterial Cells

Preparation of Competent E. coli Cells for Transfection

Competent E. coli cells were prepared using a

modification of the method described by Mandel and Higa

(107). An overnight culture of E. coli was diluted 1:100

into 100 ml YTN medium and grown at 37C with vigorous

shaking until the OD at 590 nm reached approximately 0.37.

The cell culture was chilled on wet ice for 5 minutes, split

into two 50 ml tubes and then centrifuged at 2500 rpm in an

IEC rotor (1400 X g) for 10 minutes at 4C. Following

centrifugation the supernatant was discarded and the cell

pellets vortexed gently, resuspended in 10 ml cold sterile

transfection buffer (60 mM CaCl2, 10 mM Pipes (pH 7.0), 15%

glycerol) and centrifuged at 2500 rpm in an IEC rotor

(1400 X g) for 10 minutes at 4C. Next the supernatant was

discarded and the cell pellets were gently resuspended in 10

ml cold sterile transfection buffer and incubated for

30 minutes on wet ice. Upon completion of the incubation

the cells were centrifuged at 2500 rpm in an IEC rotor










(1400 X g) for 10 minutes at 4C, the supernatant was

discarded, and the cell pellets were gently resuspended in

2.5 ml cold, sterile transfection buffer. The competent

cells were stored at 4C and used within 24 hours of

preparation.


Transfection of Recombinant Plasmid DNA

Transfection of recombinant plasmid DNA was carried out

using a modification of the method described by Mandel and

Higa (107). A fraction of a ligation mixture (1/10-1/2) and

100 gl competent E. coli DH5a (8) cells were combined in a

1.5 ml microcentrifuge tube, incubated for 10 minutes on wet

ice and then incubated for 5 minutes at 370C. Following the

incubation the cells were diluted with 1.0 ml prewarmed YTN

medium and incubated with shaking for 1 hour at 370C. An

aliquot of the transfection mixture was plated onto the

appropriate bottom agar plate and incubated inverted for

12-18 hours at 370C.



Transfection of Recombinant Bacteriophage M13 DNA

Transfection of recombinant bacteriophage M13 DNA was

carried out as described by Messing (115). A fraction of a

ligation mixture (1/10-1/2) and 200 pl competent E. coli

XL1-Blue (21) cells were combined in a 14 mm X 100 mm glass

culture tube, incubated for 40 minutes on wet ice and then

heat-shocked for 2 minutes at 420C. Immediately following









61
the incubation, 4 ml of YTN top agar (45C) was added to the

tube, vortexed gently and poured onto a NZCYM bottom agar

plate. Upon solidification of the top agar the plate was

incubated inverted for 12-18 hours at 37C to allow for

plaque formation.



DNA Sequencing
Sanger Dideoxy-Mediated Chain Termination Method

Dideoxy sequencing (146) was carried out using a

Sequenase 2.0 DNA sequencing kit according to conditions

described by the manufacturer. After the sequencing

reactions were completed, loading buffer (95% formamide,

20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol)

was added and the samples were heat denatured at 720C for

2 minutes and loaded onto a 6% polyacrylamide sequencing gel

containing 7.8 M urea (108). Following electrophoresis the

gel was soaked in 10% acetic acid, 12% methanol for 15-30

minutes to remove urea, dried under vacuum at 80C using

3 sheets of Whatman 3MM paper as backing, and placed against

preflashed Kodak XAR-5 or Cronex X-ray film at room

temperature.



Chromosomal in situ Hybridization

Chromosomal in situ hybridization was performed as

described (23). Probe DNA (871 nt Eco RI/Sac I fragment

isolated from IHHC289 (35)) was labeled with 3H to a










specific activity of 2-4 X 107 dpm/gg. Slides containing

metaphase chromosomes from normal male (46 XY) peripheral

blood lymphocytes were aged at 4C for 7-10 days and

pretreated with ribonuclease A (Sigma) for 1 hour at 370C.

The chromosomal DNA was denatured at 70C for 2 minutes in a

70% formamide: 2 X SSC mixture (pH 7.0). The probe DNA was

denatured in a hybridization mixture containing 50%

formamide, 2 X SSC and 10% dextran sulfate (pH 7.0).

Hybridization was carried out at 370C overnight. After

rinsing at 39C in 3 changes of 50% formamide: 2 X SSC and 5

changes of 2 X SSC, slides were dehydrated, air dried,

subjected to autoradiography and banded with Wright's-Giemsa

stain solution mixed with 1-3 parts of a pH 9.2 borate

buffer (23).



Mammalian Cell Culture

HeLa Cell Culture and Synchronization

HeLa cells were grown in suspension culture (at

3-6 X 105 cells/ml) in Joklik-modified Eagle's minimal

essential medium supplemented with 7% calf serum and were

synchronized by two successive treatments with 2 mM

thymidine (162). The two, 14 hour, 2 mM thymidine

treatments were spaced 9 hours apart. Rates of DNA

synthesis were monitored by measuring the incorporation of

[3H]-thymidine into TCA-precipitable material in a 30 minute

pulse (162). DNA synthesis was inhibited by treating the










cell culture with 1 mM hydroxyurea for 1 hour, beginning

four hours after release from the second thymidine block.

Cells (hydroxyurea treated or untreated) were harvested at

various times after release from the second thymidine block.



HL60 Cell Culture and Differentiation

Human promyelocytic leukemia cells (HL-60 cells) were

cultured in liquid suspension in RPMI 1640 medium

supplemented with 10% fetal calf serum. HL60 cells were

plated at a cell density of 2 X 105/ml, grown for 24 hours

to a cell density of 3 X 105/ml and harvested by

centrifugation. For differentiation of HL60 cells,

cultures at a density of 3 X 105/ml were treated with TPA at

a final concentration of 16 nM, and harvested after periods

of 1, 2, 3, 4, and 5 days.



3T3L1 Cell Culture

Mouse 3T3L1 cells were maintained as subconfluent

cultures in DMEM containing 5% calf serum in a moist,

5% C02, 370C incubator.



Transfection and Transient Expression of Recombinant DNA in
3T3L1 Cells

Transfection of 3T3L1 cells was carried out as

described by Gorman et al. (56). Four hours prior to

transfection, 3T3L1 cells in monolayer at 20-40% confluency

were refed with fresh medium. The calcium phosphate/DNA










complex was prepared as described by Graham and Van der Eb

(58) using 10 pg of plasmid DNA and 10 ug of salmon sperm

DNA as carrier. Four hours after the addition of the

calcium phosphate/DNA complex the medium was removed and the

cells subjected to a one minute shock with 15-20% glycerol

(v/v) in medium containing 10% fetal calf serum. Incubation

was continued in fresh medium for 40-48 hours. Following

incubation, the cells were harvested and total cellular RNA

was isolated and analyzed by Sl nuclease protection

analysis.



Selection of Stable Polyclonal 3T3L1 Cell Lines

3T3L1 cell monolayers were transfected as above using

20:1 molar quantities of construct and pSV2neo (158) without

using carrier DNA (20 Mg total DNA quantity). Selection of

cells was carried out as described by Southern and Berg

(158) using Geneticin (G418 sulfate). The medium plus drug

was changed every three to four days until resistant

colonies grew to approximately 1 cm in diameter. Polyclonal

cell lines were established by combining resistant colonies

as follows: the medium from each plate (containing from 2-20

resistant colonies) was removed, and the plates were treated

with 2 ml trypsin (0.25% in buffered saline) for 2-5

minutes. The cells were rinsed from the surface of the

plate and a 0.2 ml aliquot of the heterogeneous cell

suspension was transferred to a plate containing fresh








65

medium and was maintained as a subconfluent culture in DMEM

containing 5% calf serum in a moist, 5% CO2, 370C incubator.

Ten polyclonal cell lines were established from the

transfection of each of the constructs described above and

are numbered P1 through P10. Cell lines 105-P7, 110-P10,

and 109-P7 were chosen for further analysis (see chapter 5)

based upon their ability to differentiate to adipocytes.



Induction and Differentiation of 3T3L1 Cells

Induction of 3T3L1 cells to adipocytes was carried out

as described by Bernlohr et al. (7). 3T3L1 cells were grown

to confluence and 3 days post confluence were given fresh

DMEM containing 10% fetal calf serum, 0.22 AM insulin,

0.6 AM dexamethasone and 0.5 mM 3-isobutyl-l-methyl-

xanthine. Forty-eight hours later the medium was replaced

with fresh DMEM supplemented with 10% fetal calf serum.

Adipocyte conversion was detected within 8 days of

induction.



Cell Culture for Chromosome Localization

Isolation, propagation and characterization of parental

human and murine cells as well as the human-mouse somatic

cell hybrids was carried out as previously described

(81,82,136) in Dr. Carlo Croce's laboratory at Temple

University School of Medicine, Fels Research Institute,

Philadelphia, PA. The human chromosomes (either partial or










complete chromosomes) retained in the rodent-human hybrids

used in determining the chromosomal location of the human

H2B histone gene are schematically illustrated in Figure

4-10B. I would like to acknowledge Shirwin Pockwinse, Dr.

Kay Huebner, Dr. Linda A. Cannizzaro and Dr. Carlo Croce for

their work on the chromosomal localization of the H2B-GL105

gene.


THP-1 Cell culture

Acute monocytic leukemia cells, THP-1 (173), were

maintained in suspension cultures in RPMI medium 1640

supplemented with 5% fetal calf serum and kanamycin (60

Ag/ml).














CHAPTER 3

ISOLATION AND CHARACTERIZATION OF A cDNA FROM A HUMAN
HISTONE H2B GENE WHICH IS RECIPROCALLY EXPRESSED IN RELATION
TO REPLICATION-DEPENDENT H2B HISTONE GENES
DURING HL60 CELL DIFFERENTIATION



Introduction

Replication-dependent histone genes do not contain

introns (164) and their mRNAs are structurally simple; they

are not polyadenylated and have short 5' leader and 3'

trailer sequences (77). However, replication-dependent

histone mRNAs do have a characteristic 3' stem-loop motif

(164). Replication-dependent histone genes are coordinately

expressed during the cell cycle and their expression is

coupled with DNA synthesis (3,74,126,130,131,176). The

abundance of the replication-dependent human histone mRNAs

is regulated at both the transcriptional and post-

transcriptional levels (reviewed in 150,164).

In contrast, replacement histone genes may contain

introns (20,182,183) and their mRNAs, which are

polyadenylated and frequently contain long 5' leaders and 3'

trailers, are structurally more complex than their

replication-dependent counterparts (20,28,45,71,72,182,183).

Although the processes governing replacement histone mRNA

levels are not well understood, it is clear that their mRNAs

67










(10,19,25,45,71,89,152,155) as well as tissue-specific

histone mRNAs (27,34,64,83,84,93) are not always regulated

in a replication-dependent manner.

In these studies we cloned and characterized a cDNA

from a variant human histone H2B gene which has a complex

pattern of regulation with respect to the HeLa cell cycle

and HL60 cell differentiation. Our results reveal a

reciprocal relationship during the onset of HL60

differentiation between the expression of the HHC289 H2B

gene and the replication-dependent H2B genes.



Results

Isolation of an H2B cDNA Clone From a Igtll Poly A+ cDNA
Library

A 1gtll human liver cDNA library was screened with the

32P-labeled human histone probes indicated in Figure 2-1.

The library screening was carried out as indicated in Figure

2-2. Upon completion of the third round of screening 151

H3/H4 positives and 158 H2A/H2B/Hl positive plaques had been

obtained. Using the human histone probes described in

Figure 2-1, 51 H3/H4 positives and 51 H2A/H2B/Hl positive

plaques were separated according to individual histone class

by plaque dot analysis (Figure 3-1). Two H1 clones, 9 H2A

clones, 22 H2B clones, 26 H3 clones and 5 H4 clones were

identified. No clone gave a positive signal with more than

one histone probe (Figure 3-1).


























Figure 3-1. Plaque dot hybridization analysis of putative
positive clones from the Xgtll poly A+ cDNA library
screening

Upon completion of the third round of Igtll library
screening (see Figure 2-2) 51 H3/H4 positives and 51
H2A/H2B/Hl positive plaques were separated according to
individual histone class by plaque dot analysis. Spot assay
analysis was carried out using 0.5 pl aliquots of
I bacteriophage dilutions -10 pfu. Upon plaque formation
2 filter lifts were prepared from the H3/H4 plate and 3 from
the H2A/H2B/Hl plate and each hybridized to an individual
histone 32P-labeled probe (see Figure 2-1) as indicated
above each panel. The filters were then washed and
autoradiography performed using Kodak XAR-5 film.


















| *** 0e a ** **u
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I,,,0100


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71
Restriction Endonuclease Mapping of Positive Clones Isolated
From the Iqtll Poly A+ cDNA Library Screening

Twelve clones suspected of containing human histone

genes were picked for further analysis (two from each

histone class as well as two randomly picked additional

clones) and mapped using a combination of restriction

endonuclease double digestions and southern blot analysis

(using the probes in Figure 2-1) as illustrated in Figure

3-2 for clone IHHC185. A restriction map of IHHC185 is

illustrated at the top of Figure 3-2. Restriction

endonuclease maps of the 12 positive clones isolated from

the Agtll poly A+ cDNA library and chosen for further

analysis are illustrated in Figure 3-3. Insert sizes for

the positive recombinant clones ranged from 490 nt for

IHHC176 to 2160 nt for IHHC289.



Sequencing Strategy for the Positive Clones Isolated From
the Agtll Poly A+ cDNA Library Screening

To evaluate whether any of the positive recombinant

clones contained histone coding sequence, the largest clone

in each of the histone classes was chosen for sequence

analysis. The arrows in Figure 3-4 represent regions of the

various Agtll clones which were sequenced by Sanger dideoxy

sequencing. Three of the clones (AHHC289, IHHC227 and

IHHC4) were found to contain histone protein coding sequence
























Figure 3-2. Restriction endonuclease Southern mapping of
XHHC185

Each lane of a 0.8% agarose gel was loaded with 3 Ag of
IHHC185 DNA restricted with: E, Eco RI; X, Xho I; S, Sac I;
H, Hind III; EH, Eco RI/Hind III; XH, Xho I/Hind III; SH,
Sac I/Hind III; EB, Eco RI/Bam HI. Lane U was loaded with
3 pg uncut IHHC185 DNA and lane M with 3'-labeled I DNA
digested with Eco RI and Hind III and with 3'-labeled pBR322
DNA digested with Hinf I. Following separation of the DNA
fragments electrophoretically, the DNA was transferred to
Zeta-probe nylon membrane and hybridized to a 32P-labeled
H2B probe (see Figure 2-1). A photograph of the above gel
stained with ethidium bromide is shown to the right of the
figure for reference. A restriction map of AHHC185 is
illustrated at the top of the figure. Igtll arms are
represented by closed boxes at the termini of the
restriction map. Restriction enzyme abbreviations are as
follows: E=Eco RI; X=Xho I; S=Sst I.


















S E X X



U E X S H EH M XH SH EB
mom


*


a


XHHC 185 MAPPING




























Figure 3-3. Restriction endonuclease maps of positive
clones isolated from the IAtll poly A+ cDNA library
screening

Restriction endonuclease maps of 12 positive clones
isolated from the Igtll poly A+ cDNA library screening are
illustrated. Restriction endonuclease sites are as depicted
in the top right corner of the figure. Igtll arms are
represented by closed boxes at the termini of the each
restriction map. A denotes a fragment giving a positive
hybridization signal with a histone coding probe.















S= DNA FLANKING ECO RI INSERTS


Whhc 227
Hl


176
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and 3 clones (AHHC289, AHHC227 and XHHC41) to contain a

stretch of poly(A) at the terminus of their inserts (Figure

3-4).


Sequencing of a Poly A+ H2B cDNA

Of the 22 cDNA clones containing H2B histone sequences

obtained from the initial screening of the lambda library

four were mapped using a combination of restriction

endonuclease double digestions and southern blot analysis

and the clone with the largest insert (Figure 3-3, IHHC289 -

2119 nt) that also contained a poly(A) tract (Figure 3-4)

was then selected for detailed sequence analysis. Regions

of the human H2B histone cDNA IHHC289 which were sequenced

are indicated by arrows in Figure 3-5 and the nucleotide

sequence is presented in Figure 3-6(A). The AHHC289 cDNA

encodes H2B amino acids 25-125 and the translation stop

codon. Also contained in the cDNA sequence is the poly(A)

addition sequence, AATAAA, located 1776 nt downstream from

the translation stop codon, and a poly(A) stretch located

16 nt downstream of the poly(A) addition sequence. The

protein coding region is GC rich (63%) whereas the 3' non-

translated trailer is relatively GC poor (45%). Shown in

Figure 3-6(B) are pertinent restriction sites and the

AHHC289 fragments used as probes for northern blot analysis.
















0 **

0) 0 0



S-i 0 '
Q) *H 41
: c 1i 44)



0 OH
C ) to
co 0 4
N k 0 4-)
u 4J *

B > *O
Z a) k- 4.)
* H .! 0


0 0


0 ) 4J
-H5 0
0



a(0) H



4U))
H 0 0
W C u J
0 *4
C >iT *


0 X H
*H 0 X!





( 410 H
to



O 0 V

i- C HH
k *H
k 0*-













I





UV
Ut








IC
vF n
X :t I
X f
1^ I 1
I8 I S
i ^i i


























Figure 3-6. (A) Nucleotide and deduced amino acid sequence
of the human H2B histone cDNA XHHC289. (B) Restriction
map of the cDNA IHHC289

(A) The IHHC289 cDNA encodes H2B amino acids 25-125 and
the translation stop codon. Also contained in the XHHC289
cDNA sequence is the poly A* addition sequence, AATAAA,
located 1776 nt downstream from the protein stop codon and a
poly A+ stretch located 16 nt downstream from the last
nucleotide of the poly A+ addition sequence. Solid double
underlined ( ) sequences represent potential splice sites.
(B) The H2B protein coding region is illustrated by a closed
box and the Agtll arms are represented by the open boxes.
Solid bars below the restriction map illustrates IHHC289
probes used for northern analysis. Restriction enzyme
abbreviations are as follows: E=Eco RI; X=Xho I; S=Sst I.












A GAC GGC AAG AAG CGC AAG CGC AGC CGC AAA GAG AGC TAC TCC ATC
,2Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Ile
TAC GTG TAC AAG GTG CTG AAG CAG GTC CAC CCC GAC ACC GGC ATC
.Tyr Val Tyr Lys Val Leu Lys Gin Val His Pro Asp Thr Gly lle
TCG TCC AAG GCC ATG GGC ATC ATG AAC TCC TTC GTC AAC GAC ATC
-Ser Ser Lys Ala Met Gly Ile Met Asn Ser Phe Val Asn Asp lle
TTC GAG CGC ATC GCG GGA GAG GCT TCC CGC CTG GCG CAC TAC AAC
.-Phe Glu Arg lle Ala Gly Glu Ala Ser Arg Leu Ala His Tyr Asn
AAG CGC TCC ACC ATC ACA TCC CGC GAG ATC CAG ACG GCC GTG CGC
,,Lys Arg Ser Thr Ile Thr Ser Arg Glu lie Gin Thr Ala Val Arg
CTG CTG CTG CCC GGC GAG CTG GCC AAG CAC GCC GTG TCC GAG GGC
,1oLeu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val Ser Glu Gly


ACC AAG GCG GTC ACC AAG TAC ACC AGC TCC AAG TGA
..Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys ***


GGACCTGGCG CTCGCTCGCT
CCACCCACCT AATCACTAGA
AGTAAGTTAT CTTAGTGTGA
GAACTC6AGG TCCCCAGTGC
CATTGTGCTG CTTAGCCTTC
CTTTACCCGC CCCCACTCCC
TCTAAAACGA AGTGGCTGAG
CCGGGTTCCG CAAACACTGC
TGTCCGGTCA CCAGTTCTGC
ATTTTGGACG AAGGCGGCAG
CAAAATTCTG CGCCTTTTTC
TTGAAATTCC TATTTCTCAT
TGGTAACGCT ACGGCCCCAG
GTCACCTCTC AGAGACCTAC
CTCATTTTAT CTTCCTTCTA
GTATTCTCAA GCCCTTGACA
ACATTATGGA CACTTAAATA
CCACCCTTTT CTTAATCGCT
GTATGTGAAG GCACAGTGAA
TTAGTCACCT CAGTGCAACA
AGGAGGTGGC AATGGTAGAT
TTTTCATCAA ATTCAGCGTG
TCTCCCCTCA TGGTGTTCCC
CATTTGCTTT TCTGGAGGCC
CAAATGAGGC CAAGAGAAGC
TGTATTCTCA GATGCAGGAC
TACTCTTTCT GTCCAACCCT
TCAGCTGTTT TTGGCTAAGG
AGCTCTAGAC ATTATCACAG
AATGTGTATA GATGCTATTG
AAAA


B 19.6


PROTEIN
CODING
540 nt 5'


CGAGTCGCCG
AAAGAGCTTG
AGGTCATGGG
GTCATTGGAT
CCAGGAGTCG
GCCCCACACG
TTCGGCTGTC
GTGACAGCTC
CGTGCGATGG
CCGGGTCCCA
TAATTTGTAG
TTTGTTGATA
GTCACTGCGA
GTCATCCACT
GCAGCTGTCT
GACCGGCTAG
CTACGTATTG
TCCGTGGATG
AATGGAAATG
GCTGGGAGGG
CCACCCTTAT
TTGGTCACTG
CTCTTAAAGG
ATGCAATATA
CTCATTGGTT
AATTGCATTT
TGATTCTGCC
GCTTTTGGAG
ACTGAATAGA
TTATTAATAA


GCTGCTTGAC
TTCACTTATT
AAATGGCATA
TTGCTTTTGA
GTTCTCAATT
CGCCCTGGTG
ATTTAAGAGA
TGTATGACTG
GGCCTCCTGT
GCCTTGTCCT
ATTTCAGTTT
ATTTCTGCAT
GGCACTTACC
CAGGAATTCG
GAAATTGGTT
TGTGGTTTTC
ATCTAATATT
GATGAAGGGT
TTCTTGGAGC
GGCCGTGTTA
GCTTCTCAGT
GAAAGAGCCT
AGAGGAGCTT
GGCGGGACTA
CACAGTCATG
TAGTTTTATT
GAGGAAGACA
CTGATGGCAG
TCTTAACTGT
AGTTACCAAT


GTC CCT GCC


TCCAAAGGCT
CCCTTAGTTT
CGTAGCTTTT
ATCTAGAGCG
AGGCTGTTGG
GCTCCTTGGG
ACTCCAGGAC
ACGCTTGGCA
GGATACCAGC
GATTGGGCGA
CCGTCGTTCA
TTAATGGTCT
ATGTAGATAC
CGCCTCTCAT
CGTCTGTTTT
CCGTGCATCT
GTTGGGTTAA
GCTGTTCATT
TACTTCCTCA
AGATTTTTTT
TTAGCATAAC
TTTCCTTCTC
TTAATTTACA
CAGAGTTAAT
CAGCTCATAC
TTGTGGAGGT
CTGATGGTTT
GGGTTTGATG
CTCCTACATG
TAATTTAAAA


CTTTTCAGAG
CTTTTCATAA
TAACTATTTG
TGTCTTTACT
GAATCCGCCT
TCTGTTTCAT
ACAATTCAGC
GCAGCTTTTG
CGTTCTGTGT
CAAGAATATT
CTTTGAGACT
GTGCTTTAAA
GGGCTCAAAA
ACTTGCCTGT
CTTGTTTATG
TCAGCCTGGC
TTTTTCCATC
TCCATTAGAT
AAATGTATCC
TGCTACAAAG
CTCTTATGGA
CTTTTCTTAC
CTTACCACCT
CTCCTTTTTA
TGTCCACCCT
GCAGAATATT
GATGAGTGAT
AATCCAAATG
TGTGTTTTCA
AAAAAAAAAA


375
435
495
555
615
675
735
795
855
915
975
1035
1095
1155
1215
1275
1335
1395
1455
1515
1575
1635
1695
1755
1815
1875
1935
1995
2055
2115
2119


24.1
SE S E HC89


PROBE 870 nt 3' PROBE








83

Comparison of the AHHC289 Predicted Amino Acid Sequence With
Other H2B Amino Acid Sequences

In Figure 3-7(A) is shown a comparison of selected

amino acids from the predicted histone H2B sequence of

IHHC289 with a multi-species consensus H2B protein sequence

(184), a human genomic sequence (134) and several chicken

genomic sequences (27,59,70). The amino acids shown vary at

the indicated positions among the genes reported in Figure

3-7(A). These amino acids are either species-specific, that

is, they are conserved among known chicken H2B genes but may

differ between the chicken and other species reported in

Figure 3-7(A) (amino acids 18, 21, 25 and 26), or subtype-

specific which may vary among H2B protein variants within a

species or between species (amino acids 30, 31, 32, 39, 60,

94, 122 and 124). The amino acid sequence data for human

H2B histones are too incomplete to permit classification of

the HHC289 H2B protein as a replication-dependent,

replacement, or tissue-specific variant. However, the

predicted protein is 97% similar to the replication-

dependent HHG39 H2B protein (134) with amino acid

differences at positions 39, 94 and 124 (Figure 3-7(A)) and

identical to the multi-species consensus H2B sequence

compiled by Wells (184). The IHHC289 predicted amino acid

sequence is also similar to the pHh4A/pHh4C H2B sequence

reported by Zhong et al. (194), except in the region of

amino acids 27 through 33.