HOST NUCLEIC ACID SYNTHESIS IN CHICKEMBRYO FIBROBLASTS FOLLOWING

INFECTION BY ROUS SARCOMA VIRUS

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Authors DeLamarter, John Frederic, 1948-

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DeLAMARTER, John Frederic, 1948-HOST NUCLEIC ACID SYNTHESIS IN CHICK EMBRYO FIBROBLASTS FOLLOWING INFECTION BY ROUS SARCOMA VIRUS.

The University of Arizona, Ph.D., 1976 Biology

Xerox University Microfilms, Ann Arbor, Michigan 48106

HOST NUCLEIC ACID SYNTHESIS IN CHICK

EMBRYO FIBROBLASTS FOLLOWING INFECTION BY

ROUS SARCOMA VIRUS

by

John Frederic DeLamarter

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF MICROBIOLOGY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN MOLECULAR BIOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 6

THE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by John Frederic DeLamarter

entitled HOST NUCLEIC ACID SYNTHESIS IN CHICK EMBRYO FIBROBLASTS

FOLLOWING INFECTION BY ROUS SARCOMA VIRUS

be accepted as fulfilling the dissertation requirement of the

degree of Doctor of Philosophy •

1 /-•< .

Dissertation Director Date T 6

After inspection of the final copy of the dissertation, the

following members of the Final Examination Committee concur in

its approval and recommend its acceptance:-

This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination.

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to bor­rowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made,, Requests for permission for extended quotation from or re­production of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in­terests of scholarship* In all other instances, however, permission must be obtained from the author.

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. Marshall

Dinowitz for his steadfast encouragement, constructive criticisms,

and generous friendship. Dr. Harris Bernstein's critical reading of

this dissertation and the timely encouragements of Drs. Harris Bern­

stein, Charles J. Gauntt, and David Mount were most helpful to me. I

also wish to acknowledge the friendship of many graduate students,

particularly that of Eileen Nonn, that greatly enriched my graduate

years.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES vi

LIST OF ILLUSTRATIONS ..... vii

ABSTRACT o © o o ® o o o ® . ® o « o . . . . . . . . . ® . . o I X

INTRODUCTION o o o o o o o o o o o o o o o o o . o . o o o o o I

RSV Life Cycle ooooo.o.eoooo.oooooooo 1 Provirus Synthesis o.o.o.ooooooo.oooo 2 Integration of the Provirus .0.000..0.000 3 Viral RNA Synthesis 00.00000000.000.0 ^

Virus-Host Interactions ................. 5 Cellular RNA Synthesis 5 Cellular DNA Synthesis and Division . 6 Endogenous Viral Sequences . 7 RNA Tumor Virus Origins .... ..... 8

Cellular Aspects ..... ... 9 Cellular DNA Synthesis ....... . o . o . . ® o . 9 Cellular RNA .00.000 O O . O . O O O D ® . . . ® 1 1

This Investigation 00.0.000000.0000.0.0 11

MATERIALS AND METHODS ... .00.00.00000.0.00 13

Cells OO.OO...............OOOOOO 13 Infections with RSV and Labeling ...... 13 Extraction and Preparation of Host Nucleic Acids ..... 1^

Preparation of Radioactive DNA . . . . . o o . . . . . 15 Extraction of Host RNA .................. 16

Nuclear RNA . . . . . . . . . . . ^ . . . . . o o o o 16 Validation of Protocol Modifications ......... 17 Polysomal RNA Extraction . . o o . . . . . . . . . . . 2 0

DNA/DNA Reassociation ...... ... 22 Assay of DNA Annealing ......000.0000.0 23

RNA/DNA Hybridization ..... .... 2b Assay of RNA/DNA Duplexes ......... 26 Modification of RNase Assay ... 26

iv

V

TABLE OF CONTENTS—Continued

Page

RESULTS 29

Cell Transformation . 29 Host Cell DNA Synthesis Following Infection ........ 29

Labeling of Host Cell DNA «>«*««ooo»o«»oo 32 DNA/DNA Reassociation Kinetics .............. 36 Analysis of Host RNA Synthesis Following Infection . . . » bj>

Labeling of Host Cell RNA .000.0.00.00000 bb Hybridization of Pulse-Labeled Host Cell nRNA » . o . o o o bb

RNA/DNA Hybridizations of Control Cell nRNA » . . o 0 «, ^6 InfeCted Cell nRNA ooeeoeoooeo.ooooOQ ^9

Constant DNA/RNA Ratio Comparison Hybridizations . . . . . 5b Polysome-Associated "Repetitive" RNA .......so 59 Labeling and Extraction of pRNA oo.oooo....e 59 Low CQt Hybridizations ....osoeoooooo.. 60

Post-Transcriptional Processing of "Repetitive" RNA . . . « Gb

DISCUSSION 66

DNA/DNA Reassociations . . * 66 Theoretical and Practical Aspects o 66 Observations and Interpretations 69

RNA/DNA Hybridizations . . . . . . . . . . . . . « o S S . 70 Theoretical and Practical Aspects . . . . . . o . o . o 70 Observations and Interpretations ....... . . . o 7 ^ Repetitive Sequence Transcripts ...........s 75

Conclusions ......... 8l

LIST OF REFERENCES Sb

LIST OF TABLES

Table Page

1. Comparison of digestion schemes for radioactive RNA ... 28

2. Summary of pulse-labeled DNA extractions ........ 33

3. Summary of reassociation kinetics for pulse-labeled Cellular DNA o.oe..eoe.oooeeeo.o..oo ^f2

k. Summary of pulse-labeled nuclear RNA extractions .... ^5

5. Summary of pulse-labeled polyribosomal RNA extractions . 62

vi

LIST OF ILLUSTRATIONS

Figure Page

1. Sephadex chromatography of DNase-treated nucleic acids . . 19

2. Sedimentation of the cytoplasmic fraction of chick cells on a sucrose gradient 00.0.0000000000000 21

3» Liability of RNA during RNA/DNA hybridization . » . » o . 25

k. Morphological transformation of normal cells by RSV infection o o o o o o o o o o o o o o o o o o o . o o o o 30

5. Partial synchrony of cellular DNA synthesis following subculturmg o o o o o o o o o o o o o o o o o o o o o o o 35

6. Reassociation analyses of uninfected cell DNA ...«.« 37

7. Composite of control-cell DNA reassociation curves . » . . 38

8. Reassociation analyses of RSV-infected cell DNA . . » o » kO

9. Composite of infected-cell DNA reassociation curves o . » *tl

10. Hybridization of normal CEF nuclear RNA ..o.ooooo ^7

11. Hybridization of RSV-infected cell nRNA with cell DNA ® o 50

12. Hybridization kinetics for unique sequence transcripts from RSV-infected and control cell nRNA ......... 51

13. Hybridization kinetics for repeat sequence transcripts from RSV-infected and control-cell nRNA ......... 53

1^-. Low CQt hybridizations of control cell and RSV-infected cell nRNA 0000.00.00000.000.0000.0 55

15. The fraction of nRNA hybridized by a CQt of 10 for control a n d R S V — i n f e c t e d c e l l s 0 0 0 0 0 0 0 0 . 0 0 . 0 0 0 . 0 0 5 ^

16. Hybridizations of nuclear RNA at a constant DNA/RNA r a t i o . O O O . O . O O . O O O O O 0 . . . O O O . . 0 0 5 8

17. Sedimentation profiles of cytoplasmic fractions from c o n t r o l a n d R S V - i n f e c t e d c e l l s . . . . . . . . . . . . . . 6 l

vii

viii

LIST OF ILLUSTRATIONS—Continued

Figure Page

18. Ratio of infected to control cell polysomal RNA hybrid­ized at a CQt of 15 63

19. The relationship between the polysomal and nuclear "repetitive" RNA fractions as a function of infection . . 65

20. Summary of cellular events after subculturing or infec­tion with RSV 82

ABSTRACT

Host cell nucleic acid synthesis was monitored for alterations

during infection by a transforming RNA tumor virus. Nucleic acids

pulse labeled during the transformation of infected cells were analyzed

by their annealing kinetics in liquid reaction mixtures,. When suscep­

tible chick embryo fibroblasts (CEF) were infected with Rous sarcoma

virus (RSV), within 2k hours individual cells showed transformed mor­

phology and by 50 hours the entire culture was morphologically trans­

formed.

Conditions used for subculturing and infecting CEF cells re­

sulted in partial synchrony for DNA synthesis of both normal and

infected cells, VJhen normal cells stopped synthesizing DNA, the addi­

tion of fresh serum to the culture medium induced new DNA synthesis®

For DNA samples pulse-labeled at different times during the provirus

phase of RSV replication (3-12 hours after infection), DNA/DNA re-

association kinetics were very similar whether the DNA was derived

from infected or control cells.

Transcription in normal and infected cells was analyzed by

liquid RNA/DNA hybridizations of pulse-labeled RNA. The fraction of

repetitive sequence transcripts in normal cell nuclei decreases

slightly during active cell growth compared to cultures of confluent

monolayers. Similarly the repetitive sequence transcripts found on

the polyribosomes from normal cells represent a smaller fraction in

ix

X

rapidly dividing cells. However, late after infection by RSV (25-50

hours), nuclei of infected cells contained twice the fraction of repeat

sequence transcripts as control cell nuclei® By approximately 15 hours

after infection, repetitive sequence transcripts found on infected-cell

polyribosomes show half the concentration of those from control cells.

The observed changes in representation of repetitive sequence RNA in

the different cellular compartments suggests that the transport of

these transcripts may be inhibited in RSV-infected cells. Very late

(58 hours) after infection — at a time when control cells had reached

confluency and infected cells were all transformed as well as having

reached saturation density — the proportion of repeat sequence tran­

scripts were similar for control and RSV infected cells in both the

polyribosomal and nuclear RNA fractions. These data are consistent with

models which have assigned regulatory functions to the transcripts of

repetitive-sequence DMA.

INTRODUCTION

RNA tumor viruses replicate in an intimate relationship with

their host cell (Tooze, 1973)« More than host RNA and protein machinery

is required for the synthesis of their progeny (Temin, 1971a)„ Alter­

ations in cellular functions that follov; infection are indications of a

virus-host inter-relationship existing at the level of the gene0 In­

fection of susceptible cells with the RNA tumor virus, Rous sarcoma

virus (RSV), causes major inheritable changes in cell morphology (Temin

and Rubin, 1953; Temin, 1962) and metabolism (Bissell, Hatie and Rubin,

1972; Buck, Glick and Warren, 1971) including continued production of

virus. These characteristics of RSV infection of its host, chick

embryo fibroblasts (CEF), make it an appropriate system in which to in­

vestigate altered regulation of host-cell nucleic-acid synthesis. In

this study CEF cellular DNA and RNA were analyzed to determine whether

there were differences in DNA replication and transcription following

infection by RSV.

RSV Life Cycle

Replication of RSV genomic single-stranded RNA is a complex

process beginning with virus adsorption to appropriate receptors on the

cell surface (Piraino, 1967), penetration of the cell membrane by

phagocytosis (Crittenden, 1968), and probable uncoating of the virus

in the cytoplasm (Tooze, 1973)• From this point on the fate of the

virion RNA is not certain. In an electron micrographic study, Dales

2

and Hanafusa (1972) showed virion-sized parental RNA in autoradiograms

of the host cell nucleus one hour post infection. Synthesis of a DNA

intermediate, the provirus, is the next major event in RSV replication.

However, evidence establishing its existence conflicts with the nuclear

location for the parental RNA just described,,

Provirus Synthesis

Temin (1962) first postulated a DNA intermediate for RSV repli­

cation based on the Mendelian inheritance of viral information in cells

once transformed by RSV. Indirect evidence accumulated to support this

hypothesise Virus production proved to be sensitive to agents inter­

acting with DNA such as Actinomycin D and Bromodeoxyuridine (Temin,

1963; Bader, 196^; Boettiger and Temin, 1970; Balduzzi and Morgan, 1970).

Observations yielding indirect support to the DNA provirus hypothesis

culminated in the discovery of the virion associated RNA-dependent DNA

polymerase (reverse transcriptase) by Temin and Mizutani (1970) and

Baltimore (1970). Only very recently has it been possible to directly

isolate the proviral DNA. Varmus and co-workers (Varmus, Guntaka, Deng

and Bishop, 197*0 used RSV infected Duck cells from which they ex­

tracted 0o002& of the total DNA representing proviral sequences as

shown by hybridization to viral 70S RNA.

Several studies are pertinent to the provirus synthesis. Again

using the RSV-duck cell system, Varmus, Guntaka, Deng and Bishop (197*0

showed cytoplasmic synthesis of viral DNA beginning at 3 hours after

infection with transfer to the nucleus seen at 6 hours reaching a

maximum at 9«5 hours. This time frame had been indicated

by previous investigations. For example, Bader and Brown (1971) had

induced mutations in RSV (an RNA virus) by use of a DNA analogue

(Bromodeoxyuridine, BrdU) finding that only the first 12 hours follow­

ing infection proved to be sensitive to the mutagen,, During neither

the preceding nor the following 12 hour periods was viral replication

susceptible to the presence of the mutagen,. These studies implicated

the time between 3 and 12 hours after infection for synthesis of the

proviral DNA„ Rather than existing as a viral episome, however, RSV

DNA appears to integrate its sequences into the host chromosome (Varmus,

Guntaka, Deng and Bishop, 197*0 in a manner somewhat analogous to the

insertion of lambda phage into the bacterial chromosome (Hershey, 1971)-

Integration of the Provirus

Baluda and Nayak (1970) demonstrated increased viral sequences

in host DNA following transformation of cells by an avian oncornavirus.

They hybridized avian myeloblastosis virus (AMV) labeled RNA to trans­

formed myeloblast cell DNA and found an increased hybridization when

compared to control cell DNA. Using similar viral-RNA/cellular-DNA

hybridizations, Neiman (1972) demonstrated that RSV transformed cells

contained many more copies of a part of the transforming genome plus

a more complete copy of the viral sequences. While these studies

documented the integrated state of the viral genome in transformed

cells, the integration step remained unproven. Schincariol and Joklik

(1973) prepared radioactive viral DNA in vitro from the RNA template

by reverse-transcriptase and used this to probe infected cell DNAe

Their data show an increasing number of copies of viral sequences

beginning at 3 hours post infection such that by 10 hours twice as many

copies of viral DNA sequences were present as in uninfected cells<>

Samples taken at later times yielded approximately 3 copies of the

viral sequences present per cello Their work did not rule out possible

plasmids for the increased number of copies seen. Varmus, Vogt, and

Bishop (1973)? however, cleverly demonstrated viral integration into

the host chromosome. Taking advantage of the normal repeated sequences

found in eukaryotic DNA, these investigators demonstrated that network-

made by denaturing and quickly reannealing infected host cell DNA

showed increasing amounts of virus-homologous sequences as infection

proceededo Subsequently, they presented data which placed viral DNA in

the host nucleus 6 hours after infection (Varmus, Guntaka, Warner et al,

1974). The average copy number/nucleus in infected cells increased

to 0.8 by 9„5 hours after infection with 90$ of the RSV genome repre­

sented,, Taken together these investigations demonstrate that the first

12 hours after infection are the critical period for proviral integra­

tion into the host chromosome. Evidence indicates that synthesis of

viral RNA follows this step.

Viral RNA Synthesis

Studies with Actinomycin D had indicated that viral RNA was

transcribed from a DNA template. Near the end of the proviral phase

(10 hours post infection) the first detectable increases in viral RNA

following infection sire found (Schincariol and Joklik, 1973). The con­

centration of viral RNA continued to increase until late after infec­

tion (96 hours). Infectious virus production was not detected until

5

18 hours after the infection and leveled off after 72 hours. This se­

quence is consistent with the timing of synthesis and integration of

the provirus and the fact that viral transcription requires the host

nuclear RNA polymerase form II (Jaquet et al„, 197^5 Rymo et al„, 197*M

Dinowitz, 1975b)o

Virus-Host Interactions

Information concerning host-virus interdependence is limited.

The topics investigated to date have of necessity been restricted by

our understanding of virus specific and normal cell functions.

Cellular RNA Synthesis

RSV infection has been shown to increase the rate of cellular

RNA synthesis in general and of one class (ribosomal RNA, rRNA) in par­

ticular (Colby and Rubin, 1969). The synthesis of RNA in transformed

cells is also more resistant to low concentrations of BrdU and to

a-amanitan than normal cells (Levinson et al., 1970; Dinowitz, 1975a).

Recently the activation of embryonic globin genes in CEF cells by RSV

infection has been reported (Groudine and Weintraub, 1975). These in­

vestigators found 100-500 transcripts/cell of the embryonic globin gene

sequence, but none of the adult globin sequence. From these data it

can be inferred that RSV infection and transformation of CEF cells

results in a difference in the general regulation of transcription as

well as the expression of specific genes.

6

Cellular DNA Synthesis and Division

The requirements for cellular division or DNA synthesis in the

life cycle of RSV remains unresolved,, The necessity of these host

functions has been investigated both in transformed cells continuously

releasing virus and in newly infected cells becoming transformed by

RSV «

Leong, Levinson and Bishop (1972) studied the effect of the

host cell cycle on virus production in cells transformed by RSV„ When

fresh serum was added to transformed cells grown in serum-free medium,

the cells left the early "Gi" phase and progressed through the cell

cycle in a synchronous fashion0 Using the virion-associated reverse-

transcriptase product for hybridizations with cellular RNA, the authors

showed maximum viral RNA synthesis occurring in cells from early "S"

phase® Virion protein synthesis followed during the early "G^" phase.

Similarily, Paskind, Weinberg and Baltimore (1975) grew Moloney Murine

Leukemia virus transformed mouse cells in medium with reduced serum to

induce cell cycle synchrony,. Release of the cells from serum depri­

vation resulted in DNA synthesis and virus release as measured by

assaying reverse-transcriptase activity in the culture fluids® These

studies link viral production to the host cell cycle®

Temin (1967a) investigated the requirement for cellular DNA

synthesis and division in transformation of the host„ These experi­

ments produced evidence in favor of host cell division as a requirement

for RSV transformation of CEF cells® Conversely, data derived from

mitotic inhibitor studies were interpreted by Bader (1972; 1973) to

show virus-induced cell transformation without cell division® More

7

recently, Humphries and Terain (197*0 used chick cells synchronized for

cell growth by serum deprivation to study this relationship„ In these

experiments cells were infected 12 hours prior to the addition of fresh

serum which had previously been shown to induce cell DNA synthesis0

Cells treated in this manner did not synthesize detectable viral RNA,

as assayed by hybridization of cellular RNA to viral DNA until 18 hours

after the addition of serum* Furthermore, if these cells were stimu­

lated with serum in the presence of colchicine, the detection of viral

RNA was delayed 12-2^ hours over colchicine-minus controls,, The

authors interpret these results as supportive of a requirement for cell

DNA synthesis and division before infection by RSV can result in virus

replication®

Endogenous Viral Sequences

The presence of avian leukosis and sarcoma viral sequences in

most chick cells is now well documented (Rosenthal et al., 1971; Baluda,

1972; Neiman, 1972; Neiman, 1973)» Expression of these sequences in

the form of a viral protein (gs) and a helper virus activity (chf) has

been shown to be determined by inherited genes which are not coordi-

nately controlled (Hanafusa et alc, 197*0° Whether these regulatory

genes are cellular or viral in origin is not clear (Tooze, 1973)» Some

form of transcriptional control appears to exist for these nearly

ubiquitous endogenous viral sequences» Hayward and Hanafusa (1975)

have recently isolated a new Rous associated virus (RAV 60) which they

believe to be recombinant between the host's endogenous virus and the

infecting exogenous virus. These authors used reverse-transcriptase

viral DNA from RAV 60 to show partial homology with the exogenous viral

RNA, host cell RNA, and the endogenous viral DNA. Their data indicate

that infecting viruses can interact v/ith endogenous viral sequences to

yield recombinant genomes- The exchange of genetic information ob­

served shows a close relationship between some host and viral DNA®

RNA Tumor Virus Origins

Two theories attributing cellular origins to oncornaviruses are

described here as possible links between cellular and viral functions0

Temin (1970) published his protovirus theory following the discovery of

the RNA dependent DNA polymerase0 Simply put, the theory suggests that

RNA tumor viruses are one of the current evolutionary products of an

original "protovirusThe protovirus was a piece of the cellular

genome capable of being copied into either RNA or DNA and replicating

in the later form (Temin, 1971b)«, Cellular information was transferred

from cell to cell by infrequent infection v/ith this uncoated nucleic

acido Protoviruses then would be potential sources for single-

generation information transfer in a stable form,, Their evolution to

an enveloped virion with associated reverse-transcriptase can readily

be imagined in terms of the selective advantage accrued,,

Recently, Gillespie and Gallo (1975) have also argued that RNA

tumor viruses originated from normal cell transcripts via a mechanism

they term "paraprocessing«M They have integrated evidence which sup­

ports the idea that virion RITA resembles normal cell nuclear RNA which

has not undergone the usual processing to messenger RNA (mRNA), hence

paraprocessingo This process, v/hen coupled with cytoplasmic transport,

acquisition of a cellular reverse-transcriptase, and budding from the

cell membrane could account for the origins of many of the character­

istics currently associated with RNA tumor viruses,, Paraprocessing as

a possible mechanism for oncornavirus evolution is an attractive theory

for two reasons that are also valid for the protovirus theory,, Both

these hypotheses give plausable reasons for the _a priori existence of

these viruses plus the special relationship that exists between their

host and viral genomes.

Cellular Aspects

The analyses of cellular nucleic acid synthesis undertaken in

this study were done to identify virus-induced changes in normal cell

metabolism,, To provide the framework for these investigations, normal

cell functions including information about synchronous synthesis of

DNA, transcriptional control, and the nature of cellular RNA are dis­

cussed below0

Cellular DNA Synthesis

Eukaryotic DNA replication involves discontinuous synthesis

initiated at many sites (Callan, 1973). Since chromosomal replication

does not begin at a unique origin and progress in a linear manner to

completion, distinct classes of DNA sequences may be synthesized at

different times. Virus infections might then influence the timing of

synthesis for these classes. Linear replication of eukaryotic chromo­

somal DNA during a single period has been challenged by a number of

investigators (Brown and Blackler, 1972; Tobia, Schildkraut and Maio,

10

1970; Gall, 1969; Dickson, Boyd and Laird, 1971). Their evidence indi­

cates several alternative modes for replication; including over-

synthesis followed by degradation, extra-chromosomal synthesis, and/or

amplification of genetic information., Klevecz, Kapp and Remington

(197^) synchronized several different mammalian cell lines by mitotic

selection and then monitored their DNA synthesis by long (30 minute)

3 and short (2 minute) pulses with H-thymidine. Long pulses showed a

single peak of DNA synthesis, while shorter ones showed discontinuous

synthesis throughout "S" phase. These data suggest the transient syn­

thesis and catabolism of undefined classes of the cell's DNAo

Balazs, Brown and Schildkraut (1973) investigated the temporal

replication of different cellular cistrons. They labeled the DNA

replicated at different times during "S" phase of a synchronously

growing SV4o transformed mouse cell line. These investigators deter­

mined that integrated SVkO sequences were maximally replicated in

middle "S" phase at approximately tv/ice the rate of early and late "S"

phaseo In the same study they report an alteration in the time of

satellite DNA replication during the induced DNA synthesis that fol­

lows SVkO infection of permissive cells* In uninfected synchronously

dividing mouse cells, satellite DNA replicates very late in the "S"

phase, but infection by SV^O causes the replication of satellite se­

quences to be shifted to the period of bulk DNA synthesis. These data

imply that specific cistrons replicate at different times during the

"S" phase and that viral infections may cause temporal alterations in

the order in which some repetitive DNA is replicated.

11

Cellular RNA

In the nucleus of eukaryotes, large heterogeneously-sized mole­

cules of RNA (HnRNA) are rapidly synthesized which contain both repeti­

tive and unique DNA sequence transcripts including sequences homologous

to protein-specific mRNA (Lewin, 1975a)» Poly-adenylate tracts are

added post transcriptionally to the 3' end of the HnRNA and the mole­

cule is probably then cleaved to a single protein messenger size in a

manner analogous to the processing of ribosomal RNA (Greenberg, 1975;

Lewin, 1975b)» In growing normal cells only about bC#> of the unique

sequence diversity present in nuclear RNA (nRNA) is found on polyribo­

somes (Grady and Campbell, 1975a)„ On the average this nRNA is rapidly

turning over (half-life of approximately 23 minutes) compared to some

classes of mRNA which have half-lives of 2b hours (Brandhorst and

McConkey, 197^5 Perry and Kelley, 1968; Singer and Penman, 1973)«. The

existence of repeat sequence transcripts in mRNA similar to those in

nRNA has been uncertain,, Recent evidence supports the contention that

two distinct classes of mRNA exist — the major fraction being tran­

scribed only from unique sequences and a minor fraction transcribed

solely from repeat DNA (Campo and Bishop, 197^5 Klein et al., 197*0 •

This Investigation

Host cell nucleic acid synthesis was studied during the conver­

sion of normal CEF to RSV transformed cells. Secondly, alterations of

a relatively transient nature were identified by pulse labeling with

radioactive precursors® In this manner, temporal changes as a function

12

of RSV infection in both cellular DNA synthesis and transcription were

examined.

Host cell DNA pulse-labeled during the synthesis and integra­

tion of the RSV provirus was analyzed by denaturation and reassociation

of the labeled DNA. Results of this technique indicate the relation­

ship between different reiteration classes of DNA synthesized at the

time of the pulse (Britten and Kohne, 1968)0 Analogous changes to the

shift in replication of the highly repeated mouse satellite DNA by SVbO

infection were sought throughout the period of host and viral DNA

interactionso

Host cell RNA was pulse labeled during the conversion of the

RSV-infected culture from normal to transformed morphology. Cells were

fractionated into nuclear and cytoplasmic components in order to sepa­

rately investigate RNA from these two cellular compartments. The RNA

found in the nucleus and that subsequently appearing on the cytoplasmic

polyribosomes was examined by RNA/DNA hybridizations. Similar to DNA

reassociation, the RNA hybridizations carried out in this study deter­

mined the fractions of RNA transcribed from DNA templates that occur

in repeat or single copy frequency in the genome (Wilt, 1971)®

The period after infection that was investigated corresponded

to the time of probable maximum alterations for both cellular DNA and

RNA synthesis due to the virus infection. Since repetitive sequences

have been hypothesized to play a role in the regulation of growth and

differentiation in eukaryotic cells (Britten and Davidson, 19&9)»

changes in the relationship between repetitive and unique sequence

elements were sought during these periods®

MATERIALS AND METHODS

Cells

Ten to 11 day old chick embryos (Kimber Farms; Freemont, Ca„)

were decapitated, evicerated, minced, and trypsinized (0„05$> trypsin

in TBS /tris(Tris hydroxy-methyl-aminomethan)-buffered saline;(Vogt,

1969)/, 39°C) for 5 minutes, three times., The cell suspension was

plated on 100 mm plactic petri culture dishes (Falcon Plastics) and

passaged at least once before use. As a source of Rous sarcoma virus

(RSV), a second cell-type culture was maintained,, These were the con­

tinuous line of RSV transformed and virus-producing cells, RTAZ-1,

established by Dr. Marshall Dinowitz (1976).

Cell cultures were grown in standard medium (SM) consisting of

medium 199 (GIBCO) supplemented with 10^ tryptose phosphate broth, 8^

calf serum and 2% fetal calf serum,, To retard microbial growth 100

units/ml penicillin, 100 /xg/ml streptomycin, and 2.5 /ig/ml amphotericin

B (Fungizone, Squibb) were added to the medium. Cells were incubated

in a humidified CO^ incubator under an atmosphere of 95$ air and 5%

C02 at 39°C.

Infections with RSV and Labeling

Infections of CEF were carried out with tissue culture fluids

from confluent RTAZ-1 cultures. The culture medium was clarified by

centrifugation to remove cells and cellular debris (5 minutes, 2,000xg).

This supernatant routinely contained 2-k x 10^ FFU/ml (Dinowitz, 1975c)

13

when assayed for focus formation on CEF (Temin and Rubin, 1958)e Cells

were infected in suspension with a mixture of fresh medium and the

clarified tissue culture fluid in equal volumes.. Control cells were

mock infected as above by substituting conditioned medium from normal

cell cultures, ioe„, normal cell tissue culture fluids clarified and

mixed with an equal volume of fresh medium. Cells were infected at a

multiplicity of infection (moi) of ~3 focus forming units (FFU).

For experiments where DNA was to be examined, the cells were

exposed to 5 HCi/m± ^H-thymidine (TdR) (k0-60 Ci/mM; NEN) during the

indicated 3 hours of incubation. Cells in which RNA was to be examined

were pulse-labeled with 50 /iCi/ml ^H-(5?6) uridine C+0-50 Ci/mM; MEN)

for 2 hours. Incorporation of radioactive precursors were terminated

by removing the cells from the culture dish and placing them in ice-cold

TBS."

Extraction and Preparation of Host Nucleic Acids

Chick embryo fibroblast DNA was prepared from 10-11 day old

chick embryos decapitated, eviscerated, and minced in TBS. Nuclear

cell DNA was extracted from this tissue by a procedure modified from

that of Marmur's (1961)= Cells were placed in reticulocyte standard

buffer (RSB: 0.01 M tris, pH 7®^; 0.01 M NaCl; 105 mM MgC^) and

homogenized in a dounce homogenizer. The disrupted cells were centri-

fuged at 1500 x g, 5 minutes and the nuclear pellet was resuspended in

a NaCl-EDTA buffer (0.15 M, 0.1 M) which was made 1% in sodium dodecyl-

sulfate (SDS) and then 1 M in sodium perchlorate. The suspension was

15

extracted three times with an equal volume of chloroform-isopentyl

alcohol (v/v, 2^:1) by shaking for 15 minutes in a wrist-action shaker

and separating the phases by centrifugation (1600 x g for 10 minutes).

The DNA was spooled from the final aqueous phase by adding two volumes

of 9% ethanol, resuspended in IX SSC(0„15 M NaCl, 0.015M sodium

citrate), treated with bovine pancreatic RNase A (50 fxg/ml, previously

boiled for 10 minutes to eliminate DNases) and a-amalyse (50 /ig/ml) for

30 minutes at 37° C followed by pronase (250 g/ml, 15-18 hours, 37° C)0

The DNA was then extracted two more times as above, spooled, resuspended

in IX SSC, re-extracted once, spooled again, and finally resuspended in

3 mM EDTAo Normal CEF DNA prepared in this manner exhibited absorbance

ratios of 260nm/230nm of >2. and at 260nm/280nm of >1.8. For all DNA

samples the DNA concentration was determined by absorbance at a wave­

length of 260nm (21 O.D. units=lmg/ml). This DNA was then sheared by

sonication in an ice bath (10, 1 minute pulses separated by 0.5 minute

cooling periods) using a Heat Systems-Ultrasonic, sonifier (Heat Sys­

tems Inc., Plainview, N.Y.).

Preparation of Radioactive DNA

Pulse-labeled cellular DNA was extracted and prepared in the

same manner as unlabeled cell DNA except that total cellular DNA was

isolated. The concentration of DNA was too low to allow spooling of

the nucleic acid; therefore, for each spooling step in the previous

procedure pelleting of the DNA by centrifugation (27*000 x g, 30 min­

utes) was substituted. Radioactivity was monitored during the extrac­

tion by counting trichloroacetic acid (TCA) precipitatable DNA.

Aliquots were spotted onto Whatman 3MM filter dies, dried and treated

twice with cold 5^ TCA„ The filter dies were then rinsed in cold 9%

ethanol, dried and counted in a liquid scintillation fluid gm BBOT/

L of toluene) used for the liquid scintillation, spectrophotometer.

Extraction of Host RNA

Nuclear RNA

Labeled cells were fractionated into cytoplasmic and nuclear

fractions using the Non-idet NP^O procedure (Borun, Scharff and Robbins,

1967) as modified to substitute Triton X-100 (Dinowitz, 1975b) «> Cells

were washed with cold TBS, then treated with cold 0e5/e Triton X-100 in

RSB. After 5 minutes incubation at *f°C the lysed cells were removed

with the aid of a rubber policeman, placed in a tube, vortexed vigor­

ously, and centrifuged to pellet the nuclei (1500 x g, 5 minutes)» The

supernatant was retained as the cytoplasmic fraction for immediate pro­

cessing as described below. Further cleaning of the nuclear pellet was

accomplished by resuspension in RSB; making the solution 0.5^ in sodium

deoxycholate, vortexing, then 1% in Tween 0, and mixing again (Penman,

1966). Nuclei were pelleted from the wash by centrifugation as before

and the supernatant discarded. The clean nuclei were then extracted by

a modified protocol of Penman (1966). The extraction was carried out

by resuspending the nuclei in a lysis buffer (lOmM tris, 1 mM EDTA,

lOOmM NaCl, 0o5% SDS, pH 7°*0 and the suspension was extracted with an

equal volume of phenol (redistilled), chloroform, isopental alcohol

(50:^8:2, v/v/v) saturated with the lysis buffer shaken for 5 minutes

on a wrist-action shaker. The extraction mixture was centrifuged at

10,000 x g for 5 minutes and the solvent removed from the bottom, then

replaced by an equal volume of fresh solvent» After repeating the ex­

traction three more times, the aqueous phase was removed, made up to

0.15 M NaCl, 2 volumes of cold 95^ ethanol were added to precipitate

the RNA and the mixture was stored at -20°C for three or more hours®

Precipitated nucleic acids were pelleted by centrifugation at 27,000

x g for 30 minutes at it-°C0 The pellet was resuspended in a DNA diges­

tion buffer (50 mM tris, 25 mM NaCl, 10 mM MgC^, 2o5 niM CaCl^, pH 7®2),

100 /ig/ml bovine pancreatic DNase I was added and the samples were in­

cubated at 37°C for 30 minutes- These samples were then chromatographed

through a G-50 Sephadex column using 0,15 Mo NaCl as the eluent buffer.

The column had been previously washed with 2 column volumes of buffer,

brought to pH 11 by passing a 0.1 M NaOH solution through it in order

to denature RNase which may have been present, then returned to neutral

pH by flushing with 5-10 more column volumes of buffer,. Void volume

fractions were collected from the loaded column and the RNA was pre­

cipitated by ethanol and pelleted as described above.

Validation of Protocol Modifications

Coupling the DNase with sephadex chromatography is a modifi­

cation of the Penman technique suggested by Birnie et al.,(197if). In

further modifications, extractions were initially carried out with

CaCl^ present in a modified lysis buffer to allow for an earlier DNase

step as described by Penman (1966). This addition caused nearly 90$>

of the labeled nucleic acid to remain either in the solvent phase or

18

at the solvent-aqueous interface. By using the lysis buffer described

above, extractions resulted in the retention of greater than 8($ of the

total input radioactivity..

To test whether the DNase-sephadex step was to be an effective

method for removing the small DNA fragments remaining after DNase

treatment, eluent fractions were examined to determine where DNase-

treated DNA was eluted from the column,, Purified native DNA eluted

exclusively in the void volume of the G-50 Sephadex column (Fig. 1A).

The DNase-treated material eluted at about one half column volume

•z latere Nucleic acids labeled for 2k hours with H-adenosine, extracted,

DNased, and chromatographed as before resulted in radioactivity eluting

in both the void volume and the position characteristic of DNA frag­

ments (Fig® IB). Since this nucleotide is a precursor to both DNA and

RNA, an elution profile consistent with the presence of both nucleic

acids was expected,, In contrast, nucleic acids pulse-labeled for 2

3 hours with H-uridine, DNased, and chromatographed as above eluted

exclusively in the void volume (Fig. IB).

Nuclear RNA samples were resuspended in 2x glass distilled water

and the RNA concentrations measured in a spectrophotometer by absorbance

at a wavelength of 260 nm (2k 0D„ units = 1 mg/ml). Recovery of RNA

was monitored during the extraction procedure by measuring TCA-

precipitable radioactivity as described for the labeled DNA® . For use

in hybridization reaction mixtures, nRNA samples were concentrated by

precipitation with ethanol pelleting and resuspending in the required

amount of 10X SSC.

19

a o

8

7

6

5

4

3

2

I

O

4

A

o—°—. os *0

—gz §/> —©—o—o—®-

B to

o

5 CL o

t

l.Q-0-

Ul

t i

f * °X /

A---oy * '•r\ CXzzS—A—A.-- A--- ""A- DV Oy

I I I I I I I 10 20 30 40 50 60 70

FRACTION NUMBER

Figure 1. Sephadex chromatography of DNase-treated nucleic acids. — Nucleic acids were digested with DNase I, chromatographed on a G50 Sephadex column, and fractions collected as described in Materials and Methods. The radioactivity in eluent fractions was determined by counting in a scintil­lation spectrophotometer. Panel A shows the elution pro­file for purified, native DNA (s o) and for the same DNA after DNase treatment (o- - -o)«, Panel B depicts elution profiles for purified DNase-treated cellular nucleic acids, which were either labeled with 3H-adenosine for 2k hours ( • •) or 3jl-uridine for 120 minutes (A A).

Polysomal RNA Extraction

Cytoplasmic fractions isolated as described above were immedi­

ately layered onto a sucrose sedimentation gradients Linear 15-30^

(w/v) sucrose gradients with RNase free sucrose in RSB were centrifuged

for 90 minutes at 200,000xg in a Spinco SW *f0 rotor at k°Ca Fractions

were collected from the bottom of the gradient through a continuous

flow cell in the light path of a spectrophotometer set at a wavelength

of 260 nm. Figure 2 shows typical tracing from one of these gradients

demonstrating typical polysome, monosome (80S) and ribosomal subunit

(60S and hOS) peakso Fractions representing the polysomes (up to, but

not including the monosome peak) were pooled, mixed with an equal vol­

ume of 2X SDS buffer (SDS buffer is 0.1 M NaCl; 0.025 M tris, pH 7.^;

Q.OJfa SDS), precipitated with cold ethanol, and pelleted as described

before« The pellets were resuspended in a tris-EDTA-NaCl-SDS buffer

(0.01 M tris, pH lnM EDTA, 0.1 M NaCl, and 1% SDS) and extracted

by a method designed to minimize the loss from the polysomal fraction

of RNA's containing poly-adenosine tracts (Perry et alo, 1972). The

resuspended polysomes were extracted 3 times with an equal volume of

phenol-chloroform (1:1, v/v) saturated with 0.01 M Acetate, pH 6.0,

containing 0.1 M NaCl, and IrriM EDTA. Extractions included 5 minutes

of shaking on a wrist-action shaker followed by centrifugation at

10,000 x g for 5 minutes to separate the phases,, After the final ex­

traction the aqueous phase was precipitated at -20°C with cold 9

ethanol and the precipitate was pelleted as before. The pellets were

resuspended in 2X distilled water, the concentration of RNA was

Figure 2. Sedimentation of the cytoplasmic fraction of chick cells on a sucrose gradient.

The cytoplasmic fraction of CEF cells, prepared as de­scribed in the text, was centrifuged on a linear 15-3^ sucrose sedimentation gradient at 200,000 x g for 90 minutes at k°C. The gradient was collected from the bottom through a continuous flow cell placed in the opti­cal path of a spectrophotometer set at a wavelength of 260 nm. The tracing shown is a record of the absorbance at 260 nm exhibiting polysomal peaks as seen in a typical gradient.

21

80s

0.2

E c O CD CM

I-<

LLI O ;2: < CD CC O CO CD <

0

0.0

Pooled Polysomal Fractions

Bottom «s5- Top Direction of sedimentation

Figure 2. Sedimentation of the cytoplasmic fraction of chick cells on a sucrose gradient.

determined by optical density and the radioactivity measured by TCA-

precipitable counts. The RNA in these samples was concentrated by

ethanol precipitation and resuspension in 10X SSC. Radioactivity in

the samples was again determined by TCA precipitable counts.

DNA/DNA Reassociation

Reassociation analysis of denatured, pulse-labeled, host cell

DNA was performed by a modification of the original Britten and Kohne

(1967; 1968) procedure,, Reaction mixtures contained sheared, labeled

DNA and normal CEF DNA, phosphate buffer (PB: equal volumes of mono and

dibasic sodium phosphate, pH 6.8), and 0.2% SDS. The concentrations

of the excess, unlabeled DNA and the PB were of two values chosen to

readily reach low or high Cot values in short incubation times where

CQt is the initial concentration of the DNA (moles/Liter) multiplied

by the time of incubation (seconds) (Britten and Kohne, 1968). For low

2 CDt reactions (CQt <10 ) the excess, unlabeled DNA concentration was

p 0.5 mg/ml and PB was 0.12 M. High CQt reactions (CDt >10 ) on the

other hand, contained 5 mg/ml excess, unlabeled DNA and 0.'+ M PB. Mix­

tures of the appropriate concentrations of PB and unlabeled DNA plus

labeled DNA (800-5,000 cpm/reaction mixture) were sealed in microcapil-

lary tubes (2.5-25 ML), placed in a boiling water bath for ten minutes

to denature the DNA strands, plunged into an ice-water bath, and incu­

bated for the appropriate times at 60°Co Triplicate reaction mixtures

were removed from the 60°C water bath, terminated by rapid immersion in

an ice-water bath, and then stored at -20°C until assayed. The

resultant equivalent CQ s were calculated from the time of incubation

and the final concentration of total DNA present, corrected for rate

acceleration due to salt concentrations (Britten and Smith, 1970)•

Assay of DNA Annealing

The extent of reassociation was assayed by the single stranded

specific nuclease, SI (Ando, 1966) purchased from Sigma Chemical Co.

(St. Louis, Missouri)o This enzyme will digest single-stranded nucleic

acids and portions of duplexes that are not complementary (Sutton,

1971)« Before using the SI preparation, the enzyme was diluted 1:1

with a solution of 50/6 glycerol in SI digestion buffer (0.03 M Sodium

Acetate buffer, pH 5; 0.01 M ZnSO/^. and 10 /ig/ml denatured, calf

thymus DNA) and the activity was titrated with labeled single stranded

DNA. The concentration needed to completely digest ten times the DNA

present was determined and used in subsequent assays. Aliquots of the

enzyme were sorted frozen at -70°C until used.

Reassociation reaction mixtures were thawed and diluted into

1 ml of Sl-digestion buffer. The final concentration of PB present did

not exceed 2-h mM as greater concentrations inhibited the enzyme ac­

tivity (Dinowitz, 1975c). Reaction mixtures were divided equally into

two 0.5 ml aliquots. One tube received an appropriate amount of SI

nuclease and the other served as a control. After digestion at 50°

for 2 hours, 0.05 mg/ml of calf thymus DNA carrier was added to each

tube followed by k ml of TCA. After 20-60 minutes incubation of the

mixture at ^°C, the contents of the tubes were filtered through Whatman

2b

glass filter fiber dies (GF/C), washed twice with cold 5% TCA, dried

and placed in 5 ml of organic scintillation fluid and counted in a

liquid scintillation spectrophometer. The percentage of double-stranded,

labeled DNA present in the various samples was calculated by dividing

the precipitable counts in the SI digested tube by those in the un­

treated tube. Digestion of known, sheared, single stranded and native

DNA standards were included to monitor the SI nuclease activity.

RNA/DNA Hybridization

All hybridizations performed followed the essentials of a pro­

cedure described by Neiman et al„ (197*0» These reaction conditions,

termed liquid hybridization in modest DNA excess, were chosen for their

ability to show nearly complete hybridization of a single-copy sequence

RNA molecule. Reaction conditions were designed to reduce loss of RNA

by degredation over long periods of incubation at elevated temperatures.

Neiman, Purchase and Okazaki (1975) found no loss of precipi­

table CPM after 672 hours of incubation, but a halfing of the size of

the RNA every 168 hours. In this laboratory greater than 959^ of the

input radioactive RNA remained acid precipitable for 200 hours incuba­

tion under these conditions. A trend in the loss of precipitable

counts was observed following this time (Fig. 3)» Extrapolation of

this curve indicates that 1/2 the input RNA would be acid soluble

3 after 1.9 x 10 hours of incubation. However, hybridization incuba­

tions did not exceed 900 hours in any of these experiments.

Pulse-labeled RNA in 10X SSC was mixed with an equal volume of

normal CEF DNA which had been resuspended in formamide. Reaction

(3 2 2 < 5 UJ a:

1.0

q: UJ _i m <

S0.5 o LU a: a a o < 2 o i— a <0.0

.90 x I03hrs.

0 LOGj0TIMEOF INCUBATION (HOURS)

Figure 3. Lability of RNA during KNA/DNA hybridization. — Hybridization reaction mixtures made to a final concentration of 50^ in formamide and 5X SSC, contained equal volumes of cellular DNA (25.5 nag/ml) and radioactive RNA (60-300 fig/ml). The fraction of the initial labeled RNA remaining acid-precipitable is shown as a function of incubation time at 50°C.

ro VJ1

26

mixtures were drawn into micropapillary pipettes (2.5-5 /*L/pippet),

sealed, and denatured in a boiling water bath for 10 minuteso These

reaction mixtures were then incubated at 50°C for up to four weeks to

5 reach a Cct of 10 (Neiman et al., 197*0. Following the appropriate

incubations, replicate samples were removed from the 50° water bath,

immersed in an ice-water bath and stored frozen at ~20°C until a com­

plete set was ready to assay.

Assay of RNA/DNA Duplexes

Hybrid formation was assayed by a modification of the method

described by Klein et al. (197*0 • Reaction mixtures were diluted into

1 ml of 2X SSC, split into equal aliquots with one tube receiving 10

pig/ml bovine pancreatic Ribonuclease A and the other serving as an un­

digested control. The digestion mixtures were incubated at 22°C for

one hour. The method for precipitation and counting of the undigested

RNA hybrids was the same as that described for DNA/DNA reassociations.

As was previously reported (Neiman et al„, 1975; Klein et al., 197*0?

a fraction of input RNA is resistant to digestion under these con­

ditions before hybridization. To account for this resistant fraction,

duplicate hybridization reaction mixtures were frozen unincubated fol­

lowing the denaturation step. The percentage of labeled RNA remaining

in these samples after RNase digestion was subtracted from the values

obtained for the incubated samples.

Modification of RNase Assay

To obtain maximum digestion of RNA which had not hybridized

with DNA, several digestion conditions were tested. Various

27

combinations of RNase A and RNase T1 at 25°C or 37°C incubation for 1

hour were examined. The maximum digestion of non-hybridized cytoplas­

mic RNA in solution was obtained using RNase A (10 fj.g/ml) plus T1 (10

units/ml) with incubation for 1 hour at 37°C (Table 1)» Hence, RNA/

DNA hybridizations in later studies were assayed using both RNases A

and T1 under the conditions just described. Unincubated hybridization

reaction mixtures digested by this scheme averaged 3-^9° resistant

counts which were subtracted from the percent hybrid found in incubated

samples.

28

Table 1, Comparison of digestion schemes for radioactive RNA.a

RNase A (10 jig/ml)

RNase T1 (10 u./ml)

RNase A (10 g/ml)+ RNase T1 (10 u./ml)

25°C/60 min. 9.1(3)b 91.5(*0 8.1(4)

k.k(k)

37°C/60 min. 6.5CO 5^.6CO 3.25(5)

a. Approximately 3i000 cpm/sample of cellular H-RNA in 2X SSC was treated with RNase(s) as shown. The average fraction of radio­activity remaining acid-precipitable after the treatments listed is expressed as a percent of the controlT incubated without RNase present.

b. The number of samples digested.

RESULTS

Cell Transformation

The time course for conversion of susceptible cells to tumor

cells by RSV infection was established for the conditions used in sub­

sequent experimentso V/hen CEF cells were infected with RSV as de­

scribed under Methods, individual rounded cells overlaying neighboring

cells were noted within 2b hours (Fig* *fA and B) . During the next 2b

hours these characteristic morphological changes became apparent for

all cells of the culture (Fig. and D). At this time control cells

had grown to confluent monolayers and the transformed cells had reached

high population density. The analyses of nucleic acids isolated from

infected cells reported below were done with cultures that were consis­

tently transformed over the time course shown in Figure b.

Host Cell DNA Synthesis Following Infection

Host cell DNA, pulse-labeled after infection, was examined by

DNA/DNA reassociation analysis for differences in the relationship be­

tween reiterated and unique sequences. If the proportion of these

classes of DNA synthesized were altered by infection, this change should

be evident in the reassociation kinetics of the cell DNA. Experiments

designed to explore the effect that RSV infection has on the relation­

ship of newly synthesized repeat to single copy DNA are presented.

29

,{

30

ms-.: •

5 '• -V ••"•:;•••' - ; •

5 r v • ' . • " ; ; • ••

.v '• .*•

. \ \ • '• • • • o* »• , V V'V

•v; s:;

• • • :

W.

v-

-V T

\

. v ' Si-

'"v

. ;•

• " " ,\ . v,t, v -' v

: -• - • X. , A. . • *..,,y

\ ' •

•a ' ':v '•••}'•'

• ; :v;

• ' '• ''

"

Figure k. Morphological transformation of normal cells by RSV infection. — Confluent monolayer cultures of CEF cells were infected as described in Materials and Methods. Equivalent cultures 2*f hours after plating of uninfected cells (Panel A) and infected cells (Panel B) are shown. The same cultures are shown 50 hours after subculturing in Panel C, and infection in Panel D (mag. = V76X).

V.

31

ifOj/p : :V.

'• **' ' J • ' •

y J» .-..4 . tf , • > t . S • i . ' - - :

V 5 >"• • ' ' 'r W-.'t ; • :

• '>„• •' /'v y. • V - V.

D ' " r t ' ' ^ ~ yyyy'• L/ • .'• . •'••• "U'.~ : V---' '• - '-•••

• . -.'4 ? : '' ° y ,

> '•SsV :'>-v'. y ~. . ' •-'•'• i -

f; M.' .. -,•> •.-••; .: -6 ?>;.'• .-•••• - ••.•:•..•• w / ° . , ' ' • " • ;•'••••. ••••.' •••,,. ;>.i< '. •' .< J-1 ; •• ."•• v.'- ' 1 '"• '

-:.v. t f • ... '. - '••' • •

, v -

' / r - ' t ' . C , . V C I • >7, * !r," • '

-,-y •^'•ys^y.

;'' • !. '"-V* •••' '•,• .i'i "ft "r'V; . o...• • ' -"V/ • • -V y !

, •• ^ ••• : -t-. ^ J"" , >'%•«)* •

- • •• ' •--/ . r>- _ ~ ;>_ • .

Figure continued. Morphological transformation of normal cells by RSV infection.

Labeling of Host Cell DNA

The period of proviral synthesis and integration (3-12 hours

after infection; Varmus, Guntaka, Deng and Bishop (197*0 was chosen for

this study since it is during this period that the DNA copy of the

virus is synthesized and processed,, The possibility that a transient

alteration in host DNA synthesis may result from these interactions was

examined by pulse labeling the DNA«> Methods used for subculturing

cells resulted in partial synchrony in cellular DNA synthesis.. Since

expression of viral functions is host cell cycle dependent in infected

(Humphries and Temin, 197*0 and transformed cells (Leong, Levinson and

Bishop, 1972), processing and integration of the viral genome may also

be cell cycle dependent„ In such a case the partial synchrony induced

in the host cell cycle would be an advantage for isolating viral in­

fluences on cellular DNA synthesis* To specifically identify host DNA

synthesized during short periods of the proviral phase, cells were

pulse-labeled with ^H-thymidine for 3 hour intervals beginning 3 hours

after infection, then harvested and pelleted for DNA extraction as

previously outlined- The preparations are all shown to be relatively

free of contaminating protein by their 260nm/280nm absorbance ratios

exceeding 1„8,, Results of these extractions demonstrate consistent

i s o l a t i o n o f p u r i f i e d D N A ( T a b l e 2 ) „

The partial synchrony introduced into DNA synthesis by sub­

culturing CEF cells is a consistent finding under the conditions used

in these experiments® Partial synchrony in cellular DNA synthesis is

shown by the peak in specific activity seen for DNA labeled 6-9 hours

33

Table 2. Summary of pulse-labeled DNA extractions.

Time of Labeling (hrs.)

Control Cells Infected Cells Time of Labeling (hrs.)

^260nm A280nm

Specific Activity (cpm)

(fig )

^260nm A230nm

Specific Activity (cpm)

( (iS )

3-6 N.D.b NeDo 1.91 1.8%103

6-9 1.84 k

2.3^x10 1.88 1.05x10^

9-12 1.85 k

1.30x10 1.91 if.92xl05

12-15 1.86 6.11xl03 1.91 2.69xl05

15-18 1.82 5.70xlo5 N.D. N.D.

n a. RSV-infected and mock-infected (control) cells (1-2x10 cells/sample)

were pulse-labeled for 3 hour periods with 5^Ci/ml 3H-thymidine. Cellular 3H-DNA was isolated and extracted as detailed in Materials and Methods.

b. Not determined.

after plating (Table 2). These data indicate that the rate of DNA

synthesis increases then declines over the period examined,, A separate

experiment was performed to test the conditions which resulted in par-

3 tial synchrony0 Normal CEF cells were subcultured with H-thymidine

present in the media from passage until the cultures were harvested.

The rate of DNA synthesis for duplicate cultures was calculated from

the amount of DNA synthesized by the indicated harvest times (Fig. 5A).

3 Incorporation of H-thymidine into whole cell DNA increases until

approximately 10 hours post plating then declines to very low levels

by ifO hours. Under the conditions used here, the addition of fresh

serum induced new DNA synthesis in cells in which DNA synthesis had

stopped (Fig. 5A). The addition of fresh serum to cultures which were

no longer dividing has been previously shown to induce new rounds of

DNA synthesis and division (Temin, 1971b). Other experiments similar

to those reported in Table 2 and Figure 5A resulted in nearly identical

observations- These experiments were designed to further examine the

early period of DNA synthesis (i.e., the first 15 hours) following pas­

sage of the cells. For cells pulse labeled with ^H-thymidine, both

whole-cell acid-precipitable radioactivity and the specific activity

of DNA isolated from these cells showed a peak in the rate of DNA syn­

thesis 6-9 hours after plating (Fig. 5B and C). Whether or not the

cells were infected with RSV, the rate of DNA synthesis in the culture

was similar,. These results (Table 2, Fig. 5) demonstrate that in both

infected and control cells, partial synchrony in cellular DNA synthesis

occurs under the conditions used here to infect cells. This

Figure 5» Partial synchrony subculturing.

of cellular DNA synthesis following

Acid-precipitable radioactivity was measured in cells labeled with 3H-thymidine (^H-TdR) after passage and plating of the cells. Panel A shows the rate of DNA synthesis calculated from duplicate cultures of normal CEF cells where label (0.1 MCi/ml, ^H-TdR) was present continuously up to the time when cells were harvested. Panel B shows the rate of DNA synthesis for duplicate cultures of normal CEF cells (o o) and PSV-infected cells (e o), pulse-labeled with 3H-TCLR (5 Ci/ml) for the 3 hours prior to harvest of the cells. Panel C shows the specific activity of DNA from normal cells (A~ A) and RSV-infected cells (A A) isolated from the cultures described in Panel B.

3.0 - A

2.0

M I o

£ a. u

1.0 Fresh serum added

3.0 - B

0 2 0 - 4 0 6 0

Hours after plating cells

80 0 5 .10 15

.Hours after plating cells

0 5 10 .15

Hours after plating cells

< 2 O

U z a

o

t > o t- v

X < E y s-U_ w u lU CL <0

Figure 5. Partial synchrony of cellular DNA synthesis follov/ing subculturing.

VJ1

36

characteristic of subcultured CEF cells along with their response to

serum deprivation may contribute to some of the effects noted for host

nucleic acid synthesis.

DNA/DNA Reassociation Kinetics

The relationship between repeat and unique sequence cell DNA

synthesized following infection by RSV was examined by reassociation of

pulse-labeled DMA. If sequences of a particular reiteration were pref~

erentially synthesized at a given time after infection, this might be

seen in reassociation analyses of pulse-labeled DNAo The possibility

that infection might alter replication of DNA sequences occurring at

different copy numbers/genome was investigated in this manner,

Reassociation of the individual pulse-labeled control cell DNA

is shown in Figure 6. These reassociation curves demonstrate charac­

teristic second order reaction kinetics in the high CQt (unique

sequence) region (Cot>200); completing the reannealing approximately

two decades after it begins. The unique DNA C0t^ (the CQt at which the

reassociation of unique sequences is half completed) varies between

•z 1.6-2.0 x 10 as calculated from the reassociation curves. In the low

CDt (repeat sequence) region (CQt of 1 to 50) reassociation is essen­

tially completed before CQt < 1. The repetitive DNA which reassociated

before a CGt of 1 represented 9-1% of the labeled cell DNA. The dif­

ferences noted between the reassociation kinetics of the DNA samples

pulse labeled at different times after mock infection are small as can

be seen in Figure 7•

Figure 6. Reassociation analyses of uninfected cell DNA.

Pulse-labeled ^H-DNA (55-120 jig/ml) was mixed with an excess of unlabeled normal CEF DNA (0.5 mg/ml for low CQt and 5 mg/ml for high C0t analyses) in liquid associ ation reaction mixtures. After denaturation, the 3h-DNA was allowed to reanneal at 60°C until the desired C0t values were obtained. The extent of reassociation was assayed by the single strand specific nuclease, SI, as described in Materials and Methods. Panel A: re­association curves for DNA labeled 6-9 hours post-plating. Panel B: 9-12 hours post-plating. Panel C: 12-15 hours post-plating. Panel D: 15-18 hours post-plating.

—d-°-d—•-D DVd

X cl

6 - 9 H r s . p o s t p l a t i n g ( p . p . )

Log(0equivalent CQt

—Q.

B

9 - 1 2 H r s . p . p .

\

2 3 Logloequivalent CQt

a—

2 O

0

20

40

60

80 < o 0 100 CO , tn ' < UJ cc

1 0 a

z 20 UJ 0 £ 40 01

60

80

100

—D-0-

• u\

\ 12 -15 Hrs. p. p.

Log equivalent C.t 10 o

D " a

\

15 -18 Hrs.p.p.

Log|()equivalent CQt

Figure 6. Reassociation analyses of uninfected cell DNA.

50 CONTROL CELLS

6 - 9 H r s . p . i . 9 - 1 2 H r s . p . i .

12 -15 Hrs. p.i. 15 -18 Hrs. p.i.

100

LOG|0 EQUIVALENT CQt

Figure 7* Composite of control-cell DNA reassociation curves. — Curves from Figure 6 are shown together on a single graph for comparison.

oo

39

Reassociations of the DNA derived from cells infected with RSV

are shown in Figure 8. As noted for the control cell DNA associations,

all the infected cell DNA reassociation curves show second order reac­

tion kinetics for single copy sequences,, CDty2 values for unique DNA

3 lie between lol-2o0 x 10 for the various pulse-labeled DNA's,, Similar

to the reannealing of control cell DNA, infected cell DNA demonstrates

very little additional reassociation between a Cot of 1 and 50o Rapidly

reassociating DNA increased from 6o5 to approximately 10„5 percent

from the earliest to latest pulse-labeling times. Data from Figure 8

have been combined in Figure 9 in order to show that as with the con­

trol cell DNA, the differences observed between curves for each pulse-

labeling time are small„

Table 3 summarizes values for the kinetics of reassociation

from the curves of Figures 6 and 8C These reactions were kinetically

complete as demonstrated by the 75 to 85^ of the labeled DNA that re-

if association by sin equivalent CDt > 10 0 Small differences m unique

DNA C0ti£ and the fraction of repeat DNA reassociating were noted be­

tween labeling timesa The percent reassociating as repeat DNA generally

increased with time, while the values for unique DNA Coty2 were about

3 1»5-1<>9 x 10 o These fluctuations in kinetics may be due to the

preferential labeling of specific sequences during the partial syn­

chrony observed for DNA synthesis0 Differences in the temporal repli­

cation of sequences represented at reiterated frequencies in the genome

might then be responsible for these variations,.

Figure 8. Reassociation analyses of RSV-infected cell DNA.

Pulse-labeled cell DNA(60-90 jzg/ml) extracted from CEF cells infected with RSV was mixed with an excess of un­labeled normal CEF DNA (0.5 mg/ml for low C0t analyses) in liquid reassociation reaction mixtures., After de-naturation, the 3H-DNA was allowed to reanneal at 60°C until the desired CQt values were achieved. The extent of reassociation was assayed by the single strand spe­cific nuclease, SI, as described in Materials and Methods. Panel A: reassociation kinetics for infected cell DNA labeled 3-6 hours post infection. Panel B: 6-9 hours post infection. Panel C: 9-12 hours post infection. Panel D: 12-15 hours post infection.

2 O

0

20

40

60

80 •— < 0 8 100 cn i < UJ cr. <

1 0

2 20 UJ o £ 40 a

60

80

100

3 - 6 h r s . p o s t i n f e c t i o n ( p . i )

I i I

Log,, equivolent C t

_Q_

B

6 - 9 h r s . p . i .

Log|oequivolent CQt

o-~-o—

0

20

40

z 60 0 < 80 u to 100 CO { < UJ or <

1 0

z 20 UJ o £ 40 CL 60

80

100

9-12 hrs.p.i.

i

\ V

Log equivolent C.t 10 0

—O-O—O-

D

12-15 hrs.p.i.

V

\ On«o

Log|0equivalent CQt

Figure 8. Reassociation analyses of RSV-infected cell DNA.

s

0

50 INFECTED CELLS

3 - 6 H r s . p . i . 6 - 9 H r s . p . i . 9 - 1 2 H r s . p . i .

12 -15 Hrs. p. i.

100 _L

Figure 9«

2 LOG |0 EQUIVALENT CQt

Composite of infected-cell DNA reassociation curves. — Curves from Figure 8 are plotted together for comparison.

k2

Table 3« Summary of reassociation kinetics for pulse-labeled cellular DNA.a

Time of Labeling Unique DNA C 3^ oti/2 x 10 Percent Repeat DNAC

(hrs.) Control Cells Infected Cells Control Cells Infected Cells

3-6 N.D.d 1.15 N.D. 6.5

6-9 1.97 1.92 9.0 8.0

9-12 1.87 1.93 10.0 9.5

12-15 1.56 1.57 9.0 10.5

15-18 1.76 N.D. 15.5 N.D.

a. Parameters reported here for the reassociation kinetics of de­natured cell DNA were determined from data in Figures 6 and 8.

b. Got at which 5Q?o of the unique sequences are reassociated. c. Fraction of DNA reannealed at C0t = 10. d. Not determined.

k3

The unique DNA C0ty2 changes less than 5^ in value from infected

to control cells for comparable labeling periods. While variations in

the percent of repeat DNA are greater during the same labeling times,

the absolute differences are quite small» Hence, both the single copy

DNA C0ty2 and the percent repeat DNA annealed change very little as a

result of RSV infection* These data suggest that synthesis and inte­

gration of the RSV DNA provirus do not perturb host DNA synthesis

sufficiently to be measured by reassociation kinetics under the con­

ditions employed here»

Analysis of Host RNA Synthesis Following Infection

Alterations in RNA synthesis seemed likely after infection,

since the synthesis of the RSV genome and new or altered proteins are

found in transformed cells* Possible viral effects on host RNA syn­

thesis seemed most probable during the host's transition from a normal

to a transformed cello An understanding of the effects of virus in­

fection on cellular RNA synthesis might yield information indicative

of the mechanism(s) by which cells are transformed. Since budding RSV

is first detected 18 hours after infection of chick cells and the cul­

ture morphology is completely transformed by 30 hours after infection,

the period examined for transcriptional alterations was extended to

over 50 hourso During this period infected and control cells were

"T pulse labeled with H-uridine and the isolated RNA analyzed by RNA/DNA

hybridization. The relationship between repeat and unique sequence

kk

transcripts was examined in this manner to identify virus induced

changes in cell transcription.,

Labeling of Host Cell RNA

Transient differences in host cell RNA synthesis during infec­

tion could be studied by pulse labeling RNA which would allow examina­

tion of RNA synthesized only during the labeling period. Secondary CEF

3 were either mock infected or RSV infected and pulse labeled with H-

uridine for two hours at various times after replating the cells« These

pulse-labeled cells were harvested, fractionated into cytoplasmic and

nuclear samples, and nRNA was extracted as detailed in Materials and

Methods*

Results for two separate experiments of the extractions and

treatments described are shown in Table Approximately kO-5C$> of the

acid-precipitable whole-cell radioactivity was recovered after this ex­

tensive purification.. The specific activity of RNA from the control

and infected cells of the same labeling period were comparable indi­

cating that the rate of RNA synthesis in control and infected cells is

similar.. These data further demonstrate that the isolation and re­

covery of nRNA from both control and RSV infected cells is consistent.

Overall, extraction yielded uniform nRNA that had a molecular weight

greater than 50,000.

Hybridization of Pulse-Labeled Host Cell nRNA

In order to investigate differences in the populations of RNA

synthesized in RSV-infected cells, the RNA was analyzed by hybridization

Table 4. Summary of pulse-labeled nuclear RNA extractions.a

Experiment 1 J Time Time j at Percent CPM Concentration Specific Activity at Percent CPIs

Pulse Recovered (fig/ml) (cpm x 10~b/ug) Pulse Recovered] (hrs) Control Infect,, Control Infect, Control Infecto (hrs) Control Infec

5 1+8 bz 15d 21.5 2.11 1o36 12 39 U

15 52 bz 29=5 17.3 1.02 1.56 2b 39 5<

25 53 3b *fl.5 29°7 lo28 lobo b6 39 55

50 67 50 b3ol 68.0 2.03 l.6l 58 b3 4C

n a. RSV-infected and control cells (1-2 x 10 cells/sample) were pulse-labeled

with H-uridine (50 Ci/ml) for 120 minuteso Nuclear fractions were isolated and the RNA extracted from them as detailed in Materials and Methods.

b. Limited recovery of RNA in void volume of sephadex column.

if5

ed nuclear RNA extractions.3

Experiment 2

tration /ml)

Specific Activity (cpm x 10-Vug)

Time at

Pulse Percent CPM Recovered

Concentration (ug/ml)

Specific Activity (cpm x 10 /j/g)

Infect. Control Infect. (hrs) Control Infect. Control Infect. Control Infect.

21 o5 2 oil 1«. 36 12 39 l^b 10.2 7 ok 9O60 ka66

17 °3 1.02 1o56 2k 39 50 16.3 1^.1 7«>78 10A

29<»7 1O28 lo^O k6 39 55 35«8 37®3 9<>22 llo9

68o0 2o03 I06I 58 ^3 ko ^3o2 59cl 3=91 3-72

7 s (1-2 x 10 cells/sample) were pulse-labeled or 120 minutes„ Nuclear fractions were d from them as detailed in Materials and

id volume of sephadex column.

46

with cell DNA® The kinetics of these RNA/DNA hybridizations defined

the classes of unique and repeat sequence transcripts and indicated the

relative concentration of repeat sequence transcripts,. Differences in

these parameters were sought between nENA from RSV-infected and control

cellSo

Several broad classifications are used here for RNA hybridizing

in these experiments., RNA which is presumed to be transcribed from the

unique DNA sequences (termed "unique" RNA) hybridizes with apparent

second order reaction kinetics at high Coto The RNA transcribed from

reiterated DNA sequences (called "repetitive" RNA) forms duplexes at

low Cot's (C0t = 1-100) and RNA which hybridizes below a CQt of 1 is

transcribed from highly repetitive DNA templates and will also be re­

ferred to as "highly repetitive" RNA.

RNA/DNA Hybridizations of Control Cell nRNA

For hybridizations of pulse-labeled nRNA from control cells,

the representation of the three classes of RNA varies with the time at

which the nRNA was labeled (Fig® 10). "Highly repetitive" RNA is de­

tected for nRNA labeled 5 and 25 hours after plating but very little

"repetitive" RNA is seen to hybridize from these samples (Fig® 10A and

10C). nRNA labeled 15 hours after plating shows "repetitive" RNA hybrid­

ization but no "highly repetitive" RNA hybridization (Figo 10B)« The

RNA/DNA hybridization of nRNA labeled at 50 hours demonstrates hybrid­

ization of both "highly repetitive" and "repetitive" RNA (Fig 10D)«

From a low of almost no detectable hybridization for "highly repetitive"

Figure 10. Hybridization of normal CEF nuclear RNA.

Nuclear RNA (75.5 to 215*5 ug/ml) from cells pulse-labeled 120 minutes with 50 MCi/ml H-uridine was mixed in equal volume with normal CEF DNA (25.5 mg/ml for high C0t re­actions and 0.212 mg/ml for low CQt analyses) in liquid reaction mixtures containing a final concentration of 50/6 formamide and 5X SSC. The DNA/RNA ratio ranged between ' approximately 1-^ x 102 for high CQt reactions and between 3:1 to less than 1:1 for low 0ot samples. Reaction mix­tures contained 2,000 to 10,000 cpm of labeled RNA. Following incubation at 50°C for times appropriate to reach the desired Cot values, duplicate reaction mixtures were assayed for RNase A-resistent hybrids. Hybridiza­tion curves for nRNA labeled 5 hours post-plating (Panel A); 15 hours post-plating (Panel B); 25 hours post-plating (Panel C); 50 hours post-plating (Panel D) are shown.

0

5

10

1 5

20

2 5

3 0

0

5

10

1 5

20

2 5

3 0

" °oo

- A \ o

5 hrs. post plating (p.p.) X

1 1 1 1 1 1

_ B \

ay

15 hrs. p.p. 9

o*-| I | 1 I |° | 1 1 1 1 1 1 1

o-O-Q-o—o—o^o 1 I 1 1 1 1 1 1

°\ \ _ c \ _ D o.

°\ \ 25 hrs. p.p. \o 50 hrs. p.p. \

\ J QyO

V -1 1 1 1 1 1

O^Q.

1 1 1 1 I I 1 I 0 I 2 3 4 5 - 1 0 1 2 3 4 5 6

log C t 10 0

Figure 10. Hybridization of normal CEF nuclear RNA.

•P--n3

^8

nRNA labeled at 15 hours, a maximum of 2 percent of the input RNA

hybridizes for RNA labeled at 50 hours- These extremes are closely

coincident with the rates of maximum and minimum DNA synthesis observed

with increasing time after plating of the cells (Fig. 5)° "Repetitive"

RNA hybridizing by a CQt of 10 shows similar temporal differences

varying between 1 and 3 percent of the input RNA labeled at these

time So

The "unique" RNA hybridization varies very little from one

labeling time to the next hybridizing with apparent second order reac­

tion kinetics for each sample„ Estimates of the C0ty2 values for the

3 RNA annealings (approximately 2.5 x 10 ) are consistent with kinetics

for unique sequence transcripts. The total fraction of "unique" nRNA

hybridized was between 18 and 23 percent for the different RNA samples.

These differences may represent small changes in the fraction of the

genome transcribed as discussed below.

Data presented for control cell nRNA hybridizations indicate

two points about transcription in these cells. The "unique" RNA for

hybridizations of nRNA labeled at various times after passage are rela­

tively uniform and consistent. Both "highly repetitive" and "repeti­

tive" nRNA synthesized during different periods show a small increase

with time after subculturing. During active cell growth these frac­

tions of the RNA account for a smaller portion of the newly transcribed

nRNA than during the late, stationary-growth phase.

k9

Infected Cell nRNA

RSV-infected cells pulse-labeled with "^H-uridine as described

in Materials and Methods, were fractionated into cytoplasmic and

nuclear fractions and the nRNA was isolated,, Labeled nRNA was allowed

to hybridize to cell DNA in liquid hybridization reaction mixtures

identical in reactants and conditions to those of the control cell nRNA

annealings. Results of these hybridizations are shown in Figure llo

The data demonstrate a pattern similar in several ways to that seen for

control cell nRNA hybridization., The "unique" RNA hybridization kinet­

ics Eire very much alike for all time periods examined® Estimated Cotyz

3 for "unique" RNA hybridizations were approximately 2„5 x 10 « The

portion of total nRNA hybridized in the unique sequence region of these

curves ranged from approximately 20 to 2b percent0

The fraction of the nRNA which hybridized as "repetitive" RNA

increased with time after infection, as did the nRNA from control cells.

For the first two time points (5 and 15 hours post-infection) the same

fraction of nRNA represented by "highly repetitive" and "repetitive"

RNA are seen to hybridize as with control cell nRNA« Repeated sequence

RNA found in RSV infected cells with increasing time after infection

while following the trend of control cell nRNA hybridization — in­

creased beyond that observed in the uninfected controls during the

period 25 to 50 hours after infection,,

The data for "unique" RNA are compared in Figure 12 with those

of the control cell nRNA hybridizations in order to demonstrate their

similarities. Hybridizations of unique sequence transcripts for each

Figure 11. Hybridization of BSV-infected cell nRNA with cell DNA.

Pulse-labeled nRNA (76.5 to 3^0 fig/ml) was mixed with equal volumes of normal CEF DNA (25-5 mg/ml for high C0t reac­tions and 0.212 mg/ml for low CQt analyses) in liquid reaction mixtures. These reaction mixtures, the hybrid­ization condition, and the assay procedure were all identi­cal to those described in the legend to Figure 10. Hybridization curves are shown for nRNA labeled 5 hours post infection (Panel A); 15 hours post infection (Panel B); 25 hours post infection (Panel C); 50 hours post infection (Panel D).

0

5

10

15

20

25

30

0

5

10

15

20

25

30

Q-o- -o—o—O-

5 hrs. post infection (p.i.)

CT

15 hrs. p.i.

-QJ

25 hrs. p. i 50 hrs. p. I.

J L J I L 12 3 4 5

'°5,(P0t

0 I 2 3 4 5

'OflKpO*

re 11. Hybridization of RSV-infected cell nRNA with cell DNA.

CD 10

5 hrs. p.i 25 hrs.p.i

m

15 hrs. p.i. 50 hrs

l°9,oCot

Figure 12. Hybridization kinetics for unique sequence transcripts from RSV-infected and control cell nRNA„ — The unique sequence regions (between a CQt of 102 and 105) of the hybridization curves for control cell nRNA (o——o) from Figure 10 and those of RSV-infected cell nRNA (o- - -©) from Figure 11 are shown together for comparison of similar pulse-labeling times.

of the times assayed are not readily distinguishable0 The small dif­

ferences in the total amount of RNA hybridized noted earlier can also

be identified, but no consistent differences between normal and in­

fected cells have been observed,,

To demonstrate the differences between control and infected

cell "repetitive" nRNA hybridizations, the results from control cell

hybridizations (Fig. 10) and those from infected cells (Fig. 11) are

combined in Figure 13° These differences are seen at late times after

infection when transformed morphology becomes recognizable for infected

cellso Nuclear nRNA labeled 5 or 15 hours after infection or plating

hybridized with similar kinetics» Hybridizations of nRNA synthesized

25 or 50 hours after infection, however, diverge from the results

obtained with normal cells. During this period when the transformed

character of the culture is established, the fraction of "repetitive"

RNA in infected cells increases over that observed in uninfected cells

by more than twofold.

In order to verify the differences between repeat sequence RNA

transcribed in control and infected cells, a second, independent ex­

periment in which cells were mock or RSV infected and cellular RNA

pulse-labeled was carried out. The method of infection, RNA labeling,

cell fractionation, nRNA extraction, and RNA/DNA hybridization were all

done as before. In this second experiment the times of pulse-labeling

were chosen to examine the periods shortly after infection when no dif­

ferences in control and infected cell nRNA hybridizations were seen,

late after infection when differences were observed, and very late

© O O ' O Q

25 hrs.p.i 5 hrs. p. i.

O-Q-O

15 hrs. p. i. 50 hrs. p.i.

'° ioCo*

Figure 13® Hybridization kinetics for repeat sequence transcripts from RSV-infected and control-cell nRNA. — The repeat sequence region (between a Cot of 10"- and 10 of the curves for control cell nRNA hybridizations (o o) from Figure 10 and those of RSV-infected cell nRNA hybridizations (©_ - -o) from Figure 11 are shown together for comparison of similar pulse-labeling times.

vn

5*+

after infection when both infected and control cells were rarely divid­

ing. nRNA hybridizations of infected and control cells from the same

labeling times are shown together in Figure l*fo As before, nRNA from

the earliest time period examined (12 hours) demonstrated similar

kinetics for low CQt hybridizations, while during the 2k~b6 hour inter­

val nuclei from infected cells again exhibited a larger fraction of

newly-synthesized "repetitive" RNA than did control cells. By ^6 hours

after infection a maximum difference in the hybridization curves for

infected and control cell RNA is evident. Analysis of nRNA synthesized

at 58 hours after infection indicated that RNA from infected cells con­

tained the same fraction of repetitive sequence transcripts as controls,

although the sequences transcribed may not be the same as those synthe­

sized in control cells.

This independent repeat of the earlier experiment confirmed the

original observations,, Data from these experiments are summarized in

Figure 15 where the fraction of nRNA hybridized by a CQt of 10 for each

of the RNA annealing curves (Figs. 13 and 14) are shown. Between 2b

and 50 hours after infection the concentration of "repetitive" RNA in

infected cells is greater than controls. The difference in the frac­

tion hybridized by C0t = 10 reaches a maximum by 50 hours and then

declines by 58 hours after infection.

Constant DNA/RNA Ratio Comparison Hybridizations

Since the repeat DNA sequences occur at different frequencies

and their transcripts occur at unknown frequencies, specifying the mass

Figure lA- Low CQt hybridizations of control cell and RSV-infected cell nRNA.

Pulse-labeled nRNA (70.5 to 275 from a second, independent experiment (see the legend to Table k) was mixed with an equal volume of normal CEF DNA for liquid hybridization, and assayed under conditions described in the legend to Figure 10. Low CQt hybrids formed with nRNA from control cells (o o) and from RSV-infected cells (©- - -©) from the same labeling times are shown.

5 o cr 00 >

5 io < 2 cc

< z Q

1-z UJ o cc UJ Q.

0

10

o S o © ° _o 0

• •

12 hrs.p.i.

A

• < t

X cr^Q. *© ®***«».

© «°

46 hrs p.i. ®

c

1 ! 1 0J 1 I ©X 1 ' '

Gl>—? o ® 'O-® S ° ©•» _ ©

©®^8

24 hrs.p.i. 58 hrs.p.i.

B

i i i

D

i i i

Figure l*t. Low C0t hybridizations of control cell and ESV-infected cell nRNA.

56

O LLJ N

Q

CT CD >-

or

h-"Z. LiJ O £T LJ CL

7.5

5.0

2.5

0

/

/

/A / /

/4 I / \

\ \

o JA

03

o

0 20 40 60 TIME AT LABELING (HOURS)

Figure 15. The fraction of nRNA hybridized by a CDt of 10 for control and RSV-infected cells. — The percent of nRNA found in hybrids at a CQt of 10 in the curves of Figures 13 (circles) and Ik (triangles) are shown for both control (open symbols) and RSV-infected cells (closed symbols).

57

ratios of DNA to RNA does not indicate the sequence ratio for particu­

lar DNA sequences and their transcripts,, If in general the "repeat"

RNA hybridizing is less concentrated than the repeat sequences from

which they were transcribed, all the RNA should hybridize. However,

it may be possible that the RNA copies exceed in number their templates

even at high DNA/RNA mass ratioso Hence, it is necessary to specify

some constant DNA/RNA ratio for comparison of hybridizations<> To rule

out the possibility that a variable DNA/RNA ratio might account for

some of the differences observed, hybridizations in which the DNA/RNA

ratios were held constant were performed.. Although the actual sequence-

to-transcript ratio was indeterminant, a constant DNA/RNA mass ratio

implied that the extent of DNA saturation by RNA is consistent for each

hybridization,, nRNA was diluted to a constant concentration and

hybridized to normal CEF DNA to a CQt of 14-16. The hybrids formed

were assayed using the complete digestion conditions provided by the

combination of RNases A and T1 as described in Materials and Methods®

Results of constant DNA/RNA ratio hybridizations are shown in

Figure l6A. These findings confirm the pattern previously seen,, Con­

trol cell nRNA labeled at different times varies slightly in the frac­

tion of "repetitive" RNA hybridized. Hybridization of pulse-labeled

nRNA from infected cells increases with time and this increase again

surpasses the normal cells in the period 25-50 hours after infection.

Arithmetic means for the percent of infected cell nRNA hybridized for

both the k6 and 50 hour time points are statistically different from

control cell values at the 99% confidence level (t = -5.56 and -7-56

Figure 16. Hybridizations of nuclear RNA at a constant DNA/RNA ratio.

Nuclear RNA was diluted to a constant concentration (50 /ig/ml) and hybridized with an excess of normal cell DNA to a CQt of 1^ or 16 as described in Materials and Methods. Quadruplicate samples were assayed by diges­tion with both RNases A and T1 and the mean values for remaining hybrids plotted. The RNA used in this experi­ment was the same as used for the experiments described in Figure 13 (square symbols) and Figure 1^ (circles). Panel A shows the percent of control cell nRNA (open symbols) hybridized and that of RSV-infected cell nRNA (closed symbols) hybridized for the labeling times indicated. Panel B shows the ratio of hybridization of infected cell nRNA to control cell nRNA.

58

< Zio cr —

< •*-<> 2: O O •}—

S s o »-

_Q CD >>

Q_ X

5.0 - A

2.5

0

*o <D N

-Q >» X

or c

CD O

T3 CD

O a>

"O CD M

-g

-Q >* X

cc c

"a5 o

c o o

3.0

0 20 40 60 Time After Infection (hrs.)

- B

2.0

1.0

0

- •

0 20 40 60 Time After Infection (hrs.)

Figure 16. Hybridizations of nuclear RNA at a constant DNA/RNA ratio.

respectively)0 The ratios of the fraction of RNA hybridized from in­

fected cell RNA to control cell RNA are shown in Figure l6B« A two

fold increase is observed for the fraction of "repetitive" RNA found

in the nuclei of cells infected with RSV during the 25-50 hour period

after infection — a time when the majority of the cell population is

becoming morphologically transformed.

Polysome-Associated "Repetitive" RNA

Alterations observed in the newly-synthesized "repetitive" nRNA

of infected cells raised an immediate question: could changes also be

seen in mRNA, a putative product of nRNA processing (Greenberg, 1975)?

Since polyribosomal associated RNA (pRNA) is representative of the

mRNA population actively being translated, this RNA was examined for

differences in the representation of "repetitive" transcripts,, Cyto­

plasmic fractions from infected and control cells were centrifuged on

sedimentation gradients to isolate polyribosomes <> RNA extracted from

the pooled polysomes was then analyzed by means of low CQt RNA/DNA

hybridization.

Labeling and Extraction of pRNA

During the period when infected cells are converted from normal

3 to transformed characteristics, cells were pulse-labeled with H-

uridine for 120 minutes and the cytoplasms were prepared and layered

onto sucrose sedimentation gradients. The sedimentation of the cyto­

plasmic fractions routinely yielded absorbance (260 nm) profiles

6o

demonstrating intact polyribosomes (Fig. 17). Separation of polyribo­

somes allowed isolation of RNA sedimenting with diribosome complexes

or faster., Approximately 20 percent of the total acid-precipitable

radioactivity present in the cytoplasm was isolated from the polyribo­

some region of the gradients (Table 5)° Nearly complete recovery of

this pRNA followed the phenol-chloroform extraction designed to retain

polyadenylated RNA molecules by minimizing their loss in the organic

solvent (Perry et al0, 1972)„ Table 5 shows that the resulting specif­

ic activity for infected and control cell pRNA from comparable label­

ing periods are similar.

Low Opt Hybridizations

pRNA samples were adjusted to a constant concentration and

hybridized with excess normal cell DNA to a CQt of 1^-16. The results

from separate experiments show that early control cell pRNA hybridizes

to about Zfoo For later times this percentage drops to 1-1.5% hybrid­

ized. The infected cell pRNA follows and amplifies this trend.

Hybridization of "repetitive" pRNA initially represents 2% of the pRNA,

then falls to approximately 0.5% for the next kO hours. Fifty-eight

hours after infection this trend is reversed and control pRNA hybrid­

ized by a CQt of l*f-l6 surpasses that of infected cells0

The relationship between the fraction of control and infected

cell pRNA hybridizing is shown in Figure 18. At the earliest time

examined (5 hours), control and infected cells hybridize very similar

fractions of their pRNA. By 12 hours after infection hybridization of

Figure 17. Sedimentation profiles of cytoplasmic fractions from con­trol and RSV-infected cells.

Cytoplasm isolated from infected and control cells from 12 to 58 hours after infection were centrifuged on 15-30^ sucrose gradients as described in the legend to Figure 2. The gradients were collected from the bottom through a continuous flow cell placed in the optical path of a spectrophotometer set at a wavelength of 260nm. Tracings at A260 are shown for the different samples. Panel At sedimentation profile of control cell cytoplasmic frac­tion labeled 12 hours post plating. Panel B: sedimen­tation profile of infected cell cytoplasmic fraction labeled 12 hours post infection. Panel C: sedimentation profile of control cell cytoplasmic fraction labeled ^6 hours post plating. Panel D: sedimentation profile of infected cell cytoplasmic fraction labeled k6 hours post infection. Panel E: sedimentation profile of control cell cytoplasmic fraction labeled 58 hours post plating. Panel F: sedimentation profile of infected cell cyto­plasmic fraction labeled 58 hours post infection.

DIRECTION OF SEDIMENTATION

Figure 17. Sedimentation profiles of cytoplasmic fractions from control and RSV-infected cells.

Table 5* Summary of pulse-labeled polyribosomal RNA extractions,

Experiment 1 Time at Pulse (hrs) Control Infect,

Percent CPM Recovered

Time Concentration Specific Activity at

(fig/ml) (cpm x 10-3//jg) Pulse Control Infect. Control Infect. (hrs)

Percent d Recovertj

Control lnf(

5 12 16 93 78 0o4l o.6l 12 7

25 16 16 121 100 0.76 1.04 46 22

46.5 18 21 110 114 l044 1.55 58 21

n RSV-infected and control cells (1-2x10 cells/sample) were pulse labeled with 3H-uridine (50 MCi/ml) for 1-20 minutes. Cytoplasmic fractions were prepared and the polyribosomal RNA was isolated on sedimentation gradients (see legend to Fig. 17) and extracted as described in Materials and Methods.

9 62

beled polyribosomal RNA extractions»a

L 1

Experiment 2 Time

;entration Specific Activity at Percent CPM Concentration Specific Activity Ifig/ml) (cpm x 10-3/ur) Pulse Recovered (/ig/ml) (cpm x ! 10-3/ug) :ol Infect. Control Infect. (hrs) Control Infect. Control Infect, Control Infect.

5 78 O.kl 0.61 12 7 13 170 102 1=07 3*72

: 100 0.76 1.0*f ke 22 23 215 297 2.16 2.19

) Ilk lokk 1.55 58 21 25 168 269 2.89 2.55

h ells (1-2x10 cells/sample) were pulse labeled ) for 120 minutes„ Cytoplasmic fractions were lomal RNA was isolated on sedimentation gradients .d extracted as described in Materials and

I $

63

"D <D M

jg

-Q > n

< ;z or Q_

<D CJ

~o CD

*+— O <D

"O <D N

jg

_Q

X

1.5

.0

a: CL

3 0.5 "o c o o

0

0 20 40 60

TIME AFTER INFECTION (HRS.)

Figure 18. Ratio of infected to control cell polysomal RNA hybridized at a CDt of 15® — Pulse-labeled pRNA isolated from the polyribosomal fractions of RSV-infected and control cells was diluted to a con­stant concentration (250 fig/ml), hybridized with excess cell DNA to a Cot = 15, and assayed under stringent conditions as described in the legend to Figure 16. The ratio of the percent hybrid for infected-cell pRNA to that of control-cell pRNA is shown for RNA from two separate experiments (Ex­periment 1 - open symbols; Experiment 2 - closed symbols).

6k

pRNA from infected cells is reduced to less than half that of controls.

This difference continues during the period of transformation of in­

fected cells from 12-^8 hours® Late after infection when all the cells

are transformed and their population density is very high this differ­

ence diminishes.

Post-Transcriptional Processing of "Repetitive" RNA

Data presented so far indicate several interesting points about

newly synthesized "repetitive" RNAo While the fraction of repeat se­

quence transcripts from control cell nuclei increases slightly with

time after plating, the polysomal associated "repetitive" RNA decreases.

RSV infected cells also follow but accentuate this trendo Infected

cell nuclei have a larger fraction of "repetitive" RNA than controls

while the "repetitive" RNA from polyribosomes represent a smaller frac­

tion than controls,, These results from Figures 16B and 18 have been

combined in Figure 19 to show the virus induced alterationso The in­

crease compared to controls in "repetitive" RNA from the nuclei of

infected cells has been divided by the fraction found on infected-cell

polysomes to yield the ratios shown* This index suggests an alteration

in the post-transcriptional processing of these repeat sequence tran­

scripts due to the infection by RSV„ During the cells' conversion from

normal to transformed the appearance of the expected proportions of

"repetitive" RNA in the infected cell cytoplasm was inhibited.

Figure 19. The relationship between the polysomal and nuclear "repetitive" RNA fractions as a function of infection.

RSV-induced changes in the representation of repetitive sequence transcripts on the polyribosomes compared with that in the nucleus of infected cells is shown. The ratio of infected to control pRNA concentration of repetitive sequence transcripts (from Fig. 18) has been divided by the same ratio for nuclear RNA (from Fig. 16B) to yield these values plotted against the time at which the RNA was labeled.

65

o> D> c c *N N

"O jo "l— X3 JD >» >»

JZ sz

< < ;z

(Z £T CL c

"<D "<D > >

•4— cu 0) CL CL <D CD

%-

"o3 ~c5 o o o o i— c c o o o o o o •H

•O "O <D CD

-J— o o CD CD 4— «+-c c H— o o o o

'•*—

a D tr CL

2.0

1.0

0.5

0.0 0 20 40 60

Time after infection (hours)

Figure 19. The relationship between the polysomal and nuclear "repetitive" RNA fractions as a function of infection.

DISCUSSION

The results presented in this study indicate that some tran­

scriptional controls in CEF cells may be influenced by both the growth

state and RSV infection,. Transcription of repetitive sequence DNA

increased twofold in cells as a result of infection by RSV. Changes in

cellular DNA synthesized after infection were not identified when

measured by DNA/DNA reassociation techniques- As shown here, these

observations correlate with the transformed state of the cells.

DNA/DNA Reassociations

Theoretical and Practical Aspects

DNA/DNA reassociation has proved a powerful tool for investi­

gating the organization of the eukaryotic genome. Data generated by

this technique have demonstrated a high degree of complexity for the

structure of the genome of higher organisms. The assay consists of

shearing chromosomal DNA to small fragments and melting the double

helices into their component single strands. These molecules are then

incubated to allow reannealing through complementary base pairing be­

tween the two strands. The extent of reassociation is most accurately

measured by exhaustively digesting single stranded molecules or por­

tions thereof and determining the resistent double stranded fraction.

In the ideal case reassociation of DNA is a second order reaction

dependent on the concentration of the two reactants (the single

66

stranded molecules) and the time of incubation.. Britten and Kohne

(1967) used these values to create standard measurements of the extent

of the reaction by multiplying the initial concentration of the total

DNA (Co) by the incubation time (t) C0t0 The unexpected result of

Britten and Kohne®s (1968) experiments was the demonstration of multiple

copies of some DNA sequences present in the genome of eukaryotes® They

g found repeat sequences occur from a few to 10 copies per genome, repre­

senting families of similar but not necessarily identical base compo­

sitions The occurrence of multiple copies of nearly identical sequences

has also been shown for ribosomal DNA (Moore and McCarthy, 1968) and

some RNA tumor-virus integrated genes (Gillespie and Gallo, 1975)°

Investigating reassociations of repetitious DNA lead to the demonstra­

tion of the interspersion of these sequences amongst the unique se­

quences of the chromosome (Davidson and Britten, 1973)° This arrange­

ment of repeat DNA has also been shown by Kleinschmidt electron

microscopy for reassociation fragments of DNA (Davidson et alo, 1973) <•

The presence of repeated sequences in the DNA raised questions

about their function.. Very highly reiterated DNA (satellite DNA) from

mouse cells is composed of tandem repeats of 300 nucleotides which are

not transcribed (Flamm, Walker and McCallum, 1969)0 These sequences

have been hypothesized to have a "housekeeping" function in maintain­

ing the structure of the chromosome (Waiker, Flamm and McLaren, 1969)®

Other repetitive sequences, whose functions remain unknown, are tran°

scribed, though a smaller fraction of these transcripts are found in

the cytoplasm than in the nucleus (Greenberg, 1975)• Britten and

Davidson (1969) first proposed that repeat sequences represented

address sites which were differentially activated for control of growth

and differentiation,, This theory postulates that either sequence-

specific proteins or RNA binds to the address sites (repetitive DNA)

resulting in transcription of the associated unique sequences (David­

son and Britten, 1973)o Their model allows for elaborate coordinate

and integrated transcription of physically-separated sequences through

these moleculeso The possibility of regulatory repeat sequence tran­

scripts is particularly interesting in light of the finding of in­

creased "repeat" RNA in infected cells reported in this investigation.

The presence of repeat sequences in animal cell genomes is seen

in the DNA renaturation assay at low Cots. Before the unique sequences

have had an opportunity to reanneal, repetitive sequences reassociate

due to their relatively higher concentration.. The fraction of repeat

sequence DNA varies from one eukaryotic genome to another (see Britten

and Kohne, 1968), amounting to about 25-30% of the total for chick DNA

(Rosen, Liarakos and O'Malley, 1973; Schincariol and Joklik, 1973;

Neiman, 1972). DNA/DNA reassociation assays are useful for character­

izing both the fraction and redundancy of these repeat sequences. This

technique can also measure the kinetic complexity of the unique se­

quences (Davidson and Britten, 1973)»

A practical consideration germane to this study is the method

of observing these different sequences. If hyper-chromicity of DNA

or the duplex formation of uniformly labeled DNA are followed during

renaturation, then the entire polynucleotide population will be

69

monitored. However, pulse-labeling during DNA synthesis results in

incorporation of radiolabeled precursors only into DNA sequences repli­

cated during the labeling period,, For a population of cells, only

those cells synthesizing DNA and of these only the specific sequences

replicated will be labeled with a radioactive isotope0 Hence, in a

DNA/DNA reassociation where pulse-labeled DNA is assayed, only the re-

association of radioactive sequences will be monitored,, Such assays

measure the relationship between repeat and unique DNA that is synthe­

sized during the time of pulse labeling.

Observations and Interpretations

The reassociations of pulse-labeled cellular DNA from both in­

fected and control cells show very similar kinetics (Figs. 7 and 9)*

For those samples where both normal and RSV infected cell DNA was

examined, the fraction of repeat DNA reassociation varied no more than

lo% (Table 3)° Unique sequence DNA reassociated with comparable

kinetics varying no more than 3^ in their CQtyz values for similar

labeling times. Differences noted between the various labeling pulses

for these measures are relatively small and were not further investi­

gated. It may be that they are a function of both the induced partial

synchrony in cellular DNA synthesis and the differential labeling of

sequences during the pulse. These results imply that the cellular DNA

synthesized in RSV infected and control cells are very similar when

assayed by DNA/DNA reassociation techniques.

70

RNA/DNA Hybridizations

Theoretical and Practical Aspects

In order to interpret data presented in this study and the

findings derived by different means by other investigators, a general

discussion of hybridization techniques illustrating the power and

limitations of each of the various methods will be useful«,

Gillespie and Spiegelman (1965) developed the filter-bound DNA

hybridization technique which was widely used prior to the advent of

the liquid hybridization procedure,, Denatured DNA was bound to nitro­

cellulose filters which were then immersed in solutions of radioactive

RNA. Following incubations of 18-2^ hours or longer, these filters

were washed, treated with RNase and counted for radioactivity remain­

ing in RNase-resistent RNA/DNA hybridso When increasing amounts of RNA

were present in the hybridization solutions, the number of counts

hybridized to the DNA reached a maximum that did not increase with

greater quantities of RNA. The plateau which resulted was interpreted

to mean that all available DNA sites were saturated„ By knowing the

specific activity of the input RNA, the amount of RNA bound can be cal­

culated and from that the amount of DNA saturated by RNA is calculable0

(The percent DNA saturated is also defined when the amount of filter-

bound DNA remaining is known*) Although not appreciated at the time,

these plateau values reflect the complexity of the DNA transcribed;

i.e., the total number of DNA sequences which were used as template for

RNA synthesis. However, early experiments suffered from two central

problems; a reduced specificity of hybridization and an inability to

measure unique sequence transcripts (McCarthy and Church, 1970). The

low temperature and high salt concentrations used resulted in less

stringent homology requirements for hybridization. The relatively

short incubation times and low RNA concentrations employed were gener­

ally sufficient to allow only repeated sequences to hybridize, thereby

exacerbating the problem of mismatch. Early plateaus were probably

achieved due to the kinetic separation between hybridization of repeat

sequence and that of unique sequence transcripts,. For example, calf

thymus DNA repeat sequences have completely reassociated three decades

of Cot before unique sequences begin to anneal (Britten and Kohne, 1968).

Still, this technique is effectively used today when the limitations

just described are carefully considered in designing and interpreting

the experimentSo

A more sensitive method has been developed for analyzing total

genomic transcription.. High specific-activity, denatured DNA is mixed

with a vast excess of unlabeled cellular RNA in a liquid hybridization

mixture (Kohne, 1968). DNA can hybridize with the RNA much more readily

than it can reanneal with itself because the RNA is present in higher

concentration than the DNA. When the reaction is complete, i.e., when

no more DNA will hybridize, the percent of DNA in DNA/RNA hybrids is a

measure of the total portion of the genome transcribed. While results

from this type of procedure are easier to interpret than the filter

method described above, they also present problems. First, the method

is not very sensitive to small changes. A differential transcription

72

of a percent or less of the genome would be difficult to monitor by

this method, although it may represent a major alteration in the RNA

present in the cello This is true for two reasons* First, a one per­

cent difference in the genomic transcription represents a large number

of sequenceso For the chicken chromosomes this fraction would involve

expression of approximately 1„3 x 10"^ daltons out of the total 1®3 x

12 10 daltons of genetic information (Mirsky and Ris, 1950)„ Secondly,

the technique is relatively insensitive to the copy number of a tran­

script since only a few copies are necessary to form hybrids with the

7 limited amount of DNA» For example, if 10 copies of these sequences

2 were present in one cell and 10 in another, both cases would likely

result in no differences in the final percent DNA hybridized,, Both of

these limitations are found in filter-bound DNA saturation experiments

where RNA complexity is also assayed,.

An additional theoretical limitation of DNA/RNA hybridization

using excess unlabeled RNA and labeled DNA is pertinent to this study0

The use of unlabeled, whole cell RNA combined with the fact that in

some cells 1/3 of mRNA's have an average half-life of 24 hours (Singer

and Penman, 1973) reduces many results to steady-state (or time average)

considerations,. This is a direct outcome of the inability to specify

at what point the RNA was synthesized., When all the above information

is weighed, total cellular RNA isolated from cells differing 24-48

hours in age may show a similar percent of labeled DNA hybridized

though the instantaneous transcription may be very dissimilar in these

cells of differing ages.

73

RNA/DNA hybridization, where the RNA is labeled and the DNA is

in excess, is a technique that has been used recently to examine the

hybridization kinetics of RNA present in eukaryotic cells (Gelderman,

Rake and Britten, 1971; Melli et al», 1971)o Employing model systems

for this type of hybridization, Bishop has shown that the reaction

kinetics of the labeled RNA faithfully reflect the kinetic complexity

of the DNA from which it is transcribed (Bishop, 1972)o Since he ob­

served a slower rate for RNA hybridization than DNA reassociation, how­

ever, the C0ti/2 for hybridization of RNA transcribed from a particular

DNA is greater than the C0ty2 for the reassociation of that DNA. Hence,

a known standard must exist to relate any given CDty2 to the absolute

complexity of the DNA from which the RNA is transcribed.

The most outstanding drawback to this technique is the limit

to the total amount of RNA that will hybridize. While Bishop's model

systems show 10C$ hybridization, in hybridizations of eukaryote cellu­

lar RNA to DNA, 25-50?o of the total RNA hybridizes, depending on the

method of assay and the cell system used (Goldberg et al., 1973; Klein

et al«, 197^; Campo and Bishop, 197*0• Evidence reported by Goldberg

et ale (1973) and Smith et al. (197*+) supports the hypothesis that the

extent of hybridization is limited by the extremely high number of

copies of some unique sequence transcripts. Their data indicate that

a large portion of the total RNA is composed of greater than 10^ copies

of these unique sequence transcripts.

7^

Observations and Interpretations

In the study reported here, unique sequence transcripts showed

similar hybridization kinetics for both control and infected cell nRNA

(Figo 12)o Using a different technique, Grady and Campbell (1975a)

assayed transcription in both normal and polyoma transformed 3T3 cells.

By hybridizing labeled DNA to unlabeled nRNA in excess, they found a

small difference (1%) in normal cell transcription as a function of the

growth stateo They deemed this insignificant when compared to the 50%

increase over controls of transformed cell transcription regardless of

the growth state or cell population densityD It is possible that the

differences noted by Grady and Campbell are a function of the cell-line

clones that they examined,, Dissimilarities may then be a function of

the original clones, AL/N and PY AL/N clone 3» as much as they could be

due to transformation by polyoma,. Unlike the system examined by Grady

and Campbell, RSV infected and control cells used in this study were

derived from the same chick embryo cultures which were either infected

or mock infected at the same time0 Furthermore, Grady and Campbell

assayed the extent of unique sequence transcription, whereas the hybrid­

ization kinetics of unique sequence transcripts were examined in this

investigation.. Results reported here support the conclusion that dif­

ferences in "unique" RNA hybridization kinetics between normal and RSV

infected cells are small. However, changes noted in the "repeat" RNA

fraction require that differences in the "unique" RNA fraction must

exist, although they are likely to be only a few percent.

75

Repetitive Sequence Transcripts

Birnie et al„ (197*0 analyzed nuclear RNA which was labeled

during growth or non-growth states of several mouse cell systems.

Using filter bound DNA for labeled RNA hybridizations, they found that

a greater number of repeat sequences were transcribed when cells were

not growing than when they were dividingo On the other hand, Grady

and Campbell (1975b) were unable to detect any differences in the

extent of repeat sequence transcription between growing and confluent

cultures of a mouse cell line0 These investigators hybridized labeled

DNA to excess whole-cell RNA to arrive at their conclusion,. The dif­

ference in results may be due to the undetermined time of synthesis for

the hybridizing RNA used in the experiments of Grady and Campbells

Since the RNA species hybridizing were not restricted to those tran­

scribed during a particular time period, they may represent an average

of the RNA populations present at different times in the cells' growth

cycle»

The low CDt hybridizations reported in this study address the

question of the concentration of repeat sequence transcripts present,

rather than the fraction of the DNA transcribed,. Since these reactions

are driven by DNA, the kinetics are determined by the DNA concentra-

tionB Thus, it is not possible by this technique to determine the

number of different repeat sequences which were transcribed* The

values seen for these hybridizations yield the portion of the total

labeled RNA which was transcribed from repeat sequence DNA during the

pulse-labeling period.

76

Since "repeat" RNA may hybridize to DNA families of similar but

not identical base sequences, the extent of mismatch of the hybrids

formed is higher than that observed with unique sequences„ Hybridi­

zation conditions used here are quite stringent due to the combination

of high formamide concentration and high temperature plus the use of

extensive RNase digestion (see McConaughy, Laird and McCarthy, 1969;

Hayward and Hanafusa, 1975)• The potential for similar but hetero­

logous sequences hybridizing is reduced by elevated temperatures which

select against duplexes with lower "stacking-free energy" (Kennel,

1971)o The RNase assay used for hybrid formation minimizes the pos­

sibility of mismatched sequences being scored as hybrids since both

free ends and internal unhybridized loops are digestedo Where both

RNase A and T1 at 37°C are used, the amount of non-specific hybridiza­

tion can be presumed to be very small due to the complementary substrate-

specificities of these enzymes (Barman, 1969) and the higher incubation

temperature,, It should be noted that all the differences observed in

this study are derived from comparisons between identically treated

infected cell and normal cell RNA samples®

Examining "repeat" RNA hybridization in this investigation

generated two observations — one a function of the time after sub-

culturing cells (growth state) and the other a function of infection

by RSV. In separate experiments where pulse-labeled nRNA was hybrid­

ized to cell DNA, the data show comparable results for the indepen­

dently prepared sets of nRNA. A slightly larger fraction of nRNA

isolated from uninfected control cells early after plating hybridizes

to repeat DNA than that isolated during the period 15-25 hours after

plating (see Fig„ 10), nRNA isolated even later after subculturing,

hybridizes a greater portion of its total to repeat sequences (Figs®

10 and 16). Hence, it is concluded that non-growing CEF cells tran­

scribe a slightly greater fraction of their total nRNA from repeated

DNA sequences than do dividing cells« The second observation with

pulse-labeled nRNA was the presence of proportionately greater amounts

of repeat sequence transcripts within the nuclei of infected cells late

after infection,. Between 25 and 50 hours after infection by RSV cellu­

lar nRNA is demonstratably richer in repeat sequence transcripts®

Grady and Campbell (1975b), using labeled DNA in hybridization

with excess RNA, found no difference in the number of repeat sequences

used as templates for RNA synthesis between normal and polyoma trans­

formed 3T3 cells* Earlier, Neiman and Henry (1969) hybridized labeled

RNA to filter-bound DNA and showed that normal lymphocyte nuclei con­

tain "repetitive" RNA of greater complexity than leukemic cells0 Con­

versely, the same technique demonstrated nRNA from hepatoma cells had

greater complexity than the control liver cell nBNA (Church, Luther

and McCarthy, 1969)0 These filter hybridization studies contain all

the inherent problems discussed above that limit their sensitivity to

the overall RNA synthesis occurring* Without contradicting these

findings, the data presented in this study demonstrate that a greater

portion of the nRNA synthesized late after infection is transcribed

from repeated DNA than is transcribed in uninfected cells, regardless

of the number of different sequences expressed.

78

The dissimilarities seen between infected and control cell nBNA

hybridizations diminished at 58 hours after infection (Figs„ 15 and 16).

It would appear that the increase in the fraction of repeat sequence

transcripts observed during the transformation of CEF cells does not

continue indefinitely.. Infection of normal cells by RSV may be causing

a transient alteration of repeat sequence transcription that recedes

after transformation. In fact, morphological transformation of cells

begins about 2k hours after infection (see Figo k) at approximately the

same time that differences are first noted in nBNA synthesis® These

observations argue for the possibility that the transient alteration in

••repeat" RNA synthesis marks the passage of cells from normal to trans­

formed states and is in some way related to this phenomena. A second

explanation would be that transformed-cell transcription responded to

the culture conditions late after passage<, According to this hypothe­

sis, the differences between normal cells and transformed cells would

be detected when both these cell types are actively gro\ving, but not

seen when they are rarely dividing.. Data presented in this study plus

evidence cited from other investigations indicate that normal cells do

respond to this form of regulation,. It was shown here that normal

cells stopped synthesizing DNA after kO hours in culture and that fresh

serum could induce new DNA synthesis at this time* The effect of serum

on the multiplication and saturation density of RSV transformed cells

is also well documented (Temin, 1967b).. It seems plausible that the

return to control values for the amount of repeated sequence nRNA

hybridized from transformed cells could be a function of growth limiting

79

factors in the culture. This is not to say that the "repetitive" nRNA

found late after infection in control and infected cells represents the

same transcripts but only that the fraction of nRNA that is "repeti­

tive" RNA is similar for both cell types0 In fact, it seems much more

likely that these transcripts are different but that that difference

is not measured by the technique usedo

The examination of polyribosomal RNA for the amount of "repeat"

RNA showed that, while normal cells had a reduced concentration of

"repetitive" RNA associated with the polysomes, RSV infected cells

exhibit a further decrease in this proportion (Figo 18)<> This finding

is consistent with the fact that representation of total nRNA in the

polysomal fraction is known to be very limited (see Greenberg, 1975)®

Early hybridization studies with filter bound DNA demonstrated greater

complexity for repeat sequence transcripts found in nuclear compared

to that found in cytoplasmic fractions of tumor cells (Drews, Brawerman

and Morris, 1968).

One possible contribution to the observed increased concentra­

tion of repetitive sequences in infected cell nuclei would be an in­

creased synthesis of ribosomal RNA (rRNA)„ Colby and Rubin (1969) have

noted that RSV transformed chick cells do synthesize more rRNA than

normal confluent cells. Other investigators have established that

normal cells — both CEF and the 3T3 cell line — synthesize more rRNA

while growing than when contact inhibited (Emerson, 1971; Johnson et al.,

197*0• However, it is not likely that increased rRNA synthesis could

account for the higher fraction of repeat sequence transcripts observed

in this study. The labeling pulse used here was sufficiently long to

allow synthesis and transport of rRNA to the polysomes (Perry, 1967;

Dinowitz, 1975b)o If the "repeat" RNA were rRNA, one would predict

that greater concentrations of "repeat" RNA in the nucleus would also

be seen in pRNA from infected cells and this was not observed (Figo l8)o

In addition, the copy number of ribosomal cistrons/genome is incon­

sistent with the CDt at which differences in "repetitive" RNA was

observed,, Since chick ribosomal sequences occur at 100 times the fre­

quency of unique sequences (Attardi and Amaldi, 1970), the C0ti/2 for

rRNA should be 100 times faster than the "unique" RNA (0oty2 = 2„5 x 10 )

or 25° All of the differences noted for repetitive sequence transcript

hybridizations were completed before this CQt (see Figs0 13 and 1*0«

The relative distribution of "repetitive" RNA in nuclear and

polysomal fractions appears to be greatly altered by virus infection

(Fig0 19)<= It is tempting to speculate that the increased "repetitive"

RNA seen in the nucleus and the decreased "repetitive" RNA found on the

polysomes results from a reduction in processing or transport of the

RNA from nucleus to the cytoplasm — as has been observed in another

virus-infection system (Dinowitz and Green, 1972)» This hypothesis

would specify that similar proportions of "repetitive" RNA are tran­

scribed in RSV infected and control cells but that some event following

synthesis is altered in transforming cells. Late after RSV infection

both the nuclear and polysomal differences in concentration of "repeti­

tive" RNA diminish, suggesting that the degree of altered transcription

or processing reaches a peak and then is reduced. However, different

81

gene sequences may be transcribed before and after the time of altered

RNA synthesis.

Conclusions

Evidence presented in this investigation suggests that a change

occurs in RSV infected cells in the concentration of RNA transcribed

from reiterated DNA» This difference is sufficiently large to be

measured by the procedures used here beginning about 25 hours after in­

fection and reaches a maximum ^5-50 hours after infection., "Repetitive?'

RNA found on the polysomes of infected cells decreases in concentration

over controls during this same period,, These findings have been sum­

marized in Figo 20 which shows the relationship between cellular events

and the hours in culture* Observations characteristic of normal and

RSV-infected cells — as documented in the literature •— are listed

immediately above and below the time line showing the order of their

occurrenceo Moving further away from the time line, observations from

this study are shown- It is interesting to note the coincidence of

several changes in growth and morphological characteristics with bio­

chemical alterations., The decreased concentration of "repetitive" RNA

associated with infected cell polysomes occurs at approximately the end

of the provirus integration phase. The concentration of newly synthe­

sized "repetitive" RNA from infected cell nuclei increases at about the

time the culture first contains cells appearing morphologically trans­

formed. These differences all occur during the period in which RSV-

infected chick cells are converted from the normal to transformed

morphology®

Figure 20. Summary of cellular events after subculturing or infection with RSV.

Selected events occurring in normal and RSV-infected cells in culture are compared with emphasis on growth condi­tions, nucleic acid synthesis, and transformation to tumor cells. Immediately above and below the time line are observations of changes related to cell density and RSV infection reported in the literature (/—* \). Moving away from the time line are observations supported by data from this study (y—™x) concerning DNA synthe­sis and transcription.

CO o h-cn cc UJ h* O < cc < x o

UJ o

or o

CO u h-CO

or UJ t— o < cc < X o

UJ o Q UJ I— o UJ UL

Newly synthesized "repetitive" nRNAand pRNA decrease in concentration

Cellular DNA" synthesis reaches

a maximum

Newly synthesized "repetitive"nRNA and pRNA

increase in concentration

Cellular DNA synthesis is no longer detectable

/"

Growing cells A

10 Hrs. p. plating

./•

Confluent monolayer a _

30 50

0

\_

Hrs. p. i 20 40 60

Provirus phase Budding RSV detectable

N ..

TJ Q) O a>

0) O

Culture morphologically transformed

Newly synthesized "repetitive" nRNA increases in concentration over controls

Newly synthesized "repetitive" pRNA decreases in concentration over controls

Figure 20. Summary of cellular events after subculturing or infection with RSV.

oo

83

An attractive hypothesis to account for these observations is

that some repeat sequences or repeat sequence families are involved in

the regulation of changes in cell metabolism related to growth and

differentiation (Britten and Davidson, 1969; Davidson and Britten,

1973)i and that infection with RSV affects these controlling sequences.

The data presented in this investigation are consistent with such a

possibilityc, Further characterization of specific repeat-sequence

transcripts and their functional products (or primary effects) may pro­

vide some insight into the changes induced in cellular transcription

by RSV infection. However, until the role of repetitive sequence DNA

and its transcripts are better understood, the fundamental significance

of these observed alterations will remain a matter for speculation.

LIST OF REFERENCES

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