.3 NA" AGE H 7 EM". _ ., .‘ .‘wms? N . _ AA A tJfl M . :x... i312 E... éree WW 1 A ‘3" AAA 7 TM {A hr"; : the” 7_ ’6 .A. ‘i ERITY STATE \ N A: 't‘r;;: This is to certify that the thesis entitled III {CF/(6)9603 Befaoec'q' RNA T’Olymc’flrugq («Ll/\(t bN/\ bumfi~ TL! TXC‘Ci‘tf/‘PA/ILC. bC \JC'JOpnu mt presented by DONNA LORPJ‘L(HC [V164 {jpmea‘gr has been accepted towards fulfillment of the requirements for PL\' b degreein MICFO£710 [£514 (and. PmbllC/H 8.0.1 /f , 0/07“ f. 89705» Date 7A/75/ / 7 0—7639 ‘ “I BINDING‘ ‘ 800K BIND \3 BY BINDEF “ ‘fl-anv “V s I ABSTRACT INTERACTIONS BETWEEN RNA POLYMERASE AND DNA DURING T4 BACTERIOPHAGE DEVELOPMENT BY Donna Lorraine Montgomery An RNA polymerase mutation (rifR-Z) of Escherichia coli poorly supports the growth of T4 because T4 is unable to utilize the mutated RNA polymerase for at least one of its esSential functions. Mutants of T4 have been isolated which grow better than wild-type T4 on the RifR-Z. These are missing the function B-glucosyl trans- ferase and can grow better because the incoming parental DNA is partially unglucosylated. Only the 8-, not the o-, glucosyl transferase is involved, suggesting a special role for at least some of the hydroxy-methyl cytosines normally glucosylated with a B linkage. This phenomenon is independent of restriction. Only the state of glucosyla- tion of the parental, not the progeny, DNA matters for this phenomenon. RifR-Z has been shown to have a direct effect on both DNA synthesis and late gene expression. The effects of rifR-Z are partially suppressed by B-glucosyl Donna Lorraine Montgomery transferaseless mutants (Bgt-) of T4, and B-glucosyla- tion is inhibitory both early and late in infection. RifR-Z also has an effect on early gene expression, causing disproportionate synthesis of some early T4 pro- teins. However, B-glucosylation has no effect on this early gene expression defect, although we have shown that B-glucosylation inhibits an early function required for phage production. RifR-Z also causes defective host nucleoid unfolding and a delay in host DNA degradation. B-glucosylation is inhibitory for host DNA degradation, suggesting that the transcription of B-glucosylated hydroxymethyl cytosines in T4 DNA is somehow involved in this step. These data further support the model pro- posed earlier that RNA polymerase must alter the structure or association of T4 parental DNA early in infection for DNA replication and late transcription to occur. INTERACTIONS BETWEEN RNA POLYMERASE AND DNA DURING T4 BACTERIOPHAGE DEVELOPMENT BY Donna Lorraine Montgomery 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 1975 ACKNOWLEDGMENTS I am extremely grateful to my major advisor, Dr. Loren R. Snyder, for his interest, encouragement, and support throughout my research. I will always be indebted to him for the knowledge I was able to adsorb from our numerous, invaluable discussions. I would also like to thank my committee members-- Dr. John A. Boezi, Dr. Robert R. Brubaker, and Dr. Leland F. Velicer--for their helpful suggestions, with a special thank you to Dr. Velicer for allowing me to use much of his equipment. And I would like to thank Ms. Sue Rose for preparing the finished graphs for the second article. I was financially supported throughout my graduate studies from a National Science Foundation Traineeship awarded to the Department of Microbiology and Public Health, as well as from a departmental assistantship. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES; INTRODUCTION . LITERATURE REVIEW. Host Macromolecular Synthesis After T4 Infection . E. coli Nucleoids Host Nuclear Disruption, Unfolding, and Degradation After T4 Infection . Modification of Host RNA Polymerase After T4 Infection . . . Use of Host RNA Polymerase in DNA ° Synthesis. T4 DNA After Infection. Structure of T4 DNA . RifR-Z Mutation in E. REFERENCES . coli. T4 Mutants That Grow on RifR- 12 13 14 l6 17 19 ARTICLE I - A Negative Effect of B-Glucosylation on T4 Growth in Certain RNA Polymerase Mutants of Escherichia coli: Genetic Evidence Impli- cating Pyrimidine-Rich Sequences of DNA in Transcription . . . . ARTICLE II - Bacteriophage T4 Growth on an RNA Polymerase Mutant of Escherichia coZi: Effect of B-Glucosylation on Early T4 Functions. APPENDIX - Inhibition of T4 Growth by an RNA Polymerase Mutation of Escherichia coli: Physiological and Genetic Analysis of the Effects During Phage Development. . . iii LIST OF TABLES Table Page ARTICLE I 1 Map position of gor mutants . . . . . . . . 31 2 gor ARE egt'. . . . . . . . . . . . . . . . 32 APPENDIX 1 Map position of gor-Z type mutations. . . . 91 iv LIST OF FIGURES Figure ARTICLE I 1 Growth of a T4 mutant on RifR-Z 2 Wild-type dominance and inability of gar mutants to complement . . . . . . . . 3 Identity of gar and Bgt' 4 Growth of the double mutant agt , Bgt' compared to Bgt on RifR -2. . . . . . S The phenotype gar+ is subject to pheno- typic mixing. . . . . . . . . . . . . 6 Unglucosylated wild- type T4 (T4*) grows poorly on RifR -2. . . ARTICLE II 1 The rate of amino acid incorporation after T4+ infection . 2 Proteins synthesized from 4 to 12 minutes after infection of NF58 with T4 amber and deletion mutants. . . . . . 3 Proteins synthesized from 4 to 12 minutes after infection of NF58 and NF58-RifR-2 with T4 am N122 (gene 42) . . . . . 4 Protein synthesis from 2 to 5 minutes after infection of NF58 and NF58- RifR -2 with T4 am N122 . . . . . . . . 5 Protein synthesis after infection of NF58- RifR- 2 with T4+ and T4Bgt' . 6 There are greater differences in the relative rates of synthesis of various proteins in T4* and Bgt' infections of NF58-RifR-2 at later times. V Page 30 30 31 32 33 34 48 51 55 57 61 64 Figure Page 7 Alkaline sucrose density gradient analysis of E. coZi DNA from uninfected NF58 and from T4 am N122 infected NF58 and NF58-RifR-2 . . . . . . . . . . . . . . 66 8 Alkaline sucrose density gradient analysis of E. coli DNA from NF58-RifR-2 at 12 minutes after infection . . . . . . . . . . 69 9 Host nucleoid unfolding after infection of NF58 and NF58-RifR-Z with T4 am N122 . . 71 10 Host nucleoid unfolding in RifR-Z in the absence of protein synthesis. . . . . . . . 74 APPENDIX 1 The rate of T4 DNA replication after infection of K803 and K803-rifR2. . . . . . 91 2 Same as Figure 1 except that the cells were infected at 40° and diluted 1:10 into medium at 27° at the time indicated by the arrow. . . . . . . . . . . . . . . . 92 3 All of the gene products required for T4 DNA synthesis accumulate in RifRZ at the nonpermissive temperature . . . . . . . 93 4 Effect on DNA replication can be bypassed in the absence of replication . . . . . . . 93 5 Phage production after a shift from 40°- 27° at 10 min after infection . . . . . . . 94 6 RifRZ is blocked in late gene expression. . 94 7 SDS-polyacrylamide slab gel electrophore- sis of T4 proteins synthesized after a shift from 40° to 27° at 10 min . . . . . . 95 8‘ Inhibition of phage production by paren- tal B-glucosylation can be bypassed early in infection at the permissive temperature. 96 9 Inhibition by parental B-glucosylation can be bypassed in the absence of repli- cation. . . . . . . . . . . . . . . . . . . 97 vi INTRODUCTION Dr. Snyder's laboratory is studying the roles of host RNA polymerase in T4 development, with emphasis on the interactions between T4-coded proteins and RNA polymerase. The approach taken has been to use RNA polymerase mutations (rifR-Z) of E. coli which inhibit T4 growth. Characterization of RifR-Z infections has shown that RNA polymerase is directly required for T4 DNA synthesis, as well as for viral transcription. The first article in this dissertation is a pub- lished manuscript describing the isolation, mapping, and partial characterization of T4 mutants which grow better than wild type T4 (T4+) on RifR-Z. These mutants all mapped in the gene for B-glucosyl transferase, the enzyme which glucosylates about 30% of the hydroxymethyl cyto- sines in T4 DNA, with a B-linkage. It is the ungluco- sylated state of the T4 DNA, and not the malfunctioning enzyme, which allows B-glucosyl transferaseless mutants to grow better than T4+ on RifR-Z. The second article in this dissertation is a manu- script that will be submitted for publication. It describes further effects of the rifR-Z mutation on T4 1 2 development, and compares these effects in T4+ and Bgt- infections. Besides affecting late T4 gene expression and T4 DNA synthesis, the rifR-Z mutation also causes a delay in host nucleoid disruption and degradation, and affects early T4 gene expression. The Appendix contains a published manuscript (Snyder and Montgomery, 1974) which describes some effects of rifR=2 on T4development, and further describes effects of B-glucosylation. This publication was included because the experiments described in the second article rely heavily upon the data reported therein. LITERATURE REVIEW Host Macromolecular Synthesis After T4 Infection The bacteriophage T4 causes a cessation of all E. coli macromolecular synthesis within a few minutes after infection (Monad and Wollman, 1947; Volkin and Astrachen, 1956; Nomura et aZ., 1960; Hosoda and Levinthal, 1968). T4 could accomplish this by one, or any combination, of the following mechanisms. One or more immediate early T4 proteins could be required for shutoff, or the adsorp- tion and binding of T4 to the host cell wall could cause conformational changes in the membrane which lead to shutoff. Other possibilities are that the T4 protein(s) required for shutoff of macromolecular synthesis is injected with the DNA from parental T4, or that T4 DNA competes with E. coZi DNA for some critical site in the host. In fact, two mechanisms for shutoff of both DNA and RNA synthesis have been prOposed. The first mechanism requires T4 protein synthesis, since in the presence of various inhibitors (i.e., chloramphenicol, rifampin, and histidine starvation) host DNA and RNA synthesis continue 4 (Duckworth, 1971; Hayward and Green, 1965; Nomura et aZ., 1962; Nomura et aZ., 1966; Terzi, 1967). However, shut- off does occur in the presence of inhibitors at high multiplicities of infection (Nomura et aZ., 1966), and in E. coli infected with T4 phage "ghosts" (T4 protein coats without their DNA) (Duckworth, 1970). Therefore, another mode of shutoff, not requiring T4 protein synthe- sis, must also be functioning in E. coZi. Terzi concluded that only this second mode of shutoff functions in T4 infections of ShigeZZa, since the shutoff of host macro- molecular synthesis is incomplete and is dependent upon the multiplicity of infection (Terzi, 1967). It has been suggested that this second mode is due to conformational changes occurring in the host membrane when T4 adsorb to E. coli (Nomura et aZ., 1966; Duckworth, 1971). This type of shutoff is seen only in "ghost" infections or at high multiplicities of infection in the absence of T4 protein synthesis, because in normal T4 infections the damage to the host membrane is repaired by a T4-directed function(s) (Duckworth, 1971). Probably the mode of host DNA synthesis shutoff which does not require protein synthesis plays a minor role in normal T4 infections of E. coZi. However, it does show that a membrane change occurs when T4 adsorb to the cell wall, and it signifies that this change alone is capable of altering internal functions of the cell. This is 5 reminiscent of the action of colicins on E. coZi. Colicin E2 does not enter the cell, yet is capable of killing E. coli by solubilizing its DNA, even in the presence of chloramphenicol (Swift and Wiberg, 1971). Therefore, it must cause existing host enzymes to degrade the DNA by altering the membrane, and perhaps acts by releasing the host DNA from its membrane-bound site, making it more available to nuclease attack. E. coli Nucleoids Besides causing host macromolecular synthesis to cease, T4 infection causes host nuclear disruption, unfold- ing, and subsequent degradation of the DNA to nucleotides. A review of the properties of the bacterial nuclear structure is presented here so that the significance of the T4 disruption and unfolding can be better appreciated. Although the DNA of E. coli is over 1 mm in length (Cairns, 1963), it is packaged in nuclear bodies about 1 um in diameter. Cytological studies have shown these bodies to be multilobed and located in the center of the cell (see, for example, Fuhs, 1965; Kellenberger et aZ., 1958). When cells are gently lysed, DNA can be isolated from E. coli as highly folded structures, referred to as nucleoids, which correlate in size to the nuclear bodies seen cytologically (Stonington and Pettijohn, 1971). In addition to DNA, the nucloids contain nascent RNA chains 6 (about 30% by weight) and protein (about 10% by weight) (Pettijohn et aZ., 1973), with over 90% of the protein being core RNA polymerase (Worcel et aZ., 1973). At least some of the RNA found in the nucleoid is required‘Uahold DNA in the highly folded structure, as shown by the nucleoid unfolding when treated in vitro with RNase (Stonington and Pettijohn, 1971; Worcel and Burgi, 1972). In vivo treatment of cells with rifampin, a drug which inhibits RNA polymerase activity by binding to the B-subunit (Zillig et aZ., 1970), yields only unfolded DNA after lysis (Pettijohn and Hecht, 1973; Dworsky and Schaechter, 1973), suggesting that RNA polymerase must be continually functioning to keep DNA in the highly folded structure. Worce and Burgi (1972) and Pettijohn and Hecht (1973) have proposed a model for nucleoid structure. They suggest that the DNA is folded into 12-80 loops per chromo- some, with RNA molecules defining the positions of these folds by their binding to DNA. The bound RNA molecules also prevent rotation between loops, thus separating the DNA into domains of supercoiling. Nucleoids have been shown to be bound to the membrane of E. coli (Dworsky and Schaechter, 1973; Worcel and Burgi, 1974; Delius and Worcel, 1973). This membrane attachment seems to be required for synthesis since replicating DNA is isolated from E. coli as membrane-bound 7 nucleoids whereas nonreplicating DNA is isolated only as membrane-free nucleoids (Worcel and Burgi, 1974). Dworsky and Schaechter (1973) have shown that RNA polymerase is involved in the DNA attachment to the membrane, by decreas- ing the number of attachment sites (by a factor of 4) with rifampin treatment of the cells. They suggest that the role RNA polymerase plays in stabilizing the folded structure of the nucleoid may be related to the role it plays in binding DNA to the membrane; that the RNA "core" may be associated with the membrane, but can be released without damage to the structure of the nucleoid (Dworsky and Schaechter, 1973). Host Nuclear Disruption, Unfolding, andDegradation After T4 Inféction The E. coli genome is disrupted within the first two or three minutes after infection by T4, and accumulates in clumps along the cell membrane (Kellenberger, 1960; Kellenberger et aZ., 1959; Luria and Human, 1950; Murray et aZ., 1950; Snustad et aZ., 1972). Disruption requires the product of T4 gene D2b, because mutants in that gene no longer cause disruption (Snustad and Conroy, 1974). Nuclear disruption has been found to be nonessential for T4 growth, and is not required for host DNA degradation (Snustad et aZ., 1974). Tutas et a1. (1974) have shown that T4 rapidly convert the folded bacterial genome to a less compact structure 8 within the first five minutes after infection, and that this process, called nucleoid unfolding, may require an early, or immediate early, T4 protein(s). Nucleoid unfolding is independent of nuclear disruption since mutants defective in nuclear disruption do not prevent unfolding (Snustad et aZ., 1974). However, no mutants of T4 have been found which affect nucleoid unfolding, so the required T4 protein(s) has not yet been identified (Snustad et aZ” 1974; Tutas et aZ., 1974). Recently, we have found that the rifR-Z mutation of E. coli causes a delay in nucleoid unfolding (see body of dissertation), thus being the first genetic system which affects bac- terial genome unfolding. After dirsupting and unfolding the nucleoid, T4 causes subsequent degradation of the host DNA to nucleo- tides, which can be reincorporated into T4 progeny DNA (Hershey et aZ., 1953; Koch et aZ., 1952; Kozloff, 1953; Kozloff and Putnam, 1950; Weed and Cohen, 1951). Degra- dation seems to occur in two stages. First the host DNA undergoes limited cleavage to fragments with a minimum molecular weight of about 106 , then these fragments are further degraded to acid soluble pieces of DNA (Kutter and Wiberg, 1968). Bose and Warren (1969) proposed that this two-step process requires at least one exonuclease and two endonucleases that are highly specific and probably coded by T4. T4 endonuclease II is required for 9 the second stage, since mutants in that gene degrade the host DNA to fragments no smaller than 106 (Hercules et aZ., 1971; Warner et aZ., 1970). It has been proposed that T4 endonuclease IV is also involved in the second stage of degradation because it degrades DNA to fragments of 150 nucleotides, cleaves adjacent to cytosine residues, and has no effect on denatured T4 DNA, either glucosylated or nonglucosylated (Sadowski and Hurwitz, 1969). Also, some rII deletions which do not make endonuclease IV are defective in host DNA breakdown (Bruner et aZ., 1973). No T4 mutants have been found which affect the pro- posed first step of degradation, so the required T4 protein(s) is not yet known. It seems likely that nuclear unfolding may coincide with the first step in host DNA breakdown by T4, since unfolding also requires an early or pre-early T4 enzyme (Tutas et aZ., 1974). However, there is no proof of this relationship at the moment. Modification of Host RNA Polymerase After T4 Infection Besides causing injury to the cell, T4 modifies much of the cell's macromolecular machinery so that it func- tions specifically for T4 development. Only the modifica- tions of the host RNA polymerase will be reviewed, since the main emphasis of my research has been to study the roles of host RNA polymerase in T4 development. 10 Although T4 codes for its own DNA polymerase, it uses the host RNA polymerase for all of its transcription (Haselkorn et aZ., 1969; di Mauro et aZ., 1969). Numerous modifications have been found to occur to the host RNA polymerase during the course of T4 development, some of which are essential for transcriptional control. Transcrip— tion of T4 genes is a complex process which has been classified into three categories: immediate early (IE), delayed early (DE), and late (Bolle et aZ., 1968a; Grasso and Buchanan, 1969; Milanesi et aZ., 1969; Salser et aZ., 1970). Each one of these steps requires some mechanism of control, and some involve known modifications of RNA polymerase. No modification of the polymerase by T4 proteins seems to be required for immediate early transcription, since IE genes can be transcribed in the presence of chloram- phenicol (Salser et aZ., 1970). There is evidence that the polymerase uses E. coli sigma (0) factor for transcrib- ing IE genes, because it does not lose the 0 factor until two or three minutes afterinfection (Bautz et aZ., 1969). It has been proposed that loss of the factor may explain the shutoff of some early genes at two to three minutes after infection (Bautz et aZ., 1969). Delayed early transcription has been defined as that occurring between 1.5 and 5 minutes after infection. This requires a T4 protein(s) because delayed early genes 11 cannot be transcribed in the presence of chloramphenicol (Salser et aZ., 1970). The shutoff, as well as the initation, of T4 early messenger RNA occurs in different stages. Some early mRNA are produced throughout infection, while others are turned off at the initiation of DNA synthesis (Hosoda and Levinthal, 1968; Salser et aZ., 1970) or when host RNA synthesis is shut off (Hosoda and Levinthal, 1968; Matsukage and Minegawa, 1967; Nomura et aZ., 1966; Salser et aZ., 1970; Terzi, 1967). With all the transcriptional switches required for early gene expression, only one mutant of T4 has been found to have an effect on early transcription, that effect being to alter the timing of early mRNA synthesis (Mattson et aZ., 1974). Late transcription requires continuous DNA synthesis (Bolle et aZ., 1968b) and at least three viral coded gene products (Bolle et aZ., 1968b; Notani, 1973; Wu, 1973). The RNA polymerase undergoes additional modifications by the time late transcription begins. Stevens (1972) has shown that four polypeptides are bound to RNA polymerase by five minutes after infection. She has identified two of these polypeptides as the products of genes 33 and 55, both of which are required for late transcription (Bolle et aZ., 1968b; Notani et aZ., 1970). The other two poly- peptides (mol. wts. 14,000 and 10,000) have not yet been identified with a gene, so their function is unknown. 12 Recently, there has been evidence, both biochemical (Ratner, 1974) and genetic (Coppo et aZ., 1974; Snyder and Montgomery, 1974), that the gene 45 product also interacts with RNA polymerase. Since the 45 product is required for late gene expression (Wu, 1973), it is probably involved in modifying the polymerase for late transcription. Use of Host RNA Polymerase in DNA Synthesis Recently there has been much evidence supporting the idea that RNA polymerase is required directly for DNA synthesis in a number of systems, including T4 (Brutlag et aZ., 1971; Buckley et aZ., 1972; Lark, 1972; Sugino et aZ., 1972; Sugino and Okazaki, 1973). Buckley et al. (1972) have shown the existence of a transitory RNA:DNA copolymer early in T4 infection, and Speyer et al. (1972) have found low levels of RNA covalently bound to DNA in viral particles, thus implicating RNA polymerase in T4 DNA synthesis. Recent genetic evidence from our labora- tory has shown that T4 DNA synthesis requires RNA polymerase for some function(s) other than gene expression (Snyder and Montgomery,1974). It is of interest in this regard that the gene 45 product which probably binds to RNA polymerase (see above) is required for T4 DNA replication as well as late messenger RNA synthesis (Epstein et aZ., 1963). 13 These additional functions of RNA polymerase in T4 development may also require T4 coded control proteins, either to modify the RNA polymerase or the DNA template. The number and complexity of the roles that RNA polymerase play in phage production suggest that there are many T4 functions required for control. Since only a few T4 gene products are known to be required for control of RNA and DNA synthesis, there are probably others which have not yet been identified. T4 DNA After Infection RNA polymerase must interact with a DNA molecule for all of its known functions. Therefore, the structure of the DNA, and its modification during T4 development, may play an important role in regulation of transcription or DNA replication. It is known that T4 DNA binds to the host membrane early in infection (Altman and Lerman, 1970; Earhart et aZ., 1968), and that binding does not require protein or DNA synthesis, but does require RNA synthesis (Earhart et aZ., 1973). Membrane attachment seems to be required for DNA synthesis (Altman and Lerman, 1972; Earhart et al., 1968; Kozinski and Lin, 1965; Miller and Kozinski, 1970), and is terminated late in infection when phage head formation releases progeny DNA from the membrane (Siegel and Schaechter, 1973). Thus head encapsulation of T4 DNA acts as one control over DNA synthesis. Besides binding to cell membrane, DNA also begins to be 14 associated with viral-coded proteins by four to five minutes after infection (Miller and Kozinski, 1970). Structure of T4 DNA The structure of parental T4 DNA differs from E. 001i DNA even before T4-directed modification occurs. All T-even bacteriophage contain hydroxymethyl cytosine in place of cytosine in their DNA. These hydroxymethyl cytosine residues are glucosylated, with glucosylation of T2, T4, and T6 being characteristically divided among a-glucose, B-glucose, and a-gentiobiose groups (Kuno and Lehman, 1962; Lehman and Pratt, 1960). The hydroxymethyl cytosines of T4 DNA are all singly glucosylated, with about 70% containing the a-linkage and 30% the B-linkage (Lehman and Pratt, 1960). Glucosylation functions by protecting parental T4 DNA from host restriction, but seems to have no effect on DNA replication, since unglu- cosylated T4 (T4*) can grow normally in ShigeZZa and restriction- mutants of E. coli (Hattman, 1964; Hattman and Fukasawa, 1963; Luria and Human, 1952). The distribution of a- and B-glucosyl groups reflects the specificity of the a-glucosyl transferase, which is influenced by the nature of neighboring nucleotides (Lunt and Newton, 1965). It has been shown in all T-even phage that a-glucosyl transferase is unable to glucosylate those hydroxymethyl cytosines which are attached to another hydroxymethyl cytosine through their SA-carbons, 15 and is limited in its ability to glucosylate those hydroxymethyl cytosines which have a purine nucleotide on either side of it (Pu-H-Pu) (Burton et aZ., 1963; de Waard et aZ., 1967; Lunt and Newton, 1965). In T2 and T6, all of the hydroxymethyl cytosines not a-glucosylated are unglucosylated, while in T4 they are all B-glucosylated. Since a-glucosyl transferases from all T-even phage are similarly restricted in their gluco- sylation ability, it is possible that those left ungluco- sylated in T2 and T6, and B-glucosylated in T4, have a special function in phage development. Pyrimidine clusters have been found in many organisms, both procaryotic and eucaryotic, suggesting by their ubiquity that they may play a specific role in DNA or RNA synthesis (for example, see Burton et aZ., 1963; Champoux and Hogness, 1972; Szybalski et aZ., 1966). Szybalski et al. (1966) showed that pyrimidine clusters are only found on the transcribing strand of DNA from a variety of organisms, and Champoux and Hogness (1972) showed these types of clusters to be grouped on the late third of A DNA. Because they are not randomly distributed throughout the DNA molecule, and are found on transcribing strands, it is possible that they are involved in DNA synthesis- directed late messenger RNA transcription. However, no known function has yet been assigned to pyrimidine clusters. 16 RifR-Z Mutation in E. can Although much is known about the sequential steps in. T4 development, details of the molecular mechanisms involved are still few. Our laboratory is studying the roles of host RNA polymerase in T4 development on a molecular basis. Our approach to this problem has been through the host's contribution, by using RNA polymerase mutations of .E. coli which inhibit T4 growth. The mutation we have most fully characterized is referred to as rifR-Z because it was obtained as a mutant resistant to rifampicin (Snyder, 1972). Strains harboring the rifR-Z mutation grow normally, so the mutated RNA polymerase is only defective in T4 development, probably because it is unable to interact correctly with either a T4 coded protein(s) or with T4 DNA. Although the rifR-Z mutation is not temperature dependent (i.e., the strain is resistant to rifampicin at all temperatures), the effects it causes on T4 produc- tion are all cold sensitive (Snyder and Montgomery, 1974). At 40°, T4 produce normally-sized, clear plaques, while at 27°, the plaques are barely visible. The rifR-Z muta- tion causes incomplete shutoff of host transcription (Snyder, 1972), a delay and reduction in rate of T4 DNA synthesis, and defective late gene expression (Snyder and Montgomery, 1974), all of which are also cold sensitive. Therefore, RNA polymerase is required for either gene l7 expression or some other function in all of these proces- ses. We have shown that RNA polymerase is required for a function other than gene expression in DNA synthesis. This was done by using temperature-shift experiments to show that all the proteins required for replication accumu- late at the nonpermissive temperature (Snyder and Montgomery, 1974). T4 Mutants That Grow on RifR-Z We have also isolated and mapped mutants of T4 which grow better than wild type T4 (T4+) on RifR-Z. These mutations are referred to as gor, for grows on RifR-Z. The first gor mutations all mapped in the gene for B- glucosyl transferase (Montgomery and Snyder, 1973). B- glucosyl transferaseless mutants of T4 (Bgt') partially overcome the rate reduction in DNA synthesis (but not the delay), and the block in late gene expression. We have shown that these effects are not due to the B-glucosyl transferase itself, but are caused by the unglucosylated state of the T4 DNA. This implies that at least some of the hydroxymethyl cytosine residues which are normally B-glucosylated may be important sites of interaction with RNA polymerase for both DNA synthesis and late gene expres- sion (Snyder and Montgomery, 1974). Two other gor-type mutations have been found and mapped, but they have not yet been further characterized. The second gor-type mutations found map in a new gene l8 (gor-Z) located between genes 55 and agt (Snyder and Montgomery, 1974). The product of this new gene may be a nonessential protein that interacts with host RNA polymerase during infection and becomes inhibitory on RifR-Z. However, more experiments are needed to prove this point. We found that known amber mutants in gene 45 show the gor phenotype when grown on suz+ strains of E. coli harboring the rifR-Z mutation, probably because the reduced amount of 45 product produced in a suppressed amber restores some essential balance (Snyder and Montgomery, 1974). As mentioned above, this adds further genetic evidence that the gene 45 product interacts with RNA polymerase during T4 deve10pment. REFERENCES REFERENCES ALTMAN, S., and LERMAN, L. S. (1970). Kinetics and intermediates in the intracellular synthesis of bacteriophage T4 deoxyribonucleic acid. J. Mol. Biol. 39, 235-261. BAUTZ, E. K. F., BAUTZ, F. A., and DUNN, J. A. (1969). E. coli factor: A positive control element in phage T4 development. Nature 223, 1022-1024. BOLLE, A., EPSTEIN, R. H., SALSER, W., and GEIDUSCHEK, E. P. (1968a). Transcription during bacteriophage T4 development: Synthesis and relative stability of early and late RNA. J. Mol. Biol. 33, 325-348. BOLLE, A., EPSTEIN, R. H., SALSER, W., and GEIDUSCHEK, E. P. (1968b). Transcription during bacteriophage T4 development: Requirements for late messenger synthesis. J. Mol. Biol. 33, 339-362. BOSE, S. K., and WARREN, R. J. (1969). Bacteriophage- induced inhibition of host functions. II. 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Biol. 33, 43-51. 27 WU, R., GEIDUSCHEK, E. P., RABUSSAY, D., and CASCINO, A. (1973). Regulation of transcription in bacteriophage T4-infected E. coli: A brief review and some recent results. UCLA Symp. Virus Research, 181-204. ZILLIG, W., ZECHEL, K., RABUSSAY, D., SCHACHNER, M., SETTI, V. S., PALM, P., HEIL, A., and SEIFERT, W. (1970). On the role of different subunits of DNA- dependent RNA polymerase.from E. coli in the transcrip- tion process. Cold Spring Harbor Symp. Quant. Biol. 33, 47-58. ARTICLE I A Negative Effect of B-Glucosylation on T4 Growth in Certain RNA Polymerase Mutants of Escherichia coli: Genetic Evidence Implicating Pyrimidine- Rich Sequences of DNA in Transcription BY Donna L. Montgomery and Loren R. Snyder Virology 33, 349-358 (1973). Reprinted from VIROLOGY, Volume 53, No. 2, June 1973 (_.‘('l’)'nizht 57.3 1973 by Academic Press, Inc. Printed in ('.S.A. \‘lRHlADGY 63, 349 358 (1973) A Negative Effect of B-Glucosylation on T4 Growth in Certain RNA Polymerase Mutants of Escherichia coli: Genetic Evidence Implicating Pyrimidine-Rich Sequences of DNA . . . 1 In Transcription DONNA L. MONTGOMERY ANI) LORRN R. SNYDER Department of Microbiology and Public Health, .llichiguu State ('m'rcrsily, East Lansing, Michigan 48823 Accepletl February 21, 1.973 In a previous publication, an RNA polymerase mutant of Eschericlu‘o coli was described which poorly supports the growth of the bacteriophage T4. .\lut ants of T4 have been isolated which grow better than wild-type T4 on the RNA polymerase mutant. These are missing the function B-glucosyl transferase and can grow better because the incoming parental DNA is partially unglucosylated. Only the B-, not the a-, glucosyl transferase is involved, suggesting a special role for at least some of the hydroxymethyl cytosines normally glucosylated with a B linkage. This phenomenon is independent of restriction. Only the state of glucosylation of the. parental, not of the progeny, DNA matters for this phenomenon, suggesting that part, of the. parental DNA may play a special transcriptional role throughout phage infection. An argument is presented that these experiments are consistent with the idea that pyrimidine-rich sequences are in ciro recognition sites for RNA polymerase. INTROI.)U(‘ITION T4 promotes many transcriptional switches during its development. Host, RN A synthesis is shut off very early in infection as well as the synthesis of some of the earliest appearing T4 RNAs (Terzi, 1967; Nomura cl 01., 1966; Matsukage and Minagawa. 1067). Some later appearing RNAs are not. made in the presence of chloraimmcnicol (Brody et al., 1970; Grasso and Buchanan, 1969) which may indicate a separate regula— tory mechanism. Also, the synthesis of some of the later appearing RNAs continues throughout infection while the synthesis of some ceases at about the time of onset. of the, latest appearing messenger RNAs which are in turn made only after T4 DNA replication (Bolle et al., 1968). The program of T4 pro- 1This is Journal Article No. (3205 from the Michigan Agricultural l‘lxperiment Station. li-lll (‘opyright Q 1973 by Academic Press, inc. All rights of reproduction in any form reserved. t‘ciu synthesis substantially coincides with the program of RNA synthesis (Hosoda and Levinthal, 1968). Despite this spate of transcriptional switches, there are only two genes of T4 whose products are known to be involved in transcriptional regulation. These are genes 33 and :35, whose products are par- tially and completely required, respectively, for the synthesis of T4 late messenger RNA (Bolle et al., 1968). There are a number of possible explanations for the dearth of known regulatory genes. One is that they have remained undiscovered or, if they have been discovered, their role in regulation has remained unnoticed. Another possible ex- planation is that they are relatively nou- essential and thus were not included among the original conditiimal-lethal assortment. (Epstein cl (1]., 1963). Different approacl'ics 3.30 may lead to the discovery of heretofore un- (‘liscovcrcd regulatory genes. RNA polymerase mutants of the host offer another approach to this problem. The rationale is as follows. T4 uses the host RNA polymerase for all of its transcription (Hasel- korn cl (1]., 1969; (ii Mauro el al., 1969). Presumably, at least some of the transcrip- tional switches which occur during T4 dc— Velopment are due to the interaction between T4-codcd factors and host. RNA polymerase. These T4-coded factors could either bind to the polymerase or alter it. by some enzymatic mechanism. While mutant RNA polym- erases must of necessity function for the growth and replication of the cell, it is pos- sible that some may not properly interact with the responsible virus-coded factors, leading to a defective transcriptional switch and possibly defective growth of the virus. Conversely, T4 mutants may exist which overcome the effect of the mutant RNA polymerase and these may be in genes in- Volvcd in regulation. With this rationale in mind, RNA polym- erase mutants of E. coli were isolated and T4 growth on them was studied. The pheno- type of T4 growth on one such mutant has been discussed (Snyder, 1972). The RNA polymerase mutation leads to a significant delay in the production of T4 viral particles. T4 mutants do exist which grow better than wild-type T4 on the RNA polymerase mutant. Thcj are the subject of this pub- lication. .\I.-\TI'IR 1 ALS AND .\1 1‘7““ )1 )h‘ I’lnn/c strains. T4 R4 (dgt‘)'~’ and R20... (agt“)'~’ and the double mutant HA 57 (agt ", gr) were from the collection of H. Revel (('ieorgopoulous and Revel, 1071), and were obtained from J. Wiberg. The amber mu- tants used in the mapping were N122 (gene 42), N81 (gene 41), and 1'31140 (gene 62) from \V. \Vood. l’lkc was obtained from I). I’reidi‘nan. 2dgt": i‘i-glucosyl transferaseless; ”gt: (1'- glucosyl transferaseless .\l( )N'l‘( :( ).\1 1511 Y ANI) SNYDER Bacterial: The following strains were used as recipients of the Rif“—2 mutation because of the underlined property. (tun)(xc1s57): NFSSI K803 : am su+ Tl.“ am su— arg‘ met- rti—.r‘2.4— (permissive for unglucosylated T4) r‘m‘, met‘, gal‘,i(_-\Vood: \V. R., 1966) The strains N '“58-RifR-2 and 1(803-RifR-2 were constructed by transduction from (77(300A357-RifR-2. Alt't+ recombinants were selected, and in each case about ‘20 92 were also rifampicin resistant. One of each of these was chosen for the experiments. The 1(803 transduction was performed at a low multiplicity to avoid Pl lysogens which are restrictive for unglucosylated T4 (Revel and (ieorgopoulos, 1969). The UDPG ppase‘ mutant used was “(4597, obtained from J. Wiberg. Growth of virus was measured at 30° in Mtis medium: 5.54 g Nang’Ot (anhyd.), 3 g l\'H-_»l’0t (anliyd.), 5 g NaCl, 1 g NH4Cl, 4 g glucose, 10*3 mole MgS04, and 10 g casamino acids (Difco) per liter. The multiplicity of infection was determined from a careful titer of the virus and the turbidity of the bacterial suspension. The bacteria were at a concen- tration of 4 X 108 per ml for all experiments. For low multiplicity experiments (~O.1') it. was assumed that every virus infected a cell. ('s(“l-purilied T4 were used for the high multiplicity experiments (>3). Intracellular phage production was measured by disrupt- ing the. cells with CHCl3 at the time, indi— cated and plating with an exponentially growing bacterial indicator and a soft agar overlay. (lent-tic crosses were performed by absorb- ing the Virtlses to be crossed to the. permis- sive bacteria for .3 min at room tenmerature at an m.o.i. of .‘l of *ach and then diluted 1: 100 into tryptone broth (10 g tryptone, .3 g Na(‘l per liter) at 300 and shaken for 4.") min before (.‘ll(_‘l;, was added. GROWTH OF T4 figt“ 1N Hit“ E. coli It l‘ISU LTS Isolation of 71.4 .‘Uutants thic/l Gl‘on.‘ Better Than W z'td-T ype T4 on EUR-2 T4 mutants which make distinct plaques on RifR-‘Z arise spontaneously with a fre- quency of about 1 in 10‘. Three such mutants which had arisen independently were iso— lated by growing three separate lysates on RifR-Z starting from wild-type plaques and picking gar-type. plaques after plating on RifR-Z. The growth of all three mutants is enhanced on RifR-‘Z to about the same extent and is relatively independent of the mul- tiplicity of infection and the strain of E. coli harboring the. RifR-Z mutation. The growth of one mutant on Rif“-2 is compared to wild-type T4 growth on Rita—2 and on wild- type E. coli in Fig. 1. The mutant grows ISO” I00 "' 4mm cell FIG. 1. Growth of a. T4 mutant on lilfli-22'1‘4+, (n00 (Elm—D ); T4+, CHOU-RifR-‘Z (o 0); T4 gor—l, (.ViOO—llifR-Q (O! - O ); each at an m.o_i. ()f 5. 351 40” E u U (1) 13 Q) E L 20” (1) O. (D 0’ (U C (l O 1 - g 0 ‘IO 30 50 Time after Infection (min ) FIG. 2. Wild-type dominance and inability of gor mutants to complement. gor-l, gar-2 mixed (D—~—[j); gar-1 (O-—— —O); gor-l, T4+ mixed (‘ ..... A); T4 (.—-—.); gar-‘2, Tl” mixed (I- - 'l). All on CGOO-RifR-‘Z and at a total lll.(>.l. of 7. significantly better than the wild-type T4 on Ril'R-‘Z. ()n wild-type E. coli, the mutants grow about as well as wild type T4. (Data not shown.) These mutants (grow on Rif“—2). have been 'alled gor The Three Independently Isolated gm' illn- tants Hare Mutations in. the Same (i'tstron The wild type is dominant to go-r in mixed infections as shown in Fig. 2. This fact allows a simple test of whether gor mutants can complement each other since, if they occur in dif‘ferent cistrons, they should grow like the wild type in mixed infections. None of the three gor mutants can complement, so they are all mutant in the same cistron. Data for two are shown in Fig. 2. Mutations to the gor phenotype may occur in other eistrons, but there has been no thorough search for them. .\l();\"l‘( :UMI‘IRY AN I) SNYI )l'llt TABLE 1 (iross: l’laqucs Indicator bacteria ("1300 411 -l. (‘tiUO llif”-‘2 230 2.‘ NF58 ltif“—2 150 1 "} Recombination 0.36 No. of mn‘ which are our ”/20 JIa/iping go)‘ .l/Il/(Ittons Recombination frequencies between known amber mutations and gar mutations have been measured by constructing the double mutant, crossing it against wild-type T4, and measuring the frequency of gor, (on+ recombinants by plating on an (on su“ IL'. coli harboring the rif“-2 mutation. A low rm-ombination frequency (~4cg) was ob- tained with a mutation in gene 42. To ascer- tain on which side of the 42 mutation the gor mutations lie, the triple mutants gm‘, (on 41*, am 42”“ and gor, mn ~12“, (un (52‘ were crossed with (on (322‘ and am 41‘ single mutants, respectively, and the frequency of gm", (nn't' rm'ombinaut types was measured. lf gor mutations lie to the left of the an: 42“ mutation (but to the right of the an: 4]“ imitation), three crossovers are required in the first cross and only one in the second cross. The reverse is true if the mutation lies to the right of the am 4'2“ mutation (but to the left of the (on (32" mutation). The results of the crosses are shown in Table l; gor, with a fre- quency of 0.36"} and 1.18”"? in the first and second crosses, 1espectiVely, suggesting that (nn’+ recombinmxts arose the gor mutations lie between the amber I'nutations in genes 4| and 42. .-\dditional evidence for this map position was obtained by picking (on-t recombinants and testing these to see how many were also gor. Fewer \\'t)lll(l be expected to be nor in the first than in the second cross, and this was observed. Thus, the {/M' mutations lie between the (on 4] and am 4‘2“ mutations. .\l.\l' l’osi'rtox‘ or gor .\li"r.\.\Ts gar, 41'“, 42‘ X ()2~ l’l'T ml gor, 42‘, 62‘ X 41‘ .l’laqucs I’I’U/ ml X 10‘“ 406 4.66 X 10“) X 10‘" 269 2.69 X 10‘0 X 10" 548 5.48 X 105 1.18 7/‘20 6C '- PFU / ml. x109 O L Time after IDiE‘CIfOU from) FIG. 3. Identity of gor and tigt'. R4 (dgt') (D D ), yor-l (O O ). TV (. fie.), all on (‘tif)t)-—llif“-'.Z at a m.o.i. of 7. 1H + gor-l on (7600- lif"~2 at a m.o.i. of 3.5 of each (A ----- A). They are complemented by both of these mutants (data not shown), so they must lie in a distinct cistron between genes 4] and 4‘3. 'I'ln' Identity of {/m‘ and do!“ There is one. known gene which maps in this region, the gene for the enzyme [3- glllcosyl transferase (( ieol'gopoulos, 1968). A GROWTH ()F T! figt' IN ltifR E (‘olt TABLE 2 gor ARI-1 flgt‘ No. of progeny which Cross do not Spot on B r agt‘ X figt‘ «U48 agt‘ X gor-l 5/48 agt‘ X gar-2 (V48 agt‘ X gor-3 5/48 Bgt“ mutant grew like gor and did not com- plement gm' mutations, as shown in Fig. 3. It is possible that. this figt‘ mutant is a deletion which includes an adjacent cistron responsible for the gor+ phenotype. To test this, all three gor mutants were crossed against a mutant with a mutation in the cistron for a-glucosyl transferase and the frequency of the progeny which could grow only on E. coli permissive for unglucosylated T4 was measured. Only the double mutant (agt‘ .dgt‘) is completely restricted on wild— type If. coli and these should arise at a frequency of about 1 9? if the gor mutants are also figt‘. The result is shown in Table ‘2. About 1 ‘70 of the progeny of each cross are capable of growing only on permissive E. coli. Thus, all three gm' mutants are also figt‘ indicating strongly that it is an inability to make an active B-glueosyl transferase which makes them gor. [Influence of a-(ftucosnltl'ansfcl'asc on T4 Growth in [{ILTR-Z Since the strain of If. coli which was used in the original selection of gor mutants is nonpermissive for unglucosylated T4, agt— mutants and the double mutants agt‘. gt " would not have been detected even if the a-glueosylation is also inhibitory since both these mutants would be at least partially restricted. Accordingly, a. BUR-:2 strain per- missive for unglucosylated T4 was con- structed and the growth of the double mu- tant agt‘figt’ was compared to that of dgt‘. The result is shown in Fig. 4. The double mutant grew no better than the single mutant demonstrating that a-glu— cosylation is not inhibitory even though 100L 9 I / 80+ I, “4.. / E U U Q) I: col 9. E L Q: Q a, 40» (3' m c (L 2?» LIL ‘L 1“ 1" 29 3k /0 J ’ {7' I: after hfect On ll'WdiQ \ puivtll' B I"! +- [Ill —7 ‘ II/ Qt {I \J O 4 $8 | /,> ‘“ :th /'_ :12 I; E l ./: l )" '~ 4 ,,. a.» r; (L A! Q: s l; ” ‘ / r; l t ,c l x! L | “I, I It / / I 13' ,’ j, ’ X / / l / / / 1 / 1 . ,’/ ,/ / / / . -1 L? A .fv‘ _1 _ _ Afiw a l (1 3C) ‘43 TWT‘G a‘t.er Ir.‘ec‘.'or~(mn) Flo. 4. (lrowth of the double mutant ugt’, tigt 'compared todgt‘on llif“-2.T4xigt" (0,- ~- —O); Tldgt‘, ogt‘ (D C] ); T“ f.» 7.). (A): All grown on K803, m.o.i. z 7. (B) All grown on K803— llif“-‘.Z, m.o.i. 2 7. 3.34 DOr .' 30* . is I: 'e X >- i" ,' a _ ; / s .s l .' / :3 ( l / .L J / a y / tO , // t / ' / / / .- . / ./) .yfi-_L A I” ‘7' ,- _ _4__fi:1~ . 20 2‘3 30 3:-) 4'5 45 32.. Time after selector: (rrs n) Flo. 5. The phenotype gort is subject to pheno- typic mixing. T41’3gt' (O ----- 0), T4" (.77 7.); dgt‘ and T4+ mixed 1:1 before infection (1:) 7 , [3); dgt" and T4+ grown together in the previous infec- tion tA---A). .\l.o.i. = 0.1 throughout. (irowth on K81)3-Rif“~2. St) Hti‘t of the hydroxymethyl cytosines are a-glucosylated with the figt— mutant ((ieor— gopoulos and Revel, 1971.) The fact that figt“ mutants are gor eVen on permissive If. coli indicates that this effect is directly due to t3~glucosylation of the I)N.~\ and has nothing to do with restriction. I'inl/ gtia-osjnlation of Only the l’arcntal I).\'.1 Is huflicicnt to Inhibit (lron'th on It’ll/Wm.) lf dgt‘ mutants are grown with wild~type T4 in mixed infections, the progeny viruses will be fully glucosylated, even though half of them will have the figt— genotype. If these viruses are then used to infect cells at low multiplicity one-half of the infected cells will receive a wild-type virus amt one-half will receive a figt' virus. The ones that re- ceiv . a Ugt‘ will exhibit a growth curve like cells infected by the wild type, if only the state of glucosylation of the parental DNA matters for this phenomenon since the prog- eny DNA will again be only partially glu- .\l()N’1‘(‘.().\lERY ANl) SNYDER. cosylated. lf glucosylation of the progeny DNA is inhibitory, we would expect the , gt" progeny to grow better than wild type. Figure .3 shows the results of such an ex- periment. The growth is as though every bacterium received a wild~type virus even though approximately 50‘}? of the progeny of the original mixed infection exhibited the .dgt‘~ genotype. (Data not shown.) Thus only the state of glucosylation of the parental DNA matters. Shown for comparison is the growth of wild—type T4 and T4 dgt“, mixed in equal numbers, and used for infection at the same low multiplicity. '11; Growth, on li’ifk-ie ('an Be Prcrcntcd tn) fi-(I'IUcos-glation of Parental DNA after I n fcct ion Th' above experiment demonstrates that full glucosylation of parental DNA is suffi- cient to interfere with growth of the virus on Rif"~2. However, the growth of the virus might also bi affected by B—glucosylation of parental DNA after infection either by dc mn'o synthesized ti-glucosyl transferase or by enzyme conceivably en 'apsulated in the viral particle. Fughtcosylated T4 'an be prepared by growth on uridine-diphosplu)gluct)se-less (UDI’G ppase") mutants of E. coli because l’Dl’G serves as the donor of glucose in the glucosylation reaction (Hattman and Fuka— sawa, 1963). These unglucosylated T4 should grow like t3gt‘ on nonrestricting If. coli harboring the Rif"—2 mutation if only the state of the incoming parental DNA is inmortant, and they should grow as the wild type if B-glucosylation of parental DNA after infection -an inhibit phage growth. The results of one such experiment are. shown in Fig. (3. Even unglucosylated wild-type growth is inhibited and the wild type is still dominant to figt“ in mixed infections. (Data not shown.) In the experiment shown in Fig. ti, T4,dgt“ * grew less well than T4i5gt‘ but this is not generally the case. Thus, fi-glucosylation of parental DNA after in— fection can interfere with phage growth on (IROWTH OF T4 figt“ IN RifK E. coli 3 O f .° 3 . U _ F 3 . ,‘ .4 t' O , Iv ‘ j t l ‘11 I C ' ,' - I f L i ,e. L I Q. . .’ l \, ' 1’ x I .1] O , (.71 1 (IE ,’ C I, / C CL l / tw/ _._’ ._ _L._—__._._. A. A ". 2 L/ 3 \_' 4 L) Tlrrze after Infection Flo. ti. I'nglucosylated wild-type T4 (T4*) grows poorly on RifR-‘Z. T4Bgt' (O ----- O);T~1;3gt“ (a _ *D };T4+ (._— -.);T4‘ (I--— ——l) (irowth (m K803-RifR-2, m.o.i. = 7. Rif“-‘2. It should perhaps be mentioned that the UDPG ppase‘ mutant used in this experiment permits low levels of glucosyla- tion of T4 DNA (Fukasawa and Saito, 1964), and it is difficult to assess what effect. this low level of glucosylation may have on the experiment. DISCUSSION There is ample evidence to support. the contention that the glucosylation of T even phage DNAs performs no necessary func- tion other than protection against restric- tion since unglucosylated phage can be culti- 'ated in Shigella (Luria and Human, 19.32) or in E. col-2' mutant in the r6 and r2,4 restriction functions (Revel, 1967). How- ever, the possibility is not excluded that glucosylation may exert a negative effect on phage growth, interfering, in some situa- tions, with reactions involving glucosylated DNA molecules. This work demonstrates such a negative effect of glucosylation. Partially glucosylatet'l T4 tigt‘ mutants are able to grow better than wild-type fully glucosylated T4 on certain RNA polymerase mutants of E. coli. This phenomenon only depends upon the state of glucosylation of those hydroxymethyl cyto- sines normally gliu-osylatcd by the o-glu- 3 .3 5 cosyl transferase (about 30 ‘1) and is inde- pendent of the ones (about 703?) normally glucosylated with an a-linkage. Thus a special role in phage development is sug- gested for at least some of the hydroxy- methyl cytosines normally glucosylated by the fi—glucosyl transferase. Also, only the state of glucosylation of the parental DNA (or part of it) is important since the phe- nomenon is subject to phenotypic mixing. However, glucosylation of the parental DNA after infection is also sufficient to prevent growth on RifR-‘Z, since ungluco- sylated wild—type T4 (T4*) grow as poorly as the wild type and are still dominant to gt— in mixed infections. There are two possible general explana- tions for the unique role of parental DNA. One is that the parental DNA is the only DNA present during the synthesis of those RNAs which are limiting in the RNA polym- erase mutant. According to this explanation the limiting RNAs could only be made after the synthesis of B-glucosyl transferase and before the onset of T4 DNA replication. The alternative explanation is that part of the parental DNA containing the hy- droxymethyl cytosines whose glucosylation is criti ‘al for growth on the RNA polymer- ase mutant preserves its autonomy as pa— rental DNA throughout some, if not all, of the latent. period. If this explanation were true, it would not be. the first demonstration of a unique role for parental DNA during viral development. Only the parental repli- -ativc form of ¢Xl74 can direct. the synthe— sis of progeny replicative forms (Denhardt and Sinsheimer, 1965) and A parental DNA can only find its way into progeny particles vis-a-vis the recombination system (Stahl ct at., 1972). Why does the inhibitory effect of fight- cosylation only become. apparent in the RNA polymerase mutant? The effect of the RNA polymerase mutation could be either direct or indirect. Some RNA polymerase mutations are known to affect cellular mor- phology (I)oi ct al., 1970) and, in fact, the Rit'R-2 mutation does affect T4 adsorption and cellular growth of some strains of E. coli especially at lower temperatures (1.. Snyder, unpublished observations). It is possible that Rif"s2 is altered in some st ruc- tural component (e.g., a membrane binding site for DNA) so that fully glucosylated T4 DNA cannot properly interact with it either directly or because hypothetical T4 gene product(s) which are requiring for the bind— ing of fully glucosylated DNA do not [n'op- erly recognize the altered cellular compo- nent. However, we prefer models in which the effect of the RNA polymerase mutation is direct because the enhanced growth of {igt‘ mutants in Rif“-2 is relatively independent of the strain of E. coli harboring the R.if"-2 mutation and because the Rif“-2 mutation delays a change in RNA polymerase which occurs after T4 infection (Snyder, to be published). A very specific model which incorporates a direct. role. for the RNA polymerase muta- tion immediately suggests itself. The a-glu~ cosyl transferases of T2 and T4 have a sequence specificity since they will not glumsylate approximately 25 (:32, of the hy- (_lroxymethyl cytosines in their DNA. The hydroxymethyl cytosines which are left un- glucosylated have been shown to be prefer- entially those which are adjacent to other hydroxymethyl cytosines (Lunt and New- ton, 1965; de “’aard ct al., 1967). I’yrimis dine-rich regions have been detected in T4 DNA as well as many other DNAs and have been circumstantially implicated in transcription be'ause they occur on the strand of DNA which is used for transcrip- tion at a particular locus (Szybalski et (11., 1966,). The important fact for this model is that. the hydroxymethyl cytosines in these pyrimidine-rich sequences would be left largely unglucosylated by the a—enzyme. Assume that they are recognition signals for RNA polymerase. If they are left unglu- cosylated, they may closely resemble recog- nition signals of the host so that the host polymerase unmodified by T4 could recog~ .\1( )NT( it ).\1 ER Y AND SNYDER nize them and the modification which may not occur in the RNA polymerase mutant would not. be required for transcription of the genes which they serve. Ergo, with the figt— mutant the need for the RNA polymer ase modification is bypassed and the phage grow better. But why then, by this model, all some T4 signals be recognized by the unmodified host polymerase since the neces- sary modification may require T4 protein synthesis and T4 DNA can be transcribed by unmodified E. coli RNA polymerase in ritro‘.’ The answer may lie in the fact that there are two types of pyrimidine rich se- quences in T4 DNA; those which are rich in hydroxymethyl cytosin * and those which are rich in thymidine (Cuba and Szybalski, 1968). The thymidine-rich regions would always be unglucosylated and could be the recognition signals for the earliest-appearing T4 messenger RNAs whereas the hydroxy- methyl cytosine-rich regions would require an RNA polymerase modifi 'ation for their recognition if they are glucosylated, and they would then be the signals for some of the later-appearing >arly messenger RNAs. The pyrimidinturich sequences on A DNA have recently been mapped (Champoux and Hogness, 1972) and found to be sometimes internal to transcription units on A DNA. These authors have suggested that they may serve a “divider” function in transcrip- tion, a hypothesis consistent with our data thus far. Of the T-evcn coliphages, T2, T4, and T6, only T4 has fully glucosylated DNA (Leh- man and l’ratt, 1960) giving it additional protection against restriction and therefore presumably broadening its host range. How- ever, our work indicates that T4 pays a price for this increased protection. By cover- ing the remaining 30'}? or so of the hydroxy— methyl cytosines, at least some of which, according to our work, play a very special role in phage development, it interferes with a reaction in which parental T4 DNA molm-ules must participate, and very possi— bly additional gene products are required to GROWTH OF T4 figt‘ IN Rif" E. coli 3.77 o\-'ercome this interference. These may be gene products which are. required only be- cause T4 DNA is fully glucosylated and which cannot function properly in the RNA polyn‘ierase mutant. Thus, although glu— cosylation of DNA is specific for T-even coliphages of all the known viruses (and therefore is without. general significance), and although it apparently plays no role in phage development except for protection against restriction, it. may prove very useful as a probe to discover the nature and func- tion of special seqtlences on DNA. ACKNOWLEDt iM ENTS This work was supported by a grant to Loren Snyder from the National Science Fouiulation. Donna Montgomery is supported by a traineeship awarded to the Department of Microbiology and Public Health by the National Science Founda- tion. REFERENCES Home, A., Ees'rmx, R. H., S.\I.si-;n, W., and GalpUsanK, E. P. (1968). Transcriptionduring bacteriophage T4 development: Requirements for late messenger synthesis. .1. Mot. Biol. 33, 339 -3; i2 . Bunny, E., Snoiznoi-‘F, R., Roma-z, A., and Er- s'rizlx, R. H. (1970). Early transcription in T4- infected cells. Cold Spring Harbor Symp. Quant. Biol. 35, 203-211. (‘uxx-ieoex, J. J., and Hoosizss, l). S. (1972). The topography of lambda DNA: l’olyriboguanylic acid binding sites and base composition. J. .ltol. Biol. 71, 3853-405. IHZNHARDT, D. T., and Sixsni-zimzn, R. L. (1965'). The process of infection with bacteriophage ¢N174. IV. Replication of the viral DNA in a synchronized infection. J. Mot. Biol. 12.6474362. pi-2\V.\_\m), A., UBBINK, T. 1'). C. M., and BEUKMAN', W. (1967). On the specificity of bacteriophage in- duced hydroxymethyl cytosine glucosyl trans- ferases. Eur. J. Biochem. 2, 303308. oi.\l.\t'no, E., SNYDER, L., 1\'I.-\m.vo,P., L.\.\no-:n'ri, A., COPPO, A., and Toccatm-eraexrmr, G. 1’. (1969). Rifampicin sensitivity of the components of DNA dependent RNA polymerase. .\'utnrc (London) 222, 533—537. Dot, R. H., Bnown, L. R., Roooizns, G., and Hsmx Y. (1970). Bacillus snhtitis mutant altered in spore morphology and in RNA polymemsc activity. Proc. Nat. Acad. Sci. U.S. 66, 404-410. Eesricix, R. H., Bonnfz, A., S'rmxnicno, C. .\l., Ki-:l.1.I-:.\‘niznol-zn, E., Boy Dr: I..\ Torn, E., (‘iiI-:\'_\1.I.i;\', R., Epoin, R. S., Sl'sxnx, .\l., l)i;xn.\np'r, G., and l.n-.l..\lrsls, A. (1963). Physiological studies of conditional lethal mu- tations of bacteriotmage T41). ('old Spring Harbor Symp. Quant. Biol. 28, 375 394. Fekisxwx, T., and S.\I'ro, S. (1964). The course. of infection with T even phages on mutants of E. coli K12 defective in the synthesis of uridine diphosphoglucose. J. .Ilol. Biol. 8, 175-183. (iizoimoeolmos, (.7. P. (1968). Location of glucosyl transferase genes on the genetic map of phage T4. I'irologi/ 34. 3644466. til-;I)RGUP()UI.OS, (3. l’., and Raw-J., II. R. (1971). Studies with glucosyl transferase mutants of T even Inu'teriophagcs. Virology 44, 2714285. Giusso, R. J., and BI't‘H.\.\'.\N, J. .\l. (1969). Syn- thesis of early INA in bacteriophage T4-in— fected Escherichia coli B. Nature (London) 224, 882 885. (iUHA, A. and SZYBALSKI, W. (1968). Fractionation of the complementary strands of coliphage T4 DNA based on the asymmetric distribution of the poly U and poly [LG binding sites. Virology 34, 608-616. ll.\si«:i.konx, R., \'ooi;1., .\l., and Rnowx, R. I). (1969). Conservation of the rifamycin sensitivity of transcription during T4 development. .\'alnrc (London) 221 , 8364838. ll.\'r'rxi.\x, S., and ch.\s.\w.\ T. (1963). Host- induced modification of T~even phages due to defective. glucosylation of their DNA. Proc. Nat. Acad. Sci. (KS. 50, 2974300. llosolu, J., and l.i:vl.\"rn.\i., (f. (1968). Protein synthesis by Escherichia coli infected with bacteriophage T41). I'irology 34, 7011-727. LI-;n.\i.\.\', l. R., and PRATT, E. A. (1960). On the structure of the glucoslyatcd hydroxymethyl- cytosine nucleotides of coliphages T2, T4, and T6. J. Biol. Chem. 235, 3254 3259. Ll'x'r, M. R., and NEWTON, E. A. (1965). Glu~ cosylated nucleotide sequences from T-even bziictcriophage deoxyribonucleic acids. Biochem. .l. 95, 717—723. LI'IUA, S. E., and lll'M.\N, .\l. L. (1952). A non- hereditary, host-induced variation of bacterial viruses. .1. Bacteriol. (rt, 557 569. .\l.\rsirk.\oi;, A., and .\IlN.\oA\y.\, T. (.1967). Shut - off of early messenger RNA synthesis in E. coli. infected with phage T2. Biochem. Biophys. Res. ( 'oin "I n n. 29 , 394—44. Nont'tu, M., WIT'ri-zx, (.‘-., .\l \N'ri-zi, N., and Ecuons, H. (1966). Inhibition of host. nucleic acid synthesis by bacteriophage T4. Effect of chloramphcnicol at. various nmltiplieities of in- fection. .I. Hot. Biol. 17, 273-278. RI-;yI-;I.. H. R. (1967). ated T-evcn bacterirmhage: lestriction of nonglucosyl- Properties of per- 358 Mt )NT( 1( ).\1 ER Y AN 1) SNYDER missive mutants of Escherichia coli B and K122. Virology 31, (388—701. SNYDER, L. (1972). An RNA polymerase mutant of E. coli defective in the T4 viral transcription program. 1"irolog‘ll 50, 396—403. ST.\HL, F. W., .\'ICMII.IN, K. 1)., STANL, M. M., .\1Al.()NE, R. E., Nozu, Y. and thsso, V. E. A. (1972). A role for recombination in the produc- tion of “free loader" lambda bacteriiqihage particles. .1. Jill]. Biol. 68, 57-2137. SZYBALsKI, W., Kl'mxsiu, 11., and Suizmuuck, l’. (1966). Pyrimidine clusters on the transcribing strand of DNA and their possible role in the initiation of RNA synthesis. Cold Spring Harbor Symp. Quanl. Biol. 31, 123—127. 'l‘mm, M. (1967). Studies on the mechanism of bacteriophage T4 interference with host me- tabolism. J. Mol. Biol. 28, 37--44. Wool), W. B. (19613). Host specificity of DNA pro- duced by Escherichia coli: Bacterial mutations atl'ecting the restriction and modification of DNA. J. .1lol. Biol. 16, 118 133. ARTICLE II Bacteriophage T4 Growth on an RNA Polymerase.. Mutant of Escherichia coli: Effect of B-Glucosylation on Early T4 Functions By Donna L. Montgomery and Loren R. Snyder Manuscript to be submitted for publication Bacteriophage T4 Growth on an RNA Polymerase Mutant of Escherichia coli: Effect of B-Glucosylation on Early T4 Functions DONNA L. MONTGOMERY AND LOREN R. SNYDER Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824 In previous publications, we have reported that an RNA polymerase mutation, rif -2, of Escherichia coli poorly supports the.growth of T4. RifR-Z has a direct effect on both DNA synthesis and late gene expression. The effects of rifR-Z are partially suppressed by B-glucosyl transferaseless mutants of T4, and B-glucosylation is inhibitory both early and late in infection. RifR-Z also has an effect on early gene expression causing disproportionate synthesis of some early T4 proteins. However, B-gluco- sylation has no.effect on this early gene expression defect, although we have shown that B-glucose inhibits an early function required for phage production. Rif -2 also causes defective host nucleoid unfolding, and a delay in host DNA degradation. B-glucosylation is. inhibitory for host DNA degradation, suggest- ing that the transcription of B-glucosylated hydroxymethyl cytosines in T4 DNA is somehow involved in this step. Our new data further support the model proposed earlier that RNA polymerase must alter the structure or associa- tion of T4 parental DNA early in infection for DNA replication and late transcription to occur. INTRODUCTION E. coli RNA polymerase performs several functions in addition to transcription of E. coli messenger, transfer, 38 39 and ribosomal RNA. RNA polymerase has been shown to be involved in DNA synthesis (Lark, 1972; Blair et aZ., 1972; Sugino at aZ., 1972) and must function to hold DNA in a highly folded state, referred to as a nucleoid (Stonington and Pettijohn, 1971; Dworsky and Schaechter, 1973; Worcel and Burgi, 1972). In addition, Pettijohn and Hecht have shown that a functioning core RNA polymer- ase seems to be continuously required to maintain the highly folded state of the nucleoids, since cells briefly treated with rifampicin yielded only unfolded genomes upon lysis (Pettijohn and Hecht, 1973). E. coli RNA polymerase also performs several func- tions in T4 development. It is required for both early and late transcription (Haselkorn et aZ., 1969; di Mauro at aZ., 1969) and for some additional function required for DNA synthesis (Snyder and Montgomery, 1974). Modifi- cations of the host polymerase are required for some of these functions since after infection the proteins associated with RNA polymerase are changed. Two such proteins, the products of genes 55 and 33, are required for late messenger RNA synthesis (Bolle et aZ., 1968; Notani, 1973) and are known to bind to host RNA polymerase after T4 infection (Stevens, 1972; Horvitz, 1973; Ratner, 1974). In addition, the gene 45 product is required for late messenger synthesis (Wu et aZ., 1973) and there is recent evidence, both biochemical (Ratner, 1974) and 40 genetic (Snyder and Montgomery, 1974; Coppo at aZ., 1974) that it also binds to RNA polymerase. Ratner has found other T4 proteins which are retained by RNA polymer- ase columns, but it is not known if their binding is essential for some step in phage development (Ratner, 1974). We have been studying the roles of host RNA polymer- ase in T4 development, as well as the proteins which interact with the polymerase, by using RNA polymerase mutants of E. coli which inhibit T4 growth. The muta- tion we have characterized is referred to as rifR-Z. The rifR-Z mutation has a cold-sensitive effect on T4 develop- ment; at 40°, T4 production is almost normal, while at 27° T4 development is severely retarded. We have found that the rifR-Z mutation inhibits both DNA synthesis and late gene expression (Snyder and Montgomery, 1974). Several mutants of T4 which grow better than T4+ on RifR-Z were obtained with the hope of finding T4 genes coding for transcriptional control proteins. The first gor (grows on rifR-Z) mutations which were found are in the gene for B-glucosyl transferase, indicating that the glucosylated state of hydroxymethyl cytosines is important in some interactions of T4 DNA with RNA polymerase from RifR-Z cells. Two other types of gor mutations have been found in genes which may code for proteins which interact directly with RNA polymerase. Amber mutants in gene 45 41 were found to grow better than T4+ on RifR-Z strains which are sug, and mutations in a new gene (gor-Z), located between genes 55 and ogt, cause T4 to grow better than T4+ on RifR-Z (Snyder and Montgomery, 1974). We have found that B-glucosylation inhibits both late gene expression and the rate of DNA synthesis, but seems to have little or no effect on the delay in DNA synthesis (Snyder and Montgomery, 1974). In pheno- typic mixing experiments where the parental, but not the progeny, DNA is B-glucosylated, we have shown that there is a function inhibited by B-glucosylation that occurs early in infection (Montgomery and Snyder, 1973; Snyder and Montgomery, 1974). This suggests that the rifR-Z mutation has an effect on some early T4 function. This paper reports experiments done to determine the effect of riijz and B-glucosylation on early T4 functions. MATERIALS AND METHODS a) Bacterial Strains and Phage All bacterial strains used were previously described (Montgomery and Snyder, 1973). T4 am A453 (gene 32) was obtained from H. Revel. The B-glucosyl transferase mutant (Bgt-) used was R4, obtained from J. Wiberg. The double mutants T4 am N122,R4 (genes 42, Bgt) and T4 am A453, r1272 (gene 32, deleted for cistrons rII A and B) were constructed in this laboratory. 42 b) Media and Buffers M98, described previously (Snyder and Montgomery, 1974), was used for all experiments except those in which proteins were labeled. The M9S media were modi- fied for protein labeling experiments by replacing the casamino acids with an amino acid mixture. Alanine, aspartic acid, cysteine, glutamic acid, glycine, histi- dine, isoleucine, lysine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (all the L-isomers) were added to a final concen- tration of 20 ug/ml. Arginine and methionine were added to a final concentration of 50 ug/ml. The M9 buffer and tryptone broth and plates were described earlier (Snyder and Montgomery, 1974). c) Infections and Survivor Determinations Infections were Carried out as described below for all experiments except those shown in Figure l. The infection and labeling procedures for those experiments are described in the figure legend. Cells were grown at 40° to an O.D. of 0.4 at 625 mu, and infected with CsCl purified phage at a m.o.i. of 5 or 10. At two minutes after infection, cells were shifted to 27°, the nonpermissive temperature for T4 growth on RifR-Z. Cells were infected at the higher temperature because phage adsorb better and are less sensitive to ghost exclusion at 40° than at 27°. 43 Survivor samples were taken at 3 minutes after infection (1 minute at 27°) by diluting into M9 buffer on ice. Infections were terminated by pouring cultures over 2M. Only ice and NaN3 at a final concentration of 10' infections producing survivors of less than 4%, and usually less than 2%, were used for the different analyses. d) SDS-Polyacrylamide Gel Electrophoresis Cells (2.5 ml) were infected and shifted as described 14C-leucine, at a concentration of 1.3 uc/ml in "c)" and 1 ug/ml, or 3H-leucine, at a concentration of 7.7 uc/ml and l ug/ml, was added after infection for the desired times. Infection was terminated as described in "c)" and the cells were concentrated 10x by centrifuging and resuspending in 0.25 ml of .OlM Tris-HCl, pH 8.0; 0.001M EDTA; 1% SDS. 2-mercaptoethanol was added to a final concentration of 2%, the samples were heated in a boiling water bath for 3 minutes, and stored at 4°. Just before electrophoresis, dye solution (20% sucrose, 0.008% Pyronin Y) was added to the sample at a ratio of 1:3. Samples (0.01 ml) were layered onto each gel. Gels containing 1% SDS and 9% acrylamide were made according to the procedure of Fairbanks et a2. (1971), with the following modification. Acid-cleaned glass 44 tubes were coated with dimethyl dichlorosilane (Sigma) and allowed to dry for several hours. Tubes were rinsed with hot water, 95% ETOH, and water, then allowed to dry before pouring gels. Gels (8.5 cm) were run at 70 volts. Under these conditions, it took approximately 2 hours for the dye front to migrate through the gel. Slices of 1 mm were collected with a.Gilson automatic gel slicer. The samples were incubated overnight at 35° with constant shaking in 0.2 ml of 1% SDS to elute proteins from the gel before adding 5.0 ml Aquasol (New England Nuclear) to each vial for counting. e) Alkaline Sucrose Gradients Cells were labeled at 40° for 1 hour before infec- tion, with 3H-thymidine at a concentration of 230 uc/ml and 28 ug/ml. Infected cells were lysed according to the procedure of Hercules et a1. (1971) except that M98 medium was used in place of the GCA (glycerol-casamino acids) media they described. Alkaline sucrose gradients (5% to 20%) were generated andrun.as described by Hercules et al. (1971). Fractions were collected from the bottom of the tube, TCA precipi- tated, collected on glass fiber filters (Whatman, GF/A), and counted in a toluene-base scintillation fluid. 45 f) Nucleoid Disruption Cells (5 ml) were labeled at 40° for 30 minutes before infection, with 3H-thymidine at a concentration of 30 uc/ml and l ug/ml. Infections proceeded and were terminated as described in "c)". In experiments using chloramphenicol, it was added at a final concentration of 150 ug/ml at 5 minutes after infection. The lysis procedure of Stonington and Pettijohn (1971) was carried out with the modifications of Worcel and Burgi (1972). However, the Sorvall spin of the lysed mixture was omitted, and 0.1 ml was layered directly onto a 10% to 30% sucrose gradient. Gradients were generated and run as described by Worcel and Burgi (1972).‘ Fractions were collected from the bottom of the tube, TCA precipitated, collected on glass fiber filters (Whatman, GF/A), and counted in a toluene-base scintil- lation fluid. RESULTS Since the rifR-Z mutation has an effect on late protein synthesis, we wished to determine if it also has an effect on early protein synthesis. Initial studies indicated that rifR-Z does not prevent the appearance of at least some early T4 proteins. The synthesis of two early enzymes, B-glucosyl transferase and dihydrofolate reductase, are produced at nearly normal levels after only 46 slight lags, and all the proteins required for DNA synthesis are present at the early times (Snyder and Montgomery, 1974). However, a more detailed study of the effect of rifR-Z on early protein synthesis was needed. The Effect of rifR-Z and B-Glucosylation on Amino Acid Incorporation The rate of protein synthesis after T4+ infection on RifR-Z and wild-type E. coli was measured as the amount of 3H-leucine incorporated during a 2 minute pulse (Figure 1A). The rate of synthesis is not sub- Stantially affected by rifR-Z early in infection, but it is later. By 36 minutes after infection, amino acid incorporation in RifR-Z is reduced by about 30% from that in wild-type cells. The effect of B-glucosylation on early protein synthesis was also determined in the absence of DNA replication. Figure 1B shows the results obtained with continuous labeling. There is no difference between N122 and N122,Bgt' in the amount of radioactive leucine incor- porated, indicating that B-glucosylation has no effect on the rate of protein synthesis early in infection in the absence of DNA replication. The Effect of rifR-z on the Synthesis of Individual T4 Proteins Although the rate of protein synthesis is not affec- ted by the rifR-Z mutation early in infection, this does 47 Figure 1. The rate of amino acid incorporation after T4+ infection. Figure 1A - The rate of T4 protein 5 nthesis after infection of K803 (-Oe).and K803-Rif -2 (-E:]-). Cells were infected at a m.o.i. of 5 at 40° and shifted to 27° at 2' after infection by diluting 1/10 into media at 27°. Cells (1 ml) were pulse labeled for 2 minutes with 3H-leucine at 1.3 uc and l ug/ml and incorporation stopped with.0.1 ml of 50% TCA. Four milliliters of 5% TCA were added, the precipitate centrifuged and resuspended in 0.3 ml 2% KOH, reprecipitated with 5% TCA, and collected on membrane filters for counting. Figure 1B - The rate of T4 protein synthesis after infec- tion with T4 am N122 (-[:]-) and T4 am N122, Bgt' (-O-). Cells were infected and shifted as in Figure 1A. 3H- leucine was added at 4' after infection at 1.3 uc and 1 ug/ml. Samples (1 ml) were stopped'with 0.1 ml 50% TCA. Samples were precipitated and collected as in Figure 1A. I ' 3 w :99 x 10 12 3H CPH x 10‘2 20 48 l 21 IIHE AFTER INFECTION (MIN) Figure 1A 1 l I 5 8 l2 rINE AFTER INFECTION (NIN) Figure 1B 49 not necessarily mean that all proteins are being pro- duced at wild-type rates. To determine the distribu- tion of label in various proteins, early proteins were subjected to polyacrylamide gel electrophoresis. To compare the early proteins from two separate infections, 14C-leucine or 3H- proteins were labeled with either leucine and electrophoresed together. Gels were frac- tionated and counted to determine the amount of radio- activity incorporated into the various peaks during the labeling time. This mixed-label method was chosen because it is more quantitative than slab gel autoradio- grams. Small differences in the relative rates of synthesis of different proteins would be difficult to determine from band densities on autoradiograms, but could be detected by the ratio of two different radio- active labels on the same gel. Since there are probably over 100 early proteins of T4, each peak in our gel patterns undoubtedly repre- sents several proteins of similar size. To determine the degree of resolution in the gels, controls were run comparing different T4 amber mutants missing early gene products. Figures 2A and ZB show the results of both T4 am E10 (gene 45) and T4 am A453, r1272 (gene 32, dele- tion in rII A and B) compared to T4 am N122 (gene 42). The results are plotted as the percentage of the total 50 Figure 2. Proteins synthesized from 4 to 12 minutes after infection of NF58 with T4 amber and deletion mutants. The ratio of 14C/3H is given below each gel pattern. Figure 2A - (-£:]-) T4 am N122 (gene 42) infection labeled with 1 C—leucine. Survivor rate = 2.15% -0-) T4 am E10 (gene 45) infection labeled with H-leucine. Survivor rate = 3.25%. Figure 2B - (- -) T4 am N122 (gene 42) infection labeled with 1 C-leucine. Survivor rate = 0.52%. (-O-) T4 A453,r1272 (deletion in RIIA'B) infection labeled with 3H-leucine. Survivor rate = 0.55%. 51 ‘ 8 . 8 d oqi' g ‘ O---=iii:::E $3 4441 0» r4 “311110140; H(/DH OllVU 4__‘,,*rdlr“"'fl-q[ 153 “f : A v v v M ‘8 0<==E j c -' ‘ ' .J"!' . iii:::il o‘i_ : v A ‘9 . E :;:E;::. " 453 ( L41 1 I :EEEQE: 1 :7 N N r—a "([391 011V! ‘RACTIOH Figure 2B Figure 2A 52 recovered CPM found in each fraction, with the ratio of the 14C/SH per fraction plotted below each graph. The gene 42 and 45 polypeptides have similar molecu- lar weights and should co-migrate on gels. The resolution of these gels is not good enough to detect the absence of the 42 product in N122 patterns, but it is good enough to detect the absence of the 45 product in T4 am E10 (Figure 2A), as a reduced peak shoulder between fractions 48 and 51. This position correlates well to that of the 45 product identified on slab gels (O'Farrell et aZ., 1973). In Figure 2B, the large reduction in height of the‘ peak between fractions 41 and 47 in the T4 am A453, r1272 infection is due to the absence of both the 32 and rIIB gene products. These two gene products are made in large quantities during the labeling period, and migrate together on slab gels to a position corresponding to the reduced peak on our gels (O'Farrell et aZ., 1973). The rIIA product is made in smaller amounts and migrates slower than rIIB on slab gels. Its absence is not as evident in these gels, but the rIIA protein migrates to the area between fractions 20 and 24, since there is a variation here in the ratio of the two isotopes. Thus, since we can detect the absence of various gene products, the resolution of these gels is sensitive enough to detect differences in major proteins, and should be able 53 to detect the more generalized transcriptional defects which might be expected in an RNA polymerase mutant. The first question we asked was whether the rifR-Z mutation has an effect on early protein synthesis. Figure 3 shows the gel patterns obtained with T4 pro- teins labeled from 4 to 12 minutes after infection of both RifR-Z and wild-type E. coli. Cells were infected with an amber DNA zero mutant to make the two strains comparable in DNA replication. The differences in the gel patterns indicate that the rifR-Z mutation does have an effect on early transcription. Because of the overlap of the normalized 3H and 14C counts seen in our controls, the differences between wild-type and rifR-Z infections observed in Figure 3 can be considered significant. One of the reduced peaks in the rifR-Z pattern, that between fractions 48 and 51, may be the gene 45 product, as shown in Figure 2B. Thus, the rifR-Z mutation may cause the gene 45 product to be synthesized in reduced amounts. This is puzzling considering genetic experiments reported earlier (Snyder and Montgomery, 1974) and which will be mentioned in the discussion. An experiment similar to that shown in Figure 2 was done on T4 proteins labeled from 2 to 5 minutes after infection to determine if rifR-Z has an effect on gene expression at earlier times (Figure 4). Here again, 54 Figure 3. Proteins synthesized from 4 to 12 minutes after infection of NF58 and NF58-RifR-2 with T4 am N122 (gene 42). (-[:'j-) NF58 infection labeled with 14c- 1eucine. Survivor rate = 1.93% (-O-) NF58-RifR-2 infec- tion labeled with H-leucine. Survivor rate = 2.28%. 2 OF TOTAL CPM SS IL I W ‘F " IL ‘ H J ‘ . J- .. ‘I . .14. ' 2D 4D 50 8D FRACTION Figure 3 56 Figure 4. Protein synthesis from 2 to 5 minutes after infection of NF58 and NF58- RifR -2 with T4 am N122. (- [:1- ) NF58 infection labeled with 3H- leucine. Sur- vivor rate