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llllfl' [WEN llilllefl'jllljlllll

LIBRARY L
Michigan State
University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

     

 

 

 

 

 

 

 

 

 

This is to certify that the

dissertation entitled

THE GOL SITE: A BACTERIOPHAGE T4 REGULATORY
REGION THAT CAN AFFECT EXPRESSION
OF ALL THE T4 £§gfi GENES

presen

Wendy Cooley Champness

has been accepted towards fulfillment
of the requirements for

M-_degree in Microb io logy

M9 %

Major professor“

51%(7/

MSUL! an Affirmative Action/Equal Opportunily Institution O~1 2771

 

 

MSU

 

 

RETURNING MATERIALS:
Place in book drop to .

 

 

LIBRARIES remove this checkout from
up. your record. FINES will
be charged if book is
returned after the date
stamped below.
«W'
’AUG 1 3 9915’

.1 22“

 

 

THE 99L SITE: A BACTERIOPHAGE T4 REGULATORY REGION
THAT CAN AFFECT EXPRESSION OF ALL THE

T4 LATE GENES

By

Wendy Cooley Champness

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

1982

 

ABSTRACT

THE GQL_SITE: A BACTERIOPHAGE T4 REGULATORY REGION
THAT CAN AFFECT EXPRESSION OF ALL THE
T4 LATE GENES

By

Wendy Cooley Champness

Escherichia coli mutants have been isolated which prevent T4

 

late (replication coupled) gene expression at temperatures below 34°C.,
primarily by acting at the level of transcription. The E, gglj_mutants
have been mapped to 25 min., where they define a new gene, 11;,

Because of the severe block to late gene expression, wild-
type T4 does not form plaques on 113’ mutant hosts at low temperatures.
T4 mutants which have been named 391, for grow on ljt' hosts, are able
to form plaque.

In a mixed infection of a litf host by golf and golf phage,
the 991 mutation is able to restore gene expression only in £13. All
ggl_mutations map within the coding region for p23, the major T4 capsid
protein. However, ggl_mutations do not exert their effect by altering
p23. Rather, they seem to affect a site on T4 DNA, which in the wild-
type form interferes with gene expression in a lit: host. The nucleotide
sequence of several 991 mutations has been determined. However, from
the sequence data accumulated thus far, the structural significance of
the ggl_mutations is not clear. I speculate that the T4 ggl_site plays

a role in the template processing that has been proposed to be required

for T4 late gene expression.

Wendy Cooley Champness

Besides affecting T4 late gene expression in a lit? host, the T4
ggl site has an effect when cloned into plasmid DNA. If a plasmid
contains a fragment of T4 DNA which includes the wild-type ggl_site, it
cannot be used to transform a lit: host, but can be used to transform
a lit? host. However, if the plasmid contains a T4 fragment which is
gglf or is deleted for the ggl_region, it can be used to transform a

litf host. Therefore, the presence of the wild-type ggl_site in a

plasmid prevents the stable transformation of lit' E. coli.

 

The ability of the T4 ggl_site to affect both T4 and plasmid DNA
may reflect that a common structure is shared by the two DNAs; further

study of the ggl_site may reveal the ig_vivo function of such a DNA

 

structure.

ACKNOWLEDGEMENTS

I gratefully thank all who advised, supported and encouraged
me, especially Dr. Loren Snyder; my Guidance Committee members,
Drs. Jerry Dodgson, Ronald Patterson, Leonard Robbins, and Harold
Sadoff; and Drs. Paul T. Magee and Michele M. Fluck.

I acknowledge a National Science Foundation Predoctoral
Fellowship and a College of Veterinary Medicine Graduate Fellowship,

as well as funding by Department of Microbiology and the National

Institutes of Health.

ii

 

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES

INTRODUCTION .
LITERATURE SURVEY

The T4 Genome . . . . . . . . . . . . . . . . . . .
Prereplicative (Early) Transcription . . . . .....

Immediate early and delayed early

Quasi- Lates . . .....

Shutoff of early genes . . .
Postreplicative (Late) Transcription .

Regulation of late gene expression .
T4 DNA Replication . . .

T4 replication proteins

The replication process

Intracellular DNA Structure .
Replication— Uncoupled Late Gene Expression . . .

In vitro systems . . . ............

 

Effects of Nucleotide Modification on Late Gene Expression : :

The role of the glg_product in late transcription
Expression of Late Genes from Clones .
The T4 PseT Gene .
Gene 63— . .
E. Coli CTer
DNA STructure and Transcription

ARTICLE I: THE §9L_SITE: A §I§;ACTING BACTERIOPHAGE T4 REGULATORY
REGION THAT CAN AFFECT EXPRESSION OF ALL THE T4 LATE GENES .

ARTICLE II: THE BACTERIOPHAGE T4 GOL SITE: SEQUENCE ANALYSIS AND
EFFECTS OF THE SITE ON PLASMID TRANSFORMATION . . . .

SUMMARY

REFERENCES .

APPENDIX A: A NEW GENE OF ESCHERICHIA COLI NHOSE PRODUCT
PARTICIPATES IN T4 BACTERIOPHAGE LATE GENE EXPRESSION: INTER-

ACTION OF LIT WITH THE T4- INDUCED POLYNUCLEDTIDE 5' -KINASE
3' -PHOSPHATASE . . . . . . . . ......

 

APPENDIX B. NILD- TYPE T4 GENOMES INTERFERE WITH THE EXPRESSION
OF GOL MUTANT GENOMES IN MIXED INFECTIONS

1'11

Page

iv

42
73
78

88

97

 

LIST OF TABLES

Table
ARTICLE I

l. Hybridization of RNA labeled after infection of th lit?

e
host to the separated complementary strands of T4 DNA .

2. Mapping of a ggl_mutation by three factor crosses .

3. Production of ggli and ggl_mutant phage in mixed infections .

ARTICLE II
l. Strains used

2. Transformation of 11;: E, coli by plasmids .

APPENDIX A
lA. Bacteriophage and bacterial strains .
2A. Mapping of lit_mutations with P1 transduction .

3A. 3' phOSphatase activities in lit_mutants and extracts of
T4-infected E, coli B .

iv

Page

33

34
39

47

65

89
9T

94

 

LIST OF FIGURES

Figure
ARTICLE I
l. T4 protein synthesis after infectioncrfthe 113: host .

2. An expanded map of the gene 23 region of T4 showing the
position of a ggl_mutation . . . . . .........

3. Synthesis of 14 gene 23 product in mixed infections with a

gene 23 amber mutation and a gol mutation on lit- E, coli.

4. Synthesis of T4 gene l8 product in mixed infection with a

gene l8 amber mutation and a ggl_mutation in lit- E, coli.

ARTICLE II

1. Correlation of genetic and physical maps in the T4 gene 23
region . . . . . . . . .

2. Cloning strategy .

3. Sequence of the 270 base pair Hpa II fragment and changes
due to ggl- mutations . . . . . . . . . . . .

4. Restriction enzyme digests of plasmids used in
transformation studies .
APPENDIX A
IA. The rate of T4 DNA synthesis after_infection of the li __:c_+
parent E. cLli A82495 and a lit recombinant of MPH “6
with T4 wild- -type and a T4 Gol mutant . . .
2A. Alkaline sucrose gradient centrifugation of the T4 DNA .

3A. Rate of synthesis of T4 proteins after infection of the
recombinant of E, coli MPH 6 . . . . . . . .

4A. T4 late protein synthesis by wild- -type and a T4 601 mutant
on E. cLli A82495 (lit ) and E. cLli MPH 7 (lit )

SA. T4 late protein synthesis on E, coli CTer at 37 and 42°C

APPENDIX 8

l8. Rate of synthesis of T4 late proteins in mixed infections
of wild-type T4 and g_ol_ mutant on l_1'_t' E. coli

 

V

Page

32

35

55
57

6O

63

91
92.

93

94

. lOD

 

INTRODUCTION

Despite the wealth of current knowledge about transcriptional
regulation in prokaryotes, the control of the expression of the T4 late
genes remains an intriguing mystery. It has been known for some time
that T4 late gene expression is coupled to concurrent replication and
it is thought that a structural alteration of the template occurs,
during replication, which activates the late promoters. The nature
of the template alteration is not known and it has not been possible

to reproduce, 3n vitro, the in_vivo state of the "competent" T4 late

 

transcriptional template.

It is becoming increasingly evident that “promoter-operator"
models for transcriptional regulation do not completely describe all
regulation, even in prokaryotes. Mechanisms operating in T4 phage
may well also operate in E, ggli and higher organisms, as well.
Therefore, elucidation of T4 late transcriptional regulation may
add to our understanding of regulatory mechanisms in general.

Another aspect of the problem of late gene expression in T4
phage is that of coordinating transcription with other intracellular
events. The phage life cycle requires that one to two hundred phage
equivalents of DNA be replicated, an amount of DNA which is greater
than ten times that of the bacterial nucleoid. The intracellular DNA
is in a highly complex, condensed structure, in which recombination is
very active. The late transcriptional units are highly active in
order to produce the structural proteins for at least one hundred
phage. Furthermore, encapsidation of DNA into phage heads begins at a

l

2
time when all these events are actively occuring; therefore replication,
recombination, transcription and packaging all occur simultaneously.
It seems likely that mechanisms exist which regulate the coordination
of these events; analogous mechanisms might operate in the even more
complex coordination problems of higher cells.

One approach to the study of T4 late transcriptional regulation,
taken in our laboratory, has been to study T4 genes which encode
functions involved in late transcription. One such gene, gggl,
encodes the T4 5' polynucleotide kinase, 3' phOSphatase. The p§g1_gene
product is required for T4 late gene expression on the E, ggli_strain
CTer (K. Sirotkin, w. Cooley, J. Runnels and L. Snyder, l978). The
ig_vit£g_activities of the enzyme suggest that it might have the

capability, and ifl_le0 role, of altering T4 DNA structure to make it

 

competent for late transcription. Also, it seemed that E, ggli_encodes
at least one function,which is altered in CTer, which is involved in
T4 late gene expression. If so, mechanisms for transcriptional regula-
tion, similar to those operating in T4 might exist in E, 9911, this
possibility inspired further study of CTer restriction of pggl:_mutants.
In the hope of simplifying genetics and biochemical analysis of
the pggllrrestrictive locus in CTer, attempts were made to isolate host
mutants, in a K-l2 background, that would mimic the p§31::restrictive
phenotype of CTer.
The E, ggli_mutants which resulted from a search for ”CTer-like”
mutants have been called lit_and are described in the publication in
Appendix A (Cooley et al., 9, Bacteriol. 140:83-91, l978). Eit_mutants

restrict pseT- deletions but not pseT' point mutations at 37°C. At

temperatures below 34°C, even wild-type T4 is restricted. T4 fails to

3
multiply on 11;: hosts because of a severe block to T4 late gene
expression at the restrictive temperature.

Spontaneous T4 mutants arise which can form plaques on 111:
host mutants at the restrictive temperature. These T4 mutants have
been called 391. In Article I, which is published in J. Mol. Biol. 155:395
0982),evidence is presented that ggl_mutations alter a site on T4 DNA
which affects the expression of all the late T4 genes. The ggl_site
may be involved in forming or maintaining the DNA template structure
which is thought to be required for T4 late gene expression. Such a
site may be a prototype for a new class of prokaryotic regulatory
sequence.

Besides affecting T4 gene expression, the ggl site affects the
transforming capability of a plasmid which contains it, i.e. a plasmid
which contains a gglf sequence will not transform a lit: host. In
contrast, if the plasmid contains a ggl_mutant sequence or is deleted
for the ggl_region, it can transform a litf host. Therefore, the 391
sequence must affect the replication, expression or segregation of
plasmids which contain it. This work, as well as DNA sequence analysis
of ggl_mutations, is described in Article II.

The literature survey discusses work that has been done on T4
late transcription, both ig_yivg_and jg_yi§:g, Because some aspect of
T4 DNA structure is required for late transcription, studies on intra-
cellular T4 DNA are reviewed. Finally, regulatory mechanisms which
involve DNA structure and which may be relevant to T4 late transcrip-
tional regulation are discussed.

In the final section, the results presented in Articles I and II
and the appendicies are discussed, and recommendations for further study

are made.

LITERATURE SURVEY

The T4 Genome

 

The T4 genome is l.66 kilobase pairs, enough to code for
approximately 160 genes. The linear molecules from phage particles are
slightly longer because they are terminally redundant. They are also
circularly permuted, giving use to a circular genetic map. About l40
of the T4 genes have been characterized. (Wood and Revel, l976)

The T4 transcripts can be classified into three major groupings:
l) those which are expressed prior to DNA replication; 2) those which
are expressed after DNA replication and 3) those which are expressed
throughout infection. The first category, the "early genes,” includes
the genes whose products function in phage DNA metabolism. Genes in
the second category, the "late genes,” encode phage assembly functions.
The late gene products begin to appear 2.5 to 3.5 minutes after DNA
replication begins, which usually occurs at 5 to 6 minutes after
infection at 300C. Most early protein synthesis starts to be shut off
at about the time late synthesis begins, but there is a class of genes,
the quasi-lates, which are expressed both early and late. The only
quasi-lates functions identified are the T4 tRNAs. (Young et al., l980).

The T4 genes are arranged on the genome such that the early
genes are clustered into two major groups and the late genes are clustered
into three major groups, separated by the early clusters. (Wood and Revel,
1976). Within each group are multiple transcription units; many genes are
expressed as both mono and multicistronic transcripts (Young and Menard,

l98l). The early genes are all transcribed with the same polarity, off

4

5
the "l” strand (Notani, l973). The late genes are transcribed off the
”r" strand (Guha et al., l97l).
The rate of chain elongation in T4 is llOO nucleotides per
minute at 300C, about one half the rate in uninfected E. coli. In

general, but with some exceptions, the mRNAs are immediately translated.

Prereplicative (Early) Transcription

 

Immediate early and delayed early

 

Within the prereplicative period, the first 5-6 minutes after T4
infection, many species of early proteins appear in a defined temporal
sequence (O'Farrell and Gold, l973). More than one regulatory class of
prereplicative messages exists, based on the following evidence (reviewed
in Rabussay and Geiduschek, l977): l) the mRNA synthesis of a small number
of early proteins cannot be initiated within the first one to two minutes
after infection (O'Farrell and Gold, T973), and the synthesis of these
proteins is sensitive to the addition of rifamycin added immediately after
infection; 2) the transcription of some genes does not occur in the
absence of protein synthesis; and 3) the relative abundance of mRNAs
changes between five and twenty minutes (Salser et al., l970). The
different regulatory classes have generally been termed immediate early
(IE), delayed early (DE), and quasi-late.

Immediate early genes are proximal to promoters; delayed early
genes are distal to promoters and appear after a time lag of l.5 minutes
at 30°C (Salser et al., l970). Synthesis of the delayed early genes is
sensitive to chloramphenicol addition (Brody et al., 1970). The chloram-
phenicol block is probably due to induced polarity (Young, l975) mediated

by :39_(Caruso et al., l979; cf Young and Menard, l98l).

 

6

Recently, Young et al (l980) measured the levels of early
mRNAs which hybridized to selected early gene clones. They found that
messages for genes 30, 39, 52, 4l, 42, Bgt and the tRNAs appeared first,
were made at their highest rates between zero and four minutes and were
insensitive to the addition of chloramphenicol. These genes would conform
to the designation "immediate early genes." Messages for genes 43, rIIA
and rIIB appeared after those for the IE genes, peaked during four to
eight minutes, and were not made in the presence of chloramphenicol
(CAM); these genes would be the "delayed early genes."

The CAM sensitivity of the switch—on of the delayed-early genes
suggests that a phage-coded factor plays a role in the switch from
immediate-early to delayed-early synthesis. Mutants have been isolated
which may be in such a factor (Mattson et al., l974). These mutants,
called mg}, reduce the expression of tRNA, 32, r118, 43, 45 and 46, but
delay the shut—off of 44, rIIA and 52. An ig_yi§§9_RNA synthesizing
system has been developed (Thermes et al., l976) in which the mg£_pheno-
type and the chloramphenicol effect of restricting transcription to
promoter-proximal regions can be reproduced.

The proposal has been made (cf Pulitzer et al., l979) that m9§_
acts as an antiterminator. However, this proposalremainsunproven and
the assignments of genes to the DE versus IE categories by mg3_effect
do not entirely correspond to the kinetics of mRNA production, as
measured by Young et al (l980)

Sizing of early mRNAs has shown that some of the messages
are synthesized in multiple transcripts, lending support to the model
that DE mRNAs are Synthesized by readthrough from IE genes (Young and

Menard, l98l). However, attempts to size the CAM RNA of early genes

 

7
in order to show that CMA RNA is shorter than normal transcripts, were
unsuccessful (Young and Menard, l98l) because extensive degradation of
RNA, both in the presence and absence of CAM, makes sizing RNAs diffi-
cult. Thus, the early regulatory switch between IE and DE synthesis is

not yet well understood.

ansi-Lates

 

The prereplicative transcripts which are present at five minutes
but increase at twenty minutes have been called the "quasi-lates.” Based
on the presence of the gene product both before and after replication,
several genes have been identified as quasi-lates, including 32, 4l, 43,
44, 63, 57 and the internal head proteins (cf Hood and Revel, l976).
However, by following the kinetics of hybridization of pulse-labeled
mRNA to cloned early genes, Young et al. (l980) did not find that the
mRNA of genes 41 and 43 increased at twenty minutes; they did not study
the mRNA of other putative quasi-lates. They did find that the synthesis

of the tRNAs showed quasi-late behavior.

Shutoff of early genes

 

The synthesis of most early gene products, with the exception of
the quasi-lates, ceases after l2 to 14 minutes of infection at 300C
(Hiberg et al., l962; Hosoda and Levinthal, 1968). The synthesis of many
early genes is not shut off synchronously (O'Farrell and Gold, l973) and
some of the genes are autogenously regulated (reviewed by Rabussay and
Geiduschek, l977). Thus, a variety of regulatory mechanisms are important
in early gene shutoff.

To a large extent, early gene shut off occurs as the result of

transcriptional regulation (Bolle et al., l968a; Salser et al., l970).

 

8
However, post transcriptional mechanisms also operate because, for many
early proteins, the stability of early mRNAs decreases at the time early
shutoff occurs (Sauerbier and Hercules, l973). Furthermore a gene has
been identified, Egg A, which can affect the stability of early messages.
In :gg_A_mutant infections, many early messages have greater stability,
an effect which is enhanced if replication does not occur.

In general, when replication does not occur, the early gene
products continue to be synthesized (Hiberg et al., l962; Bolund, l973).
Also, iriinfectionsin which gene 55 is mutant (and therefore late protein
synthesis is not turned on) early gene expression does not cease
(Bolund, l973). Because late genes are also not expressed in replication-
defective infections, these observations could be interpreted to mean
that a late gene product plays a role in early shutoff. But recently,
several situations have been described in which early gene shutoff occurs
even though no late gene expression occurs. These situations are l) in
an infection in which cytosine DNA rather than hydroxymethyl cytosine,

DNA is produced (Snyder et al., l976); 2) RNA ligase or pseT mutant

 

infections of the host CTr5x (Runnels et al., l982); and 3) wild-type T4
infections of 115: hosts at 300C (Champness and Snyder, Article I of this
dissertation). It therefore seems that Uwasimple explanation that a late

gene product shuts off early gene expression must not be correct.

Postreplicative (Late) Transcription

 

The post-replicative gene products begin to appear about 2.5 to
3 minutes after the onset of replication; i.e., at about 8.5 to 9.5
minutes after infection at 300C. (Bolle et al., l968a; O'Farrell and Gold,
l973). The temporal regulation of late gene expression is at the level

of mRNA transcription (Young et al., l980).

9

The late genes are distributed into three clusters which are
separated by regions encoding early genes (Hood and Revel, l976).
Because most of the late genes are transcribed from the ”r” strand,
with an opposite polarity to the early genes (Guha et al., l97l; Notani,
1973), they cannot be transcribed by readthrough from early promoters.
Rather, many new promoters must be utilized. Information about the
location and utilization of late promoters comes from a recent study of
transcription of the contiguous late genes 2l, 22 and 23 (Young et al.,
l98l). The late genes 2l, 22 and 23 are co-transcribed as two minor
polycistronic messages and gene 23 is also transcribed as a major
monocistronic message. Because the three messages have the same distri-
bution in both steady-state and pulse-labelling conditions, it seems
likely that the multiple messages result from the use of multiple

promoters and not from processing of a single large precursor.

Regulation of late gene expression

 

Transcription of the T4 late genes requires the association
of several T4-specified proteins with the host RNA polymerase. Early
after infection, the RNA polymerase is modified by ADP-ribosylation
of the 6 subunits (Goff, l974). Several low molecular weight polypep—
tides bind to the modified RNA polymerase. These include the products
of genes 33, 55 and 45 (Horvitz, l973; Ratner, T974), and lSK and a 12K
dalton protein (Stevens, 1972). The products of genes 33, 55 and 45 are
directly and continuously required for late transcription (Bolle et al.,
1968b, Pulitzer, 1970; and Wu et al., l975). The product of gene 45 is
required directly, in transcription, and indirectly, in replication

(Wu et al., 1975).

10

In addition to modification of RNA polymerase, T4 late gene
expression is thought to require processing of the DNA template. This
idea has resulted from the observation that late gene expression is
normally coupled to concurrent DNA replication (Riva et al., T980), but
in the absence of replication, mutations in genes which lead to the
formation or stabilization of interruptions in DNA permit late gene
expression (Bolle et al., l968b; Hosoda and Levinthal, l968; Wu et al.,
l975). Therefore, it has been proposed (Riva et al., l970b) that nicks
or gaps in DNA might be required for late transcription.

Because late gene expression is replication-coupled it is

appropriate at this point to discuss T4 DNA replication.

T4 DNA Replication

 

T4 replication proteins

 

The replication complex consists, at least, of the products of
genes 43 (DNA Polymerase), 32 (helix-destabilizing protein), 44 and 62
(a complex with DNA-dependent ATPase activityL 4l (DNA-dependent ATPase
activity), 45 (which stimulates the activity of the products of gene 44
and 62) and 6l. The above complex can be reconstituted to carry out
replication ifl_yit:g_(Hibner and Alberts, l980). The ifl_yi§59_system
mimics in_yjy9_replication with respect to rate of chain elongation,
RNA priming of Okazaki fragments and the general structure of the
replication fork. In addition to the above proteins, other T4 proteins,
including the products of gene 1 (deoxynucleotide Kinase) and 42 (cCMP
hydroxymethylase) may have structural roles in replication.

Recently, a T4 Type II topoisomerase has been identified as the

product of genes 39, 52 and 60. Mutants in these genes had been

 

ll
characterized as the DNA-delay mutants because DNA replication rates in
39', 52' or 60' infections are lower and delayed relative to wild-type
(Naot and Shalitin, l973; Mulfi and Bernstein, l974). The T4 topoisomer-
ase probably acts at replication forks (McCarthy, l979). Topoisomerase
mutants (DNA-delay) are cold-sensitive and vary in the severity of their
replication defects on B versus K-lZ strains. These phenomena may
reflect that an E, ggli_enzyme (probably gyrase) can substitute for the

T4 topoisomerase under some conditions.

The replication process

 

The first event after T4 injection into the cell is attachment
of the DNA to membrane, at what will be the origin of replication (cf
Siegel and Schaechter, l973): DNA-membrane attachment occurs without
phage protein (Earhart, l970) or DNA synthesis (Earhart et al” l973),
but some RNA synthesis is required. A candidate for the required RNA
is an RNA-DNA copolymer (Buckley and Kozinski, l972) which is synthesized
early, hybridizes to the “l” strand and is an obligatory intermediate in
DNA synthesis.

Attachment of the DNA to membrane is sensitive to energy poisons
but this sensitivity is reversible after removal of the poison (Earhart
et al., l973).

Within five minutes after infection, deoxynucleotides are added
to the parental DNA, increasing the size of the parental molecules by 6%
(Murray and Mathews, l969a). This addition occurs in DD mutants and is
apparently mediated by E, ggli_enzymes. Surprisingly, this "early DNA"
does not hybridize to mature T4 DNA at a detectable level (Murray and

Mathews, l969a), but does hybridize to vegetative DNA. A possible

 

explanation, which has not been investigated, is that the early DNA

is copied from a sequence which becomes amplified in vegetative DNA.

In this regard, Kozinski et al., (l980) have recently found that, in the
early stages of replication, the replication origins are repeatedly
amplified by replication. The amplification extends through much of the
major late cluster of genes.

T4 replication initiates at multiple sites, primarily in the
regions of genes 50-5 and 25-29 (Halpern et al., 1979), and proceeds
bidirectionally until 20 to 25 phage equivalents are produced. These
DNA molecules recombine (Tomizawa, l967), through the action of the gene
products 46, 47, and 59, to produce concatemers (cf. Broker and Doermann,
l975). DNA synthesis is not a prerequisite for concatemer formation
because concatemers are formed in a gene 44- (DNA replciation negative)
infection (Murray and Mathews, l969b). Replication continues in a
complexly interconnected structure (Huberman, l968) which is distributed
throughout most of the intracellular volume (Hamilton and Pettijohn,

l976).

Intracellular DNA structure

 

The complex structure of T4 DNA late in infection is poorly
understood, despite considerable experimentation designed with the
purpose of describing intracellular DNA structure. The main experimental
approach has been to gently lyse infected cells and study the sedimenta-
tion behavior of the released T4 DNA. Because different workers have used
a variety of lysis procedures, the reported results are difficult to
compare. There is agreement among workers (cf. Siegel and Schaechter,
1973) that both rapidly sedimenting and slowly sedimenting forms are
seen, but the actual S values of the slow and fast forms vary from

report to report.

l3

Altman and Lerman (1970), using a low salt, lysozyme, SDS
lysis procedure, reported four differently sedimenting species: l) ”slow"
or <l005; 2) "fast” or 170-6505; 3) mature phage 650-9505; and 4) "bottom"
or >950-35008. Within the first five minutes after infection the slow
form, consisting of parental DNA, appears. Half of the parental DNA is
rapidly converted to >950-35005, the form in which new DNA appears.

This "bottom" DNA has membrane associated with it. Later, some of this
DNA is converted to the "fast" form, l70-6SOS. From this pool, mature
phage (650-9508) are drawn. Some of the "bottom" DNA goes to a slow
form (<l005) and never is encapsidated. Also, 40% of the DNA in the
bottom fraction neither moves to the slow form nor is encapsidated into
mature phage.

Cox and Conway (l975a, b) attempted to determine the template
properties of the differently sedimenting forms of vegatative T4 DNA.
They found that a lSOS form appeared at 12 minutes after infection (at
30°C). When DNA from this fraction was recovered from a sucrose gradient,
it was an active template for protein synthesis; the protein products
were identified as structural (late) proteins. In contrast, the DNA
from their fast fraction, l000 to 2000 S, was an active template for
early proteins. They found that the slow form was missing when the
gene 55 product is missing (the product of gene 55 is normally required
for late protein synthesis). The absence of a slowly sedimenting form
in a gene 55 mutant infection has also been observed by Snyder and
Geiduschek (l968). The "slow" DNA could be created by the action of a
late gene or could be the template from which late genes are expressed;
Cox and Conway (l975b) favored the latter possibility. But the proteins

synthesized in their gene 55 mutant infections closely resembled those

l4
produced from their slow DNA template; because it is known that gene 55
mutant infections do not produce late proteins, this discrepancy raises
doubt about their identification of ”late" proteins.

Mutants which are defective in recombination do not make fast
sedimenting DNA (Cox and Conway, 1975b; Shah, l976) because they are
defective in concatemer formation. Nevertheless, they do make
significant amounts of late gene products. Concatemers are also not
formed in infections by gene 52 mutant (a component of the T4 topoisomer-
ase) infections, which also produce late gene products (Naot and Shalitin,
l973). Therefore, the fast sedimenting form pg§_§§_must not be required
for late synthesis.

In summary, the studies that have been done do not lead to

conclusions about which DNA fraction is the jn_vivo template for late

 

gene expression.

Many phage mutants which are defective in head-filling accumulate
a 2005 form of DNA. Phage which are mutant in gene 49 also do not package
DNA into capsids; they accumulate DNA in a ”very fast sedimenting“ form
of l-2000 S (Kemper and Janz, l976). The DNA, when visualized by elec-
tron microscopy, consists of tangles, loops and free ends (Kemper and
Brown, 1976). The DNA can be activated for head-filling if an infection
with a ts mutant in gene 49 is returned to the permissive temperature.

Curtis and Alberts (l976) further studied the role of the gene 49
product and found that it is involved in creating single-stranded gaps
in the DNA at an average length of one to two genome distances. The gaps
are probably protected by the gene 32 product. Curtis and Alberts also
studied the structure of the fast-sedimenting complexes and found that

they were unaffected by Pronase, Proteinase K or pancreatic RNase treatment.

l5
Also, the sedimentation rate was higher than could be accounted for by
the amount of DNA, They concluded that the T4 DNA must be condensed
into a compact formation, a conclusion that is in agreement with the
electron micrographs of intracellular DNA (Huberman, 1968; Kemper and
Brown, 1976; Hamilton and Pettijohn, l976). Curtis and Alberts
suggested that the DNA was cross-linked or branched, at least partially
because of recombination forks. In light of current knowledge about
the ability of T4 topoisomerase to concatenate and knot DNA (Liu et al.,
T980) it is likely that some of the compactness of the intracellular DNA
may be due to condensation by topoisomerase. This hypothesis is consis-
tent with the finding of Hamilton and Pettijohn (1976) that gp 39 and gp
52 (now identified as topoisomerase components) are associated with
compact DNA.

Hamilton and Pettijohn (l976) also attempted to quantitate the
amount of supercoiling in T4 DNA, They measured the sedimentation, after
ethidium bromide intercalation, of condensed complexes isolated ten
minutes after infection. The bulk of the DNA was not supercoiled but
one eighthof the DNA appeared to be supercoiled with a superhelical
density similar to that of other naturally occurring supercoiled DNAs.
The supercoiled DNA seemed to be segregated into domains which were

somehow held physically separate from the bulk of unsupercoiled DNA.

Replication-Uncoupled Late Gene Expression

 

Although late gene expression normally requries concurrent
replication, some late gene expression can occur in the absence of
replication, for example, in an infection by DD mutant phage (phage

which do not synthesize DNA). Such “replication-uncoupled"

 

transcription is delayed with respect to that which occurs in a wild-

type infection, is temperature-sensitive, and depends on high multi-
plicity of infection (Wu et al., 1975). However, the normal replica-
tion dependence of late transcription is not due simply to the need to
accumulate DNA or to achieve a high rate of replication because
mutations in several genes, including 30 (DNA Ligase) or 46, 47 (re—
quired for recombination and concatemer formation), restrict DNA
replication but allow significant late transcription (Wu et al., 1975).

Wu and Geiduschek (1977) attempted to clarify the relationship
between replication-coupled and replication-uncoupled late transcription
by studying the protein requirements for the two modes of late trans-
cription. They found that proteins required for replication-uncoupled
transcription accumulate later than the proteins required for normal
late transcription. It is known that, in the absence of replication,
breaks accumulate in the parental DNA. As discussed earlier, inter—
ruptions in the primary DNA structure may be important in late trans-
cription. If so, a protein which is synthesized relatively late may
create some of the breaks which are normally created during replication.
Nuclease activities which appear late have been described (Altman and
Meselson, 1970; Kemper and Hurwitz, 1973); and one of these nucleases
could activate the template. There may be an analogy to the T5 bacterio-
phage system. There is a T5 exonuclease, the product of the D15 gene,
which is essential for T5 late transcription (Chinnadurai and McCorquo-
dale, 1973).

Wu and Geiduschek (1977) also found that the product of gene 55
(which is an RNA polymerase-binding protein and required for both repli—

cation-coupled and -uncoupled late transcription) interacts differently

 

17
with replication-coupled and replication-uncoupled transcription units,

possibly through interaction with the DNA polymerase.

ln_vitro systems

 

Because of the complexity of the requirements for T4 late
transcription, ifl_yj;§g_late transcription systems have been difficult
to develop. The first systems were crude preparations from T4-infected
cells which could synthesize late RNA ig_yi£:9_only from an endogenous
template~(Snyder and Geiduschek, 1968). More recently, Rabussay and
Geiduschek (1977), have developed an ig_yi§:g_system in which a T4-
infected cell lysate is gently lyzed and suspended on a cellophane disc.
This system initiates late RNA transcripts, yields a high proportion of
late transcripts and is active for at least one hour after lysis.

The development of such an ig_yi§:g_transcription system has
allowed preliminary analysis of the in_yi§rg_relationship between
replication and late transcription. In an attempt to distinguish between
actual involvement of the DNA replication proteins in late transcription
and a requirement for replication-coupled processing of the late trans-
cription template, Rabussay and Geiduschek (1979) compared the effects on
late transcription of metabolically blocking DNA replication by adding
FUdR (which blocks DNA synthesis at the level of thymidylate production)
and by thermally inactivating the products of the replication genes 42
and 43. These two different ways of interfering with the normal replica-
tion process have both quantitatively and qualitatively distinguishable
effects on late transcription which mimic their effects jg_yiyg,

In vivo, temperature-sensitive mutations in the two DNA replica-

 

tion genes, gene 43 (DNA polymerase) and gene 42 (dCMP hydroxymethylase),

18

reduce late transcription at the non-permissive temperature (Rabussay
and Geiduschek, 1979). However, although both mutations reduce DNA
replication equally, they have different quantitative effects on late
transcription, i.e., at high temperature the temperature-sensitive gene
43 mutation reduces late transcription more than the temperature-
sensitive gene 42 mutation. The ig_yi§:g_effects of ts gene 42 and 43
mutations are similar. An in_yi§:g_system prepared from a temperature-
sensitive gene 43 lysate synthesizes less late RNA than a temperature-
sensitive gene 42 system. However, if DNA replication is limited ig_
yiE:g_by the absence of dNTPs, although the temperature-sensitive gene
43 infection produces negligible late transcription, the temperature-
sensitive gene 42 system yields as much late transcription as it does
at the permissive temperature. This result suggests that the temperature
sensitivity of the gene 43 system may be due more to the absence of
functional gene 43 product than to the absence of replication.

The addition of FUdR blocks DNA replication severely, but does

allow a residual amount of synthesis. I vivo, FUdR addition as early

 

as six to eight minutes after infection at 370C blocks most DNA replica-
tion but allows appreciable late transcription. The in_yigrg_effect of
FUdR is similar to the ig_yiyg effect.

The RNA synthesis which occurs in the presence of FUdR may be due
to the residual DNA synthesis. Alternatively, the reduced levels of late
RNA synthesis in temperature-sensitive gene 43 infections may reflect a
requirement for DNA polymerase involvement in late transcription, either
by processing DNA to create a "late-transcription competent" template,
or by physical interaction with the transcription proteins.

In summary, considerable evidence has accumulated for the

interaction of the late transcription complex with DNA replication

19
proteins including at least the DNA polymerase and the gene 45 product.
In addition, it has been recently shown thatoneof the previously
unidentified small molecular weight polypeptides which binds to RNA
polymerase is the product of gene 60 (C. Goff, Cold Spring Harbor Phage
Meeting, 1981). The product of gene 60 is also a subunit of the T4

topoisomerase (liu et al., 1979).

Effects of Nucleotide Modification
on Late Gene Expression

T4 DNA normally contains hydorxymethylcytosine (HMC) instead of

 

cytosine. In addition, the HMC residues are a or B glucosylated.
Appropriate phage and host strains can be used to force T4 to incorporate
C instead of HMC into its DNA (Revel and Georgopolous, 1969; Runnels and
Snyder, 1978), but the substitution of C for HMC prevents late gene
expression. Some r-strand RNA is transcribed but has an unknown defect
which prevents its translation (Wu and Geiduschek, 1975). The gene
responsible for prevention of late transcription from C-DNA (cytosine—
containing DNA) has been identified; mutations in this gene, gig, allow
late transcription on C-DNA (Snyder et al., 1976).

The role of the alc gene product
in late transcription

 

 

Besides preventing late transcription from C—DNA, the T4 gig gene
product is responsible for unfolding the host nucleoid shortly after T4
infection (Snustad et al., 1976; Sirotkin et al., 1977). Alg:T4 are also
defective in the shutoff of some host RNA synthesis. (Sirotkin et al.,
1977; Tigges et al., 1977). Because the supercoiled domains of the E,
gglj_nucleoid (Worcel and Burgi, 1972) are partially maintained by RNA

(Stonington and Pettijohn, 1971; Pettijohn and Hecht, 1973), the gig

 

20

gene product could act to shut off host RNA synthesis and thereby
unfold the nucleoid, or could unfold the nucleoid directly and thereby
shut off host RNA synthesis which depends on the nucleoid structure.

The latter hypothesis is supported by experiments reported by
Pearson and Snyder (1980). They studied the effect of T4 superinfection
on x- producing cells and found that the gig_gene product shuts off
the late transcription of 1, including that which is already underway.
They proposed that the gig function does not act simply as an RNA
polymerase-binding protein which is effective on cytosine—containing
promoters, but rather, acts on specific DNA structures and thereby
prevents the utilization of promotors and causes premature termination
of transcription. By this model, T4 late transcription, x late
transcription and some E, ggii_transcription share a common DNA template
structural requirement; the T4 gig function normally unfolds the host
nucleoid but cannot distinguish T4 C-DNA from E, ggii_C-DNA and therefore
destroys the T4 DNA structures which are required for T4 late gene

expression.

Expression of Late Genes from Clones

 

As an approach to simplify the study of the expression of T4 late
genes, attempts have been made to study the expression of cloned late
genes after superinfection by T4 phage. A cloned gene will efficiently
complement an infecting amber mutant phage (for example, amber 923),
providing that the phage contains mutations to prevent breakdown of the
plasmid DNA (Jacbos et al., 1981). The effects of infecting with three
genotypes of phage have been examined (Jacobs and Geiduschek, 1981):

1) the infecting phage are gig? and HMC-containing; 2) the phage are

a c+ and C-containing, and 3) the phage are gig: and C-containing.

 

21

In cells uninfected by T4, some plasmid-mediated expression of
gene 23 occurs, presumably from a plasmid promoter. This expression is
shut off after infection by HMC-containing gigf phage but not after
infection by C-containing gigf phage.

After T4 infection, plasmid—mediated expression of gene 23
requires the gene 55 product, as do the chromosomal late genes. However,
the three cases have different requirements for DNA replication and DNA
replication proteins. In case 1 (infecting phage have HMC-DNA and are
gigf) expression of plasmid gene 23 requires the functioning replication
proteins, DNA replication, topoisomerase activity (gene 52 product), and
functioning gene 46 activity (46 is normally required for recombination).
These requirements are similar to the requirements for the expression of
the chromosomal late genes, with the exception that late chromosomal
replication occurs in the absence of the gene 46 product. It is not known
whether the required replication must take place on the chromosome or on
the plasmid, for example, to incorporate HMC into one strand of the
plasmid which then undergoes a gene—46 mediated reaction. In case 3,
(phage have C-DNA and are gig?) replication, replication proteins and
gene 46 product are not required and significant plasmid-mediated synthe-
sis occurs in their absence. Finally, in case 2 (phage produce C-DNA and
are gigf), no plasmid gene expression occurs; no chromosomal late

expression occurs under these conditions either.

The T4 PseT Gene
More clues to the requirements for T4 late gene expression have
come from the study of two T4-induced enzymes, the products of the pseT
gene and gene 63. Both of these enzymes are required, under some

conditions, for T4 late gene expression.

22

The T4 gggi_gene encodes an enzyme which has two distinct ig
yiggg_activities, a 5' polynucleotide kinase activity and a 3' phospha—
tase activity (Cameron and Uhlenbeck, 1977; Sirotkin et al., 1978). The
3' phosphatase removes terminal 3' phosphates from DNA or deoxyribonu-
cleotides or, less preferentially, from RNA or ribonucleotides (Becker
and Hurwitz, 1967). The 5' polynucleotide kinase phosphorylates 5'—0H
terminated DNAs or RNAs by transferring the gamma phosphate from ATP.

It will phosphorylate single-stranded molecules, molecules with
protruding 5' ends, and, less efficiently, nicks or gaps in double
stranded DNA (cf. Kleppe and Lillehaug, 1979).

Clues to the biological role of the gggi_gene product come from
the phenotype of gggif mutants on the E. coli strain CTr5x,on which they
are nonviable (Depew and Cozzarelli, 1974; Sirotkin et al., 1978). In
a gggi_mutant infection of CTr5x, no phage are produced because DNA
replication (Depew and Cozarelli, 1974) and late gene expression are
defective (Sirotkin et al., 1978). The reduced rate of replication caused
by a gggif mutation occurs before late gene expression begins, suggesting
that the gggi_gene product may act directly in DNA replication.

Mutants in the gggi_gene usually lack both of the ig_yiggg activi-
ties. However, one mutant, pggi_1 lacks the phosphatase activity but
retains the kinase activity and another mutant, gggi_47, lacks the kinase
activity but has a low level (about 3%) of the phosphatase activity
(Soltis and Uhlenbeck, personal communication). Eggi_47 and gggg I have
the same phenotypes as other gggi_mutants and do not complement each other,
suggesting that either it is the lack of phosphatase that is critical to

the mutants loss of viability or that, i vivo, the kinase and phosphatase

 

act in concert.

23
Gene 63

Gene 63 maps very near to the gggi_gene and codes for a protein
product which acts both in tail fiber attachment during phage morphogene-
sis (Wood and Bishop, 1973) and as an RNA ligase (Snopek et a., 1977).
The RNA ligase activity and the tail fiber attachment activity of gene
63 are probably unrelated (Snopek et al., 1977, Runnels et al., 1982).

The RNA ligase activity of gene 63 catalyzes the joining of
single—stranded polynucleotides ighyiigg. It will cyclize an RNA
polymer by joining the 3' hydroxyl—terminated ”acceptor" end to a 5'
phosphate ”donor” end and it will join 3' hydroxyl and 5' phosphate
termini of separate polynucleotides. The enzyme shows a strong
preference for RNA as the acceptor molecule, but almost any ribo or
deoxyribo oligonucleotide which has been activated by the addition of
AMP in a 5'-5' linkage can serve as the donor molecule (England and
Uhlenbeck, 1978). Therefore, the enzyme can join not only RNA to RNA
but also DNA to RNA and also stimulates “blunt-end joining" by T4 DNA
ligase ig_yiigg (Sugino et al., 1977).

Mutants in the RNA ligase activity are viable on most E. ggii
laboratory strains but, on E, ggii_CTr5x, RNA ligase mutants have pheno-
types that are indistinguishable from pggif mutant phenotypes (Runnels
et al., 1982). The similarity in phenotype observed in RNA ligase- and
pggif mutants, and the observation that mutants in both genes are
suppressed on CTr5x by the same T4 extragenic suppressor has led to the
hypotheSis that the two enzymes interact ig_yiyg_in some reaction which
is required for normal replication and which activates the DNA template

for T4 late gene expression (Runnels et al.,l982).

24
Not enough information is currently available to propose
precisely at what step in DNA metabolism and transcription the gggi
and RNA ligase functions are involved. Possibilities include removal
of nicks in DNA formed during the removal of RNA primers, formation of
DNA structures required for replication and late transcription, or

processing of an RNA which is involved in the formation of T4 "nucleoids."

E; ggii CTr5x

CTr5x is a strain derived from CT196, a clinical isolate of
E, ggii, The original CT196 isolate restricted the growth of gggi
mutants but also restricted wild-type T4. CT196 was crossed with various
Hfr donor strains until a recombinant was obtained which restricted gggi
mutants but not wild-type T4 (Depew and Cozzarelli, l974).

CTr5x restricts T4 mutants in gggi and RNA ligase as described
above. A derivative of CTr5x which contains an efficient amber suppres-
sor no longer restricts any gggi_mutants, even those which are deletions.
It seemed likely that CTr5x might contain an amber mutation in a gene
which is required for the growth of gggif and RNA ligase' phage and which
is suppressed in the amber-suppressing CTr5x (cf. Sirotkin et al., 1978).
This hypothesis was the inspiration for the search for E, ggii mutants
in a K12 background which would emulate the restrictive behavior of CTr5x.
Eii_mutants, which resulted from this search, are described in Appendix A
(Cooley et al., 1979).

Recently, it has proven possible to map the locus in CTr5x which
is responsible for the restriction of gggif and RNA ligase mutants
(A. Jabbar and L. Snyder, personal communication). The locus,named Egg,
lies between gig_and ggg_on the E, ggii_map at about 29 min. Interest-

ingly, when transferred to a heterologous genetic background, the Egg

25
recombinant strain no longer restricts Eggif or RNA ligase- mutants at
370C, only at lower temperatures. However, when the pgg_locus is
transferred to a iii: host, the restriction of gggif and RNA ligase‘
mutations is complete at 370C, suggesting an interaction between the
Egg locus and the iii_gene. This observation is more fully discussed

in the Summary section.

DNA Structure and Transcription

 

Evidence is accumulating that gene expression can be differen-
tially affected by DNA structure. The best documented structural
influence on transcription is supercoiling, i.e., the degree of super—
helical density can differentially activate promoters.

In Escherichia ggii, the omega protein, Topoisomerase I, relaxes
negatively supercoiled DNA. The structural gene for Topoisomerase I
has recently been identified (Trucksis and Depew, 1981) as iggA_in E.
ggii_and gggX in Salmonella typhimurium. The ggg_X locus was discovered
some time ago as a suppressor of promoter mutations in the igg_operon of
Salmonella (Mukai and Margolin, 1963). §gg_X mutations also restored
expression of mutant igg_operons which had inactive CAP—binding sites,
and affected many other promoters as well (cf. Smith, 1981). In view of
the ig_yiiig_activity of omega, a likely explanation for the ggg X pheno—
type is that, in the absence of Topoisomerase I, intracellular DNA is
more negatively supercoiled (by DNA gyrase) and the increased supercoil-
ing allows some mutant igg_and igg_promoters to be utilized.

Further evidence that supercoiling can differentially affect E.
ggii promoters comes from experiments on the ig_viyg effects of naladixic

acid and coumermycin. These antibiotics are inhibitors of DNA gyrase

26
and when they are added to cells they inhibit transcription from some
operons but not others (Sanzey, l979).

ig_yiiig, supercoiled DNA is often transcribed more readily
than relaxed DNA. For example, purified RNA polymerase transcribes 1
RNA five times more efficiently if the DNA is supercoiled (Botchan, 1976).

The condensed E, ggii_genome is separated into supercoiled do—
mains. DNA gyrase, presumably the topoisomerase which supercoils E, ggii
DNA, introduces about forty-five breaks per chromosome in the presence
of oxolinic acid (Snyder and Drlica, 1979). If breaksites correspond
to DNA-gyrase binding sites, there may be one binding site per loop.
Perhaps differential supercoiling within domains functions to modulate
the transcriptional activity of certain genes clustered in a domain.

A variety of topoisomerases have been isolated from different
cell types (Baldi et al., 1980; Hsieh and Brutlag, 1980). However, no
enzyme other than E, ggii_gyrase has been reported to introduce negative
supercoils into DNA. For example, the T4 topoisomerase does not exhibit
any gyrase activity ig_yiigg (Liu et al., 1980). Nevertheless, domains
of supercoiling apparently do exist in intracellular T4 DNA (Hamilton an
Pettijohn, 1976). E, ggii_gyrase can substitute for T4 topoismerase
in replication (McCarthy, 1979); it may be able to introduce the super-
coils observed in T4 DNA. Alternatively, an unidentified T4 or E, ggii
subunit may interact with the T4 topoisomerase ig_yiyg, giving it gyrase
activity.

Some ig_vivo activity of T4 topoisomerase plays an important role

 

in T4 late gene expression. In the absence of T4 topoisomerase, the E.
coli gyrase can provide a substitute activity, but in the absence of both
enzymes, T4 late gene expression is abnormal (Gisela Mosig, personal

communication).

ARTICLE I

THE GQE_SITE: A Ei§fACTING BACTERIOPHAGE T4 REGULATORY
REGION THAT CAN AFFECT EXPRESSION OF ALL

THE T4 LATE GENES

Wendy Cooley Champness and Larry Snyder

This article is publishedin Journal of Molecular Biology,
155 395, 1982.

 

27

 

 

 

 

Reprinted from J. Mol. Biol. (1982) 155, 395—407

The gol Site: A Cis-acting Bacteriophage T4
Regulatory Region that can affect Expression
of all the T4 Late Genes

WENDY (foowy (‘HAMPNEss AND LARRY vamm

 

J. Mol. Biol. (1982) 155, 395—407

The gol Site: A C is-acting Bacteriophage T4
Regulatory Region that can affect Expression
of all the T4 Late Genes

WENDY COOLEY CHAMPxEss AND LARRY SNYDER

Department of Microbiology and Public Health
Michigan State University. East Lansing. Mich. 48824. USA.

(Received 31 March 1981. and in revised form 21 September 1981)

We have shown that mutations in the Escherichia coli lit gene can prevent the
expression of the late genes of bacteriophage T4 at temperatures below 34°C. The
defect in late gene expression occurs, at least partially. at the level of transcription,
and neither DNA replication nor DNA encapsidation into phage heads is
significantly affected. Rare. T4 “901” mutations overcome the defect in late
transcription. Refined mapping experiments place go] mutations within gene 23.
but an altered gene 23 protein is not responsible for the phenotype. Rather. gol
mutations seem to alter a cis-acting site in T4 DNA. the wild-type form of which
interferes with late transcription in lit“ hosts.

1. Introduction

The intracellular structure of the DNA of even simple procaryotes and viruses has
proved relatively intractable to biochemical analysis. The DNAs are highly folded
and often supercoiled. but beyond this little is known. It seems reasonable to
suppose that there exist periodic sites on DNA that are involved in the formation
and/or maintenance of DNA structures. However. it is difficult to predict how one
might identify such sites. since it is not clear what effect mutations in the sites
would have on phenomena such as replication. recombination and transcription.

It has been proposed that a DNA structure created during replication is required
for the transcription of the true-late genes of bacteriophage T4 (Riva et al.. 1971)).
The genes are called the “true—late” genes to distinguish them from those early
genes that are transcribed early as well as late after infection. but whose
transcription. at least at early times. is not coupled to the replication of the DNA.
The DNA structure is probably fragile because infected cells must be lysed and
treated in particular ways to achieve true—late transcription in. ritro (Rabussay &
Geiduschek. 1977). There may be alternative ways of creating the DNA structures
other than replication. since some transcription ofthese genes occurs in the absence
of replication, but it is delayed and dependent on the multiplicity of infection (Wu
& Geiduschek. 1975).

While most studies of T4 true—late gene expression have focused on the role of T4

395
0022—2836/82/080395—13 $02.00/0 (“1 1982 Academic Press Inc. (London) Ltd.
19

 

396 W. (‘. (‘HAMPNESS AND L. SNYDER

gene products. it seems likely that host genes are also involved. Such host genesi
would be of particular interest because they could be involved in transcriptional
regulation in uninfected cells. We have identified a candidate for such in
Escherichia coli gene. the [it gene, at 25 minutes on the genetic map (Cooley elal.
1979). The wild-type (111+) allele of this gene is required for normal T4 late gene
expression. and cells with the mutant allele (litT) fail to support late gene
expression at temperatures below 3400. Only late gene expression and not T4 DNA
replication are affected. so the effect is specific to gene expression.

Rare T4 mutants exist that can multiply in a lit‘ host ((‘ooley et al., 19'79).Thcc1I
“gm-lit" or “got” mutants exhibit. normal rates oflate gene expression in lit‘ hosts. ‘
We showed that go] mutations are (co—dominant for late gene expression with wild-
type T4 and that they are closely linked to an amber mutation in gene 23 (Cooley
cl al.. 1979). In this paper. we present evidence that got mutations define a site on
T4 DNA rather than a (liffusible gene product. In a lit— host something happens (or
doesn't happen) at this site that prevents the late transcription of T4
bacteriophage.

2. Materials and Methods
(a) Bacteria/(1nd phage strains

(i) T4 strains

The amber mutants were obtained from R. Vanderslice and W. B. Wood or were from 0111
stock collection. The mutations will be referred to by gene and by name. For example. amber
mutation H11 in gene 23 is 23amHll. T4 gol6B was isolated as a spontaneous plaqut
forming mutant on E. coli MPHD’. a tit” host (Cooley el al.. 1979).
(ii) E. coli strains

CR63 (supD) was used for crosses and B834 (su') (Wood. 1966) was used to detect 411ml
recombinauts and as the lit+ host for physiological experiments. In the original experimellleiv
the effect of host lit‘ mutations on T4 development was studied using the K-12 strains from
which lit' mutants were first isolated (Cooley et al.. 1979). It is difficult to achlef'e
synchronous infections of K~12 strains. so we transferred a lit‘ mutation into the B strallli
E. roll B834. as follows. An Hfr strain. RHO41. which is purB‘ and transfers clockwise from
97 min. was obtained from B. Bachmann and the Yale Stock Collection. A lit _ mutationlitili
was transduced with bacteriophage P1 into this strain from MPH6 using par+ as the 86190
marker. An Hfr tit' recombinant was then mated with E. coli B834 made his' 11."
mutagenesis with nitrosoguanidine (Adelberg at al.. 1965). Two out of 80 his+ recombinantS
of the mating were lit'. One of these. E. coli B834Jit403. was the lit' host used for all ill"
experiments to be described. Since only one [11' host was used it will be referred to simplyfls
“the lit‘ host".

(1)) ("rose-es amt infections for labeling

(‘rosses and infections were performed in .119 medium as described previously (Sllyder
cl al.. 1976). To measure phage yields the cells were grown in tryptone broth at 37°C and
infected at a total multiplicity of infection (moi) of 10. After 5 min, T4 antiserum (Cappell
was added and at 13 min the cells were diluted 103 times. At 90 min chloroform was added'
and the phage were diluted further and plated. In some experiments it was necessflV'to
determine accurately the ratio of got' and go/I phages in mixed infections. To accomP‘llsh
this. the mixture of phage to be used or the phage released were plated on lll+ E40011"
sector of the plate that contained about 100 plaques was chosen and the percentage Oflhe
plaques due to gol‘ mutants was determined by toothpicking all of the plaques in this seem
onto a plate with lit" indicator.

, . 4

A cis-ACTING SITE IN T4 GENE EXPRESSION 397

him (0) Analysis of T4 protein and RNA synthesis

”Li-‘7 To label proteins, [3SS]methionine (1000 Ci/mmol, 10 pCi/ml) was added to cells infected at
iir.‘ 30°C in M9 medium without Casamino acids. The procedures and buffers of Studier (1973)
'00), were used for the processing of the samples and slab gel electrophoresis. The gels were 11%
n ,1; (w/v) acrylamide, with the exception of the gel shown in Fig. 3, which was 950/0 acrylamide.
“ Gels were stained (to ensure that total protein per column was uniform), dried, and used to
[ la: expose XRP-l Kodak X-ray film for varying lengths of time. To label RNA, [5-3H]uridine
ml! (26 Ci/mmol, 10 ”Ci/ml) was added to cells infected at 30°C as above. The RNA was
extracted with phenol as described by Bolle et at. (1968), except that the RNA was
979- precipitated with alcohol after 4 extractions and treated with 10 pg RNAase—free
l‘" DNAase/ml (Worthington) for 200 min at 37°C before the final 2 extractions with phenol.
I l' ' The purified “l” and “r” separated complementary strands of T4 RNA were a very generous
““1 gift from Dietmar Rabussay, who also helped with the hybridization assays. Hybridization
1’3 was by the liquid method in 2 x SSC (SSC is 0-15 M-NaCl, 0-015 M-sodium citrate) for 8 h at
H" 60°C. The hybrids were collected on nitrocellulose filters and washed with 0-5 M-KCl, 00] M-
a)" TI‘iS‘HCl (pH 7'9).

)ll
3. Results
(a) T4 gene expression in the lit— host

We reported previously that host lit- mutations prevent T4 late gene expression
(Cooley et al., 1979). Figure 1 (columns A and a) shows the synthesis of T4 proteins
late in infection of the lit+ and lit' host, respectively. At this labeling time, some
3.. T4 late protein synthesis occurs in the lit’ host, but it is reduced in rate compared
~; to the 121+ control. At later labeling times, T4 protein synthesis has almost totally

ceased in a lit“ host (Cooley et al., 1979).

Host lit" mutations only prevent T4 late gene expression at temperatures below

. 34°C (Coo’ley et al., 1979). Furthermore, we have found that the temperature at.
~ which the cells were grown prior to infection is important. More late protein
-‘ symthesis occurs in lit" cells grown at 37°C and infected at 30°C than in cells grown
‘ and infected at 30°C (data not shown). This indicates that the temperature
dependence of late gene expression in a lit" host is not due to the temperature
E dependence of a particular reaction, but rather to differences in cells grown at
a. different temperatures.

It is interesting to contrast the block imposed on T4 late gene expression by host
lit mutations with that imposed by most T4 mutations, which prevent the synthesis
of true-late proteins; for example, DNA-negative or gene 55 mutants. In the latter
cases. early protein synthesis continues and the product of T4 gene 32 is sometimes
grossly overproduced (cf. Gold et al., 1976). After infection of the lit' host by wild-
t-ype T4, true-late proteins are made in reduced amounts. but early protein
synthesis also ceases, probably somewhat earlier than usual, and the gene 32
product is not overproduced. This is demonstrated in Figure l, where we show the
results of infecting with T4 having amber mutations in genes 44 and 42. the
products of which are required for DNA replication. Now, as mentioned above. the
synthesis of the products of many early genes, including 42 and 43. continued late
into infection (Fig. 1, lanes b and c). In fact, the program of gene expression in the
lit~ mutant was not very different from that in the [27+ host (con‘ipare with Fig. 1.
lanes B and C). A similar result was obtained with a T4 gene 55 mutant. except. that

398 W. C. CHAMPNESS AND L. SNYDER
p?—
" ...-r ~ ...—p43
v... ~— ...-u ~ -.
I...“ a w
V“

         

”
“
i—p42
'/?‘ ’42
ff; m. 7’7” mm? W
A 0 B b C C

\Wl‘l’

FIG. 1. T4 protein synthesis after infection of the lit" host. Proteins (identified as gene products
labeled from 18 to 21 min. and electrophoresed and automdiographed as described in Materials and
Methods. Lanes A. B and C. lit+ E. mli: a. b and e. lit‘ E. coli. A and a. T4: B and b. JZIIMNIZZZZ: (7‘ undo.
44(I7IIN82.

in this instance the gene 32 product was overproduced in both hosts (data not

shown). Therefore the phage mutations that prevent true—late gene expression art‘
epistatic over the host lil mutation in that they prevent the shutoff of early gene
expression even in the lit‘ host.

In an earlier paper ((‘ooley 01:11.. 1979). we proposed that host lit mutations block
T4 late gene expression at the level of transcription. To prove this we performed The
experiment shown in Table 1. We radioactively labeled RNA late in infection oftlll‘l
HF“ and lil‘ hosts and hybridized the RNA to the complementary r and l strands Ol
T4 DNA. If the defect occurs at the level of transm‘iption. we would not (“PM

A cis-ACTING SITE IN T4 GENE EXPRESSION 399

TABLE 1
Hybridization of RNA labeled after infection of
the lit‘ host to the separated (complementary strands of T4 DNA

 

 

(‘ts/min (‘ts/niin (‘ts/min % Of input
RNA input hybridized hybridized RNA hybridized
r strand I strand to r strand
E. coli lil+ gol+ T4 637 334 60 52
E. roli lil‘ gol+ T4 941 {I9 86 10
E . coli Iil‘ T4 9016 B 1064 759 63 71

 

RNA was labeled from 28 to 31 min after infection The specific activities of the RNAs were. from top
to bottom: 26. 3'4 and 2-4 cts/inin per ng.

much hybridization to the r strand with the RNA made in the lil‘ host since T4
late RNA hybridizes mostly to the r strand of T4 DNA. With RNA prepared after
infection of the [11+ host. 52% of the input RNA hybridized to the 7' strand of T4
DNA. while in the lll_ case only 100/0 hybridized to the r strand. Not only did the
percentage of RNA hybridizing to the r strand drop. but the total hybridization to
the separated strands also dropped. The latter result is reproducible and suggests
that the distribution of the RNA may be grossly altered. or that the RNAS may be
much smaller. or that some host RNA may be synthesized. In any case. it seems
clear that the host lil_ mutation affects T4 late transcription and this is probably
sufficient to explain the defect in late gene expression and phage production.

(b) Effccl of 7'4 gol mutations on T4 late, gene expression,

T4 gol mutations are single point mutations that overcome the defect in late
protein synthesis in 11'! _ hosts ((‘ooley et al.. 1979). Ifthe defect in late transcription
is causing the defect in protein synthesis. we would also expect gol mutations to
overcome the effect on late transcription. In Table 1 we have included
hybridization results from experiments with RNA labeled in the HF host after
infection with the go] mutant. This late RNA hybridized with an efficiency of 77%
t0 the separated strands of T4 DNA and 71% ofthe input RNA hybridized to the r
strand. Thus the RNA labeled after infection by the gol mutant was more like
normal T4 late RNA in that most of it hybridized to the r strand of T4 DNA. \Ve
conclude that T4 go] mutations overcome the effect on late RNA synthesis caused
by host [it mutations.

(c) (Janelle analysis of T4 gol Inn/alloys

In an earlier paper. we reported that go] mutations are closely linked to T4 gene
23 (Cooley cf al.. 1979). The 200 go! mutants we have tested so far all had mutations
in this region. If mutations to the (lol phenotype can occur elsewhere. they must be
less frequent by at least two orders of magnitude.

To localize further a particular gol mutation. we performed three—factor crosses.
The results are shown in Table 2. This particular gol mutation. gold/f. apparently
lies within gene 23. clockwise (or carboxy—terminal) to the very i1llllllllrlfll'llllllal

400 W. (‘. FHAMPNESS AND L. SNYDER

TABLE 2

Mapping of a go] mutation, by three—factor crosses

 

Frequency of

 

 

 

Cross got mutants 0/0 Order deduced
among am+ gol mutants
recombinants
1. amHl 1, 90163 x (171113270 13/96 14 B270-Hll—gol
got
2.amH11,gol6er1mBl7 117/126 93 H11 -Bl7
got
3. amBl7. golé'B XamHll 6/80 8 H11 -B17
got
4. amB17. golfiB xamB26 17/148 12 BI? ——825
gal
5. (1171317. gotfiB X amE389 13/93 H BIT ———l‘13h‘!1
gol
6. (1an389, 90168 x amBl7 138/146 94 Bl? grass)
7. amE389. golb'B X (1mB270 105/143 73 liZTthnl—vlifml
Composite map order: 9016]?
o l o.
R : 5 g s
m :1: m {:2 a:
| L | l l 1
...... | r , | . . . . . .
gene 22 gene 23 gene 24

NH2———COOH

Double mutants consisting of the gol6B mutation and amber mutations in genes 22. 2.3 and :34 were
crossed against other single am mutants and am + recombinants were tested for growth on the lit/ host
The crosses unambiguously place the 90] mutation between the gene 23 mutations (III/HI l and {1111133351-
very close to amBl7. The crosses do not clearly show on which side ofamBl7 the got mutation lies. “9
have indicated this by brackets.

23amH11 and close to the 23amB17 mutation. A. H. Doermann (personal
communication) has confirmed this map position using “marker rescue" mappinzq
techniques and has further concluded that this got mutation lies clockwise (0r
carboxy-terminal) to 23amB17. An expanded map of this region is shown ill
Figure 2. The product of gene 23 is the major capsid protein. During head
formation, the N-terminal portion of the polypeptide is removed by proteolfi'tlf
cleavage (Laemmli. 1970) and the site of the cleavage lies between 23:1);11111 and
23amBl7.

((1) Evidence that gol mutations define a cis-acttng site on T4 DNA
In spite of the map position of got mutations. the G01 phenotype is not due 1'03“
altered gene 23 product. In the experiment shown in Figure 3(b) (lane 3) a doublt‘
mutant. having both the go] mutation and 23amE389. was used t'o infect the lit’
host. The gel mutation stimulated the synthesis of all the T4 late proteins.
including the amber fragment of gene 23. Thus the (101 phenotype is unchangt’d

A sis-ACTING SITE IN T4 GENE EXPRESSION 401

 

43
82
r
‘39
63
\90
/6‘&
v;
0
’1)
6
as“

 

FIG. 2. An expanded map of the gene 23 region of T4 showing the position of a 90! mutation.

even in the absence of a functional gene 23 polypeptide. We repeated this
experiment with the mutation 23amHl ] . which. as mentioned above. is N—terminal
to the gol mutation and in the part of gene 23 coding for the fragment removed
during head formation. Now the amber fragment was too small to be detected. but
the gel. 23amH11 double mutant synthesized all the late proteins except the gene
23 product (data not shown). We conclude that. since the go! mutation could
stimulate the expression of the other late genes in the absence of the gene 23
product or even the N—terminal fragment. the G01 phenotype is not due to an
alteration of either the gene 23 protein or the N—terminal fragment of the gene 23
protein.

The possibilities remain that the G01 phenotype is due to altering the gene 23
mRNA rather than protein: or to altering a gene whose sequence overlaps that of
gene 23 or is on the opposite DNA strand. Alternatively. gal mutations may define a
site. The evidence presented immediately below suggests that go] mutations define
a site on the DNA rather than a diffusible gene product.

lfgol mutations define a site on the DNA. they may be more Closely linked than if
their target is a diffusible gene product. Of the five spontaneous go] mutations we
have tested. four seemed to be in the same base—pair. since they gave recombination
frequencies of less than 001%. the frequency of recombination between mutations
in adjacent base—pairs. The other mutation gave a frequency of about 0-0400 with
the other gol mutations, which is consistent with its being about four base—pairs
away. Therefore. the “target size" for gal mutations seems to be very small.

402 W. C. CHAMPNESS AND L. SNYDER

 

FIG. 3. Synthesis ofT-l gene 23 product (p23) in mixed inti-etions with a gene 23 amber mutation all?la
gal mutation on [if E. coli. An electrtmhcrogrnm ofT4 proteins labeled from 28 to 31 min after infection
is shown. (a) lit+ E. coli: (In/17F E. coli. Lanes 1. T4 gulb’B; 2. 23mnE389: 3, the double mutant

LA

.B‘Q

 

A cis-ACTING SITE IN T4 GENE EXPRESSION 403

If gal mutations define a site on T4 DNA they should be sis-acting for the
expression of T4 late genes in mixed infections. To test this, we performed the
experiment shown in Figure 3. lanes 5 and 6. We coinfeeted cells with the T4 gal
mutant and gal+ T4 with the 23amE389 mutation (lane 5) and, conversely, with
gal+ T4 and a double mutant having the gal mutation and 23amE389 (lane 6). In
Iil+ E. coli. gene 23 was expressed from both the gal mutant and 901+ DNA since
the am fragment and complete polypeptide were synthesized at approximately
equal rates (Fig. 3(a), lanes 5 and 6). In contrast. in the lit‘ E. coli gene 23 was only
expressed from the DNA with the gal mutation. In other words. if the gal+ DNA
had the amber mutant allele. only the complete gene 23 polypeptide was
synthesized (Fig. 3(b). lane 5). but if the gal mutant DNA had the amber allele,
only the amber fragment was synthesized (Fig. 3(b), lane 6). Thus, the gal mutation
stimulated the synthesis of the gene 23 product only in cis.

We attempted a semivquantitative determination of the stimulation of the
complete polypeptide and the am fragment in each experiment by determining the
area under each band, using a scanning and integrating densitometer. A
background was subtracted that was taken to be the area in the same region of the
gel when the (1m mutant or wild—type T4. respectively. had infected the HF host
without the coinfecting gal mutant. Subject to the limitations of this method, it
appears that the prcsencc of a gal mutation on the same DNA stimulated the
synthesis of either the complete polypeptide or the amber fragment at least 300-
l'old without stimulating gene expression from the gal+ DNA in the same cell. The
synthesis of the um fragment appears to the eye to be stimulated more by the gal
mutation than the complete polypeptide. but the densitometer reveals that this is
an illusion bascd on the width of the bands in different parts of the gel.

To determine if the cls effect of gal mutations extends to late genes other than 23.
we repeated the experiment shown in Figure 3. but with an amber mutation in gene
1N.Thc rcsults arc shown in Figure 4. Like the gene 23 product. the gene 18 product
was only synthcsizcd from the DNA with the gal mutation. We tried the same
cxpcrimcnt with gcnc 34. The results were less clear but there did seem to be some
I‘I'N cffcct on this gcnc as well (data not shown). Apparently. the (is effect of gal
imitations cxtcnds some distance. and in both directions. from the site of the gal
mutation.

(inc could arguc. from the experiments shown in Figures 3 and 4, that gal
mutations are totally recessive. but that a subset of the cells received gal mutant
phagc exclusively. Since these were the only cells in which late proteins were
synthesized. only one allele of gene 23 or gene 18 was expressed. because this was
the only allclc in those cells that were making any late proteins. This seems
unlikely. Thc rate of late protein synthesis in the mixed infections was about 30%
that ol'thc li/+ control. so 30% 0f the cells could not have been infected by gal+ T4.
But to climinatc this trivial explanation for the ('is effect. we designed the
cxpcrimcnt shown in Tablc 3. The experiment is based on the observation that host

Z-EmHICZlSll. gal/ill; 4. wildrtlvpc T4 plus T4 golfilf: 5. 2311”] F138” plus T4 gnllili: 6. the double mutant
Zilrnul-IRSH. gum/f plus wild-type T4. In the singlc infections (lancs | to 3) the m.o.i. was [0. ln the mixed
infections (lanes 4 to 6) tlic m.o.i, was ll) of each. The gene 23 product and the amber fruginent left by
Zvllllllll‘llixll (l’) arc irlcntilicd

   

404 W. C. CHAMPNESS AND L. SNYDER

(a)

| 2 3 4 5 6

FIG. 4. Synthesis of T4 gene. 18 product in mixed infections with a gene 18 amber mutation and a gal
mutation in HF E. coli. shown in an electropherogram ofthe T4 proteins labeled from 28 to 31 min after
infection. (a) lit+ E. coli: (b) lil‘ E. coli. Lane 1. IRUIIIElS: 2. the double mutant ISamElS. golfiB plus
wild-type T4: 3. T4 wild—type: 4. 18r1mElR:5. the double mutant 1841111 EIS. gal63; 6. the double mutant
18amE18. gal6B plus wild-type T4. For single infectimis (lanes 1. 3. 4 and 5) the m.o.i. was 10. For the
mixed infections (lanes 2 and 6). the m.o.i. was 10 of each. The T4 gene 18' product (p18) and the amber
fragment left by ISIIMIL‘IS (F) are identified.

A sis-ACTING SITE IN T4 GENE EXPRESSION 405
TABLE 3

Production of gol+ and go] mutant phage in mixed infections

 

 

Phage per
Phage Bacterium infected cell l'70 galb‘B input 0/0 galb‘B output
T4+ E. coli lit+ 55 0 0
T4 gnlb‘h’ E. coli lit+ 4-3-5 100 100
T4 golfiB+23umHll E. coli lit+ 15 51 53
(unHll E.coli lil+ 4X10'4 0 0
T4 + E. coli lil' 0'75 0 0
T4 {10168 E. coli llt‘ 135 100 100
T4 galliB+23umHll E. coli HF 27 51 67
2.3" m H1 1 E. coli lit‘ 45 x u)‘ 4 0 0

 

At least 100 plaques were tested for the Go] phenotype to determine the percentages of gal mutants
produced. In this experiment the yield of T4 galfiB was higher in the lit“ mutant than in mi E. coli.
This is not generally the case.

[it mutations do not substantially affect T4 DNA replication : so. in a lil‘ host. both
gal+ and gal mutant DNA is replicated. Thus. even if gol mutations are (it's-acting
for gene expression. those late proteins synthesized from the gal mutant DNA could
package gal+ DNA in the same cell. Therefore. in a mixed infection of the lil- host.
such as that shown in Figures 3 and 4. both gal+ and gal mutant phage should be
produced if gal mutations are cls—acting. In contrast. if the explanation for the
apparent ris effect is that gal mutations are recessive and the only cells synthesizing
late proteins are those infected exclusively with gal mutants. then only gal mutant
phage will be produced in mixed infections. Even if gal mutations are cis—acting we
expect some bias toward the gal mutant phage because those cells that are infected
mOstly with go] mutants will produce more phage. since they will synthesize more
late proteins. In the experiment shown in Table 3. the gal+ phage had an amber
mutation. 23(11/1Hl I. so any gal+ phage produced must obtain their major coat
protein from the gal mutant genomes in the same cell. The results were that in the
mixed infection about nine phage per cell or 330/0 of the total phage released were of
the gal+ genotype. This represents a stimulation in the production of gal+ phage of
about 20.000 times more than that produced after infection by the gal+ phage
alone. It is also many more phage than are produced after gal+ phage without an
amber mutation infect the lit" host (see Table 3). We conclude that late proteins
are synthesized in cells infected by both gal+ and gal mutant phage. and that gal
mutations are not simply recessive. However. there is some interference from gal+
genomes on the expression of gal mutant genomes in the same cell. In mixed
infections ofgal+ and gal mutant phage. such as those shown in Figures 3 and 4. the
rate of late protein synthesis was less than half that of normal even if half of the
infecting phage had the gal mutation.

4. Discussion
Host ll! ' mutations severely alter the transcription of the T4 genome late in
infection and thereby prevent the expression of the late genes. One site. the gal site
on T4 DNA. is responsible for the defect in late gene expression. and T4 with

 

406 W. ('. (‘HAMPNESS AND L. SNYDER

mutations in this site multiply normally on lit' hosts. Our evidence that gal
mutations define a site rather than a diffusible gene product is twofold. First. the)"
are very closely linked. Second. they are cis-acting for the expression of the late
genes. e.g. when lit‘ cells are mixedly infected with gal+ and gal mutant phage
having different alleles of a late gene. only the allele on the gal mutant DNA is
expressed. There is no ('ls effect on DNA packaging. however. and both gal+ and gal
mutant DNA are packaged in late proteins made from the gal mutant DNA.

We have demonstrated a cis effect on the expression of genes 23 and 15’. both at
which are closely linked to the gal mutation. Somewhat unexpectedly. we alsn
observed some cis effect on the expression of gene 34. which is about 100' away an
the circular map (or about 10 pm on DNA) from the site of the gal mutation.
although the results were less dramatic because of the lower rate of expression of
gene 34 than that ofeither gene 23 or 18. Because T4 recombination is so active. one
might not expect to be able to demonstrate a cis effect on the expression of a gene
so far away. since the gal mutation will often be separated from the gene 34 amber
mutation by recombination. Nevertheless. this result need not vitiate our
interpretation. Most recombination occurs late in T4 development (Levinthal ti
Visconti. 1953). perhaps later than the times at which we labeled the proteins.

Models of the function of the T4 gal site must await information on the nature of
go] mutations and of the function ofthe host lit gene product. In a lil ' host. T4 late
gene expression is temperature—dependent. This temperature dependence is at least
partially due to differences in E. coli cells that have been grown at different
temperatures and not to the thermal sensitivity of a protein or reaction. Also. the
effect of host lit mutations on T4 gene expression is qualitatively different from
that of most types of phage mutations that block late gene expression. \Vhen true
late gene expression is prevented by mutations in replication genes or gene 55 or
33. the synthesis of many early proteins continues late into infection. However. late
into infection of a lil‘ host. we see. the cessation of synthesis of even tlmse early
gene products that are normally synthesized throughout infection. ()ther situations
are known in which true—late gene expression is prevented and the synthesis of the
early gene products also ceases. These occur when T4 DNA replicates to contain
cytosine and the T4 are alr+ (Snyder ct al.. 197(5). and when polynucleotide kinase
or RNA ligase-deficient mutants of T4 infect E. coli CTr5x (Sirotkin cl al.. 1978:
Runnels et al.. 1982). It is interesting that the alc gene product also unfolds the
bacterial nucleoid after infection (Sirotkin et al.. 1977: Tigges et al.. 1977) and that
some of the original lit' mutants restricted polynucleotide kinase~deticient
mutants of T4 (Cooley ct al.. l979). Perhaps all these gene products are involved in
a common pathway.

We can only speculate on how the wild-type gal+ site can interfere with late gene
expression in a. lil‘ host. Host Iii mutations might prevent the utilization of the
K10“ site. and gal mutations alter the site so it. can function in a lil' host.
Altwna‘m9131inalilfi host. something may happen to the gal+ site that prevents tllt'
expression of the genome harboring the site. For example. host llt_ mutations may
“Twat“ ll l'esti'ictimi~like nuclease. which makes a break specifically at the gal site.

H this is true. then late gene expressitm must be particularly sensitive to cleavage
at this site, since after infection by gene 46 or 47 mutants. for example. double-

A ci.s-»A(‘TING SITE IN T4 GENE EXPRESSION 407

stranded breaks accumulate in T4 DNA (Hosoda e! (11.. 197]) but late gene
expression is not affected. Furthermore. the observation that {101+ genomes can be
packaged in mixed infections of lil’ hosts argues against the "I'estriction~sit.e"
hypothesis. since packaging requires concatameric genomes.

We have recently observed that plasmids with T4 DNA "inserts" containing the
wild»type (gol+ ) site can be used to transform lil+ but not lil’ E. coli. In contrast. if
the T4 DNA insert contains the go] mutant site or deletions of this site. then either
[ifr or [if E. coli can be transformed. Apparently. something happens at this site
even in the absence of the rest of the T4 genome. Note that these observations
support the latter of the two possibilities mentioned above: that something
happens at the 901+ site in a Iil‘ host that is deleterious to the expression of
genomes harboring the site. This raises the possibility that the go] site is not
normally required for late gene expression in rim but can act as a ”spoiler” under
some conditions. It may not be required for late transcription because other similar
sites can substitute for it. or because its real role is to (co—ordinate some other event.
such as DNA packaging, with late transcription. In any case. even if the gel site is
not normally involved in transcriptional activation. an understanding of how an
event at one place in the DNA can affect the expression ofgenes so far away should
enhance our understanding of the intracellular structure of 'I‘-l DNA and how this
structure activates the transcription of the late genes.

We thank Dietmar Rabussay for help with the hybridization experiments and Peter
Geiduschek for critical discussions. This work was supported by National Science
Foundation grant I’(.‘MZ4422 and National Institutes of Health grant (JMZ800l-OI. W.(‘.
acknowledges an NSF predoctoral fellowship.

REFERENCES

Adelberg. E. A.. Mandel. M. & (‘hcn. (l. (‘. (‘. (l965). Bloc/Jeni. Biophys. Res, (‘mnnzun. 18.

788—795.
Bolle. A., Epstein, R.. Salser. W. & Geiduschek. E. l’. (1968). J. 1110/. Biol. 33. 339—362.
Cooley. W.. Sirotkin. K.. Green. R. & Snyder. L. R. (1979). J. Bacteriol. 140. 834)].
Gold. L.. O’Farrell. P. Z. & Russel. M. (l976). J. Biol. ('hem. 251. 725l77262.
Hosoda. J.. Mathews. E. & Jansen. B. (l97l). J. l'irol. 8. 372 4387.
Laemmli. U. K. (1970). Ali/luv (London). 227. 680—68.").
Levinthal, (l. & Visconti. N. (l953). Genetics. 38. 50(FSI l.
Rabussay. D. & Geiduschek, E. l’. (1977). Prov. Na]. :1 rail. Sri.. ('.SA. 74. 530475309.
Riva. S.. Cascino. A. & Geiduschek. E. 1’. (I970). J. Mol. Biol. 54. 85— l()2.
Runnels. J. M.. Soltis. D., Hey. T. & Snyder. L. (1982). J. Mol. Biol. 154. 273—286.
Sirotkin. K.. VI'ei. J. & Snyder. L. (1977). Nalurc (Lam/on). 265. 28732.
Sirotkin, K.. Cooley. VV.. Runnels. J. & Snyder. L. R. (l978). J. .110]. Biol. 123. 2217233.
Snyder, L., Gold. L. & Kutter. E. (l976). I’mc, .Val. Arm]. Sci. USA. 73. 3098—3102.
Studier. F. W. (1973). J. Mol. Biol. 79. 2377248.
Tigges. M. (.‘.. Bursch. J. H. & Snustad. I). l). (1977). J. l'irol. 24. 7757785.
Wood. W. B. (l966). J. Mol. Biol. 16. ll87l33.
Wu. R. & Geiduschek. E. I). (l975). J. .110]. Biol. 96. 5l37538.

Ediletl by M. (lollcsmun

 

ARTICLE II

THE BACTERIOPHAGE T4 GOL SITE: SEQUENCE ANALYSIS AND

EFFECTS OF THE SITE 0N PLASMID TRANSFORMATION

Wendy Cooley Champness and Larry Snyder

Department of Microbiology and Public Health
Michigan State University

East Lansing, Michigan 48824

42

ABSTRACT

T4 gol- mutations overcome, in gig, the block to T4 late gene

expression imposed by Escherichia coli lit mutants. We have proposed

 

(Champness and Snyder, g, M91. Biol., l982) that 391: muta-

 

tions alter a site on T4 DNA that has a regulatory function in T4 late
gene expression. This work reports nucleotide sequence analysis of

gglf mutants; the sequence data supports the proposal that ggl_mutations
do not alter a protein product, but rather, alter a regulatory site on

T4 DNA. Besides affecting T4 gene expression, when cloned into a plasmid
the T4 ggl site has an observable effect on plasmid behavior. If a
plasmid contains the ggl+ T4 sequence, it cannot transform a 1137 host
although it can transform a litf host. But if the plasmid contains a
921: sequence, or is deleted for the 391 region, it can transform a lit:

host. Therefore, the presence of the gol+ DNA interferes with the

ability of a plasmid to stably transform litf g, coli.

43

 

44
INTRODUCTION

Escherichia coli lit- mutants prevent T4 late gene expression at

 

temperatures below 340C. T4 gol mutations were discovered because they

overcome the block to T4 development imposed by E, coli lit_mutants

 

(Cooley et al., l979). Evidence has been presented that ggl_mutations
affect gene expression only in gig in a mixed infection of a lit: host
by gglf and 991i phage and therefore do not alter a diffusible gene
product. Rather, it has been proposed that gol mutations alter a site,
the wild-type form of which interferes with normal gene expression in a
111: host.

Expression of the late genes of T4 seems to require template
”processing," (Rabussay and Geiduschek, l977) which is normally coupled
to DNA replication. The nature of the processing events remains unde-
fined but evidence has been accumulated for the involvement of single-
stranded nicks or gaps (Riva et al., l970b; Wu and Geiduschek, l974),
concurrent DNA replication (Riva et al., l970a), and replication protein—
transcription complex interactions (Wu et al., T975; Wu and Geiduschek,
T977); Rabussay and Geiduschek, l979).

There is also a requirement for some activity of the T4
topoisomerase type II (Liu et al., l980) in T4 late gene expression
(Gisela Mosig, personal communication). Because the E, ggli_gyrase can
substitute for the T4 topoisomerase, the topoisomerase could be
required for supercoiling. Although there is some evidence that domains
of supercoiling exist in T4 DNA (Hamilton and Pettijohn, l976), it is
not known if the T4 topoisomerase is capable of catalyzing supercoiling;

1Q vitro, the enzyme can not introduce supercoils. Therefore, the

 

45
topoisomerase may create some other kind of tertiary conformation in
intracellular T4 DNA.

It is tempting to speculate that the ggl_site plays a role in
the template processing required for late gene expression. But, because
of the complexity of T4 DNA organization at times when ggl mutations
exert their effect, it is difficult to study the effects of 991 mutations
on intracellular T4 DNA.

However, we have found that ggl_DNA has an effect when cloned
into plasmids; this paper describes the effects of the presence of 991+
and ggl mutant DNA on plasmid transformation.

It also reports the sequence of some 991: mutations, both
spontaneous and mutagen-induced, as a beginning to the molecular

characterization of the gol site.

 

46

MATERIALS AND METHODS

Bacterial and Phage Strains

 

The strains used, their relevant characteristics and their

source or reference are listed in Table l.

Hydroxylamine mutagenesis

Mutagenesis was performed as described by Tessman (l968).

Growth of Cytosine-Containing T4 Phage

 

Because T4 DNA contains glucosylated 5-hydroxymethyl cytosine
(HMC) in place of cytosine, the phage DNA is insensitive to most restric-
tion endonucleases. However, by using appropriate phage and host
mutants, T4 can be grown with mostly cytosine in its DNA. T4 which
is mutant in the genes 56 (deoxycytidine triphosphatase); den A (endo
II, a double—stranded-DNA-specific nuclease which degrades the host
chromosome); den B (endo IV, a single-stranded DNA-specific nuclease
which destroys T4 progeny DNA molecules which contain cytosine); and
alc (a gene whose function prevents true-late transcription from a
template containing cytosine) will have up to 95% cytosine in its DNA.
(Snyder, et al., l976). Growth of these phage on the E, £911 strain
B834 galU56 will ensure that the residual HMC residues are not glucosy-
lated and that HMC—containing revertants will not accumulate (Runnels
and Snyder, l978). This host strain is partially deficient in uridine
diphosphoglucose, which is the source of glucose found in phage DNA; in
this host mutant, an intact :gl restriction system recognizes unglucosy-
lated HMC residues in phage DNA, but unmodified C-containing DNA is not

degraded because the strain lacks a functioning rB restriction system.

 

 

47

 

 

Table I
A. E, coli Strains
Strain Relevant Characteristics Source or Reference
++ - —
K803 Su II rk mk
8834 rB'mB- rgl+,su_ Snyder et al., l976

8834 galU56
8834 lit 403
MPH 7
JMlO3

B B t i h

_ _ + _ _
rB mB , rgl ,su ,UDPG
lit—, su-
lit', suI+

- +
A .
lac pro, rk mk ,

F' traD35,proAB,lach,
ZAMl5

Runnels and Snyder, 1978
Champness and Snyder, 1982
Cooley et al., I979

Bethesda Research
Laboratories

 

Strain Relevant Characteristics Source or Reference
Dec 8 56'(amE§l), den A_,
den B , alc our laboratory
Gol 6B gglf, spontaneous Champness and Snyder, I982
Dec 8, HI gg_:, hydroxylamine
mutagenesis our laboratory
Dec 8, H2 gglf, hydroxylamine

mutagenesis

our laboratory

 

48

In order to digest ggl_mutant DNA with restriction enzymes, it
is necessary that the mutant be in a genetic background which allows the
phage to multiply with cytosine rather than hydroxymethyl cytosine in its
DNA. Therefore, ggl_EE, was crossed into a EEE, g§fl_A', g§fl_Ef, gig?
background selecting on B834 golU56 for cytosine, and on a 1137 host for
the ggl_mutation. Other ggl_mutants were obtained by mutagenizing a EEE,
ggg_flf, ggfl_Ef, glgf strain with hydroxylamine.

Cytosine-containing phage were purified on a CsCl step gradient
as described previously (Sirotkin et al., l978) then phenol extracted

and the DNA was dialized against IM NaCl overnight, then H20 overnight.

Enzymes

Eco RI, Hind III, Hpa II, DNA Polymerase I large fragment
(Klenow fragment) and T4 DNA ligase were from Bethesda Research Labora-

tories. Acc I was from New England Biolabs.

DNA Manipulations

Restriction enzymes were used according to the supplier's
recommendations. DNA restriction fragments were separated on a 0.7%
(w/v) agarose gel in 0.09 M Tris-borate, pH 8.3 and the desired fragment
was recovered by electrophoresis onto filter paper, followed by centri-
fugation to elute the DNA and finally, two ethanol precipitations.
Ligation of T4 restriction fragments to plasmid DNA was done with an
equimolar ratio of plasmid to restriction fragment, at a total DNA con-
centration of 40 ug/ml and incubated overnight at l80C. For ligation of
Acc I-cleaved Ml3mp7 DNA to Hpa II-cleaved T4 restriction fragments the
DNAs were mixed in a l to 3 molar ratio at a concentration of l2 ng/S HI

and incubated for 24 hours at 9°C.

 

 

 

49
Cloning in Ml3mp7

The MI3 lg£_vector which was used (Messing et al., I98l) carries
an insert coding for the first I45 amino acids of the B-galactosidase
gene. The phage is therefore a donor for alpha complementation. The
135 DNA contains a 42 base pair Eco RI fragment which does not inhibit
a complementation and which contains cloning sites for Eco RI, Bam Hl,
Pst I, Sal I, Acc I and Hinc II. The intact phage forms blue plaques
when plated under conditions of IPTG induction with X-gal as a substrate.

DNA insertion into any of the cloning sites disrupts s-galactosidase

production and causes the appearance of white plaques.
Transfection

Exponentially growing JMl03 was pelleted by centrifigation at
7000 g for 5 min, resuspended in one half the growth volume in 50 mM
CaClz, then incubated on ice for 20 minutes. The cells were centri-
fuged again, then resuspended in one tenth the growth volume in 50 mM
CaClZ. 0.3 ml of cells were then added to l2 ng of a ligated mixture
of Ml3mp7 and T4 DNA, incubated on ice for 40 minutes, then heat
shocked at 42°C for two minutes. Immediately, 0.2 ml of exponentially
growing JMl03, 3 ml of YT soft agar, IPTG (final concentration, 0.3 mM)
and x—gal (final concentration, 0.03%) were added and then plated on YT
agar plates. (YT agar contains, per liter, 8 gm tryptone, 5 gm NaCl,
5 gm yeast extract and I8 gm agar). After overnight incubation at 37°C,
intact phage gave blue plaques and recombinant phage gave white plaques.
White plaques (putative recombinants) were plaque—purified on JMI03,
then an individual plaque was suspended in 0.5 ml 0.85% saline for

marker rescue (see below).

 

 

'31-; 5‘7 Ella/:21?" ’

50

Identification of Clones by Marker Rescue

 

If T4 infects a cell containing a clone of T4 DNA in a plasmid
or Ml3, recombination can occur between the cloned DNA and the infecting
DNA. If the cloned DNA contains a ggl_mutation some of the progeny virus
will be ggl_mutants and will multiply on a liE_host. This allows one to
determine if the cloned DNA contains the ggl_mutation. The actual
method used is somewhat different for plasmid and MI3 cloning vectors.

I. Plasmids. First, exponentially-growing MPH 7 su+l (lit-) was

plated with top agar. Then, approximately l06 cells of a culture of B834

 

containing the putative recombinant plasmid were spotted onto the plate.
Finally, about l07 wild-type T4 were spotted directly onto the first

spot and plates were incubated overnight at 28°C. Under these conditions,
wild-type T4, when spotted alone or spotted onto B834 containing pACYCl84,
gave no plaques within the spot. A clone which contained a T4 Eco RI 991:
fragment produced discrete plaques within the spot, as a result of marker
rescue of wild-type T4 by the ggl_mutation on the clone.

2. Ml3 clones. Exponentially growing MPH 7 su+l was plated as
above. Spotting was done in the following order: first JMI03 (~I06 cells)
was spotted onto the MPH 7 su+l lawn, then I pl of the recombinant MI3
phage suspension, then l07 wild-type T4. After overnight incubation at
280 the spots were examined for plaque formation. Under these conditions
MI3 spotted onto JMI03 gave no plaques; wild-type T4 spotted onto non-
recombinant MI3 plus JMI03 gave a background of no more than l-S plaques;
and test spots in which marker rescue occurred gave discrete plaques at

a level at least IO-fold over background.

5T

Transformation

Transformation by plasmid-T4 DNA ligated mixtures was done by
the method of Selzer et al. (l978). Cells were spread on tryptone plates
(l0 gm tryptone, 20 gm agar, 5 gm NaCl, I0 gm Casamino acids per liter)

with 25 ug/ml ampicillin or ID ng/ml tetracycline.

Preparation of MI3 DNA for Sequencing

 

Recombinant plaques which tested positively for the presence of
ggl_mutant DNA were plaque purified as above, then a single white plaque
was transferred with a sterile toothpick into 5 ml of YT medium. 50 pl
of anexponentiallygrowing culture of JMI03 was added and the culture was
incubated at 37°C with shaking for 7 hours. One ml was microfuged for
ID minutes, the supernatant was poured off into another tube and the virus
were precipitated by incubation in 4% PEG 6000 and 0.5 M NaCl at room
temperature for 30 minutes. After microfuging for 5 minutes, the super—
natant was discarded, the inside walls of the tubes were wiped clean and
the virus were resuspended in TES buffer (20 mM Tris pH 7.5, l0 mM NaCl,
0.l mM Naz EDTA). The virus were then extracted with phenol saturated
with ID mM Tris HCl pH 8.0, l mM Na2 EDTA, for 5 minutes. After micro-
fuging for 5 minutes, the aqueous layer was removed and the DNA was
precipitated by the addition of O.l M Na Acetate and two volumes of
ethanol and overnight incubation at -ZOOC. The precipitated viral DNA
was collected by microfuging for ID minutes, washing with cold ethanol
and then microfuging again. After drying, the DNA was resuspended in
50 ul TES buffer. This procedure routinely gave enough DNA for ID sets

of sequencing reactions.

52

Selection for Loss of Tetragycline Resistance

 

Cultures were grown in the absence of tetracycline for a number
of generations. Cells which had lost the tetracycline resistance
carrying plasmid were selected using fusaric acid (Maloy and Nunn,

l98l).

Sequencing Reactions

 

Sequencing reactions were adapted from the procedures of Sanger
et al., (I977) as adapted for MI3 (Schrier and Cortese, l979). l pg of
MI3 template DNA and 5 ng primer (a 26 base pair fragment which is
complementary to the 133 DNA immediately adjacent to the Eco RI site)
were annealed at 90°C for 5 minutes in 70 mM Tris HCl pH 7.5, 70 mM MgCIZ,
500 mM NaCl in a volume of 0.0l5 ml, then slowly cooled to room tempera—
ture. Deoxynucleoside triphosphates, dideoxynucleoside triphosphates were added
in the concentrations given by Sanger et al., (l977). o-32P-dATP, l
unit DNA Pol I large fragment and 5 mM dithrothreitol were added and
incubated for IS minutes at 300C. 0.025 mM dATP was added and incubation
continued for another l5 minutes. Reactions were stopped by addition of
IO pl of formamide dye mix 0.l% (w/v) xylene cyanol FF, 0.l% (w/v) bromo-

phenol blue, l0 mM Na EDTA, 95% (v/vldeionized formamide. 6%

2

sequencing gels were prepared as described by Sanger and Coulson (l978).

Materials
o-32P-dATP was from New England Nuclear (400 Ci/mmol) or Amersham
(800 Ci/mmol). Deoxynucleoside triphosphates and dideoxynucleoside
triphosphates were from Bethesda Research Laboratories. Ml3mp7 RF DNA

and 26 bp primer were from Bethesda Research Laboratories. Fusaric

acid, ampicillin and tetracycline were from Sigma.

 

53
RESULTS

Cloning gol,Mutants Into pACYCl84

 

The ggl_§E_mutation maps between 23amBl7 and 23am8272 (Champness
and Snyder, I982; Figure I) in the coding region of the gene 23 product.
Other ggl_mutations have not been so precisely mapped, but all are very
close to 23amBl7 (w. Champness, unpublished data).

Much of the T4 genome has been cloned as Eco RI restriction
fragments and gene 23 is completely included within a 3.5 Kb Eco RI

fragment (Figure l). Therefore, cytosine-containing DNA from EE-,

 

 

den AT, den 8', alc’, gol' mutants constructed as in Methods were
digested to completion with Eco RI and run on an agarose gel. Fragments
of the size 3.5 Kb were recovered and ligated to pACYCl84 (Figure 2).
Recombinant clones were screened genetically, by marker rescue, for the
presence of a ggl_mutation. Clone pG6B was derived from ggl_§E_and

clones pGHl and pGH2 were derived from the hydroxylamine-induced mutants,

gol HI and gol H2, respectively.

Cloning qol mutants Into Ml3mp7

 

Figure I shows the location and size of Hind III restriction
sites within the large Eco RI fragment including gene 23. The ggl site
is undoubtably included within the I.l Kb fragment.

The DNA sequence of this fragment has been determined (A.
Christensen, personal communication). The sequence predicts that four
Hpa II sites occur within the 1.1 Kb Hind III fragment (Figure l),
generating fragments of 460, l80, 75, 270, and l55 bases. It is also
possible to assign the probable positions of many of the gene 23 amber

mutations. The relative positions of the Hpa II sites and 23amBl7 and

54

Fig. l. Correlation of genetic and physical maps in the T4 gene 23

region

A. Gentic map. The locations of gene 22 and 23 amber mutations
with respect to Hind III restriction sites are taken from
Young et al. (I980). The distances between amber mutations
are determined by recombination frequency (Celis et al.,
I973). The position of ggl with respect to gene 23 ambers
was determined previously (Champness and Snyder, l982).

Hpa 11 sites were predicted from the sequence of gene 23

(A. Christensen, personal communication).

B. Physical map of clones derived from pBR322 (pLAI, pLA4, pLAS,

pLA3A2) is taken from Jacobs et al. (l98l).

C. Structure of ggl_mutant DNA-containing clones derived from
pACYCl84, pG6B, pGHl and pGH2; and the gg_f-DNA-containing

clone pA67.

Abbreviations: H3, Hind III; Rl, Eco RI; H2, Hpa II; Apr,
ampicillin resistance; Cmr, chloramphenicol resistance; Tcr,

tetracycline resistance; F’TFTTT indicates deleted region.

55

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Anomm.umxru01~.u>m~v

 

 

 

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56

Fig. 2. Cloning strategy. T4 cytosine-containing gglf DNA was first
digested with Eco RI, then the 3.5 Kb fragment which contains
the ggl_region was cloned into the plasmid pACYCl84 (Chang and
Cohen, l978). The 270 base pair Hpa II fragment which contained

ggl (see Figure l) was then cloned into the ACC'I site in Ml3mp7.

 

      
    
     
 

  

57
Eco RI
m
pAcvcuu
sJiKb
8 T4 col-DNA
" mum
digest with Eco RI
I at Mill mu m
digest wlth Eco RI Isolsts 3.6 Kb trsgmont I—i—L-—i

Ilgsto wlth T4 DNA ligase

select Tc". Cm°

screen for gof‘by marker rescue

   

dlaost with Hlndlll

Hlll
NIH

Htl
"II
H”
"II

digest with Acct Isolate 1.1 Kb Hindlll fragment h—A—‘A—l—J—i

llgstc

select recombinant plaques ,msrker rescue for gol-

   

lelllAccl Npolllecl

58
23am8272 predict that the 270 base pair fragment might include the ggl_EE
mutation. Therefore, plasmids pGGB and pGHl and pGH2 were digested with
Hind III and the l.l Kb Hind III fragment was then isolated and digested
with Hpa II. Hpa II ends are compatible with Acc I ends so the fragments
could be cloned in the Acc I site of MI3. Following ligation, recombinant
MI3 plaques (Figures 2) were selected and tested by marker rescue for the
presence of a 991 mutant DNA insert. By this method, Ml3mp7 clones mp7G6B,
mp7GHl, and mp7GH2 (containing ggl mutations 68, HI and H2 respectively)
were obtained. As shown below, all three of the ggl_mutations were

located within the 270 bp Hpa II fragment.

Sequence Analysis
MI3 clones mp7G6B, mp7GHl and mp7GH2 were sequenced by the

dideoxy chain termination method. The sequence changes due to mutations
are indicated in Figure 3. In each case, there was only one base change
within the insert sequence. Because each insert was shown genetically

to contain a g91_mutation, these changes found must be the biologically
relevant changes. The spontaneous mutant, ggl_EE, changes T/A to C/G

at position 207. The two hydroxylamine-induced mutants HI and H2 are
both changes of C/G to T/A at position I67. We have observed similar
clustering of 991 mutations when mapped genetically; most spontaneous
ggl mutations gave little or no recombination as if they were in the same
base or within a few base pairs of each other, but one mutation (hydroxy-
lamine-induced) gave a recombination frequency with ggl_§§ consistent
with its being close but at a distinct site from the ggl_§E_mutation

(L. Snyder, unpublished data).

 

 

59

Fig. 3. Sequence of the 270 base pair Hpa II fragment and changes due
to gglf mutations. The mutant gol 68 changes T/A to C/G at

position 207. The mutants gol HI and gol H2 change C/G to T/A

at position I67.

 

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60

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6l

Transformation Studies

 

To determine if the ggl_site exerts an effect when cloned in
plasmids we tested the ability of the clines to transform 113' E, 9911,
selecting for antibiotic resistance on the plasmid. Both pACYCl84 and
pBR322 can be used to transform liEf E. 9911, However, as shown in
Table II, a plasmid which contains the 3.5 Kb T4 DNA insert which is
991+ cannot be used to transform a 113: host. Such a plasmid will
transform a 113% host. The failure to transform occurs whether the
insert is cloned into pACYCl84 (plasmid pA67) or pBR322 (plasmid PLAI)
and regardless of orientation. In contrast, plasmids which contain an
insert with a ggl_mutation, p668, pGHl, or pGH2, can transfbrm the 1137
host indicating that the failure to transform is due to the ggl_site.

Occasionally, rare antibiotic resistant colonies arose following
attempted transformation of the 11:. strain with a 991+ plasmid. These
transformants could be grouped into two classes. The transformants in
one class were phenotypically liEf. To test whether these colonies were
revertants of the liEf mutation or if the presence of the plasmid was
affecting the liE_phenotype, loss of the plasmid was selected for by
growing cells in the absence of antibiotic and then selecting for fusaric
acid resistance (see Methods). EiEf cells remained 11;? after loss of
tetracycline resistance and presumed loss of the plasmid. Therefore,
these transformants were true liEf revertants and the Iii? phenotype was
not an effect due to the presence of the plasmid.

The second class of antibiotic resistant transformants were
Ii}: and resulted from the acquisition of a plasmid which had
undergone a deletion of the region which contains 991, One such deleted

plasmid, pLAl l, is shown in Figure 4. Digestion of the plasmid by

 

Fig. 4.

62

Restriction enzyme digests of plasmids used in transformation

studies.

Shown is an ethidium bromide stained 0.7% agarose gel on which
were run restriction enzyme digests of the plasmids shown in

Figure IB.

Lane I and 6, DNA digested with Eco RI; lanes 2, 3, 4, 5, 7,
DNA digested with Eco RI and Hind III; lane 8, DNA digested with
Hind III.

Lane I and 2, pLAI; lane 3, pLA4; lane 4, pLA3A2; lane 5, pLAS;
lanes 6, 7, 8, pLAIAl.

.‘sb—u "a _

63

 

 

12345678

64

Table II. Transformation of lit' E, coli by plasmids.

Transformation ability of the plasmids shown in Figures I and
4. "+" means greater than l03 transformants per pg DNA; "-"

means zero transformants per pg DNA.

65

transforms E. coli

 

 

 

plasmid T4 insert _ii_t"’ ii;
‘pBR322 - m+ +
pLAi gol+ + —
iii-AIM AQOI .+ +
pLA4 gol+ + _
PLA5 AQOI' + . +
pLA3A2 gol+ + +
pACY0184 — + +
pA67 got+ + _
pGGB gor + +
pGH1 goF +
pGH2 90F + +

66
by both Eco RI and Hind III shows that the plasmid contains the intact

I.0 Kb Eco Rl-Hind III Fragment, but the l.l and l.4 Kb bands are
missing. Because no new band appears, the major portion of these two

fragments has been deleted.
Taken together, these data indicate that the presence of the gglf

T4 DNA sequence in a plasmid prevents the stable transformation of lit-

E, coli.

 

To further define the exact region on the 3.5 Kb Eco RI fragment
which prevents transformation, transformation studies were done with
subclones of the 3.5 Kb Eco RI fragment. A series of plasmids which

14-

are derived from pLAl (gg__) have been constructed and described (Jacobs

et al., I98I). These were tested to see if they could transform liEf E,
9911, The structure of these plasmids is shown by restriction digestion
in Figure 4 and drawn schematically in Figure I. pLA4, which contains
the l.l Hind III and l.4 Kb Hind III-Eco Rl fragments and therefore
includes gglf DNA, does not transform the 113: strain. pLA5, which contain
only the l.4 Kb Hind III-Eco RI fragment and therefore does not include
gglf, is capable of transforming the 11;: strain. pLA3A2 has a large
portion of the l.4 Kb Hind III-Eco RI fragment deleted as the result of
exonuclease III digestion. The precise endpoint of the deletion within
the l.4 Kb Hind III-Eco RI fragment is not known but it ends short of
the Hind III restriction site at 2.l Kb. Therefore, this plasmid
contains the gglf sequence. Nevertheless, this plasmid is capable of
transforming the 11:? strain. We do not know if this plasmid contains

a small deletion or some other change within the ggl_region. If it does
not, it would appear that another region on the plasmid besides the gglf

site is involved in the failure of transformation by pLAl, pLA4 and PA67.

67
In general, however, there is a strong correlation between the presence
+
of 991. T4 DNA in a plasmid and the failure of such a plasmid to transform

a lit' host strain.

68

DISCUSSION

Nucleotide sequencing of the mutational lesions in ggl_mutants
has confirmed the genetic mapping; all the mutations sequenced so far lie
close to 23amBl7 and all are single base changes. The results also confirm
that the ggl_phenotype is not due to altering a protein.

From the mucleotide sequence (this work and work communicated
by A. Christensen), it is evident that the only open reading frame on the
"r" strand is that which encodes gp23. Two of the three reading frames
on the "I" strand are blocked by nonsense codons, but the third is open
for some distance in the region of 23amBl7-23am3272. But mutations HI
and H2 would not change the amino acid which could be encoded in this
frame. We have previously presented evidence that although ggl_mutations
are located within the coding region for the gene 23 product, the ggl_
phenotype does not occur as the result of altering p23 (Champness and
Snyder, l982). Therefore, the sequence data confirms the conclusion
that ggl_mutations do not exert their effect by altering a protein
product.

The structural significance of the mutational changes in creating
a 991: phenotype is not obvious from the accumulated sequence data.
Clearly, it would be necessary to sequence more mutants, both spontaneous
and mutagen-induced, in order to define the limits of the region which
can be mutated and the variety of changes within that region which
can create a gglf phenotype. The necessity of maintaining a functional
p23 places constraints on the kinds of gglf mutations that will be
obtained using the selection for phage growth on a liEf host. But it
may be possible to obtain a wider range of 391? mutants by selecting

plasmid mutants which can transform lit' hosts. (see below)

69

It is interesting to note some of the unusual characteristics
of the DNA sequence in the region where ggl_mutations lie. The DNA has
an unusually high G-C content; although the average G-C content of T4
DNA is about 30%,the region around 991 mutations has a G-C content of
about 70%. Also, this region is recombinationally more active than
average. The average recombination frequency for most of gene 23 is
0.0ll% per nucleotide, but there is a "hotspot" for recOmbination in the
amino terminus, apparently in the region where 991 is located. (Celis
et al., l973). We do not know if the ggl_site is responsible for the
high recombination frequency but we have not found any effect of ggl_
mutations on recombination between amber mutations in this region (data
not shown). 2

The studies on the transformation ability of ol-DNA-containing
plasmids show that, besides affecting T4 late gene expression, the T4
991 site also affects the transforming ability of plasmids, i.e., in
general the presence of the wild-type ggl_site in a plasmid prevents
transformation of a 11;: host by that plasmid. We do not yet know if
11;: restriction of T4 gglf phage and the failure of liEf cells to be
transformed by gglf plasmids result from the same mechanism, but gglf
mutations selected in phage overcome both restrictions. In this regard,
it is important to note the anomolous behavior of pLA3A2, which trans-
forms 113: cells although its deleted region ends short of where ggl_
point mutations are located. Several explanations can be proposed which
could account for the anomolous behavior of pLA3A2: the ggl_site is much
larger than the point mutations studied thus far would indicate; another
element close to ggl_interacts with 391; or the transformation block is

not strictly analogous to the T4 development block and another close-by

70
element is involved in the transformation block. Selection for mutations
that allow a gglf plasmid to stably transform would allow further study
of the relationship between these two phenomena.

We do not yet know at what level 991? plasmids are blocked in
transformation, i.e., breakage of the plasmid DNA; failure of the plasmid
to be expressed, replicated or segregated; or lethality of the plasmid to
the recipient cell. Further knowledge about the ggl_effect on plasmid

DNA may give us our best insights into the ifl_VIVO function of the T4

ggl_site.

7l

REFERENCES

Celis, J. E.; Smith, J. 0.;and Brenner, S. (I973). Nature (London)
New Biol. 24l:130-l32

 

Champness, W. Cooley and Snyder, L. (l982). J, Mol. Biol., l55z395.

 

Chang, A. and Cohen, S. (l978). g, Bacteriol. l34:ll4l-ll56.

Cooley, W.; Sirotkin, K.; Green, R.; and Snyder, L. (l978). g,
Bacteriol. l40:83-9l.

 

Hamilton, 5., and Pettijohn, D. E. (l976). g, Virol. 12:1912-1927.

Jacobs, K. A.; Albright, L. M.; Shibata, D. K.; and Geiduschek, E. P.
(l98l) g, Virol. §233I-45.

Liu, L. L.; Liu, C-C.; and Alberts, B. M. (l980). ‘Cell 123697-707.
Maloy, S. R., and Nunn, W. D. (l98l). g, Bacteriol. lfiEglllo-IIIZ.

Messing, J.; Crea, R.; and Seeburg, P. H. (l98l). Nucleic Acids Res.
2: 309-321.

 

Rabussay, 0., and Geiduschek, E. P. (l977). In Comprehensive Virology,
vol. 8, Frankel-Conrat and R. R. Wagner (eds.). (Plenum Press).

 

Rabussay, 0., and Geiduschek, E. P. (l979). Virology 29:286-30l.

Riva, 5.; Cascino, A.; and Geiduschek, E. P. (l970a). g. m], Biol.
fizBS-IOZ.

Riva, S., Cascino, A.; and Geiduschek, E. P. (l970b). g, flgl, Biol.
§43l03-ll9.

Runnels, J. R., and Snyder, L. (l977). J. Virol. Elg8l5-8l8.

 

Sanger, F., and Coulson, A. R. (I978). FEBS LETT. E13l07-ll0.

Sanger, F.; Nicklen, S.; and Coulson, A. R. (l977). Proc. Natl. Acad.
_S_gj_. USA 15:5463-5467.

 

 

Schreier, P. H., and Cortese, R. (l979). J, Mol. Biol. l29:l69-l72.

 

Selzer, G.; Bolle, A.; Krisch, H.; and Epstein, R. (I978). M21, Egg.
Genet. l59:30l-309.

 

Sirotkin, K.; Cooley, W.; Runnels, J.; and Snyder, L. (l978). g, Mol.
Biol. _l_2_3_:22I-233.

 

Snyder, L.; Gold, L.; and Kutter, E. (l976). Proc. Natl. Acad. Sci.
USA. 1;;3098-3IO2.

 

72

Tessman, I. (l968). Virology 35:330-333.

Young, E. T.; Mattson, T.; Selzer, G.; and Bolle, A. (l980). .E.

Biol.'l§E:423-445.

Wilson, G. 0.; Tanyashin, V. 1.; and Murray, N. E. (I977). 591,

Genet. lEE;2-3-2l4.

Mol.

Gen.

SUMMARY

Probably the most interesting aspect of this work is the
discovery of ggl_mutations, which affect T4 late gene expression.
Evidence has been presented that ggl_mutations define a site on T4 DNA
which affects, in gié, the expression of the T4 late genes from the
entire genome.

Not enough information is currently available about T4
intracellular structure to define the role the ggl_site plays in late
gene expression and T4 development. However, the observation that ggl_
affects both T4 gene expression and some aspect of plasmid growth
suggests that both plasmids and T4 require some common structure. If
the relationships between the ggl_effect on T4 and the ggl_effect on
plasmid DNA can be clarified, then knowledge about the defect in plasmid
growth will be important in understanding the defect in T4 development.

Based on our current knowledge, speculative models for the
effects of ggl_on T4 and plasmid DNA structures can be proposed. One
model invokes the supercoiling of DNA. Increased superhelical density
could be required for activation of the T4 late promoters. Supercoiling
could also be required for plasmid transcription or replication. Plasmids
are known to undergo a transient decrease in superhelical density during
the growth cycle (Timmis et al., l976; Crosa et al., I976). Eglf DNA
might interfere with the relaxation, maintenance, or introduction of
supercoils at the proper time in the plasmid growth cycle.

Besides, or instead of, activation of promoters, supercoiling

might be required because it forces the DNA into a conformation, for

73

74
example, a hairpin loop (Lilley, l980), which is required in some
reaction. Panayotatoes and Wells (l98l) have observed that supercoiled
plasmid DNA is cut by SI nuclease at a preferred site because super-
coiling forces the formation of a hairpin structure at that site; the jg.
yjyg_significance of that particular hairpin is not presently known. In
contrast, if an artificial, large hairpin is introduced into a plasmid,
it is lethal to the cell (Lilley, l98l). It is not known what aspect of
plasmid growth is affected or why the hairpin is lethal. The large
hairpin could reduce or eliminate supercoiling or could have a direct
negative effect on some process. Perhaps the ggl_sequence, when intro-
duced into a plasmid, alters the tertiary confbrmation of the DNA.

Another possibility is that ggl_is a site at which a topoiso-
merase acts. As defined by the sequenced mutations, the ggl_site seems
large for a protein-nucleic acid interaction site (at least 40 base pairs).
But precedents for Such large interaction sites do exist; for example,
RNA polymerase recognizes at least 50 base pairs in the igg_promoter
(Reznikoff and Abelson, 1978).

An important observation about the ggl_site is that the wild-type
ggl_site "poisons" at high multiplicity, as if it strongly competes with
the mutant site (Appendix 8, Figure lB). This "competitive" effect
suggests that when wild-type T4 infects a 113: mutant, the 991? sequence
is not simply ignored but participates in a deleterious reaction.

Considering that ggl_has an effect on both plasmid and T4 DNA,
it may be possible to learn something about what happens at the ggl_
site by learning what happens to gglf plasmid DNA when it enters a 113:
cell. Because plasmid transformation is such a low frequency event, it

would be difficult to follow the fate of the transforming plasmid DNA or

75
of the recipient cells. One approach to this problem would be to clone
ggl_DNA into bacteriophage lambda and after infection of litf cells at
a high multiplicity, follow the fate of the lambda DNA.

A possible interpretation of the data that have been accumulated
on ggl_mutants, which has not been previously mentioned, is that the
ggl_phenotypes results from changes of an RNA which is encoded in the
ggl_region. In light of the data on the sis effect of ggl_mutations,
such an RNA would have to be gi§7acting. Recently, Lacatena and Cesareni
(l98l) have shown that a gi§_RNA interaction is important in determining
plasmid Col El incompatability. A possible function of a "gg_f RNA
might be to base pair with the complementary DNA and form T4 "nucleoid-
like" structures. .

Konarska et al. (l98l) have recently described a wheat germ RNA
ligase which forms a 2' phosphomenoester, 3'5'-phosphodiester linkage,
which may be either an intermediate or end product of the ligation
reaction. If T4 RNA ligase can also generate such a structure, the 2'
phosphate might provide a branch through which an RNA can anchor DNA.

The chain of evidence that connects ggl_with the psgl_and RNA ligase
gene products makes it tempting to speculate that an RNA encoded in the
ggl_region is processed by the psgl_and RNA ligase functions to create
a mature RNA which forms and/or maintains T4 DNA structure.

We do not yet have very much information about the function of the
lit_gene product in g, 9911, Li}: mutants do not have any phenotype that
we have observed except in the ljt_double mutants discussed in Appendix A.
However, an interesting observation has been made which may be relevant

to the regulation of the lit function.

76

When wild-type T4 infects in 11;: host at 300C, less than
one percent of a normal burst of phage is produced (Article I, Table 3).
When infective centers are measured, it is seen that l0 percent of the
11;: cells produce an infective center; this subpopulatidn of cells
produces a significant burst, about five to ten phage per cell (W.
Champness, data not shown). In a ggl_mutant infection, the number of
infective centers produced is normal.) The percentage of semi-permissive
cells which produce a small burst is independent of the multiplicity of
infection. The permissivity of 113? cells for T4 may depend on what
stage of the cell cycle the cells are in (Cooper and Helmstetter, l968).
Thus, the synthesis or activity of the lit_function may be coordinated
with the cell cycle.

It is interesting to speculate that the lit_function plays a
role in regulating some E, gglj_transcription which has structural
requirements similar to those for T4 late transcription, although we
do not now have evidence for such a type of transcriptional regulation
in E, 2911, However, an unusual E, ggli_locus, Egg, has recently been
described (Froelich and Epstein, l98l) which has an interesting effect
ontranscription. In temperature sensitive rdg_mutants, the rgg_A_gene
product is required for cell viability at high temperature; the simul-
taneous loss of rgg_A_and rgg_prevents most RNA transcription. Although
lit_and rdg_are different genes and we have no reason to suspect that
the :dg_locus is even related to 11;) this observation by Froelich and
Epstein points out that there are many aspects of transcriptional regu-

lation which we do not yet understand, even in prokaryotes. Further

study of the E, coli lit_gene and the T4 gol_site should add to our

77

understanding of transcriptional regulation, especially, how intra-

cellular DNA structure can effect regulation.

 

 

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Wood, W.; and Revel, H. 1976. The genome of bacteriophage T4. Bact.
Reviews 993847-868.

Worcel, A.; and Burgi, E. 1972. On the structure of the folded
chromosome of Escherichia coli. J. Mol. Biol. 113127-147.

 

Wu, R.; and Geiduschek, E. P. 1975. The role of replication proteins
in the regulation of bacteriophage T4 transcription. I. Gene
45 and hydroxymethyl-C-containing DNA. J. Mol. Biol., 993513-
538.

Wu, R.; Geiduschek, E. P.; and Cascino, A. 1975. The role of replica-
tion of bacteriophage T4 transcription. II. Gene 45 and late
transcription uncoupled from replication. J. Mol. Biol. 993539.

Wu, R.; and Geiduschek, E. P. 1977. Distinctive' protein requirements
of replication-dependent and uncoupled bacteriophage T4 late gene
expression. J. Virol. 293435-443.

Young, E.; Mattson, T.; Selzer, J.; Van House, G.; Bolle, A; and Epstein,
R. 1980. Bacteriophage T4 gene transcription studied by hybridi-
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445.

Young, E. T.; and Menard, R. 1981. Sizes of bacteriophage T4 early
MRNA's separated by preparative polyacrylamide gel electrophoresis
and identified by 19_vitro translation and by hybridization to
recombinant T4 plasmids. J. Virol 40:772-789.

 

Young, E. T.; Menard, R. C.; and Harada, J. 1981. Monocistronic and
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790-799.

APPENDICES

 

APPENDIX A

A NEW GENE 0F ESCHERICHIA COLI WHOSE PRODUCT PARTICIPATES

 

IN 14 BACTERIOPHAGE LATE GENE EXPRESSION: INTERACTION
0F L11_WITH THE T4-INDUCED POLYNUCLEDTIDE
5'-KINASE 3'-PHOSPHATASE

This article, published in Journal of Bacteriology, vol. 140,

 

pp. 83-91, Oct. 1979, appears as an appendix instead of an article in

the body of the thesis because of University regulations.

 

[*i,

OURNAL or BAcrERIOLocY, Oct. 1979, p. 83-91
0021-9193/79/10-0083/08s0200/0

Vol. 140, No. l
A New Gene of Escherichia coli K-12 Whose Product
Participates in T4 Bacteriophage Late Gene Expression:
Interaction of lit with the T4-Induced Polynucleotide 5’-
Kinase 3’-PhosphataseT
WENDY COOLEY, KARL SIROTKINgt ROBERT GREEN, AND LARRY SNYDER‘
Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824
Received for publication 22 May 1979

We isolated five Escherichia coli mutants deficient in their ability to support
the late (replication-coupled) gene expression of T4 bacteriophage at 30°C. These

phosphatase activity in crude extracts.

Some types of bacteriophage transcription
may require covalent alterations of the DNA
template. For example, T5 induces a DNA 5’-
exonuclease which enhances its late transcrip-
tion (5, 9). Also, late transcription of T4 is greatly
enhanced by phage DNA replication (12), and
DNA ligase mutations relieve the requirement
for DNA replication (13) presumably by pre-
venting the repair of nicks in DNA.

It would be of interest to know whether the
types of bacteriophage transcription which re—
quire DNA alterations have common features,
and whether an analogous type of transcription
eXlSts 1n uninfected bacteria. A first step in ad-
dressmg this question is to isolate and study host
mutants which cannot support a type of bacte-
riophage transcription that requires DNA alter-
ations. Presumably, some of these host mutants
would be deficient in a function which partici-
pates in the phage transcription and, hopefully,
in an analogous type of host transcription. For
the selection, it may be best to start with phage
mutant in a known function thought to be in-
volved in transcription, since using the wild—type
bacteriophage for the selection is technically
dlfficnlt and would reveal little about the actual
functions involved. The host mutants of interest
are those that can propagate wild-type bacterio-
Phage but not the phage mutants.

T Article no. 9027 of the Michigan Agricultural Experiment
Station.

iPresent address: Division of Chemistry, California Insti-
tute of Technology, Pasadena, CA 91125.

 

mutants, which we call Lit mutants, define at least one novel gene at 25 mm on
the E. coli map. They were selected in an attempt to obtain mutants which
restrlct the growth of T4 mutants deficient in polynucleotide 5’-kinase 3’-phos-
phatase but not that of wild-type T4 at 37°C. Some of the mutants do have these
phenotypes under some conditions. Studies of the block in T4 development in
some of the E. coli mutants suggest that Lit mutants are affected in a gene
product involved in the metabolism of deoxyribonucleic acid nicks or single-
strand gaps. None of the Lit mutants is deficient in the major, bacterial, 3’-

We have proposed that the T4 polynucleotide
5’-kinase (10, 11) is required, under some condi-
tions, for T4 late transcription (14). This enzyme
is also a 3’-phosphatase (3, 14), so it will hence-
forth be referred to as the T4 polynucleotide 5’-
kinase 3’-phosphatase. This enzyme is normally
not required for phage development on standard
laboratory strains of Escherichia coli, since T4
pseT‘ mutants, which do not induce it (7, 14),
multiply almost normally (7, 4, 14). However,
T4 pseT" mutants are restricted on E. coli
CTr5x, a hybrid of E. coli K-12 and E. coli
CT196 (a clinical isolate) (7), because subnormal
amounts of late gene products are made (14) and
also, possibly, because of a DNA packaging de-
fect (7). Presumably, E. coli CTr5x has an amber
mutation in a gene whose product is required to
support the multiplication of pseT‘ T4 because,
with the acquisition of an efficient amber sup-
pressor, it becomes permissive for all pseT’ mu-
tants, even deletions (14). It is tempting to spec-
ulate that the host gene with the amber muta-
tion codes for an enzyme which is analogous to
the phage polynucleotide 5’-kinase 3’-phospha-
tase and can normally substitute for it during T4
development. However, other explanations are
possible.

Rather than try to map the putative amber
mutation in E. coli CTr5x which makes it re-
strictive for pseT’ T4 (which could be difficult
because the strain is distantly related to E. coli
K-12), we isolated similar restricting mutants of
a convenient laboratory strain of E. coli K-12.

 

 

84 COOLEY ET AL.

These mutants, which we call Lit‘ mutants, for
late inhibitors of T4, are similar in some, but not
all, respects to E. coli CTr5x. They define at
least one, and possibly two, new genes of E. coli.

MATERIALS AND METHODS

Bacterial and phage strains. The strains used,
their relevant characteristics, and their source or ref-
erence are listed in Table 1.

Isolation of Lit mutants. Lit mutants were iso-
lated as mutants which plated wild-type T4 but not a
T4 pseT deletion mutant, ApseTl, at 37°C. Potential
mutants were subsequently screened to determine
whether the restriction originates from the absence of
the pseT gene or from the absence of another T4 gene
included in the deletion.

The mutants we were seeking would not propagate
the T4 pseT deletion mutant but would be killed by it.
The latter presents problems for the selection, which
can be dealt with in a number of ways. The first Lit‘
mutant, E. coli MPHS, was isolated using a variation
of the “tab" procedure of Takahashi et al. (16). In this
procedure, a carefully predetermined number of phage
and mutagenized bacteria are plated together, and E.
coli mutants that survive are tested for the desired
phenotype. This method is very sensitive to the
amount of phage and bacteria as well as the condition
of the plates, and often MPH5 did not survive a
reconstruction of the selection conditions. Therefore,
we designed a type of sibling selection procedure by
which we isolated the remaining four Lit‘ mutants.

This procedure succeeds because, on an undisturbed
plate, the descendants of a mutant bacterium will be
clustered and will protect each other by failing to
produce the T4 pseT deletion mutant progeny. E. coli
ABZ495 were mutagenized with nitrosoguanidine by
the procedure of Adelberg et al. (1), and about 10“
were spread on a tryptone plate with 107 T4 ApseT 1.
The plate was incubated overnight at 37°C. This plate
was then replicated onto another plate on which about
109 T4 ApseTl had been spread, and this plate was
also incubated overnight. About 500 to 1,000 colonies
appeared, mostly due to resistant mutants. The MPH5
prototype survived a reconstruction of this procedure
even when mixed with 10" of the unmutagenized pa-
rental cells beforehand. MPH5 mutant colonies grew
with a characteristic “lumpy" appearance, like a bunch
of grapes, which helped distinguish them from resist-
ant colonies, which tended to be round and often
slimy. The lumpy colonies from the mutagenized
plates were picked under a dissecting microscope and
streaked across dried streaks of both T4 ApseTl and
T4 wild type, in that order, both at 109/ml, and the
plates were incubated at 37°C. The mutants that
seemed to be cleared by wild-type T4 but not by
ApseTl were purified by two cycles of “streaking out"
and used as indicator bacteria to plate T4 Apse’I‘l and
wild-type T4 at 37°C. We found about one Lit‘ mutant
for every 200 lumpy colonies tested, and MPHG, -7,
-21, and -24 were isolated by this procedure. All five of
the Lit' mutants were probably due to independent
mutational events because they either originated from
different mutagenized stocks or had different pheno—
types.

J. Bacraiuox.

TABLE 1. Bacteriophage and bacterial strains

 

 

lime
Strain Relevant characteristics 01' ref-
erence
T4 ApseTl Deletion including pseT I4
pseT2 Point mutant in pseT 7
BL292 Gene 55 amber 8
N82 Gene 44 amber 8
N81 Gene 41 amber 8
NG576 Gene 52 amber 8
N134 Gene 33 amber 8
M69 Gene 63 amber 8
B17 Gene 23 amber 8
E727 Gene 49 amber 8
N54 Gene 31 amber 8
E. coli CT196 Clinical isolate 7
CTr5x Hybrid K-l2: CT196 7
ABZ495 F‘ multiple auxotroph CGSC‘
trp—35 his-4 supE44
Hfr Broda 8 Transfers clockwise from 8 CGSC
mm
Hfr KL99 Transfers clockwise from CGSC
22 min
Hfr KL96 ansfers CGSC
counterclockwise from
49 min
MA 1003 per46 CGSC
W3110 hp“
PC0254 purB51 CGSC

 

" CGSC, E. coli Genetic Stock Center, Department of Hu-
man Genetics, Yale University School of Medicine, New HI-
ven, Conn.

Mapping Lit mutants. The procedure of Curtis
et al. (6) was used for Hfr crosses. P1 transduction was
with Plvir in tryptone broth supplemented with 2.5
mM CaClg. The presence or absence of the Lit phe:
notype among recombinants was determined by
streaking across T4 ApseTl and T4 wild type as in the
isolation procedure.

Assays of E. coli 3’-phosphatases. The assay
conditions were essentially those of Depew and Coz-
zarelli (7). The preparation of 3’-[32P]dTMP was from
500 ml of E. coli cells labeled for about six generations
in Tris medium with 1073 M P04 and 15 mCi of ”POI.
The cells were centrifuged and incubated overnightin
1% sodium dodecyl sulfate and 0.5 M NaOl-I at 37°C
to degrade the RNA. The extract was neutr '
blended in a Vortex mixer, extracted twice with
phenol, and dialyzed against 1 M NaCl and then water-
The dialysate was precipitated with 5% (wt/vol) tli-
chloroacetic acid and washed with 80% ethanol and
then ether before drying. The pellet was suspendedin
2 ml of water and was digested with micrococcal
nuclease (Worthington) to 40 pg/ml in 0.01 M Tris
(pH 8.7)—2.5 mM CaC12 for 1 h at 37°C, neutralized.
and further digested by adding spleen phosphodiester-
ase (Worthington) to 50 pg/ml three times, 1 h 8pm
The 3’-dTMP from the digest was purified by papa
electrophoresis in 0.05 M ammonium acetate (pH 3.5).
followed by paper chromatography with 1.8% NH4OH1
62% isobutyric acid, and 10’3 M EDTA and by a repeat
of the paper electrophoresis (14). Very high back-
grounds were observed if the 3’-dTMP was contami‘
nated with either 5'-dTMP or 3’-UMP. Any 3’-UMP

 

__._____.-_cv=lfi§§‘l§'

is

VOL. 140, 1979

left over from RNA after dialysis will be separated
from 3'-dTMP by the above procedure. However, 5’-
dTMP is not separable, so the micrococcal nuclease
and spleen phosphodiesterase must be free of conM<
inating DNase activities.

To prepare extracts for the assays, cells were grown
to 4 X 105/ml in tryptone broth (10 g of tryptone and
5 g of NaCl per liter of water), and, if infected, phage
was added at a multiplicity of infection of 10 for 15
min at 37°C. The cells were chilled, centrifuged, re-
suspended at one-tenth the volume in 0.01 M Tris (pH
7.5) with 1 mM mercaptoethanol, and lysed by soni-
cation. E. coli B extracts (0.1 ml) could be assayed
directly, but E. coli K-12 extracts first needed to be
diluted about 1:100 with resuspension buffer. For fur-
ther purification, the extracts were cleared by centrif—
ugation for 20 min at 75,000 X g and precipitated with
an equal volume of 0.5% protamine sulfate. The su-
pernatant fluid was assayed then or purified further
by (NH4)2S04 precipitation and DEAE chromatogra-
phy by the procedure of Becker and Hurwitz (2).
Approximately 30 to 40% of the activity in crude
extracts from E. coli B or K-12 strains was recovered
in the DEAE fractions.

Labeling of proteins and DNA. Because of the
multiple auxotrophies of the E. coli AB2495 parent,
Lit' mutants were difficult to infect synchronously
With T4, necessitating the following procedure. First
thyi revertants were used. To label proteins, the cells
were grown at 37°C to 4 X 108ml in M9 (5.5 g of
NazHPOh 3 g of KH2P04, 0.5 g of NaCl, 1.0 g of NH4Cl,
0.5% glucose, 10"3 M MgSOi) supplemented with thia-
mine at 50 rig/ml and all 19 amino acids at 100 ag/ml
except methionine. The cells were concentrated 10
tlmes by centrifugation and infected at a multiplicity
of 10 with T4 which had been purified on CsCl step
gradients to remove ghosts and prevent ghost exclu-
sion. After 4 min for absorption, the infected cells were
diluted 1:10 into fresh medium at 30°C and labeled
with 10 aCi of [”S]methionine per ml (865 Ci/mmol)
at the times and for the periods indicated.

To label DNA, M9 medium supplemented with 1%
casein hydrolysate, tryptophan (50 [lg/ml), and thia-
mine (50 g ml) was used, and the cells were labeled
w1th [methyl-3H]thymidine at 10 pCi/ug (1 rig/ml) to
measure the rate of incorporation and 100 pCi/ng (1
Mg/ml) to measure the size of DNA. For the measure-
ments of the rate of incorporation, cells were precipi-
tated with 5% trichloroacetic acid, resuspended with

. ,and .— ‘r"‘ w' t'” "
acid before being collected on glass fiber filters.

aline sucrose gradients. The procedures of
Depew and Cozzarelli were followed (7), layering 0.1
ml of an unconcentrated lysate on the gradient.

Slab gel electrophoresis and autoradiography.
The apparatus and procedure of Studier (15) were
used. Gels were stained with Coomassie blue to check
the total amount of protein applied to each well.

RESULTS
Isolation of Lit mutants. We isolated five
E. coli mutants which restrict the growth of the
T4 ApseTl mutant but not that of wild-type T4
at 37°C. Three of the five mutants (MPH5, - .

 

E. COLI lit MUTANTS AND T4 TRANSCRIPTION 85

and -21) were indistinguishable in some respects.
They multiplied with the same generation time
as their parent and absorbed T4 normally. Wild-
type T4 produced plaques on them with an
efficiency of about 0.2 at 37°C, whereas T4 pseT
deletions such as ApseTl plated with an effi-
ciency of less than 10“. It was perhaps fortunate
that we carried out the selection at 37°C because
there was no difference between the plating ef-
ficiencies of the pseT deletions and wild-type T4
at either higher or lower temperatures. At 30°C
both wild-type T4 and T4 ApseTl were almost
totally restricted, plating with efficiencies of less
than 10“. At 42°C, even the T4 pseT deletions
plated normally. The effect of varying incuba-
tion temperature on wild-type T4 plating effi—
ciency of Lit' mutants was remarkably abrupt,
the plating efficiency going from 10'8 to 0.2 in
the temperature range of 34 to 37°C. None of
these three Lit‘ mutants was particularly re-
strictive for the growth of T4 pseT point mutants
even at 37° .

The other two E. coli mutants (MPH6 and
-24) were different in some respects. MPH6 was
noticeably restrictive for T4 pseT point mutants
at 37°C, giving very small plaques at a frequency
of about 10.2. It absorbed T4 more slowly than
the parental strain, made slimy, opaque colonies,
especially on minimal media, and did not plate
bacteriophage P1. It was cold sensitive for T4
multiplication, as were the other Lit' mutants.

MPH24 grew poorly, particularly at 42°C. It
was more permissive for wild-type T4 at 30°C
than the other Lit mutants. It absorbed T4
slowly and did not plate P1. It was not particu-
larly restrictive for the growth of T4 pseT point
mutants.

Mapping mutations responsible for the
Lit phenotypes. MPH5, -7, and -21 behaved
similarly to each other in Hfr crosses and P1
transduction, as though they have mutations in
the same gene. With Hfr Broda 8 as donor,
approximately 80% of the Trp‘r recombinants
were Lit+, whereas only 34% of the His+ recom-
binants were Lit*. With Hfr KL208, only 30% of
the Trp+ recombinants were Lit‘. Thus, the
conjugation data with these donors suggested
that the Lit mutation lay counterclockwise and
within about 10 min of trp. This conclusion was

KL99, which transferred the region of the Lit
mutation very early, placing
min. P1 transduction was used to further localize
the Lit mutation (Table 2). We found no cotrans—
duction with per or with trp. There was, how-
ever, ahout 70% cotransduction with purB and
the Lit’ marker in all three Lit mutants, indi-
cating that all three had mutations at about 25

 

 

86 COOLEY ET AL.

TABLE 2. Mapping of lit mutations with P1
transductio

 

 

Per-
Se- Unse- N cent
Donor Recipient lected lected 0' co-
tested trans
marker marker
duc
tion
MPH5 MA 1008 per‘ [it 110 0
W3110 MPH5 trp‘ lil‘ 148 0
MPH5 PC0254 purB ' lit 63 78
MPH7 PC0254 purB ‘ lit 76 66
MPH21 PC0254 purB ‘ lit 39 51

 

min on the E. coli map. Since the mutations
were closely linked and had similar phenotypes,
we think they arose in the same gene, although
we have not established this point with comple—
mentation tests. We have tentatively assigned
the name lit to the gene and given the mutations
in MPH5, -7, and -21 the names lit-5, lit- 7, and
lit-21, respectively. The one Lit‘ transductant of
PC0254 we tested was similar to the original
MPH5 mutant in all of its phenotypes. We take
this as additional evidence that a mutation in
one gene was causing the multiple phenotypes
of MPH5, -7, and -21.

In contrast, the two other mutants, MPH6
and MPH24, were probably double mutants,
having lit mutations and at least one other un-
linked mutation in a cistron whose product in-
teracts with the lit function. The evidence for
this is as follows. When MPH6 and -24 were
crossed with Hfr Broda 8 and Trp+ was the
selected marker, most of the recombinants made
very poor lawns on plates. If, as suggested by its
map position, the mutation crossed out of MPH6
and MPH24 was a lit mutation, we would expect
the recombinants to accumulate lit mutations
upon culturing. This prediction was fulfilled, at
least for the MPH6 mutant. Furthermore, we
were able to isolate a lit single mutant as a
recombinant from the original MPH6 mutant.
This mutant was similar to our other Lit" mu-
tants, and we call its mutation lit-6. It was used
for some of the experiments discussed below.

Because of their similarities, we assume that
both MPH6 and MPH24 carried lit mutations
as well as second—site mutations in the same
other gene. We think that it is the mutation in
this other gene that caused MPH6 and -24 to be
restrictive for T4 pseT point mutants, because
the recombinants, when first isolated (at least in
the case of MPH24), restricted T4 pseT point
mutants but were completely permissive for
wild—type T4 at any temperature.

Effect of lit mutations on T4 develop-
ment. Since at least some of our lit mutations
did not affect bacterial growth or T4 absorption,

J. BACTERIOL.

we could study their effect(s) on T4 development
at 30°C to determine why T4 does not multiply
on them. The rate of T4 DNA synthesis was not
significantly affected (Fig. 1A). Also, alkaline
sucrose gradient centrifugation revealed little
difference in the single-strand length of the T4
DNA that was made (Fig. 2A). The remaining
parts of Fig. 1 and 2 will be referred to below.

Slab gel electrophoresis of proteins and auto-
radiography can be used to analyze T4 gene
expression during infection because only T4 pro-
teins will be labeled after infection, since T4
shuts off host protein synthesis. It can be seen
that T4 early gene expression is not significantly
affected by the lit mutation (Fig. 3A and E).
However, there is a dramatic effect on T4 late
protein synthesis, especially very late in infeC-
tion (Fig. 3B, C, and D and F, G, and H). It can
be seen that T4 late protein synthesis began
normally on the mutant host but then decreased

 

103 cpm

 

 

 

 

I0 2'0 3'0 4'0
mun after inf.

FIG. 1. The rate of T4 DNA synthesis after infec-
tion of the Lit+ parent, E. coli 1182495, and a Lil-
recombinant of MPH6 with T4 wild-type and a T4
Gol mutant. E. coli AB2495, open figures; MPH6
recombinant, closed figures. (0, O) Wild-type T4;
(I, El) T4 Gol mutant; (A, A) mixed, T4 Gal plus
wild-type T4 at a. multiplicity of infection of 5 of each
The wildJype T4 experiments shown in (B) and (C)
are replots of the data in (A) for comparison. Surviv-
ing bacteria were less than 0.1% at 2 min after infec-
tion in all cases.

m

§§§§§

§1Em¥

VOL. 140, 1979

 

Fraction no,

AIL r J' x

F] . . " "
T4 DNA pulse-labeled from 14 to 16 min after infec-

\

(A) O, wild-type T4 on E. coli ABZ495; and 1:1, wild-
type T4 on the E. coli MPH6 recombinant. (B) 0, T4
Go! on E. coli ABZ495; and Cl, T4 Gol on the E. coli
MPH6 recombinant. The arrow shows the approxi-
mate position of a viral T4 DNA marker on a similar
gradient. Shown are the trichloroacetic acid-precip-
itable counts.

in rate relative to the Lit+ control. We assume
that the effect on late gene expression is suffi-
cient to explain the inability of T4 to plate on a
th‘ mutant host at 30°C. T4 pseT deletion
mutants, such as ApseT 1, were no more deficient
In late gene expression than wild-type T4 at
30°C (data not shown). We have not yet ana—
lyzed protein or DNA synthesis at 37°C.
.Physiological and genetic characteriza-
tion of T4 mutants that multiply on E. coli
Lit mutants. Plaques formed at a low frequency
when wild-type T4 was plated on Lit‘ mutants
at 30°C or when T4 pseT deletions were plated
at 30°C or 37°C. These plaques were almost
normal size and were due to the growth of T4
mutants because, when the phage from them
were isolated, they gave plating efficiencies of
100% on Lit' mutants. They also gave 100%
plating efficiencies on the Lit+ parent. We call
the mutants T4 Gol mutants, for grow on Lit. A
T4 G01 mutant isolated from any Lit‘ mutant
plated on any other Lit‘ mutant. This was one
Of the first indications that the E. coli Lit'
mutants all had a common molecular basis for
their phenotypes.

The frequency of Gol mutants in lysates of
some T4 pseT deletion mutants was three orders
0f magnitude higher than in wild-type T4 ly-

E. COLI lit MUTANTS AND T4 TRANSCRIPTION 87

sates. There are three possible explanations: the
pseT deletions may be mutagenic; the G01 phe-
notype may require two mutations, one of which
is in the region included in the pseT deletion; or
G01 mutants may be selected when the pseT
deletion mutants multiply, even on a Lit+ host.
We think the third explanation, that the G01
mutants ‘ t A ' t“ ‘
the pseT deletion mutants did not accumulate
mutations to other phenotypes and because the
G01 phenotype did not depend on the pseT
deletion and could be separated from it by re-
combination.

In an attempt to map the T4 mutation re-
sponsible for the G01 phenotype, we plated the
T4 amber mutants shown in Table 1 on E. coli
MPH5 (supE). The phage that made plaques
were amber, 601 double mutants; they were
crossed with wild-type T4, and the progeny were
plated on a supE + but Lit‘ derivative of MPH6.
Only the G01, amber“ recombinants should
plate, and the recombination frequency between
the G01 mutation and the amber mutation can
thus be determined. We found no linkage with
T4 genes 55, 44, 41, 52, 33, and 63. We had
difficulty selecting G01 mutants with amber mu-
tants in genes 23, 49, and 31, so we crossed a G01
mutant with amber mutants in these genes and
tested the progeny for wild-type recombinants,
since we knew these would be viable. We found
very close linkage with gene 23, which is in the
late region of the T4 map. Four other independ-
ent G01 mutants we tested showed the same
linkage to gene 23. We are presently undertaking
more precise mapping studies.

Since Gol mutations can almost completely
alleviate the affect of the host lit mutations on
T4 development, studies of T4 Gol mutant de-
velopment should give us insights into the func-
tion of the host lit gene product. In particular, if
the effect of lit mutations on T4 late gene expres—
sion is responsible for the inability of T4 to grow
on a Lit’ host, then Gol mutations should over-
come this effect. Figure 4 shows the results of
slab gel electrophoresis and autoradiography of
the proteins made late in infection by a T4 G01
mutant. The G01 mutation had no effect on late
gene expression in the parental host strain (Fig.
4A and B). However, the G01 mutation did affect
late gene expression on the mutant host, com-
pletely overcoming the effect of the host lit
mutation (Fig. 4D and E). It should be noted
that some of the T4 late proteins affected (e.g.,
p37) are coded by genes which are some distance
from the site of the G01 mutation and which are
separated from it by early genes.

Also shown are the results of a complemen-
tation test in which cells were infected by wild-

k 4" Ian
Uyuuua

 

 

88 COOLEY ET AL.

 

we» «-

Q.—
”may

ABGCD

w ... em a: w,

J. BACTERIOL.

1..

Hi

77.!

FIG. 3. Rate of synthesis of T4 proteins after infection of the recombinant of E. coli MPH6, Slab sodium
dodecyl sulfate-12% polyacrylamide gels and autoradiograms of proteins pulse-labeled with [““Sflnethionine
after T4 infection at 30° C, multiplicity of infection of 10. Labeling period: (A) and (E), 6 to 9 min; (B) and (F),
18 to 21 min; (C) and (G), 28 to 31 min; (D) and (H), 38 to 41 min. (A to D) E. coli/132495 Lit”; (E to H) E36011
Lit". The products of some late genes are identified. P23‘ is the processed form of the product of T4 gene 23‘

type T4 and the G01 mutant simultaneously and
the effect on T4 late gene expression was deter-
mined. The Gol mutant was codominant with
wild-type T4 for late gene expression on the Lit—
host (Fig. 4F). This is not a special property of
this Gol mutation, because two other mutants
we have tested were also codominant. It is sur-
prising that codominant mutations could en-
hance T4 late gene expression since T4 late
transcription undoubtedly occurs from many
promoters simultaneously and there are many
T4 genomes present late in infection. We shall
return to possible explanations in the Discus-
s10n.

The only T4 Gol mutant we tested also
showed a defect in T4 DNA replication, and this
occurred on any host. Figure 1B and C show
that the rate of T4 DNA synthesis was sharply
reduced when a T4 Gol mutant infected the
parent E. coli AB2495 (Fig. 1B) or a Lit‘ mutant
(Fig. 1C). Again, the effect of the G01 mutation
on the rate of T4 DNA replication seemed to be
codominant, because in mixed infections the rate
of T4 DNA synthesis was intermediate between
the T4 G01 and wild-type T4 rates.

The T4 G01 mutation affected not only the
rate of DNA synthesis but also the size of the

T4 DNA that was made. In Fig. 2B, alkaline
sucrose gradient centrifugation revealed that the
single strands of the T4 Gol DNA labeled in a 2-
min pulse of [3H]thymidine were significantly
shorter in either a Lit‘ mutant or its parent. A1]
of the experiments above were done with a T4
G01 mutant which we had shown to have a
mutation close to gene 23 in the late region. We
have not absolutely ruled out the possibility of
two mutations in the G01 mutant, of which one
is responsible for the effect on late protein syn-
thesis and the other for the effect on replicat10n~
However, this seems very unlikely, since the 001
mutant was a spontaneous mutant. and both the
effects on late gene expression and replication
were codominant.

Effect of lit mutations on host 3'-phos-
phatase activities. We began the isolation 0f
E. coli Lit‘ mutants with the thought that we
might find a host gene that codes for a produCt
analogous to the T4 phage-induced p01ynucleO-
tide 5Q-kinase 3'—phosphatase. If so, Lit’ mutantS
may be deficient in a similar enzyme. NOIOD.e
has been able to detect a polynucleotide 5'1“
nase activity in uninfected bacteria (10, 11)- I“
1967, Becker and Hurwitz (2) reported a 3"Ph°5'
phatase from E. coli which was nonspecific for

 

VOL. 140, 1979

 

A B

FIG. 4. T4 late protein synthesis by wild typ

MPH7 (Lit‘). Shown are

E. COLI lit MUTANTS AND T4 TRANSCRIPTION

89

 

D E F

_ e and a T4 Gal mutant on E. coli AB2495 (Lif) and E. coli
sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiograms as in

Fig. 3. Time of labeling, 35 to 38 min after infection. (A) and (D) Wild-type T4, multiplicity of infection of 10;

(B) and (E) T4 Gal, multiplicity of 10; (C) and (F) T4 Golplus wild-type T4, multiplicity of5fo

E. coli AB2495 (Lit *) ; (D to F) MPH7.

3'- _0r 5’-phosphates on mononucleotides, but
whlch, .0“ polynucleotides, would only remove
3 -term1nal phosphates. This enzyme was re-
ported to be much more active in extracts of E.
coli C than E. coli B. We found that not only E.
coli C, but also E. coli K-12 and E. coli CTr5x,
displayed almost 100 times as much 3’-phospha-
tase activity in crude extracts as E. coli B. The
{major activity in E. coli K-12 was not deficient
In any of our Lit‘ mutants or in E. coli CTr5x
(See Table 3). We think the differences in activ-
Ities shown in Table 3 are not significant and are
due to the difficulty of assaying the enzyme in
crude extracts, because they were not reproduc—
Ible and disappeared upon further purification
of the enzyme.

We do not know if the major 3'-phosphatase
detected at much lower levels in B strains was
due to the same or a different enzyme. It purified
much the same through the DEAE chromatog-
rE1p2hy‘step of Becker and Hurwitz and could use
CO Instead of Mg2+ as a divalent cation, as
could the K-12 enzyme.

We consistently observed an inhibition of the
E. coli B enzyme after T4 infection, as indicated
by the lower level of 3’-phosphatase activity in
T4 pse‘TZ-infected cells (i.e., in the absence of
phage-Induced enzyme) compared to uninfected
cells (see Table 3). We think this inhibition is
real because it has appeared dozens of times in

r each. (A to C)

TABLE 3. 3’-Phosphatase activities in Lit mutants
and extracts of T4Ainfected E. coli B

3'-Phos-
F32“ Source of crude extract phatase
‘ activity"
I E. coli AB2495 4.8
MPH5 5.4
MPH6 3.3
MPH7 7.7
MPH21 5.1
MPH24 3.2
CTr5x 8.7
11 E. coli B 0.11
T4‘-infected E. coli 0.17
T4 pseT2-infected 0.04
E. coli B

" Nanomoles of PO. released from 3’-dTMP in 30
min at 37°C per 4 X 10" cell equivalents.

our assays. The inhibition of the host enzyme
was more apparent after partial purification of
the enzyme with protamine sulfate (see Materi-
als and Methods), when almost no activity could
be detected (data not shown). Thus, the activity
which persisted in T4 pseTZ-infected cells in
Table 3 was probably due to other competing
activities in crude extracts. In contrast, the ma-
jor 3’-phosphatase activity in E. coli K-12 did

 

90 COOLEY ET AL.
not seem to be inhibited after T4 infection (data
not shown).

DISCUSSION

Some Lit" mutants of E. coli K-12 restrict T4
pseT" point mutants more than wild-type T4 at
37°C. The Lit" mutants that behave this way
seem to have mutations in another, as yet un-
characterized, gene. It appears that a deficiency
in this other gene was the direct cause of the
restriction of pseT" mutants of T4 in some of
the Lit" mutants. There must be a very close
interaction between the products of this other
gene and the lit gene because Lit" recombinants
made poor lawns and accumulated lit mutations
as suppressors. Apparently, a deficiency in this
other function must be compensated by a defi-
ciency in lit function. It is tempting to speculate
that this other function is analogous to the 5’-
polynucleotide kinase 3’-phosphatase of T4.
None of the Lit" mutants was deficient in the
major 3’-phosphatase activity in E. coli K-12.
However, there may be other 3’-phosphatases in
E. coli, as suggested by our observation that an
activity in E. coli B, which is present at much
lower levels, is inhibited after T4 infection,
which is not true of the major K-12 activity.

A comparison of Lit" mutants and E. coli
CTr5x is interesting. In both E. coli CTr5x and
Lit" mutants, T4 late gene expression is defec-
tive. Furthermore, the effect on late gene expres-
sion in both is temperature dependent, since E.
coli CTr5x allows normal synthesis of T4 late
proteins after infection by a T4 pseT" mutant
at 42°C (see Fig. 5). However, T4 pseT G01
mutants do not plate on E. coli CTr5x. In spite
of this, it seems likely that the restriction of T4
pseT mutants by E. coli CTr5x and by E. coli
Lit mutants is related.

We think that lit mutations define a new
cistron of E. coli K-12. The only other known
mutations that map in this region and restrict
T4 are galU mutations. However, galU muta-
tions cotransduce with tip and not purB and do
not exert an effect on T4 in the first generation,
and the progeny T4 grown on a galU host will
plate on rgl but not rgl+ E. coli. Those T4
progeny that escaped the lit restriction plated
equally well on rgl’r and rgl cells (data not
shown).

The E. coli lit mutations prevented late gene
expression and the development of bacterio-
phage T4 at temperatures below 34°C, suggest-
ing that T4 needs the product of the host lit
gene for its late gene expression, at least at lower
temperatures. The plating efficiency of T4 on
host lit mutants increased abruptly from 34 to
37°C. This abrupt change occurred in all of our

J. BACTERIOL.

     

 

.. .. 7
by” m ..

fl ‘v‘ w a

”“3" m c.a

“a ’

gum-no «.4

A B C D

FIG. 5. T4 late protein synthesis on E. coli CTr5x
at 37 and 42°C. Shown are sodium dodecyl sulfate
polyacrylamide gel electrophoresis autoradiogramC
as in Fig. 3. (A) and (C) Wild-type T4; (B) and (D) T4
pseT2; (A) and (B) 37°C; (C) and (D) 42°C. Time of
labeling: (A) and (B), 38 to 41 min; (C) and (D), 28 to
31 min. Multiplicity of infection of 10 throughout.

lit mutants, even when the lit mutation had
been transduced into a different genetic back-
ground, and is probably a reflection of the func—
tion of the product of the lit gene.

The participation of the lit function in T4 late
gene expression presumably occurs at the level
of transcription and is the result of altering T4
DNA. Some of the evidence for DNA alteration
by the lit function comes from the results on the
effect of a T4 G01 mutation on T4 DNA reph'
cation, since it caused a reduction in the rate 0
T4 DNA replication and in the length of the
newly synthesized DNA even though early gene
expression was not affected. .

T4 Gol mutations have unusual CharacterIS-
tics. They map in the late region but can affect
T4 DNA replication early. They are also codom‘
inant with wild—type T4 both in their defect 1n
T4 DNA replication and in their ability to en-
hance T4 late gene expression on a Lit" hOSl-
Explanations for codominance usually 'mVOlie
either gene dosage effects or cis-acting proteinS
or sites in DNA. The gene dosage explanatIOn
for codominance assumes that the G01 functlon
is present in limiting amounts for the phenotYPe~

 

 

3CD

nlhesis on E. will
T4 late protein ”50111111111dO'lW’lil

lil mutationl
nt even when the
nsduced into a different Emilie:
and is proba ably a reflection o
h roduct of thel 1.1
aitii-ipation of thel litfliilclioninw

in t e 1e
though 9““

Mat
eednusllfll Ch
nsl‘lav V9 11 but c3313

1 mutatiionS
W11 late regionemml

4|;

VOL. 140, 1979

so that making one-half as much allows one- -half
as much late protein synthesis. The gene dosage
explanation seems unlikely, but the cis- acting
site (or protein) explanation is in some ways
more radical, since the site of the G01 mutation
must influence the expression of all the late
genes of T4 even though they are expressed from
manyp por omoters, most of which are to the 5’ side
of the Go] mutations or separated from them by
an early region which is transcribed in the re-
verse direction. Experiments can be designed
which should allow us to distinguish between
these two possible explanations for the codomi-
nance of T4 G01 mutations.

ACKNOWLEDGMENTS
rm. 1- rn‘cvrvLI L . .- -- -

and B. Bachman on E. coli genetics is gratefully acknowlv
edged. We thank Michele Fluck for a careful reading of the
manuscrip

ork was supported by National Science Foundation
grant PCM77- 24422 and by National Science Foundation pre»
doctoral fellowships to W. C. and K. S.

LITERATURE CITED

1. Adelberg, E. A., M. M ndel, andG. C. C. Chen. 1965.
Opt tirnal conditions for mutagenesis by N- met hyl- N'-
nitro N- n1trosoguan1d1ne 1nE coli K— 12 Biochem. Bio»
phys Res. Commun. 18: 788 -795.

2. Becker,A., andJ. Hurwitz. 1967. The enzymatic cleav-
age of phosphate termini from polynucleotides. J. Biol.
Chem. 242: 96—3 .

3 Cameron, V. an e.nbeck 1977. A 3 phos-
phatase activity in T4 polynucleotide kinase. Biochem-
istry 16: 5120-512

4' Chan, V L. K. Ebisuzaki.1970.Polynucleotide

09asle6 mutant of bacteriophage T4. Mo. Ge n. Gen

Chinmidurai, G., and D. J. McCorquodale, 1973. Re-

8.

...
O

...
U!

16.

.Frenke...,1CvD

. Novogro o.,dsky,A andJ .Hurw1

. Riva,

. Studier, F. W.1

E. COLI lit MUTANTS AND T4 TRANSCRIPTION 91

quirement of a phage-induced 5l exonuclease for the
expression of late genes of bacteriophage T5. Proc. Natl.
Acad. Sci. USA 70:3

.Curtiss. R; III, 9EG Carol D. P. Allison, and D. R.

Stallion Early stages of conjugation in Esche-
richia coli. J. 96Bacteriol. 100: 1091 1—10.
De eewp wR. E. an N. R. Cozzarelli. 1974. Genetics and
physiology of bacteriophage T4 3 phosphatase: evi-
dence for involvement of the enzyme in T4 DNAm
tabolism. J. Virol. 13:888—89
Epstein ., A.B 0,1e1 C. M. Steinberg, E. Kellen-
berger, E. Boy de la Tour, R. Cheval1ey, R. 85.
Edgar,M. Susm an..G H. Denhar dt, an.dA
sis. 1963. Physiological studies of conditional lethal
mutations of bacteriophage T4 dSpring Harbor
Symp. Quant BiolZ
C. C. 5Richardson. 1971 The de-
oyrbo nuclease induced after infection of E. coli by
bacteriophage T5. J. Biol Chem. 246: 4848—485.
t.z 1966. The enzymatic
phosphorylation of ribonucleic acid and deoxyribonu-
cleic acid. J. Bio. Chem. 241: 2923— 2932

. Richarfson, C. C. 1965 Phosphorylation of nucleic acid
by fe

nzyme from T4 bacterioplhage- 1ne cLed E. oli.
Proc.Nat1 Acad. Sci. USA. 54:
P.Geiduschek.'1970.

.. A. ci E. P. Geiduschek. 19
Uncou pling ofla te transcription from DNA replication
in bacteriophage T4 development. J. Mol. Biol. 54:1 103—

. Sirotkin,K., W. Cooley,J. Runnels, andL R. Snyder.

A role in Lru e-al te gene expression for the T4
bacteriophage 5' polynucleotide kinase 3 phosphatase
J. Mol Biol. 123: 221—

713. Analysis of bacteriophage T7 early
RNAs and proteins on slab gels. M..ol Biol. 79: 237—
Takahashi, H, A. Coppo, A. Manzi, G. M0 rtir
J. F. Pu lit 1.975 Design ofas system of conditional
lethal mutations (tab/k/com) affecting protein- protein
interactions in bacteriophage T4- infected E. coli
Mol. Biol. 96: 563—5

 

 

 

 

APPENDIX B

WILD-TYPE T4 GENOMES INTERFERE WITH THE EXPRESSION OF
§QL_MUTANT GENOMES IN MIXED INFECTIONS

In experiments such as those shown in Article I, figures 3 and
4, the rate of T4 late protein synthesis is less than one-half of normal,
even if one-half the infecting phage have the ggl_§§ mutation, suggest-
ing that wild-type T4 DNA can interfere with the expression of ggl
mutant DNA in the same cell. To investigate this further, we studied
the effect on late protein synthesis of varying the ratio of wild type
to ggl_mutant T4, keeping the total multiplicity conStant. We scanned
the autoradiograms and determined the area under the peaks due to seme
T4 late proteins. We then standardized the results by dividing by the
area under the same peak in the same experiment when wild-type T4
infected wild-type g, 9911, The latter is necessary because the density
of bands varies somewhat from experiment to experiment. The results of
the above calculation applied to three T4 late proteins are shown in
Figure Bl. The rate of synthesis of the T4 late proteins p23, p18, and
p37 expressed as a percentage of the normal rate is plotted against the
percentage of the infecting phage which were wild-type T4. If the rate
of synthesis of the T4 late protein is proportional to the percentage of
genomes which can synthesize that protein, then the results should fall
on the dashed straight line. The results vary
somewhat from experiment to experiment, but in all cases, the results of
the mixed infection of the llif host by a ggl_mutant and wild-type T4
deviate significantly from the line. In the experiment shown, the

97

98

the deviation was less than we normally observe and the insert shows
the spread in the highest and lowest values we have obtained from
different experiments. Most results fall somewhere in between. We
conclude that wild type genomes can "poison" the ability of ggl_mutant
genomes to express their late genes in a litf host. It is worth noting
that one of the late proteins shown, p37, is coded by a gene almost

l0 microns on DNA from the site of the gol mutation.

Fig.

lB.

99

Rate of synthesis of T4 late proteins in mixed infections of
wild-type T4 and a ggl_mutant on 11;: E, coli, Infections
were done at 300C with wild-type T4 and ggl_§§ in varying
ratios. Total multiplicity of infection was 24.

(A) The synthesis rates of various T4 late proteins are
expressed as a percentage of the normal protein synthesis
rate when wild-type T4 infects wild-type E, 9911,

E3-- gene l8 product, 0 - gene 23 product, .A- gene 37 product.

(8) *+ indicates range in values obtained for the gene 23

product in different experiments.

 

 

 

 

100

100

 

 

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