Illllll Ill 1 This is to certify that the thesis entitled The Transcription of Cytosine T4 DNA lg Vitro and the Effects of the T4 Alc Gene on Transcription presented by Robert Edwin Pearson has been accepted towards fulfillment of the requirements for M947 Major professor 0 Date 254%? 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book retu turn to remove charge from circulation records THE TRANSCRIPTION OF CYTOSINE T4 DNA I VITRO AND THE EFFECTS OF THE T4 AL£_GENE ON TRANSCRIPTION By Robert Edwin Pearson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for DOCTOR OF PHILOSOPHY Genetics Program 1979 ABSTRACT THE TRANSCRIPTION OF CYTOSINE T DNA IN VITRO AND THE EFFECTS OF Tfie T4 AL§_GENE ON TRANSCRIPTION By Robert Edwin Pearson The bacteriophage T4 normally synthesizes DNA with 5-hydroxy- methylcytosine in place of cytosine. When T4 replicates with cytosine no phage are produced because no T4 true late mRNA is synthesized demonstrating that 5-hydroxymethylcytosine is required for normal T4 true late transcription. Recently, T4 mutants, termed alg_mutants, were isolated because they allow T4 to grow with cytosine containing DNA. Alg_mutants permit cytosine T4 develop- ment by allowing almost normal T4 true late transcription from cytosine T4 DNA. Experiments were performed to investigate tran- scription from cytosine T4 DNA in_gitrg_and the §1g_gene product in_ 3139. The first article describes studies of transcription from cytosine T4 DNA, 5-hydroxymethylcytosine T4 DNA, and calf thymus DNA jn_gitrg, Experiments were performed to determine if T4 induced modifications of the host RNA polymerase prevent transcription from cytosine T DNA. The results demonstrate that the T4 modified RNA 4 polymerase transcribes cytosine T4 DNA ig_vitro. Thus, the T4 Robert Edwin Pearson induced modifications did not prevent transcription from cytosine T4 DNA. In addition, the T4 modified RNA polymerase synthesizes more RNA from cytosine T4 DNA or calf thymus DNA suggesting that hydroxymethylcytosine impedes transcription. The second article describes studies of the jn_yjyg_ affects of the alg_gene product on the bacteriophage Lambda. It was found that the alg_gene product shut off late Lambda transcrip- tion but did not affect the supercoiling of intracellular Lambda DNA. Therefore, the alg_gene product does not act by directly removing supercoils from DNA. Furthermore, the results demonstrate that normal molecular weight Lambda mRNA is synthesized after T4 superinfection, but not translated. Thus, a T4 mechanism blocks translation of non-T4 mRNAs. DEDICATION This project is dedicated to my mother and father who have supported me in many ways throughout my life. ii ACKNOWLEDGEMENTS I would like to thank Drs. L. Robbins, A. Revzin and R. Brubaker for helpful suggestions and serving on my committee. Special thanks to Drs. L. Robbins and A. Revzin for useful writing suggestions and to Drs. R. Patterson and L. Velicer for use of their laboratories for some of this work. I am also extremely grateful to Dr. Larry Snyder for his personal support, the opportunity to work with him, and his exemplary approach toward scientific problems. I would like to acknowledge support from the Genetics Pro- gram, graduate assistantships from the Biological Science Program, and financial support from the National Science Foundation. TABLE OF CONTENTS List of Tables List of Figures . INTRODUCTION AND LITERATURE REVIEW The Bacteriophage T4 The T Genome T4 Transcription . . T4 Induced RNA Polymerase Modifications . Regulation of T4 Early Transcription . Regulation of T4 Late Transcription . Synthesis of T4 Nith Cytosine T4 DNA . T4 Induced Alterations of Host Nucleic Acid Metabolism . . . . . Nuclear Disruption . . Degradation of Host DNA . . Unfolding the Host Nucleoid . . Shutting Off Host DNA Synthesis Shutting Off Host RNA Synthesis . Shutting Off Host Protein Synthesis . . . The Bacteriophage Lambda as a Model System for Investigating the alc Gene Product . . . . The Early Period of Lambda Development . Early Period Transcription . . Early Lambda DNA Replication . . The Late Period of Lytic Development Late Period Transcription . Late Lambda Replication REFERENCES ARTICLE I . The Transcription of Cytosine T4 DNA, 5- Hydroxymethyl- cytosine T4 DNA and Calf Thymus DNA In Vitro by T4 Modified RNA Polymerases iv Page vi vii ARTICLE II . The Shutoff of Lambda Transcription by Bacteriophage T4: Role of the T4 Alg_Gene APPENDIX The Shutoff of Lambda Monomeric Circle Replication_ . The Shutoff of Lambda Transcription After Alt Mod Mutant Infection . Page 62 100 101 103 LIST OF TABLES Page ARTICLE II Table l: Lambda DNA Synthesis After T4 Infection . . . 82 APPENDIX Table l: Lambda DNA Synthesis After T4 Infection . . . lOZ vi LIST OF FIGURES Page ARTICLE I Figure l. In Vitro Transcription of Cytosine, HydroxymethchytOSTne, and Calf Thymus DNAs by Uninfected and the Wild Type T4 Modified RNA Polymerase . . . . . . . . 52 Figure 2: In Vitro Transcription of Cytosine, Hydroxymethchytosine, and Calf Thymus DNAs by Alt Mod Mutant RNA Polymerase and Alc Mutant RNA Polymerase . . . . . . . . . 56 ARTICLE II Figure l: Hybridization of X RNA Synthesized After T4 Infection To A DNA . . . . . . . . 76 Figure 2: Ethidium Bromide Cesium Chloride Gradient Centrifugation of Supercoiled A DNA Without T Infection, with 935} T Infection and with a_l_c Mutant T4 Infection . . . . . . . . . . . . . 79 Figure 3: Slab Gel Electropherograms of Proteins Synthesized in an Uninduced Culture of a X Lysogen and in Heat Induced Lysogen Infected by T4 . . . . . 83 Figure 4: Sucrose Gradient Centrifugation of Heat Induced Late Period RNA Synthesized Without and With T4 Infection . . . . . . . . . . 86 APPENDIX Figure l: Hybridizations of A RNA Synthesized After T4 Infection to A DNA . . . . . . . 105 vii INTRODUCTION AND LITERATURE REVIEW The regulation of gene expression is central to many complex cellular processes including cell division and cell differentiation. An approach to understanding such complex phenomena is to first investigate a simple model. The model selected for analysis was the development of the bacteriophage T4 because it is genetically and biochemically more pliable than other systems. It is hoped that a better understanding of T4 development will provide insight to other complex biological processes. T4 is a large virulent bacteriophage of E._gglj with enough DNA to code for approximately 200 genes of which around 60% have been identified. A number of T4 genes are directly involved in regulat- ing transcription during infection. Recently, a T4 mutant termed .al_ has been shown to regulate both host and T4 transcription from cytosine T4 DNA. This gene is also involved in unfolding the nucle- oid and may be associated with an alteration of the host RNA polymerase. Clearly, a better understanding of the T4 gig gene would provide useful information about the regulation of transcrip- tion. The following discussion will be separated into three major sections. The first section reviews literature pertinent to the gig gene and the bacteriophage Lambda which was used to investigate the gig_gene. The second section describes experiments which show that T4 induced RNA polymerase modifications do not prevent transcription from cytosine T4 DNA. Also, the experiments suggest that S-hydroxy- methylycytosine directly impedes transcription from T4 DNA. The final section will examine the affects of the T4 gig gene product on the development of the bacteriophage Lambda. These experiments demonstrate that the gig_gene product shut off Lambda late peridd _ transcription, but did not affect the supercoiling of intracellular Lambda DNA. Therefore, the gig product does not block transcription by directly removing the supercoils from DNA. In addition, other experiments show that normal length Lambda mRNAs are made during an al mutant superinfection, but not translated. Thus, a T4 mechanism exists which prevents translation of non-T4 mRNAs. The Bacteriophage T4 The T4 Genome The T4 genome contains about 170 kilobases and has a mole- cular weight of approximately 120 x lo6 daltons. T4 DNA is termi- nally redundant with approximately two percent of the total DNA repeated at both termini of the molecule (Kim and Davidson, 1974; Thomas and Rubenstein, 1964). In a population of molecules, the terminally redundant sequences are not identical, but consist of a permuted collection of all T4 sequences (Streisinger et al., 1967; Thomas and Rubenstein. l964). Therefore, some genes may be adjacent to each other on some molecules and located at the antipoles on other molecules. Consequences of the circularly permuted terminal redundancies include a circular genetic map and intramolecular het- erzygotes. T4 DNA is unusual because it contains the base 5-hydroxy- methylcytosine in place of cytosine (Wyatt and Cohen, 1953). This base protects T4 DNA from at least one host restriction system (Snyder et al., 1976) and more than likely protects T4 from other restriction systems as well. In addition to this function, hydroxy- methylcytosine is involved in T4 gene expression because it is required for normal T4 true late gene expression (Kutter et al., 1975; Wu and Geiduschek, 1975). Another effect, as shown by experi- ments to be presented below, is an alteration of the template acti- vity of phage DNA jg XiEEQ- T4 DNA contains glucose as well as hydroxymethylcytosine. Glucose is attached to hydroxymethylcytosine through either an a or 8 linkage (Lehman and Pratt, 1960). Glucosylation protects T4 DNA from several host-mediated restriction systems suggesting that it is involved in extending the host range of T4 (Revel, 1967; Revel and Georgopoulos, 1969) and it effects T4 gene expression on host strains with the giiflg_mutation (Snyder, 1972; Snyder and Montgomery, 1974). The giiflg mutation, a mutation that confers resistance to the anti- biotic rifampicin, causes an alteration in the 8 subunit of the host RNA polymerase. In addition, glucosylation may alter phage transcrip- tion directly, since it effects the template activity of T4 DNA ig vitro (Cox and Conway, 1973). in Transcription w The T4 genome can be separated genetically and biochemically into four transcriptional segments (Wood, 1974). Two segments are transcribed before replication and two after. Transcription of these segments depends on the host RNA polymerase which is modified during development. For the purposes of this discussion, T4 RNA will be separated into early and late RNA. The early RNAs are syn- thesized before DNA replication and code for products involved in T4 DNA replication, T4 DNA recombination, T4 host shutoff functions, and T4 late gene expression. The late RNAs are synthesized after the onset of DNA replication and they code for functions involved in maturation of DNA and synthesis of the virion. i,l Induced RNA Polymerase Modifications.--T4 uses the host RNA polymerase for its transcriptional program (di Mauro et al., 1969; Haselkorn et al., 1969). During the course of infection, the phage induces several modifications of the host RNA polymerase. These modifications include adenylation of the RNA polymerase and addition of T4 induced polypeptides to the RNA polymerase. Two separate adenylation reactions, termed "alteration" and "modification," are induced during infection. Both reactions result in the covalent addition of adenosine-phosphate moeities to sub- units of the host RNA polymerase (Goff, 1974; Seifert et al., 1971). In the absence of protein synthesis, alteration very rapidiy causes adenylation of subunits of the host RNA polymerase (Horvitz, 1974a; Seifert et al., 1969) in a reaction requiring the gig_gene product (Horvitz, 1974b). This product has been found to be part of the phage particle (Rohrer et al., 1975), so it is probably injected into the host along with T4 DNA. The second adenylation reaction, termed "modification", occurs approximately three minutes after infection (Horvitz, 1974a; Seifert et al., 1969). Modification requires the Egg gene product and results in further adenylation of the host RNA polymerase (Goff, 1974). ig.gigg, neither alteration nor modifica— tion is essential for T4 development (Horvitz,1974b). However, ig giigg_experiments suggest that either alteration or modification might be involved in the shutoff of some host transcription. For example, Mailhammer et a1. (1975) found that adenylation of the poly- merase reduces the transcriptional activity of the RNA polymerase. They proposed that the reduction in activity was sufficient to account for the shutoff of host transcription. Whether either the gii_or ggg_gene product is involved in the shutoff of host transcrip- tion ig_giig_is not known. A number of T4 induced polypeptides bind to the RNA poly- merase of the host. As an example, Ratner (1974a) found that several T4 induced polypeptides bound to RNA polymerase immobilized on a column. Among these polypeptides were the gene 45, 33, and 55 pro- ducts, all of which are required for T4 late gene expression. Employ- ing more stringent purification criteria, Stevens (1972) reports that four T4 induced polypeptides bind tightly enough to copurify with the RNA polymerase. These polypeptides have been numbered 1, 2, 3 and 4 in the order of decreasing molecular weight. Of these polypeptides, polypeptides number 1 and number 3 have been identi— fied as the products of genes 55 and 33 respectively (Horvitz, 1973; Ratner, 1974; Stevens, 1972).. Both of these products are required for T4 true late expression, so, presumably, they alter the specifi- city of the host RNA polymerase. Recently, polypeptide number 2 of Stevens (1972) has been found to be associated with the T4 gig_gene. The gig gene of T4 is involved in shutting off transcription from host DNA and T4 cytosine DNA. Sirotkin et al. (1977) have reported that polypeptide number 2 was missing from the RNA polymerase of cells infected with an gig_mutant. However, the relationship of the gig gene with polypeptide number two is not known. At present, poly— peptide number 4 of Stevens (1972) has not been associated with any T4 gene. Regulation of T,I Early Transcription.--The early transcripts of T4 are synthesized asymmetrically from T4 DNA originating predomi- nantly from the i_stand of DNA (Guda et al., 1971; Notani, 1973). Based on the kinetics of transcription, the early transcripts have been separated into immediate early, delayed early, and quasi-late transcripts (Salser et al., 1970). Immediate early RNA is synthesized at the beginning of infec- tion and its synthesis continues until it is shut off during T4 DNA replication (O'Farrell et al., 1973; Salser et al., 1970). Imme~ diate early transcription proceeds in the absence of T4 protein synthesis. Thus, it is resistant to chloramphenicol added at the time of infection (Lemback and Buchanan, 1970; Patterson et al., 1972; Salser et al., 1970). Furthermore, immediate early transcrip- tion is resistant to the antibiotic rifampicin added one minute after infection (O'Farrell and Gold, 1973a). O'Farrell and Gold (1973a) have concluded that the immediate early promoters are recog- nized almost immediately after infection. In support of these obser- vations are reports that transcription of the immediate early T4 genes ig_iiggg_resembles immediate early transcription ig_!igg, Both asymmetric transcription of the immediate early genes and resistance to the antibiotic rifampicin added after the start of transcription can be demonstrated ig_iiigg_(Brody and Geiduschek, 1970; Black and Gold, 1971; O'Farrell and Gold, 1973b; Milanesi et al., 1969). The delayed early transcripts first appear 1.5 minutes after infection at 30°C (Salser et al., 1970) and are separable into two classes. One class is like the immediate early transcripts because it is shut off during phage replication, while the other class of delayed early transcripts is different because it is synthesized throughout infection (Salser et al., 1970; O'Farrell et al., 1973). Transcription of both classes of genes is blocked by inhibitors of protein synthesis, such as puromycin and chloramphenicol, added at the time of infection (Grasso and Buchanan, 1969; Lembach and Buchanan, 1970; Petterson et al., 1972; Salser et al., 1970). In addition, prior amino acid and potassium starvation block delayed early transcription (Boros and Witmer, 1975; Petterson et al., 1972). One interpretation of these observations is that delayed early tran— scription requires a T4 gene product. However, other experiments suggest that at least some delayed early transcription is indepen- dent of T4 protein synthesis. For example, both Black and Gold (1971) and Lemback and Buchanan (1970) report that some delayed early transcripts are synthesized in the presence of amino acid analogues which presumably cause protein dysfunction. As an alter- native to a T4 gene product being required for delayed early transcrip- tion, Black and Gold (1971) proposed that the inhibition of protein synthesis causes premature termination of T4 transcription. The precedent for this is a report that chloramphenicol induces premature termination of transcription of the tryptophan operon (Morse, 1971). Whether chloramphenicol, puromycin, potassium starvation, and amino acid starvation cause premature termination of T4 transcription has not been directly tested. Transcriptional termination aside, several reports suggest that some delayed early transcripts are synthesized as part of large polycistronic RNA molecules containing immediate early sequences. For example, Black and Gold (1971), Brody and Geiduschek (1970), and Milanesi et a1. (1969) report that some delayed early transcripts contain immediate early sequences. In accord with these experiments, Sauerbier et al. (1970) report that the ultraviolet light target size of the delayed early genes was greater than might be expected if the promoters for the delayed early transcripts were located close to the delayed early genes. Furthermore, O'Farrell and Gold (1973a) found that the promoters for some delayed early RNAs were recognized almost immediately after infection and Milanesi et a1. (1970) and Black and Gold (1971) found that the promoters for some delayed early RNAs could be physically separated from the delayed early genes by shearing. In summary, a number of experiments suggest that a subclass of delayed early genes are regulated like the immediate early genes and are more than likely transcribed by extension of the immediate early RNAs. The other subclass of delayed early genes is apparently transcribed indepen- dently of the immediate early genes suggesting that it has a differ- ent regulatory control. The third class of early RNA, the quasi-late RNA, first appears in low amounts after 1.5 minutes of infection at 30°C. Like some delayed early transcripts, this class is synthesized throughout infection (O'Farrell and Gold, 1973a) making it kinetically analogous to a subclass of delayed early RNAs. The distinctive feature of quasi-late transcription is its sensitivity to the antibiotic rifampicin added one minute after infection. By following the syn- thesis of T4 gene products with polyacrylamide gel electrophoresis, O'Farrell and Gold (1973a) observed that the quasi-late gene prod- ucts failed to appear when rifampicin was added one minute after infection at 30°C. In contrast, the immediate early and some delayed early gene products were refractive to the same antibiotic treatment. They concluded that the promoters for the immediate early transcripts and for some delayed early transcripts were used almost immediately after infection. while the quasi-late promoters were used after one minute of infection. In addition, they proposed that the quasi-late promoters were made accessible or opened by 10 transcription of the immediate early and certain delayed early genes. In the past, several limitations have restricted the investi- gation of T4 early transcription. One limitation has been the lack of a suitable ig_yiigg_system for classes of some early transcription. For example, only recently has a suitable system been developed for quasi-late transcription ig_giggg_(Thermes et al., 1976). A second limitation has been the dearth of genes which have been shown to alter early transcription. Only one gene, termed Egg, has been shown to reduce delayed early transcription (Mattson et al., 1974). Clearly, the discovery of additional mutations affecting early tran- scription and more useful iguiiggg_systems would be helpful in understanding the regulation of early transcription. Regulation of T4 Late Transcription.--Late T4 RNA is synthe- sized after the start of DNA replication. This RNA synthesis begins approximately seven minutes after infection at 30°C and new RNA sequences appear that are not synthesized early. Unlike the early transcripts, the late transcripts originate predominantly from the .g strand of DNA (Guda et al., 1971; Notani, 1973). These molecules can be separated into quasi-late RNA, anti-late RNA, and true late RNA. A discussion of quasi-late RNA has been presented above. Anti-late RNA first appears approximately two minutes after infection at 30°C and is synthesized from the i strand of DNA making it complementary to the true late RNA (Notani, 1973). As yet, the function of anti-late RNA is unknown, but its regulation is similar 11 to those delayed early transcripts which are analogous to the quasi- late transcripts. For example, Frederick and Snyder (1977) report that anti-late transcription was blocked by rifampicin added one min- ute after infection and it was altered by a mutant in the Egg gene. Normal true late transcription requires continuous T4 DNA replication, a number of T4 gene products, and the unusual base hydroxymethylcytosine. The dependence of true late transcription on continuous replication has been investigated with temperature sensitive phage DNA polymerase mutations (Riva et al., 1970a). When shifted to a non—permissive temperature, both DNA replication and true late transcription abruptly stop suggesting that T4 true late transcription was coupled to T4 DNA replication. A number of years ago, Riva et al. (1970b) and Cascino et al. (1970, 1971) found that true late transcription could be uncoupled from viral DNA replica- tion and reported that uncoupling requires a T4 DNA ligase mutation and a mutation in a T4 induced exonuclease. Since these mutations might be expected to stabilize gaps, they pr0posed that the tem- plate involved in T4 late transcription contained gaps. Furthermore, they proposed that replication was required to continuously synthe- size the gapped template. Very recently, Sirotkin et al. (1978) reported that the T4 induced 3' phosphatase-5' polynucleotide kinase, an enzyme that might be expected to act at gaps, was required for true late gene expression on certain §i_ggii_strains, again, sup- porting the role of gaps in T4 true late transcription. Besides a 12 competent template, a number of T4 gene products are essential for T4 true late transcription. One essential gene product is the gene 45 product (Epstein et al., 1963; Bolle et al., 1968). This gene product binds to the host RNA polymerase (Ratner, 1974) and is continuously required for both T4 DNA replication and true late transcription (Wu and Geidushek, 1975) suggesting that some of the replication machinery is involved in T4 true late transcription. Besides the gene 45 product, the gene 33 and gene 55 products, although not required for DNA replication, are required for true late transcription (Bolle et al., 1968; Epstein et al., 1963; Pulitzer and Geidushek, 1970). Both of these gene products bind tightly to the host RNA polymerase (Horvitz, 1973; Stevens, 1972) and they may alter the specificity of the host RNA polymerase. Regardless of their function, normal true late transcription requires the unusual base S-hydroxymethylcytosine (Kutter et al., 1975; Wu and Geidushek, 1975). The base is not required for normal length viral replica- tion (Kutter et al., 1975), but it has a direct role or roles in T4 true late transcription. One role is protective because it blocks the T4 gig_gene mediated shutoff of transcription from cytosine T4 DNA (Snyder et al., 1976). Also, some hydroxymethylcytosine may be directly required for T4 transcription (Morton et al., 1978). Perhaps the greatest limitation to the investigation of T4 late transcription has been the development of suitable in__v_i_t_r_9_ systems. Currently, only crude ig_vitro systems are available for 13 examining late transcription (Snyder and Geiduschek, 1968; Rabussary and Geiduschek, 1979) As a consequence, the template necessary for true late transcription, the activities of the T4 gene products, and the function of hydroxymethylcytosine all remain ill defined. Synthesis of i4 With Cytosine T,I DNA.--T synthesizes DNA 4 containing 5-hydroxymethylcytosine. The primary mechanism excluding cytosine from T4 DNA involves a viral induced dCTPase. The enzyme is responsible for degrading both dCTP and dCDP to dCMP (Warner et al., 1966) and is coded for by the T4 gene 56 (Wiberg, 1967). Because of this activity, dCTP is effectively removed from the nucleotide triphosphate pool used for DNA replication. With a muta- tion in gene 56, some cytosine will be incorporated into T DNA. 4 However, most, if not all, cytosine T DNA is degraded by T4 induced 4 nucleases involved in degrading host DNA (Kutter and Wiberg, 1968). These nucleases consist of two phage encoded endonucleases that fragment cytosine DNA and an exonuclease that degrades the fragments (Kutter et al., 1975; Kutter and Wiberg, 1969; Wu and Geiduschek, 1975). With mutations in the genes responsible for these nucleases, T4 will synthesize cytosine containing T4 DNA (Kutter et al., 1975;! Kutter and Wiberg, 1968; Wu and Geiduschek, 1975), some of which is of normal length (Kutter et al., 1975). Even though apparently normal cytosine containing T4 DNA is made, no phage are produced because no true late gene products are synthesized (Kutter et al., 1975; Wu and Geiduschek, 1975). 14 When T4 replicates with cytosine in its DNA, spontaneous mutations accumulate that allow T4 to develop with cytosine T4 DNA (Snyder et al., 1976; Takahashi et al., 1979; Wilson et al., 1977). These mutations, called gig mutations, allow T4 development with cytosine DNA because they permit almost normal T4 true late gene transcription from cytosine T4 DNA (Snyder et al., 1976). Therefore, the normal gig gene product must block transcription from cytosine containing T4 DNA. At present, it is not known if the gig_gene product affects early T4 transcription nor is its of action under- stood. In regards to the synthesis of T4 with cytosine DNA, it has been reported that at least some hydroxymethylcytosine is required for T4 development (Snyder et al., 1976; Morton et al., 1978) which may, or may not, be significant. i4 Induced Alterations of Host Nficleic Acid Metabolism During the course of infection, a number of gene products are induced that alter host nucleic acid metabolism. Examples of these alterations include disruption of the host nucleus, degrada- tion of the host nucleus, unfolding of the host nucleoid, and shut off of host protein synthesis. Several of the T4 gene products responsible for altering the host nucleic acid metabolism are related to the gig gene product. Therefore, it is pertinent to review some relevant T4 gene products. Nuclear Disruption.--In the uninfected host, the DNA, or chromosome, is located in the middle of the cell. After five 15 minutes of T4 infection, the chromosome position has changed from its central location to a more peripheral position adjoining the cell wall (Luria and Human, 1950; Murray, 1950). This T4 induced phenomena, termed "nuclear disruption," occurs in the absence of the phage nucleases that degrade host DNA, but requires viral gene expression (Snustad et al., 1972). Several years ago, Snustad et al. (1974a) isolated mutants which fail to disrupt the nucleus. These mutations, termed Eggf mutants, fail to induce the change in chromosome position (Snustad et al., 1974b). Nggf mutants have two additional phenotypes which may be related to the failure to disrupt the nucleus. One pheno- type is the failure of Eggf mutants to shut off host DNA replica- tion normally and the second phenotype is enhancement of T4 induced host degradation (Snustad et al., 1976a). As yet, it is not known whether the ggg_gene product affects either cytosine T4 DNA repli- cation or T4 gene expression from cytosine containing DNA. However, it should be pointed out that 49g? T4 synthesize normal length cytosine T4 DNA (Kutter et al., 1975). Thus, the gene product does not completely block phage cytosine replication, but it might affect the kinetics of replication. Dggradation of Host DNA.--T4 degrades the DNA of the host to mononucleotides which are re-utualized for phage DNA replication. This process is not essential because mononucleotides are synthe- sized gg_novo after infection. Host DNA degradation is a two step process. During the first step, phage coded endonucleases degrade 16 host DNA into fragments, while during the second step a phage coded exonuclease degrades the fragments to mononucleotides. As mentioned above, the nucleases involved in the breakdown of host DNA are also involved in the breakdown of cytosine T4 DNA. Two different T4 induced endonucleases are involved in frag- menting host DNA. One endonuclease, termed endonuclease II, is the product of the ggg_A_gene (Hercules et al., 1971; Warner et al., 1970) and it has a major role in the breakdown of host DNA, while playing only a minor role in the breakdown of cytosine T4 DNA (Hercules et al., 1971; Kutter et al., 1975; Warner et al., 1970). As characterized ig_yiigg_by Sadowski and Hurwitz (1969), endonucle- ase II breaks native double stranded and single stranded §i_ggii_and Lambda DNAs into fragments with 3' OH and 5' P04 termini. The second endonuclease involved in the breakdown of host DNA, termed endonuclease IV, requires the ggg§_gene product (Bruner et al., 1972; Sadowski and Vetter, 1973) and has only a minor role in the break- down of host DNA, except when the infecting phage also has an Eggf mutation. However, in contrast to endonuclease II, endonuclease IV plays a major role in the breakdown of cytosine T4 DNA (Kutter et al., 1975; Wu and Geiduschek, 1975). ig_yiigg, the nuclease breaks native double stranded and single strand E. coli and Lambda DNAs leaving fragments with 3' 0H and 5" P04 termini and it is ten times more active with single stranded gaps suggesting that it acts at gaps (Sadowski and Hurwitz, 1969b). 17 Exonuclease degradation of fragments is the second step in the breakdown of host DNA. Exonuclease breakdown depends on the gene 46 and the gene 47 products because mutations in either gene 46 or gene 47 fail to solubilize host DNA (Kutter and Wiberg, 1968; Wiberg, 1967). Not only do these gene products degrade host DNA. but they affect T4 DNA metabolism because mutations in either gene 46 or gene 47 cause premature termination of T4 DNA replication (Epstein et al., 1963). Unfolding the Host Nucleoid.--The chromosome of the unin- fected cell can be isolated as a highly folded complex called the nucleoid (Stonington and Pettijohn, 1971). The isolated complex has dimensions resembling those of the chromosome ig_yiyg_(Hecht et al., 1975) and contains approximately 80% DNA by weight with nascent RNA and protein accounting for the remainder (Stonington and Pettijohn, 1971; Worcel and Burgi, 1972). The DNA of the nucle- oid is arranged in supercoiled domains which are rotationally inde- pendent of each other (Pettijohn and Hecht, 1972; Worcel and Burgi, 1972). As an aside, some observations suggest that host DNA may be supercoiled ig_yiyg. Drlica and Snyder (1978) report that the drug coumermycin, a drug which inhibits the host DNA gyrase (Gellert et al., 1976a, 1976b), reduces the superhelical content of the DNA of the nucleoid suggesting that host DNA is supercoiled. In any case, the supercoiled domains of DNA are stabilized by a core which more than likely contains RNA. The RNA bound to the nucleoid is composed in part of nascent rRNA and mRNA, some of which be associated with 18 the core (Hecht and Pettijohn, 1976). When the RNA of the nucleoid is either released or destroyed, the supercoiled domains are destroyed causing the nucleoid to become much less compact (Petti- john and Hecht, 1973; Worcel and Burgi, 1972). One interpretation of these observations is that RNA stabilizes the supercoiled domains of the nucleoid. Other observations support this role for RNA in maintaining the nucleoid. For example, Dworsky and Schachter (1973) and Pettijohn and Hecht (1973) found that nucleoid is unfolded by rifampicin, again, suggesting that RNA has an important structural role. In contrast to RNA, the proteins of the nucleoid do not have an essential role in maintaining the supercoiled domains (Stonington and Pettijohn, 1971; Worcel and Burgi, 1971), although they may be involved in attaching the nucleoid to the cellular membrane (Portalier and Worcel, 1976). After T4 infection, the nucleoid becomes much less compact or "unfolded". As reported by Tutas et a1. (1974), the sedimentation coefficient of the nucleoid decreased from greater than 10005 to less than 3005 after T4 infection. Unfolding requires neither the T4 induced nuclear disruption nor the T4 nucleases which degrade host DNA (Tutas et al., 1974). Recently, mutations in the gig gene have been shown to be defective in unfolding the nucleoid (Sirotkin et al., 1977). Another mutation, termed ggi_39, also fails to unfold the nucleoid after infection (Snustad et al., 1976b). gig_mutations and ggi_39 map in the same region and are allelic, so they are more than likely mutations in the same gene (Sirotkin et al., 1977); Tigges et al., 19 1977). Thus, the normal gig gene product is required for unfolding the host nucleoid, although the mechanism for unfolding the nucleoid is not known. Shutting Off Host DNA Synthesis.--The bacteriophage T4 shuts off host DNA replication within the first five minutes of infection at 30°C (Duckworth, 1971; Nomura et al., 1966. Snustad et al (1976a) have shown that the Egg gene product is required for.the shutoff of host DNA synthesis. It is unclear how the Egg_gene product functions, nor is it clear whether the ggg_gene product is the only phage pro- duct which shuts off host DNA synthesis. In fact, some evidence suggests that a second viral mechanism inhibits cytosine DNA replica- tion. For example, Snustad et al. (1976a) report that host DNA repli- cation is blocked at ten minutes during gggf mutant infection. This delayed shutoff may represent a second shut off mechanism. Additional evidence, which is presented below, supports this interpretation. Since the ggg_function, and perhaps other functions, alters cytosine DNA metabolism, it might interact with cytosine T4 DNA replication and it may effect gene expression from cytosine DNA. Shutting Off Host RNA Synthesis.--Host RNA synthesis is blocked after T4 protein synthesis (Rouviere et al., 1968; Nomura et al., 1966; Terzi, 1967). Transcription of soluble RNA, ribosomal RNA and messenger RNA is shut off (Nomura et al., 1962; Hayward and Green, 1965). As first reported by Sirotkin et a1. (1977), the gig gene of T4 is involved in the shutoff of some host transcription. 20 They observed that gig mutants were defective in shutting off at least some host transcription. Recently, a similar observation was reported by Tigges et a1 (1977). At present, the mechanism for the giggmediated shutoff is not known, nor is it clear if the gig gene product blocks all host transcription or just some host tran- scription. Besides the gigfmediated shutoff, other T4 functions may shut off host RNA synthesis because even during an gig mutant infection some host RNA synthesis is shut off (Sirotkin et al., 1977; Tigges et al., 1977). Further support for alternate T4 RNA shutoff mechanisms from cytosine DNA will be presented below. Shutting Off Host Protein Synthesis.--T4 prevents the syn- thesis of host proteins after infection. Explanations for this shutoff include the possibilities that either the T4 induced tran- scriptional shutoff prevents host protein synthesis or, alterna- tively, a T4 coded function prevents translation of host mRNA. In support of the first explanation, Kaempfer and Magasanik (1967) report that after T4 infection the decay rate of B-galactosidase was similar to the rate of decay of B-galactosidase after actino- mycin 0 treatment. The authors conclude that the transcriptional shutoff by T4 probably accounts for the shutoff of host protein synthesis. Others have concluded that a T4 mechanism disrupts translation of host mRNA. For example, Rouviere et al. (1968) and Kennel (1970), from experiments similar to Kaempfer and Magasanik's, concluded that a T4 mechanism disrupts translation of host mRNAs. Recently, Svenson and Karlstrom (1976), in experiments analogous to 21 Kaempfer and Magasanik's, concluded that a T4 mechanism dependent on the multiplicity of infection disrupted host translation. In conjunction with these reports, Kennel (1970) showed that after T4 infection the mRNA for B-galactosidase was present, but excluded from polysomes suggesting that a phage function blocks translation. Experiments that are presented below provide additional support for a phage induced translational shutoff function. The Bacteriophage Lambda as a Model System for Investigating thegig Gene Product The gig gene product of T4 is interesting because it shuts off transcription from cytosine T4 DNA as well as some host tran- scription. This the gene product is involved in unfolding the host nucleoid and may be associated with a T4 coded RNA polymerase bind- ing protein. One approach to investigating the gig_gene product and its associated phenotypes is to investigate the effects of the gig_gene product on a more pliable model. The model selected was the bacteriophage Lambda. This bacteriophage was chosen because a great deal of genetic and biochemical information is available and because Lambda DNA is smaller and more tractable biochemically than either T4 DNA or §;.£Qli DNA. The bacteriophage Lambda is a temperate bacteriophage of E; ggii_with both a lysogenic life cycle and lytic life cycle. Only the lytic cycle will be reviewed because it more closely resembles the development of T4. For the purposes of this discussion, the lytic cycle will be separated into early and late periods. The 22 early period begins with the initiation of lytic development and terminates with the end of monomeric circle replication. This period includes the first fifteen minutes of lytic development at 37°C. The late period extends from the early period to cellular lysis which begins approximately 45 minutes after the initiation of lytic development at 37°C. The Early Period of Lambda Development During the course of the early period, Lambda DNA replicates as monomeric circles and RNA is synthesized from both strands of DNA by the host RNA polymerase (Takeda et al., 1969). The early period transcripts code for products that are responsible for Lambda media— ted recombination, Lambda DNA synthesis, Lambda late gene expression and Lambda late gene products. Based on the kinetics of early tran- scription, early RNA can be separated into immediate early RNA, delayed early RNA, and late RNA. Early Period Transcription.--Immediate early RNA is synthe- sized almost immediately following the initiation of lytic develop- ment. This RNA, originating from both strands of Lambda DNA, codes for the N_and gig_gene products (Kourilsky et al., 1968; Taylor et al., 1967). Immediate early RNA is controlled by two regions of Lambda DNA and Lambda gene products. One region of Lambda DNA is involved in regulating g strand transcription, while the other seg- ment is involved in regulating i strand transcription. Both control segments have promoter and operator sites. Mutations in either 23 promoter site cause a gigydominant reduction in transcription from the control segments both ig_yiig_and ig_yiigg_(Cohen and Hurwitz, 1968; Roberts, 1970; Taylor et a1, 1967), while other mutations in either operator cause gigfdominant constitutive transcription from the control segments (Sakakibara et al., 1971, 1972). In the absence of protein synthesis, immediate early transcription is limited to regions of DNA coding for the N_and gig_gene products (Heineman and Speigelman, 1970; Kourilsky et al., 1970; Kumar et al., 1969). Very recently, Salstrom and Szybalski (1979) identified three sites on Lambda DNA involved in the termination of immediate early transcrip- tion. Besides the control segments, at least two Lambda gene pro- ducts regulate immediate early transcription. Both the Lambda repres- sor protein and the gig_gene product block immediate early transcrip- tion by acting at the operators (Sakakibara et al., 1971; Takeda et al., 1975). The repressor physically binds to the immediate early operators preventing transcription (Ptashne and Topkins, 1968) as does the ggg_gene product (Folkmanis et al., 1976; Takeda et al., 1977). Delayed early RNA is synthesized from both strands of Lambda DNA beginning approximately two minutes after initiation of lytic development (Kourilsky et al., 1968; Taylor et al., 1967). These transcripts code for proteins that are involved in DNA replication, DNA recombination, and late gene expression. Delayed early tran- scription requires the Lambda N_gene product. Also, several observa- tions suggest that delayed early transcription is controlled directly by the immediate early control segments. These observations include 24 the reduction of delayed early transcription caused by immediate early promoter mutations (Cohen and Hurwitz, 1969; Nijkamp et al., 1970; Taylor et al., 1967) and constitutive delayed early tran- scription caused by immediate early operator mutations (Sakakibara et al., 1971, 1972). Furthermore, delayed early transcription is blocked by the Lambda repressor (Thomas, 1970; Wu et al., 1972) and by the gig_gene product (Takeda et al., 1975). Besides being con- trolled by the immediate early control segments, the delayed early transcripts contain immediate early sequences (Portier et al., 1972). One interpretation of these observations is that delayed early RNAs are initiated at the immediate early promoters and synthesized as part of large polycistronic mRNAs. After a series of transcription experiments ig_yiigg, Roberts (1969) proposed that the N_gene pro- duct was responsible for extending immediate early transcription into the delayed early genes. He also proposed that the N_gene product acts as an antagonist to [gg_dependent transcription ter- mination, thereby allowing extension of immediate early transcrip- tion. The EDQ factor of the host is involved in some types of tran- scription termination (Roberts, 1970; Korn and Yanofsky, 1976). A number of reports support the anti-termination model for the gene product activity. For example, Korn and Yanofsky (1976) and Das et al. (1976) report that NT mutants propagate lytically on §gA_mutant hosts. Host strains with §gfl_mutations have altered §gg_factors and are defective in termination of some transcription. Along with these observations, Adhya et al. (1974) report that the N_gene 25 product suppresses a Egg dependent termination signal in an inser- tion sequence. They conclude that the N_gene product acts at the promoters because the N_gene product suppressed Egg dependent ter- mination only when transcription was initiated from Lambda promoters and not when it was initiated from other promoters. Lambda late RNA is synthesized beginning approximately 8 minutes after initiation of lytic development. Late RNA is synthe- sized predominantly from the g_strand of DNA (Oda et al., 1969; Taylor et al., 1967) and codes for products involved in maturation of DNA and synthesis of the virion. Normal late transcription depends on a segment of Lambda DNA, the Q gene product, and DNA repli- cation. The segment involved in late transcription is different than the immediate early control segments (Thomas, 1970). Herskowitz and Singer (1970) fbund that a deletion of the segment of DNA caused a gigfdominant reduction of late transcription. They termed the segment 93g and concluded that it acts as a promoter for late tran- scription. Because deletion of pR§_pleiotropically reduces late transcription, they proposed that all of the late genes are tran- scribed as part of one large polycistronic mRNA. Supporting this hypothesis, are reports by Green (1970), who showed that an unusual amount of time was required for rifampicin inhibition of late tran- scription, and by Gariglio and Green (1973), who isolated an unuau- ally large polycistronic mRNA containing late RNA sequences. How- ever, studies on the polarity of amber mutations suggest that there are multiple promoters for late transcription (Murialdo and 26 Siminovitch, 1972). Promoter of promoters aSide, the Q_gene product is required for normal late transcription because g_mutants synthe- size reduced amounts of late RNA (Dove, 1966; Eisen et al. 1966; Joyner et al., 1966; Oda et al., 1969). Explanations for the failure of QT mutants to completely block late transcription include the pos- sibilities that either all Q_mutants are leaky or, alternatively, there are g_independent pathways for late transcription. Some experi- ments by Herskowitz and Singer (1970), which show that Q_independent transcription is regulated like delayed early transcription, support the second alternative. For the stimulatory effect of the Q_gene product on late transcription, Roberts (1975) proposed that Q_acts as an anti-terminator like the N_gene product. However, other modes of action are also possible. In addition to the g_gene product and 93g, Lambda DNA replication is required for normal late transcription. Replication at least increases the number of genomes available for Lambda late transcription (Stevens et al., 1970). But, normal late transcription may depend on, or be coupled to, continuous DNA repli- cation. Observations supporting this proposal include experiments by Takeda (1970) which showed that thymine deprivation blocked late transcription and experiments by Grzesiuk and Taylor (1977) which showed that late transcription was blocked in minicells. Whether Lambda phage replication coupled transcription is similar to T4 phage replication coupled transcription is not known. Early Lambda DNA Rgplication.--Upon initiation of lytic development, Lambda DNA first cyclizes (Ogawa and Tomizawa, 1968; 27 Young and Sinsheimer, 1968) and replicates as monomeric circles beginning approximately 8 minutes after initiation of lytic develOp- ment at 37°C (Carter et al., 1969; Ogawa and Tomizara, 1968; Young and Sinsheimer, 1968). Monomeric circle replication is initiated in the Q_gene of Lambda at a site on DNA termed ggi (Furth et al., 1977) which can be mutated resulting in cis-dominant reduction of monomeric circle replication (Dove et al., 1971; Rambach, 1973). Recently, ggi_has been isolated and sequenced (Denniston-Thompson et al., 1977). It contains palindromic sequences that may form hairpin loops involved in the initiation of replication. From this site, most monomeric circular replication proceeds bidirectionally (Schnos and Inman, 1970). The replication machinery responsible for DNA replication is composed in part of both host and phage pro- teins (Fangman and Feiss, 1969; Georgopoulos and Herkowitz, 1971) and the entire complex of proteins and DNA is associated with the cellular membrane (Hallick et al., 1969; Nishimota and Matubara, 1972). The two Lambda gene products essential to monomeric circular replication are the Q_and P_gene products (Eisen et al., 1966; Joyner et al., 1966). As yet, the role of both proteins is undefined, although they may be involved in nicking DNA (McMachen et al., 1975). The Late Period of Lytic Development The late period of lytic development consists of the matura- tion of Lambda DNA, synthesis of virus, and release of virus begin- ning around 45 minutes after initiation of lytic development. 28 During the late period, Lambda late transcription predominantes and Lambda DNA replicates as concatamers. Late Period Transcription.--During the course of the late period, immediate early RNA, delayed early RNA and late RNA are all synthesized. But, in contrast to the early period, immediate early transcription and delayed early transcription are drastically reduced during the late period. The Lambda function responsible for this reduction is the gig_gene product. The gig_product reduced immedi- ate early and delayed early transcription by binding to the operator sites of the immediate early control segments (Takeda, 1975; Folkmanis et al., 1976; Takeda et al., 1977). As a conseguence, Lambda late RNA synthesized from the g_strand predominants (Oda et al., 1969; Taylor et al., 1967). Late Lambda Replication.—-Late period Lambda DNA replicates as fast sedimenting concatameric DNA (Ogawa and Tomizawa, 1968; Smith and Skalka, 1966; Young and Sinsheimer, 1968). Concatameric molecules can be formed by either recombination or by DNA replica- tion (Enquist and Skalka, 1973; Greenstein and Skalka, 1975). The structures contain a mixture of linear concatamers, linear concata- mers with circular termini and concatameric circular molecules (Takahashi, 1974). To account for the variety of concatamers, it has been proposed that late period DNA replicates by a rolling circle mechanism, although other mechanisms are also feasible. Whatever the replication mechanism, the fast sedimenting DNA behaves 29 as if it contains single stranded regions because the replicative intermediate is more sensitive to shearing than linear DNA and it binds to columns which retain gapped DNA (Kiger and Sinsheimer, 1969). Consistent with the presence of single stranded regions is the extreme sensitivity of the replicative intermediate to the ggg_ BC_nuclease ig_yi!g, This nuclease must be inactivated by the Lambda ggg_gene product during the synthesis of concatameric DNA (Enquist and Skalka, 1973; Greenstein and Skalka, 1975). As yet, it has not been determined whether the single stranded regions have an essential role during develOpment. Whatever their role, concata- meric replication appears to be associated with the membrane (Hallick et al., 1959; Niskimato and Maturbara, 1972) and depends on a com- posite structure of host and Lambda proteins (Fangman and Feiss, 1969; Georgopoulos and Herskowitz, 1971. Part of the replicative complex contains the Q_and P_gene products (Eisen et al., 1966; Joyner et al., 1966), although the P_gene product is not essential for concatameric replication during the later stages of replication (Klinkert and Albercht, 1978). REFERENCES Adhya, S. M. Gottesman and B. De Crombruggle (1974). Release of polarity in E. coli by gene N of phage Lambda. Termination and anti- termination of transcription. Proc. Natl. Acad. Sci. U. S. A. 71. 2534- 2538. Black, L. W. and L. M. Gold. 1971. Pre- -replicative development of the bacterTophage T RNA6 and protein synthesis in vivo and ig_vitro. J. Mol. Biol. 365- 388. Bolle,.A., R. H. Epstein, W. Salser, and E. P. Geiduschek. 1968. Transcription during bacteriophage T4 development: Require- ments for late messenger synthesis. J. Mol. Biol. 33: 339- 362. Boros, A. and H. J. Witmer. 1975. Effect of chloramphenicol and starvation for an essential amino acid on the synthesis and decay of T4 bacteriophage-specific messengers transcribed from early and quasi-late promoters. Arch. Biochem. Biophys. 196: 415-427. Bruner, R., R. Souther, and S. Suggs. 1972. Stability of cyto- sine containing deoxyribonucleic acid after infection by certain T4 r11- 0 deletion mutants, J. Virol. 10: 88— 92. Brody, E. H. and E. P. Geiduschek. 1970. Transcription of the bacteriophage T4 template. Detailed comparison of ig vitro and ig_vivo templates. Biochem. 9: 1300-1309. Carter, 8. J., B. 0. Shaw, and M. G. Smith. 1969. Two stages in the replication of bacteriophage XDNA. Biochem. et Biophys. Acta. 195: 494-505. Cascino, A., S. Riva, and E. P. Geiduschek. 1970. DNA ligation and the coupling of late T4 transcription to replication. Cold Spring Harb Symp. Quant. Biol. 35: 213-220. Cascino, A., E. P. Geiduschek, R. L. Cafferato, and R. Haselkorn. 1971. T4 DNA replication and viral gene expression. J. Mol. Biol. 61: 357. 30 31 Cohen, S. N. and J. Hurwitz. 1968. Genetic transcription in bacteriophage X: Studies of X mRNA synthesis ig_vivo. J. Mol. Biol. 37: 387-406. ' Cox, G. S., and T. W. Conway. 1973. Template properties of glucose- deficient T-even bacteriophage. J. Virol. 12: 1279-1287 Das, A., P. Court, and S. Adhya. 1976. Isolation and characteriza- tion of conditional lethal mutants of §g_coli defective in transcriptional termination factor ghg, Proc. Natl. Acad. Sci. U.S.A. 73: 1959-1963. Denniston-Thompson, K., Moore, 0., Kruger, K. E., Furth., M. E., and F. R. Blattner. 1977. Physical structure of the replica- tion origin of bacteriophage Lambda. Science 198: 1051- 1056. di Mauro, E., L. Snyder, P. Marino, A. Lambertii, A. Coppo and G. P. Tocchini-Valentini. 1969. Rifampicin sensitivity of the components of DNA dependent RNA polymerase. Nature. 282: 533-537. Dove, W. F. Action of the Lambda chromosome. J. Mol. Biol. 19: 187—201. Dove, W. F., Inokuchi, and W. F. Stevens, 1971. Replication con- trol in phage Lambda. In the Bacteriophage Lambda. ed. by A. D. Hershey. Cold Spring Harb. Lab. 747-771. Drlica, Km and M. Snyder, 1978. Superhelical Escherichia coli DNA: Relaxation by coumermycin. J. Mol. Biol. 145-154? Duckworth, D. H. 1971. Inhibition of host deoxyribonucleic acic synthesis by T4 bacteriophage in the absence of protein synthesis. J. Virol. 8: 754-778. Dworsky, P. and M. Schaechter. 1973. Effect of rifampicin on the structure and membrane attachment of the nucleoid of Escherichia coli. J. Bact. 116: 1364-1374. Eisen, H. A., C. R. Fuerst, L. Siminovitch, R. Thomas, L. Lambert, L. P. Da Silva, and F. Jacob. 1966. Genetics and ph siology of defective lysogeny in K12: Studies of early mutan s. Virology 30: 222—241. Enquist, L. W. and Skalka. 1973. Replication of phage lambda DNA depends on the function of host and viral genes. 1. Inter- action of Egg, ggm_and rec. J. Mol. Biol. 75: 185-212. 32 Epstein, R. H., A. Bolle, G. M. Steinberg, E. Kellenberger, E. Boy de la Tour, R. Chevalley, R. 5. Edgar, M. Susman, G. H. Denhardt, and A. Lielausis. 1963. Physiological studies of conditional lethal mutation of bacteriophage T40. Cold Spring Harb. Symp. Quart. Biol. 28: 375-390. Folkmanis, A., Takeda, Y., Simuth, J., Gussin, G., and H. Echols. 1976. Purification and properties of a DNA-binding protein with characteristics expected for the gig_protein of bacteriOphage A. A repressor essential for lytic growth Proc. Natl. Acad. Sci. U.S.A., 73: 2249-2253. Fangman, W. L. and M. Feiss 1969. Fate of Lambda DNA in a bacterial host defective in DNA synthesis. J. Mol. Biol. 44: 103-116. Frederick, R. J. and L. Snyder. 1977. Regulation of anti-late RNA synthesis in bacteriophage T4. J. Mol. Biol. 114: 461-476. Furth, M. E. Blather, F. R., McLeester, and W. F. Dove. 1977. Genetic structure of the replication origin of bacteriophage Lambda. Science. 198: 1046-1051. ‘ Gariglio, P. and M. H. Green. 1973. Characterization of polycis- tronic late lambda messenger RNA. Virology. 53: 392-404. Gellert, M., K. Mizuuchi, M. H. O'Dea, and H. A. Nash. 1976a. DNA gyrase: An enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci. U.S.A. 73: 3872-3876. Gellert, M., M. H. O'Dea, T. Itoh, and J. Tomizawa. 1976b. Novo- biocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. U.S.A. 73: 4474- 4478. Georgopoulos, C. P. and I. Herskowitz. 1971. Ei_coli mutants blocked in Lambda DNA synthesis. In the Bacteriophage Lambda. ed. A. D. Hershey. Cold Spring Harb. Lab. New York, pp. 553-564. Goff, C. G. 1974. Chemical structure of a modification of the Escherichia coli ribonucleic acid polymerase induced by T4 infection. J. Biol. Chem. 249: 6181-6191. Grasso, R. J. and J. M. Buchanan. 1969. Synthesis of early RNA in bacteriophage T4 infected Escherichia coli 8, Nature. 224: 882-885. 33 Green, M. H., N. S. Hayward, and P. Gariglio. 1970. A method for localization of active promoters. Cold Spring Harbor Symp. Quant. Biol. 35: 295-303. Greenstein, M. and A. Skalka. 1975. Replication of bacteriOphage lambda DNA: ig_vivo studies of the interaction between the viral gamma protein and the host rec BC DNAse. J. Mol. Biol. 97: 543-559. Grzesiuk, E. and K. Taylor. 1977. Block between early and late transcription of coliphage in mini cells. Virology. 83: 329-336. Guda, A., W. Szybalski, W. Salser, H. Bolle. E. P. Geiduschek, and J. F. Pulitzer. 1971. Controls and polarity of transcrip- tion during bacteriophage T4 development. J. Mol. Biol. 59: 329-349. Hallick, L., R. P. Boyce, and H. Echols. 1969. Membrane association by bacteriophage DNA. Possible direct role of regulator gene N. Nature 223: 1239-1242. Haselkorn, R., M. Vogel, and R. D. Brown. 1969. Conservation of the rifampicin sensitivity during T4 development. Nature 221: 836-838. Hayward, W. S. and M. H. Green. 1965. Inhibition of Escherichia coli and bacteriophage Lambda messenger RNA synthesis by T4. Proc. Natl. Acad. Sci. U.S.A. 54: 1675-1678. Hecht, R. M. and D. E. Pettijohn. 1976. Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli. Nucleic Acids Res. 3: 767-788. Hecht, R. M., R. T. Taggart, and D. E. Pettijohn. 1975. Size and DNA content of purified E; coli nucleoids observed by fluores- cence microscopy. Nature 253: 60-62. Heineman, S. F. and W. G. Spiegelman. 1970. The role of the gene N product in phage Lambda. Cold Spring Harb. Symp. Quant. Biol. 35: 315-318. Hercules, K., J. L. Munro, S. Mendelshon and J. S. Wiberg. 1971. Mutants in a nonessential gene of bacteriophage T4 which are defective in the degradation of Escherichia coli deoxyribonucleic acid. J. Virol. 7: 95-105. Herskowitz, I., and E. Singer. 1970. Control from the g_strand of bacteriophage Lambda. Cold Spring Harb. Symp. Quant. Biol. 35: 355-368. 34 Horvitz, R. H. 1973. Polypeptide bound to the host RNA polymerase is specified by T4 control gene 33. Nat. New Biol. 244: 137-140. Horvitz, R. H. 1974a. A control of bacteriophage T of the two sequential phosphorylations of the alpha subunit of Escheri- chia coli RNA polymerase. J. Mol. Biol. 90: 727-738. Horvitz, R. H. 1974b. Bacteriophage T mutants deficient in altera- tion and modification of the alpha subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 90: 739-750. Joyner, A., L. H. Isasacs, H. Echols, and W. W. Szybalski. 1966. DNA replication and messenger RNA production after induction of wild type and mutant Lambda. J. Mol. Biol. 19: 1744- Kaempfer, R. O. R. and B. Magasanik. 1967a. Effect of infection with T even phage on the inducible synthesis of B-galastosi- dase in Escherichia coli. J. Mol. Biol. 27: 453-468. Kaempfer, R. O. R. and B. Magasanik. 1967b. Mechanism of B galac- tosidase induction in Escherichia coli. J. Mol. Biol. 27: 475-494. Kennel, D. 1970. Inhibition of host protein synthesis during infec- tion of Escherichia coli by bacteriophage T4. J. Virol. 6: 208-217. Kiger, J. A. and R. L. Sinsheimer. 1969. Vegetative Lambda DNA. IV. Fractionation of replicating Lambda DNA on benzoylated- naphtoylated DEAE cellulose. J. Mol. Biol. 40: 467-490. Kim, J. S. and N. Davidson. 1974. Electron microscope hetero- duplex study of sequence relation of 12, T4 and T6 bacterio- phage DNA. Virology. 54: 93-111. Klinkert, J. and A. Klein. 1978. Roles of A gene products Q_and P_during early and late phases of the infection cycle. J. Virol. 25: 730-737. Korn, L. J. and C. Yanofsky. 1976. Polarity supressors increase expression of the wild type tryptophon Operon of Eg_coli. J. Mol. Biol. 103: 395-410. Kourilsky, P., and M. F. Bourguignon, M. Bouquet, and F. Gros. 1970. Early transcription controls after induction of prophage A. Cold Spring Harb. Symp. Quant. Biol. 35: 305-314. 35 Kourilsky, P., L. Morcaud, P. Sheldrick, D. Luzzati, and F. Gros. 1968. Studies on the messenger RNA of bacteriophage X. Various species synthesized early after induction of the pro- phage. Proc. Natl. Acad. Sci. U.S.A. 61: 1013-1020. Kumar, 5., E. Calef, and W. Szybalski. 1970. Regulation of the tran- scription of Escherichia coli phage X by its early genes N_and 59:. Cold Spring Harb. Symp. Quant. Biol. 35: 331-339. Kutter, E., A. Beug, R. Sluos, L. Jensen, and 0. Bradley. 1959. The production of undegraded cytosine-containing DNA by bacterio- phage T4 in the absence of dCTPase and endonuclease 11, IV and its effect on T4 directed protein synthesis. J. Mol. Biol. 99: 591-607. Kutter, E. M. and J. S. Wiberg. 1968. Degradation of cytosine-con- taining bacterial and bacteriophage DNA's after infection of E; coli B with bacteriophage T 0 wild type and with mutants defective in genes 46, 47 and 6. J. Mol. Biol. 38: 395- 411. Kutter, E. M. and J. S. Wiberg. 1969. Biological effects substi- tuting cytosine for 5-hydroxymethycytosine in deocyribo- nucleic acid of bacteriophage T4. J. Virol. 4: 439-453. Lehman, I. R. and E. A. Pratt. 1960. On the structure of the glucosylated hydroxymethylcytosine nucleotides of coliphage T2, T4 and 16. J. Biol. Chem. 235: 3254-3259. Lembach, K. J. and J. M. Buchanan. 1970. The relationship of pro- tein synthesis to early transcriptive events in bacterio- phage T infected Escherichia coli B. J. Biol. Chem. 245: 1575-1587. Luria, S. E. and M. L. Human. 1950. Chromatin staining of bacteria during bacteriophage infection. J. Bact. 59: 551-560. Mailhammer, R., H. L. Yang, G. Reiness, and G. Zubay. 1975. Effects of bacteriophage T4 induced modification of Escherichia coli RNA polymerase on gene expression ig_vitro. Proc. Natl. Acad. Sci. U.S.A. 72: 4928-4932. Mattson, 1., J. Richardson, and D. Goodin. 1974. Mutant of bacterio- phage T D affecting expression of many early genes. Nature. 250: 4 -50. McMacken, R., S. Kessler, and R. Boyce. 1975. Strand breakage of phage X supercoiled in infected lysogens. 1. Genetic and biochemical evidence for two types of nicking processes. Virology. 66: 356-371. 36 Milanesi, G., E. N. Brody, and E. P. Geiduschek. 1969. Sequence of the ig_vitro transcription of T4 DNA. Nature. 221: 1014- 1016. Milanesi, G., E. N. Brody, O. Grau, and E. P. Geiduschek. 1970. Transcription of the bacteriophage T4 template ig_vitro: separation of "delayed early" from "immediate early“ trans- cription. Proc. Natl. Acad. Sci. U.S.A. 66: 181-188. Morse, D. E. 1971. Polarity induced by chloramphenicol and relief by ggfl, J. Mol. Biol. 55: 113-118. Morton, D., E. M. Kutter, and B. S. Guttman, 1978. Synthesis of T DNA and bacteriophage in the absence of dCMP hydrocymethy- lase. J. Virology. 28: 262-269. Murialdo, H., and L. Siminovitch. 1972. The morphogenesis of bacteriophage lambda: Identification of gene products and control of the expression of the morphogenetic information. Virology. 48: 785-823. Murray, R. G. E., D. H. Gillen, and F. C. Heagy. 1950. Cytological changes in §g_coli produced by infection with phage 12. J. Bact. 59: 603-615. Nijkamp, H. J. J., K. Bdure, and W. Szybalski. 1970. Controls of rightward transcription of coliphage X. J. Mol. Biol. 54: 599-604. Nishimoto, T. and M. K. Matubara. 1972. The correlation between transcription and membrane association of A DNA. Biophem. Biophys. Res. Comm. 46: 349-356. Nomura, M., K. Okamoto, and K. Asano. 1962. RNA metabolism in Escherichia coli infected with bacteriophage T4: Inhibition of host ribosomal and soluble RNA synthesis by phage and the effect of chloromycetin. J. Mol. Biol. 4: 476-387. Nomura, M., C. Witten, N. Montei, and H. Echols. 1966. Inhibition of host nucleic acid synthesis by bacteriophage T : Effect of chloramphenicol at various multiplicities. J. Mol. Biol. 17: 273-278. Notani, G. E. 1973. Regulation of bacteriophage T4 gene expression. J. Mol. Biol. 73: 231-249. O'Farrell, P. Z. and L. M. Gold. 1973a. Bacteriophage T gene expression: Evidence for two classes of prerepli ative cistrons. J. Biol. Chem. 248: 5502-5511. 37 O'Farrell, P. 2. and L. M. Gold. 1973b. Transcription and Trans- lation of prereplicative bacteriophage T4 genes jg_vitro. J. Biol. Chem. 248: 5512-5519. ~ O'Farrell, P. 2., L. M. Gold, and W. M. Huang. 1973c. The identi- fication of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 248: 5499-5501. Oda, K., Y. Sakakibara, and J. Tomizawa. 1969. Regulation of tran- scription of the X bacteriophage genome. Virology. 39: 901-918. Ogawa, H. and J. I. Tomizawa. 1968. Bacteriophage A DNA with different structures found in infected cells. J. Mol. Biol. 23: 265-276. Petterson, R. F., P. S. Cohen, and H. 2. Ennis. 1972. Properties of phage T4 messenger RNA synthesized in the absence of pro- tein synthesis. Virology. 48: 201-206. Pettijohn, D. E., and R. Hecht. 1973. RNA molecules bound to the folder bacterial genome stabilize DNA folds and segregate domains of supercoiling. Cold Spring Harb. Symp. Quant. Biol. 38: 31-41. Portalier, R. and A. Worcel. 1976. Association of the folded chromosome with the cell envelope of §i_coli: Characteriza- tion of the proteins at the DNA-membrane attachment site. Cell. 8: 245-255. Portier, M. M., L. Moncaud, A. Cohen, and F. Gros. 1972. Mechanism of transcription of the N_operon of bacteriophage A. Mol. Gen. Genet. 117: 72-81. Ptashne, M. and H. H0pkins. 1968. The operators controlled by the phage repressor. Proc. Natl. Acad. Sci. U.S.A. 60: 1282-1287. Pultizer, J. F. and E. P. Geiduschek. 1970. Function of T gene 55. 11. RNA synthesis by temperature-sensitive geng 55 mutants. J. Mol. Biol. 49: 489-507. Rmabach, A. 1973. Replication mutants of bacteriophage X: Charac- terization of two subclasses. Virology. 54: 270-277. Rabussary, D. and E. P. Geiduschek. 1979. Construction and pro- perties Of a cell free system for bacteriophage T4 late RNA synthesis. J. Biol. Chem. 254: 339-349. 38 Ratner, D. 1974. Bacteriophage T transcriptional control gene 55 codes for a protein bound To Escherichia coli RNA polymerase. J. Mol. Biol. 89: 803-814. Revel, H. R. 1967. Restriction of nonglucosylated T-even bacterio- phage: Properties of permissive mutants of E; coli B and K 12. Virology. 31: 688-701. Revel, H. R. and C. P. Georgopoulos. 1969. Restriction of Non- glucosylated T-even bacteriophage by prephage Pl. Virology. 39: 1-17. Riva, S., A. Cascino, and E. P. Geiduschek. 1970a. Coupling of late transcription to viral replication in bacteriophage T4 development. J. Mol. Biol. 54: 85-102. Riva, S., A. Cascino, and E. P. Geiduschek. 1970b. Uncoupling of late transcription from DNA replication in bacteriophage 14 development. J. Mol. Biol. 54: 103-119. Roberts, J. W. 1969. Termination factor for RNA synthesis. Nature. 1168-1174. Roberts, J. W. 1970. The y factor: Termination and anti-termina- tion in A. Cold Spring Harb. Symp. Quant. Biol. 35: 121- 127. Roberts, J. W. 1975. Transcription termination and late control in phage X. Proc. Natl. Acad. Sci. U.S.A. 72: 3300-3304. Roher, H., Zillig, W., and R. Mailhammer. 1975. ADP-Ribosylation of DNA-dependent RNA polymerase of Escherichia coli by an NAD+: Protein ADP ribosyltransferase from bacteriophage T4. Eur. J. Biochem. 60: 227-238. Rouviere,.J., J. Wyngaarden, J. Constant, F. Gros and A. Kepes. 1968. Effect of T4 infection on messenger RNA synthesis in Escherichia coli. Biochem. et Biophys. Acta. 166: 94-114. Sadowski, P. 0., and J. Hurwitz. 1969. Enzymatic breakage of deoxyribonucleic acid. I. purification and properties of endonuclease II from T phage-infected Escherichia coli. J. Biol. Chem. 244: 182-6191. Sadowski, P. D. and J. Hurwitz. 1969. Enzymatic Breakage of deoxyribonucleic acid. II. Purification and properties of endonuclease IV from T infected Escherichia coli. J. Biol. Chem. 244: 6192-6 98. 39 Sadowski, P. D. and D. Vetter. 1973. Control of T endonuclease IV by the 02a region of bacteriophage T4. Virology. 54: 544-546. Sakakibara, Y. and J.-I. Tomizawa. 1971. Regulation of transcrip- tion of Lambda phage operator mutants. Virology. 44: 463- 472. Sakakibara, Y. and J.-I. Tomizawa. 1972. Transcription and repli- cation of Lambda bacteriophage virulent derivatives. Virology. 47: 354-359. Salser, W. A. Bolle, and R. Epstein. 1970. Transcription during bacteriophage T4 development: A demonstration that distinct subclasses of the "early" RNA appear at different times and some are "turned off" at late times. J. Mol. Biol. 49: 271-295. Salstrom, J. and W. Szybalski. 1978. Transcriptional termination sites in the major leftward operon of coliphage Lambda. Virology. 88: 252-262. Sauerbier, W., R. L. Millette, and P. B. Hackett. 1970. The effects of ultraviolet irradiation on the transcription of T4 DNA. Biochem. Biophys. Acta. 209: 368-375. Schnos, M. and R. Inman. 1970. Position of branch points in repli- cating A DNA. J. Mol. Biol. 51: 61-74. Seifert, W., D. Rabussay, and W. Zillig. 1971. On the chemical nature of alteration and modification of DNA-dependent RNA polymerase of §g_coli after T4 infection. F.E.B.S. letters. 16: 175-179. Seifert, W., P. Oasba, G. Walters, P. Palm, M. Schachner, and W. Zillig. 1969. Kinetics of the alteration and modification of DNA-dependent RNA-polymerase in T4 infected E; coli cells. J. Bioch. 9: 319-324. Sirotkin, K., J. Wei, and L. Snyder. 1977. T4 bacteriophage-coded subunit blocks host transcription and unfolds the host chromosome. Nature. 215: 28-32. Smith, M. and A. Skalka. 1966. Some properties of DNA from X phage infected bacteria. J. Gen. Physiol. 49, suppl. 2: 127-142. Snyder, L. 1972. An RNA polymerase of Escherichia coli defective in the 14 viral transcription program. Virology. 50: 396- 403. 40 Snyder, L., L. Gold, and E. Kutter, 1976. A gene of bacteriophage T whose product prevents true late transcription of cyto- STnc-containing T4 DNA. Proc. Natl. Acad. Sci. U.S.A. 73: 3098-3102. Snyder, L. R. and D. L. Montgomery. 1974. Inhibition of T4 growth by an RNA polymerase mutation of Escherichia coli: Physio- logical and genetic analysis of the effect during phage development. Virology. 62: 184-196. Snustad, D. P. and L. M. Conroy. 1974a. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: Isolation and genetic characterization. J. Mol. Biol. 89: 663-673. Snustad, D. P., D. A. Parson, K. R. Warner, D. J. Tutas, J. M. Wehner and J. F. Koerner. 1974b. Mutants of bacteriophage T deficient in the ability to induce nuclear disruption. II: Physiological state of the host nucleoid in infected cells. J. Mol. Biol. 89: 675-687. Snustad, D. P., C. J. H. Bursch, K. A. Parson, and S. H. Hefender, 1976a. Mutants of bacteriophage T deficient in the ability to induce nuclear disruption: Shquff of host DNA and pro- tein synthesis, gene dosage experiments, identification of a restrictive host, and possible biological significance. J. Virol. 18: 268-288. Snustad, D. P., M. A. Tigges, K. A. Parson, D. J. H. Bursch, F. M. Carson, T. F. Koerner, and D. J. Tutos. 1976b. Identifi- cation and preliminary characterization of a mutant defec- tive in the bacteriophage T -induced unfolding of the Escherichia coli nucleoid. J. Virol. 17: 622-641. Snustad, D. P., and H. R. Warner, K. A. Parson, and D. L. Anderson. 1972. Nuclear disruption after infection of Escherichia coli and a bacteriophage T mutant unable to induce endo- nuclease II. J. Virol. l : 124-133. Stevens, A. 1972. New small polypeptides associated with DNA- dependent RNA polymerase Escherichia coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 69: 603-607. Stevens, W. F., Y. A. Saturen, and W. Szybalski. 1970. .Relationship between DNA replication and transcription in coliphage A. Fed. Proceed. 29: 466. Stonington, O. G. and D. E. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc. Natl. Acad. Sci. U.S.A. 68: 6-9. 41 Streisinger, G., J. Emrich, and M. M. Stahl, 1967. Chromosome struc- ture in phage T . III. Terminal Redundancy and length determination. Proc. Natl. Acad. Sci. U.S.A. 57: 292-295. Svenson, S. B. and O. H. Karlstrom. 1976. Bacteriophage 1 induced shutoff of host specific translation. J. Virology. 17: 326-334. Takahashi, H., M. Shimizu, H. Soito, and Y. Ikeda, 1979. Studies of viable T4 bacteriophage containing cytosine substituted DNA (T4dc Phage). Mol. Gen. Genet. 168: 49-53. Takahashi, S. 1974. The rolling circle replicative structure of bacteriophage X DNA. Biochem. Biophys. Res. Comm. 61: 657-663 Takeda, Y. 1971. Control of late messenger RNA synthesis during X phage development. Biochem. Biophys. Acta. 228: 193-201. Takeda, Y., Folkmanis, A., and H. Echols. 1977. gig_regulatory protein specified by bacteriophage X. J. Biol. Chem. 252: 6177-6183. Takeda, Y., K. Matsubara, and K. Ogata. 1975. Regulation of early gene expression in bacteriophage lambda: Effect of ggi. mutation on strand specific transcription. Virology. 65: 374-384. Takeda, Y., Y. Oyama, K. Nakajma, and T. Yura. 1969. Role of host RNA-polymerase for phage deve10pment. Biochem. Biophy. Res. Comm. 366: 533-538. Taylor, K., 2. Hradecna, and W. Szybalski, 1967. Asymmetric_distri- bution of the transcribing regions on the complimentary strands of coliphage DNA. Proc. Natl. Acad. Sci. U.S.A. 57: 1618-1625. Terzi, M. 1967. Studies on the mechanism of bacteriophage T4 inter- ference with host metabolism. J. Mol. Biol. 28: 37-44. Thermes, C., P. Daegelen, V. De Francisus, and E. Brody. 1976. ig_ vitro system for induction of delayed early RNA of bacterio- phage T4. Proc. Natl. Acad. Sci. U.S.A. 73: 2569-2573. Thomas, C. A. and I. Rubenstein 1964. The arrangement of nucleo- tide sequences in T and 15 bacteriophage DNA molecules. Biophy. J. 4: 93-i05. Thomas, R. 1970. Control of development in temperate phages. 111. Which prophage genes are and which are not transactivable in the presence of immunity? J. Mol. Biol. 45: 393-404. 42 Tigges, M., C. J. H. Bursch, and D. P. Snustad. 1977. Slow switch- over from host RNA synthesis after infection of Escherichia coli with T mutant defective in the bacteriophage T induced unfolding of the host nucleoid. J. Virol. 2 : 775-785. Tutas, D. J. Whener, J. M., and J. F. Koerner. 1974. Unfolding of the host genome after infection of Escherichia coli with bacteriophage T4. J. Virol. 13: 548-550. Warner, H. R. and S. E. Barnes. 1966. Evidence for a dual role for the bacteriophage T -induced deoxycytidine triphosphate nucleotidohydrolase. P 0c. Natl. Acad. Sci. U.S.A. 56: 1233-1240. Warner, H. R., D. P. Snustad, S. Jorgensen and J. F. Koerner. 1970. Isolation of bacteriophage T4 mutants defective in the ability to degrade host deoxyribonucleic acid. J. Virol. 5: 700-708. Wiberg, J. S. 1967. Amber Mutants of bacteriophage T defective in deoxycytidine diphosphatase and deoxycytidifie triphos- phatase. J. Biol. Chem. 242: 5824-5829. Wilson, G. G., V. I. Tonyashin, and H. Murray. 1977. Molecular cloning of fragments of bacteriophage T4 DNA. Mol. Gen. Genet. 156: 203-214. Wood, W. B. 1974. Bacteriophage T4, in Handbook of Genetics, ed. R. C. King, Vol. 1, pp. 327—331. Plenum Press, New York. Worcel, A., and E. Burgi. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 71: 127-147. Wu, A. M., S. Ghosh, H. Echols, and W. G. Spiegelman. 1972. Repression by the cI protein of phage A: ig_vitro inhibi- tion of RNA synthesis. J. Mol. Biol. 67: 407-421. Wu, R. and E. P. Geiduschek. 1975. The role of replicative proteins in the regulation of bacteriophage T4 transcription. J. Mol. Biol. 96: 513-538. Wyatt, G. R. and S. S. Cohn.' 1953. The bases of the nucleic acids of some bacterial and animal viruses: The occurence of 5- hydroxymethylcytosine. Biochem. J. 55: 774-782. ARTICLE I THE TRANSCRIPTION 0F CYTOSINE T4 DNA, 5-HYDROXYMETHYLCYTOSINE T4 DNA, AND CALF THYMUS DNA ifl_iiiflg BY T MODIFIED RNA 4 POLYMERASES 43 ABSTRACT When T4 replicates DNA using cytosine, no phage are produced because no T4 true late mRNAs are synthesized from cytosine T4 DNA. An explanation accounting for the dependence of T4 true late gene expression on hydroxymethylcytosine is that hydroxymethylcytosine is required as a template for transcription after T4 modification of the host RNA polymerase. To investigate this possibility, tran- scription from cytosine T4 DNA was examined ig_yiigg, The experi- ments demonstrated that the T4 modified RNA polymerase transcribed cytosine T4 DNA ig_iiggg, Thus, hydroxymethylcytosine is not required in the template for transcription. In addition, we found that cytosine T4 DNA is transcribed more efficiently than either hydroxymethylcytosine T4 DNA or calf thymus DNAs. 44 INTRODUCTION T4 DNA contains the base 5-hydroxymethylcytosine in place of cytosine (Wyatt and Cohen, 1953). This base protects T4 DNA from host restriction systems (Snyder et al., 1976) and is involved in normal T4 true late gene expression because no 14 true late gene products are synthesized when T4 replicates DNA with cytosine (Kutter et al., 1975; Wu and Geiduschek, 1975). One possible explanation accounting for this observation is that hydroxymethylcytosine protects against a T4 gene product which prevents transcription from cytosine DNA. Another possible explanation is that the base is required by the 14 modified RNA polymerase for transcription of T4 DNA. T4 uses (di Mauro et al., 1969; Haselkorn et al., 1969) and modifies the host RNA polymerase during infection. One form of modification is adenylation of the RNA polymerase and another form is addition of T4 induced polypeptides to the host RNA polymerase. Adenylation occurs in two separate reactidns which are dependent on the gig_and mgg_gene products (Horvitz, 1975a). Although neither adenylation reaction is required for T4 development (Horvitz, 1975b), adenylation of the polymerase reduces transcription from cytosine DNAs ig_yiigg_(Mailhammer et al., 1975). Besides adenylation, four T4 induced polypeptides bind tightly enough to c0purify with the host RNA polymerase (Horvitz, 1973; Stevens, 1972). These 45 46 polypeptides have been termed numbers 1, 2, 3 and 4 in the order of decreasing molecular weight (Stevens, 1972). Polypeptides #l and #3 have been identified as the products of T4 genes 55 and 33 (Horvitz, 1973; Stevens, 1972) and both are required for T4 true late transcription (Bolle et al., 1968; Epstein et al., 1963). Pre- sumably, both gene products alter the transcriptional characteris- tics of the host RNA polymerase enabling T4 true late transcription. Recently, another RNA polymerase binding polypeptide has been asso- ciated with a T4 gene named gig (Sirotkin et al., 1977). 14 gig mutants were isolated because they allow transcrip- tion from cytosine T4 DNA and, therefore, they permit production T4 phage containing cytosine. As might be expected, because the host has cytosine DNA, T4 gig mutants permit some host transcription after infection (Sirotkin et al., 1977; Tigges et al., 1977). In addition, gig mutants fail to "unfold" the host "nucleoid" (Sirotkin et al., 1977). The term "nucleoid" refers to the isolated compact complex of host nucleic acids (Stonington and Pettijohn, 1971). After T4 infection, the nucleoid normally becomes much less compact or "unfolded" (Tutus et al., 1974). Besides unfolding the nucleoid, the gig gene product may be related to an RNA polymerase binding polypeptide because the RNA polymerase of cells infected with an gig mutant lacks an RNA polymerase binding polypeptide (Sirotkin et al., 1977). This polypeptide corresponds to polypeptide #2 of Stevens (1972). 47 There are two interesting questions about transcription from cytosine T4 DNA. The first is, does the T4 modified host RNA poly- merase transcribe cytosine T4 DNA? The second is, do T4 induced modifications affect transcription from cytosine T4 DNA. One approach to answering these questions is to investigate the T4 modi- fied host RNA polymerase jg 21229- The following experiments show that the T4 modified RNA polymerase transcribes cytosine T4 DNA ig_giigg, Therefore, hydroxy- methylcytosine is not required by the modified RNA polymerase tran- scription for transcription of cytosine T4 DNA. In addition, the experiments show that more RNA is synthesized from cytosine T4 DNA than from hydroxymethylcytosine T4 DNA suggesting that hydroxymethy- cytosine directly impedes transcription. MATERIALS AND METHODS Bacteriophgge and Bacteria.--Four different bacteriophage strains were employed in the following experiments. One strain was wild type T40 and it was from a stock maintained in this laboratory. This second strain was an gig_mutant isolated in this laboratory by K. Sirotkin. The RNA polymerase of cells infected with this mutant fails to have the T4 induced RNA polymerase binding polypeptide #2 of Stevens (1972). The third strain was an giif mggf mutant T4. This mutant T4 was from the stock of R. Horvitz and it fails to adenylate the host RNA polymerase. The final strain was a multiple mutant T4 with gene 56', endo II', endo IV' and gig mutations and was constructed by K. Sirotkin. When this strain propagates on appropriate host strains, it will develop with cytosine in its DNA. The bacterial strains employed were E; ggiiBe (Su') from the stock of L. Gold and §i_coli 8834 (Su', r'm') from the stock of H. Revel. Synthesis of Cytosine T4 and 5-Hydroxymethylcytosine T4.-- For growing cytosine T4, the T4 strain with gene 56', endo II-, endo IV', and gig_ mutations was pr0pagated on 8834 in liquid cul- ture (Snyder et al., 1976). For growing hydroxymethylcytosine T4, wild type T40 was propagated on 8834 in liquid culture. 48 49 Purification of Bacteriophage.--Both hydroxymethylcytosine T4 and cytosine T4 were purified by procedures outlined by Pearson (this dissertation). Extraction of DNA.--DNA was extracted from purified bacterio- phage with cold phosphate buffered phenol (Bolle et al., 1968) and the concentration determined spectrophotometrically. ‘Mggigg.--The medium used for growing cells was M95. A recipe for M95 has been published by Snyder and Montgomery (1974). Isolation of RNA polymerases from Uninfected and T4 Infected .Cgii§,--§g.ggii B6 was grown at 37°C in M95 supplemented with 10 ug./ml. tryptophan to a cell concentration of 4x108 cells/ml. Two liters of cells were either mock infected or infected with T4 (m.o.i. 8) for 15 min. at 37°C. Fewer than 1% of the cells survived the infection. The infected cells were concentrated, resuspended in 50 m1. of Buffer A, and sonicated. Buffer A is 0.01 M Tris 7.9, 0.01 M MgClz,‘lmM EDTA, 0.1 mM dithiothreital, 5% glycerol. The method of Burgess (1969) was used to purify RNA polymerases. The procedure includes ammonium sulfate precipitation, DEAE cellulose chromotography, low salt glycerol gradient centrifugation and high salt glycerol gradient centrifugation. Peak fractions from the high salt glycerol gradients were dialyzed overnight against buffer A at 4°C. After dialysis, glycerol was added to 50% for storage. ig_vitro Transcription Assay.--The RNA polymerases were assayed with the method of Burgess (1969). ig vitro reactions were performed in 0.4 ml. solution (0.01 M Tris-Hcl pH 7.5, 0.01M MgCl) 50 containing 0.125 mM CTP, 0.125 mM ATP, 0.125 mM GTP, 0.1 mM UTP, lOuC 5' -H3 UTP, and 20 ug./ml. DNA. The reactions were performed at 37°C for 10 minutes. Following the incubation, the reactions were TCA precipitated, collected on filters and counted with toluene base scintilation fluid. Chemicals.--CTP, ATP, GTP, and UTP were purchased from Sigma and 5' -H3 UTP (l mCi./0.025 mg) was purchased from Schwarz/Mann. RESULTS in_yitro Transcription of Cytosine T4 DNA, 5-Hydroxymethylcytosine T4 DNA, and Calf Thymus DNA by the Bacterial and T4 Modified RNA Polymerases As a control for the wild type 14 modified RNA polymerase, we first decided to investigate the RNA polymerase of uninfected cells. The results of the experiment are presented in Figure 1A. It is clear from the data that the two types of T4 DNAs resemble each other as templates for transcription. This was not a supris- ing result because both DNAs contained T4 sequences. Also, the bacterial polymerase synthesizes less RNA from calf thymus DNA than from either of the T4 DNAs. This result is in accord with those reported by others (Bautz and Dunn, 1969). To examine the wild type T4 modified RNA polymerase, the RNA polymerase was extracted from cells 15 minutes after phage infec- tion. We choose this time because both adenylation of the polymerase (Horvitz, 1974a) and addition of the T4 induced polypeptides to the RNA polymerase are completed during this period (Stevens, 1972). After purification, the enzyme was studied in an experiment analogous to that done with the uninfected RNA polymerase. The results of the experiment are presented in Figure 18. The data demonstrate that unlike the uninfected polymerase, the wild type T4 modified poly- merase synthesizes more RNA from cytosine T4 DNA than from either 51 52 Figure l.--ig_yiggg transcription of cytosine, hydroxymethylcytosine and calf thymus DNAs by uninfected RNA polymerase (A) and wild type T4 modified RNA polymerase (B). _§g.ggii Be was grown and either mock infected (A) or infected with wild type T40 (B) as described in the text. The RNA polymerases were extracted and assayed as described in an earlier section. Each assay contained either cytosine T4 DNA (o———————o), hydroxymethylcytosine T4 DNA (o———————o), of calf thymus DNA (A—-—————A). Background counts fol- lowing incubation on ice were 300 c.p.m. and 168 c.p.m. for uninfected and T4 modified RNA polymerases respec- tively. Background counts after incubation without DNA were less than 98 c.p.m. In the figure, one relative unit of polymerase is equal to 10 ul. of enzyme solution added to the reaction. 53 A r b P H | 4 3 2 no. x 33693:. Ebro: .Edo IO 7.5 2.5 O o n C o L R. gm nu e m y z n o I E 9 mu m 9 Dn 0.4 B F P p _ 4 3 2 l Edd no. x 38898:. denim: . Relative Enzyme Units Figure l 54 5-hydroxymethylcytosine T4 DNA or calf thymus DNA. Therefore, 5- hydroxymethylcytosine is not directly required for transcription by the T4 modified RNA polymerase. Indeed, the wild type T4 modified RNA polymerase synthesizes less RNA from the T4 DNAs than did the uninfected RNA polymerase. Some of this result was in part expected because others have reported similar observations about the acti- vity of T4 modified RNA polymerases on 5-hydroxymethylcytosine T4 DNA and on calf thymus DNA (Bautz and Dunn, 1969). However, the activity of the modified RNA polymerase on cytosine T4 DNA was not expected because the uninfected RNA polymerase synthesized similar amounts of RNA on the two T4 DNAs. in Vitro Transcription of Cytosine T4 DNA, 5-Hydroxymethylcytosine I4 DNA and Calf Thymus DNA by the T4 alt' mod' Mutant RNA Polymerase and an gig_Mutant RNA Polymerase Explanations for the differences in activity of the T4 modi- fied RNA polymerase on the two types of T4 DNAs include the possi- bility that either some of the T4 induced modifications affect the activity of the modified RNA polymerase or, alternatively, hydroxy- methylcytosine directly impedes transcription. These alternatives can be investigated ig_yiigg_by employing T4 RNA polymerases that lack modifications. One 14 employed for these experiments was an gig: Egg:_double mutant. This mutant T4 fails to adenylate the host RNA polymerase during infection (Horvitz, 1974a). The second 55 T4 used was an gig mutant. After infection with this gig mutant, the RNA polymerase extracted from the cells lacks polypeptide #2 of Stevens (Sirotkin et al., 1977). Experiments analogous to that done with the wild type RNA polymerase were performed with these two T4 mutant polymerases. The results of the experiment with the gii:_mgg:_mutant RNA poly- merase are presented in Figure 2A and the results of the experiment with the gig mutant RNA polymerase are presented in Figure 28. The data demonstrate that both the gii:_mgg:_mutant and the gig mutant RNA polymerases synthesize more RNA from cytosine T4 DNA than from either hydroxymethylcytosine T4 DNA or calf thymus DNA. Also, similar amounts of RNA are synthesized from both hydroxymethyl- cytosine T4 DNA and calf thymus DNA by the T4 mutant RNA polymerases. It is clear from the data, that neither adenylation of the RNA polymerase nor addition of the gig mutant associated RNA polymerase binding polypeptide are responsible for the activity differences of the wild type T4 modified RNA polymerase on the two types of T4 DNA. 56 Figure 2.--ig-yiggg transcription of cytosine, hydroxymethylcytosine and calf thymus DNAs by giii'mgg:_mutant RNA poly- merase (A) and gig mutant RNA polymerase (B). E,_ggii Be was grown and either infected with gii:_mgg:_mutant 14 (A) or infected with gig_mutant T4 (8) as described in the text. The RNA polymerases were extracted and assayed as described earlier. Each assay contained either cytosine T4 DNA (0———————o), hydroxymethylcytosine T4 DNA (o—————o) or calf thymus DNA (A-———————A). Background counts fol- lowing incubation on ice were less than 180 c.p.m. and background counts after incubation without DNA were less than 100 c.p.m. As in Figure 1, one relative unit of polymerase is equal to 10 ul. of polymerase solution added to the reaction. 57 IO .5 7. s s .n .6." n n U U 3 e m m y y m m 5 E E 2 .m .m m m m .m. e II F. R m 5. 2 A B F C p p T — b b L 4 3 2 l 4 3 2 I no. x 33398:. denim: .Edd no. x 38898:. PSI»: .Edd DISCUSSION The experiments reported here demonstrate that the T4 modi- fied RNA polymerase transcribes cytosine T4 DNA. Thus, hydroxymethyl- cytosine is not a template requirement for cytosine T4 DNA tran- scription by the T4 modified RNA polymerase. As alternative expla- nations, either other undetected RNA polymerase modifications pre- vent transcription from cytosine T4 DNA ig_yiyg_or hydroxymethyl- cytosine protects against functions, such as the gig_gene product, that shut off cytosine DNA transcription. In addition, the experi- ments demonstrate that the T4 modified RNA polymerase synthesizes more RNA from cytosine T4 DNA than from hydroxymethylcytosine T4 DNA.~ This property of the T4 polymerase is probably not dependent on either adenylation of the RNA polymerase or addition of one of the T4 coded RNA polymerase binding polypeptides. However, it should be pointed out that the RNA polymerases were not examined for adenylated subunits nor the addition of T4 proteins to the poly- merase. Nevertheless, the results suggest that either other undetected T4 induced modifications are responsible for the differ- ences in activities of the polymerases on the DNAs or hydroxymethyl- cytosine directly impedes transcription. Because hydroxymethyl- cytosine is normally glucosylated, either the base itself or the glucose residues could be responsible. 58 59 All of the T4 modified RNA polymerases examined synthesized less RNA from the three types of DNAs than the unmodified RNA poly- merase. This result might have been anticipated, because others have shown that the T4 modified enzyme synthesizes less RNA from the three types of DNA than does the unmodified RNA enzyme (Bautz and Dunn, 1969). This loss of RNA polymerase synthetic activity after T4 infection has been correlated with the loss of some sigma subunits from the RNA polymerases during purification. Presumably, the loss of some of the sigma subunits during purification of the T4 modified RNA polymerases used in the experiments is responsible for the loss of activity of the T4 modified RNA polymerases on cytosine, hydroxymethylcytosine T DNA and calf thymus DNA reported 4 here. REFERENCES Bautz, E. K. F. and J. J. Dunn. 1969. DNA-dependent RNA poly- merase from phage T4 infected §i_coli: An enzyme missing a factor required for transcription of T4 DNA. Biochem. Bi0phys. Res. Comm. 34: 230-237. Bolle, A., R. H. Epstein, W. Salser, and E. P. Geiduschek. 1968. Transcription during bacteri0phage T deve10pment: Require- ments for late messenger synthesis. J. Mol. Biol. 33: 339-362. Burgess, R. R. 1969. A new method for the large scale purifica- tion of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid. J. Biol. Chem. 244: 6160-6167. di Mauro, E. L. Snyder, P. Marino, A. Lambertii, A. Coppo. and G. P. Tocchini-Valentini. 1969. Rifampicin sensitivity of the components of DNA dependent RNA polymerase. Nature. 282: 533-537. Epstein, R. H., A. Bolle, G. M. Steinberg, E. Kellenberger, E. Boy de la Tour, R. Chevalley, R. 5. Edgar, M. Susman, G. H. Denhardt, and A. Lielausis. 1963. Physiological studies of conditional lethal mutation of bacteriophage 140. Cold Spring Harb. Symp. Quant. Biol. 28: 375-390. Haselkorn, R., M. Vogel, and R. D. Brown. 1969. Conservation of the rifampicin sensitivity during T4 development. Nature 221: 836-838. Horvitz, R. H. 1973. Polypeptide bound to the host RNA polymerase is specified by T4 control gene 33. Nat. New Biol. 244: 137-140. Horvitz, R. H. 1974a. A control of bacteriophage T of the two sequential phosphorylations of the alpha subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 90: 727- 738. Horvitz, R. H. 1974b. Bacteriophage T mutants deficient in altera- tion and modification of the aIpha subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 90: 739-750. 60 61 Kutter, E., A. Beng, R. Sluos, L. Jensen, and 0. Bradley. 1975. The production of undegraded cytosine-containing DNA by bacteriophage T in the absence of dCTPase and endonucl- ease 11, IV and its effect on T4 directed protein synthesis. J. Mol. Biol. 99: 591-607. Mailhammer, R., H.-L. Yang, G. Reiness, and G. Zubay. 1975. Effects of bacteriophage T4 induced modification of Escherichia coli RNA polymerase on gene expression jg vitro. Proc. Natl. Acad. Sci. U.S.A. 72: 4928-4932. Sirotkin, K., and J. Wei, and L. Snyder. 1977. T bacteriophage- coded subunit blocks host transcription and unfolds the host chromosome. Nature. 215: 28-32. Snyder, L., L. Gold, and E. Kutter. 1976. A gene of bacteriophage T4 whose product prevents true late transcription on cyto- STne containing T4 DNA. Proc. Natl. Acad. Sci. U.S.A. 73: 3098-3102. Snyder, L. R. and D. L. Montgomery. 1974. Inhibition of T4 growth by an RNA polymerase mutation of Escherichia coli. Virol. 62: 184-196. Stevens, A. 1972. New small polypeptides associated with DNA- dependent RNA polymerase Escherichia coli after infection with bacteriophage T4. Proc. Nat. Acad: Sci. U.S.A. 69: 603-607. Stonington, O. G. and D. E. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc. Nat. Acad. Sci. U.S.A. 68: 6-9. Tigges, M., C. J. H. Bursch, and D. P. Snustad. 1977. Slow switch- over from host RNA synthesis after infection of Escherichia coli with a T mutant defective in the bacteri0phage T4 induced unfolding of the host nucleoid. J. Virol. 24: 775-785. Tutas, D. J. Wehner, J. M. and J. F. Koerner. 1974. Unfolding of the host genome after infection of Escherichia coli with bacteriophage T4. J. Virol. 13: 548-550. Wu, R. and E. P. Geiduschek. 1975. The role of replicative pro- teins in the regulation of bacteriophage T4 transcription. J. Mol. Biol. 96: 513-538. Wyatt, G. R. and S. 5. Cohen. 1953. The bases of the nucleic acids of some bacterial and animal viruses: The occurence of 5-hydroxymethylcytosine. Biochem. J. 55: 774-782. THE SHUTOFF 0F LAMBDA TRANSCRIPTION BY BACTERIOPHAGE T4 ROLE OF THE T gig GENE 4 Robert E. Pearson and Larry Snyder Department of Microbiology and Public Health Michigan State University East Lansing, MI 48824 Running Title: T4.Al£ and Lambda 62 ABSTRACT Bacteriophage T normally has 5-hydr0xymethylcytosine 4 instead of cytosine in its DNA. Mutants of T4 which synthesize DNA with cytosine do not transcribe their late genes due to the function of the T4 gig gene (Snyder et al., 1976) which is also responsible for shutting off at least some of the host transcription and for unfolding the host nucleoid after T infection (Sirotkin et al., 4 1977; Tigges et al., 1977). In order to examine how gig works, we have studied the affects of the T4 gig_function on X bacteriophage late transcription and on the structure of intracellular A DNA. After T4 superinfection, the T4 gig function shuts off the late transcription of X but does not affect the supercoiling of intra- cellular circular A DNA. Therefore, gig_does not unfold the nucle- oid by directly removing supercoils from DNA. We also conclude from these experiments that T4 infection alters the translational machinery in a way which causes almost 100% discrimination against non-T4 messenger RNA. Those late A mRNAs which are made after superinfection by a T4 gig mutant are of normal length but are not translated into A late proteins. 63 INTRODUCTION The shutoff of host RNA synthesis by bacteriophage T4 is an example of general genic control because many genes, regulated in many different ways, are affected. The molecular basis for the shutoff is not understood. Possible explanations include either altering the transcriptional specificity of the host RNA polymerase to prevent host transcription or altering the structure of the bacterial chromosome so it is no longer functional for transcrip- tion. Recently, a T4 gene involved in the shutoff of host tran- scription has been identified and named gig_(Snyder et al., 1976). T4 gig mutations were first isolated because they permit the syn- thesis of true late RNA from cytosine containing 14 DNA (Snyder et al., 1975). Thus, they allow the production of T4 with cytosine in place of the normal T4 base hydroxymethylcytosine (Snyder et al., 1976; Takahashi et al., 1979; Wilson et al., 1977). In contrast to wild type T T .gig_mutants permit the synthesis of at least 4’ 4 some host RNA after infection (Sirotkin et al., 1977; Tigges et al., 1977) which is perhaps expected, since the host DNA contains cytosine. Also, T4 gig_mutants often fail to "unfold" the host nucleoid after infection. To explain what is meant by unfolding the host nucleoid, from uninfected bacteria, the nucleoid of the 64 65 host can be isolated as a folded complex of nucleic acids and pro- teins (Stonington and Pettijohn, 1971). The structure of the nucleic acids in the nucleoid consists of DNA arranged in super- coiled domains around a core which probably contains RNA (Stonington and Pettijohn, 1971; Worcel and Burgi, 1972). Each supercoiled domain is stabilized by the core and is rotationally independent of every other supercoiled domain (Worcel and Burgi, 1972). After T4 infection, the host nucleoid becomes much less compact or "unfolded" (Tutas et al., 1974). A 14 mutation, ggf_39, prevents the unfolding of the nucleoid (Snustad et al., 1976b) and is allelic with gig_ mutations (Sirotkin et al., 1977; Tigges et al., 1977). Therefore, the gig gene product, which is required for the shutoff of at least some host transcription, is required for the unfolding of the host nucleoid after infection. Considering the role of RNA in nucleoid structure and the possible dependence of some types of transcription on nucleoid structures, it seems likely that the above phenotypes are causally related. There are two questions we would like to answer concerning the affects of the gig_gene product on transcription and nucleoid structure. The first question is, does the gig_gene function shut off all RNA synthesis from cytosine containing DNA or only those types of transcription analogous in some way to T4 true late RNA synthesis? The second question is, does the gig_gene product act either on the promoters for RNA synthesis or on the structure of DNA required for transcription? The first question could be examined 66 either by comparing the affects of the gig_gene product on early T4 RNA synthesis with the affects on T4 true late RNA synthesis, to see if only late transcription is affected, or by analyzing the affects of gig_mutations on the shutoff of specific host transcripts. There are difficulties with both approaches. For example, studying the affect of gig_product on T early RNA transcription from cytosine 4 DNA is complicated since the gig_gene product itself is an early function. To examine specific host transcripts, either individual E. coli genes must be isolated or care must be taken to assure that only one type of transcript is being studied. To answer the second question, how gig_acts, we could examine the affect of the gig_gene product on either the intracellular structure of T4 cytosine contain- ing DNA or host DNA. However, the intracellular structure of both DNAs is very complex. To avoid the problems mentioned above, we decided instead to investigate the action of the 14 gig gene product on A transcription and on the intracellular structure of the DNA of the bacteriophage X. It has been shown that T4 superinfection shuts off A transcription (Hayward and Green, 1965; Kennel, 1970) and gig_ could be involved. For simplicity throughout the following discussion, the lytic cycle of A will be segregated into two periods, early and late. The early period begins with the initiation of lytic develop- ment and extends through the first 15 minutes at 37°C. The late period extends from the end of the early period to cellular lysis. During the early period, A DNA replicates as monomeric circles 67 (Schnos and Inman, 1970; Ogawa et al., 1968; Young and Sinsheimer, 1968) and the transcripts synthesized, the "early" RNAs, code for products involved in viral recombination, viral DNA synthesis and late gene expression (Dove, 1966; Eisen et al., 1966; Joyner et al., 1966. At 15 minutes, the early RNA molecules are originating from both strands of the virus and can be separated into three classes: the immediate early, the delayed early, and the late RNA molecules (Oda et al., 1969; Taylor et al., 1967). During the late period, DNA replicates as concatamers (Takahashi, 1974, 1975; Young and Sinsheimer, 1968) and, like the early period transcription, three classes of RNA molecules are synthesized from both strands. How- ever, one class of transcripts now predominates: the late RNA molecules transcribed from the g_strand (Herskowitz and Singer, 1970; Nijkamp et al., 1970; Oda et al., 1969; Taylor et al., 1967). The following experiments demonstrate that the T4 gig gene product is required for the shutoff of A late period RNA synthesis, but does not alter the structure of closed circular supercoiled A DNA. We also found that those A late mRNAs which were being syn- thesized after infection by a T4 gig mutant were not being tran- slated into X proteins. This is direct evidence for a very effi- cient T4 translational shutoff function for non-T4 mRNA. MATERIALS AND METHODS Bacteriophage and Bacteria.--Two types of T bacteriophage 4 were used in the following experiments. One type, subsequently referred to as gigf T4, was constructed in this laboratory and was the parent of the gig mutant T4. The gigf parent was deficient in two phage endonucleases which nick cytosine containing DNA; in a function that degrades dCTP to dCMP; and in a function which shuts off host DNA synthesis. The T4 alterations responsible for the deficiencies are ng51_(dCTPase'), 5442_(end0 IV-, ndd-), and ggg_fi;_ (endo II'). The second type of T4, subsequently referred to as gig mutant T4, was gig_95. It was a spontaneous mutant derived from the gigf parent on 8834 galU56 (Runnels and Snyder, 1978) by a published procedure (Snyder et al., 1976) and it had all of the other muta- tions of the parent. The bacteriophage X has the mutations gi§§i and ggg_§i. The gi§§i mutation makes the prophage thermally inducible and gggD§i prevents lysis. Several different bacterial strains were used in the experi- ments to follow. E; coli 594 c1857susS7 was from the stock of Gerald Smith and is called 5655. §i_c01i 594 was an isolate of $655 cured of the prophage X. §i_coli A19 with a C01 II plasmid was from the stock of Robert Brubaker. E. coli DG75, from Worcel 68 69 and Burgi (1972), is leu‘, thy” and Ei_coli DG75 clB57susS7 is a X lysogen of DG75 which was constructed in this laboratory. Media and Buffer.--Three types of media were employed in the experiments. A recipe for M9 media was given previously by Snyder and Montgomery (1974). M9A is M9 medium supplemented with 10 ug./m1. of thiamine and 50 ug./ml. of each amino acid except methionine. KMT is KM medium of Ross and Howard-Flanders (1977) supplemented with 10 mg./ml. of tryptone (Difco). Lambda buffer was described by Ross and Howard-Flanders (1977). Purification of Bacteriophage.--To purify X, a A lysogen (5655) was induced with heat; concentrated; lysed with lysozyme and chloroform; and treated with RNAse and DNAse. To purify T4, a lysate was concentrated at 4°C with polyethylene glycol (10% w/v) at 0.5 M NaCl. Both X and T4 were further purified on cesium chlo- ride step gradients. After centrifugation, X was dialyzed in the cold against two changes of A buffer. 14 was dialyized against successive changes of NaCl (2M-lM-O.5M-0.25M) followed by a final dialysis against M9 salts with M9504. Radioisotope labelling of T,I infected induced iysoggns.-- 3 A similar protocol was employed for labelling RNA with 5-H uridine, DNA with methyl-H3 thymidine, and proteins with Sgg-methionine. After the cell concentration had reached 4 X 108 cells/m1., a lysogen and the strain cured of the prophage were heat pulsed at 70 42°C for 15 minutes. Aliquots of cells were infected with T (m.o.i. 4 of 12) at either an early time (10 min.) or a late time (50 min.) after the initial elevation of temperature. Radioisotope was added 4.5 min. after T4 infection and the incorporation was stopped 3 min. later with ice. The efficiency of T4 infection was monitored by the survival of the non-lysogen in the culture 2 min. after T4 infection, since the X lysogen will be killed by the induction of X. Unless otherwise noted, fewer than 1% of the non-lysogen survived the T4 infection. Nucleic acid extractions.--RNA was extracted with hot phenol by the technique of Frederick and Snyder (1977). Concentrations were determined spectrophotometrically (Bolle et al., 1968). DNA for hybridization was extracted from CsCl purified bacteriophage with phosphate buffered phenol in the cold (Bolle et al., 1968). Pulse labelled DNA was extracted by TCA precipitation, boiling in NaOH and neutralization with HCl (Oda et al., 1969). Nucleic acid hybridizations.--RNA-DNA hybridizations were performed in 0.01M Tris-citrate pH 7.5 with 0.25M EDTA pH 7.5, 0.5M NaCl, and 10% of saturated phenol. After five hours at 60°C, the hybrids were diluted with 0.5M NaCl, 0.2M EDTA pH 7.5; treated with RNAse A; collected on nitrocellulose membrane filters (Schleicher and Schuell); and washed with 0.5M NaCl, 0.2M EDTA pH 7.5 (Wu et al., 1972). RNA-DNA hybridizations of sucrose gradient fractions were performed in 6mM Tris-HCl pH 7.5. with 2XSSC (0.30M NaCl, 71 0.03 M Na Citrate). Hybrids were treated with RNAse A, collected on nitrocellulose membrane filters, and washed with 0.5M KCl, 0.01M Tris Cl pH 7.5 (Bolle et al., 1968). DNA-DNA hybridizations were done on nitrocellulose membrane filters by the methods of Warnaar and Cohen (1956). Protein slab gel electrophoresis.--Samples were treated as described by Frederick and Snyder (1977) and 12% slab gel electro- phoresis was performed according to Studier (1973). The gels were stained with Comassie Blue to ensure that the total protein per column was uniform. Preparing T4 infected cells containing supercoiled Lambda DNA,--A A lysogen ($655) was used to repress the lytic development of the superinfecting X and allow the accumulation of supercoiled circular A DNA. Cells were grown in KMT at 32°C to 4 X 108/m1., 9 cells/ml. in 1.5 ml. of A buffer centrifuged, and resuspended at 10 with H3-thymidine labelled X (m.o.i. of 5). After 20 min., the superinfected lysogen was diluted with 13.5 ml. of KMT and incubated at 32°C for an additional 40 min. before infecting with T4. The conditions for infecting 5 ml. aliquots with T4 were the same as those employed for infecting a heat induced A lysogen. Six min. after T4 infection, the cells were chilled and washed twice with 10 mM Tris-HCl pH 8 and resuspended in 0.2 ml. solution of 12% sucrose in 10 mM Tris-HCl, 0.25 mM EDTA pH 7.5. 72 The following procedures, which are similar to those of Ross and Howard-Flanders (1977), were used to lyse the cells. First, 0.1 ml. of a lysozyme solution (1 mg./ml. lysozyme in 10 mM Tris- HCl pH 8) was added. The extract was incubated for 10 min. on ice. Proteinase K (0.05 ml. of 1 mg./m1. preincubated for l h. at 37°C) and sarkosyl (0.2 ml. of a .5% solution V./V.) were added to the extract before incubating at 37°C for 1 hr. A portion of the lysate (0.2 ml.) was subjected to centrifugation as described below. Preparation of C01 II DNA.--A supercoiled DNA marker was prepared by the method of Blair et a1 (1971). An overnight culture of A19 was diluted one to twenty into 10 ml. of M95 supplemented ‘4 thymidine. After 3 h. of incubation at 37°C, with 10 uCi . methyl-C solid chloramphenical (Sigma) to a final concentration of 150 ug./ m1., and 10 uCi. methyl-C14 thymidine were added before continuing the incubation for another hour. The supernatant fraction from a Brij-deoxycholate lysate of the labelled cells was added to each centrifugation tube. Ethidium bromide-cesium chloride-gradient centrifugation.-- Density gradient centrifugation was performed as follows. Each 5 m1. gradient was made up in 10 mM Tris, 2.25 mM EDTA pH 7.5 and contained 0.33 mg./ml. ethidium bromide with cesium chloride to a final density of 1.54 g./ml. Centrifugation was carried out in a Beckman SW 50.1 rotor for 60 h. at 35,000 r.p.m. at 15°C. 73 Fractions of approximately 0.075 ml. were collected after centrifu- gation from the bottom of the tube and counted in PCS (Amersham/ Searle). Chemicals and Enzymes.--Amino acids, ethidium bromide, and S35 sucrose (RNAse free) were purchased from Sigma. -methionine was purchased from Amersham/Searle. Cesium chloride (optical grade), methyl-C14 thymidine, methyl-H3 thymidine, and 5-H3 uridine were all purchased from Schwarz/Mann. Proteinase K was purchased from Beckman and lysozyme from Worthington Biochemicals. RESULTS The effect of the T4 alc gene product on late period and early period A RNA snythesis.--Because the T4 gig gene product is required for the shutoff of T4 late RNA snythesis from DNA with cytosine, we investigated the effect of the gig gene product on A RNA synthesis late in A development. We needed first to decide at what time after T4 superinfection to label the A RNA. Previous evidence has demonstrated that the nucleoid has been unfolded and host transcription has been shut off by 4.5 min. after T4 infection (Sirotkin et al., 1977; Tigges et al., 1977), so, we assume, that the gig protein is functioning by this time. Accordingly, RNA was pulse labeled from 4.5 to 7.5 min. after 14 infection of a heat induced A lysogen. These RNAs were then hybridized to A DNA. As indicated in Fig. 1A, the synthesis of A RNA is barely detectable after an gigf T4 infection. In contrast, substantial amounts of X RNA are synthesized after the gig mutant infection. Therefore, the gig_function is required for the shutoff of A late transcription. The experiment has been repeated with two other independent gig_ mutants with essentially the same results (R.E.P., unpublished observations). Infection by the gig mutant did reduce the efficiency of hybridization of late A RNA. Presumably, at least some of this reduction is due to labelled T4 RNA which does not hybridize to DNA. .74 75 Since A early period transcription and late period transcrip- tion may differ in their requirements, it is of interest to deter- mine if A early period transcription is affected by the gig gene product. RNA was pulse labelled and hybridized to A DNA in an experiment like that shown in Fig. 1A, but with A in the early period of development (Fig. 18). Both gigf T4 and gig_mutant T4 superinfections substantially reduce early period RNA synthesis. There is a difference in the amount of A RNA synthesized during the _igf and gig_mutant T4 infections which may represent the gig-medi- ated shutoff of the late A RNA, since it is already being synthesized by time time after A induction (Nijkamp et al., 1970; Oda et al., 1969; Skalka et al., 1967; Taylor et al., 1967). Since both gig? and gig_mutant T4 shut off most of the A transcription in the early period, some other mechanism independent of gig_blocks early A tran- scription. Because of this other mechanism, the effect of the gig_ gene product on early period transcription could not be determined unequivocally. The alc geneyproduct did not alter the superhelical content of closed circular A DNA.--There is evidence that supercoiling of DNA enhances at least some types of transcription ig_yiyg_(0e Wyngaert and Hinkle, 1979; Puga and Tessman, 1973; Ryan, 1976). It is conceivable that the T4 gig function shuts off host transcription and unfolds the host nucleoid by directly removing supercoils from DNA. If so, then the gig function may shut off A late RNA synthesis, by directly removing supercoils from A DNA. The DNA of A becomes 76 Figure 1.--Hybridizations of A RNA synthesized after T4 infection to A DNA. Heat induced A lysogen (5655/594) in M95 supple- mented with 10 ug./ml. tryptophan was infected with T4 at 50 (A) and 10 min. (B) after induction as described in the text. The RNA was pulse labelled with s uCi./ml. 5-H3 uridine, extracted, and hybridized to 25 pg. of A DNA. Each point represents the amount of RNA hybridized to A DNA minus the amount of RNA hybridized without DNA which was always less than 70 c.p.m. The specific activities of the input RNAs were in part A, uninfected late RNA at 6.6 c.p.m./ng.; gigf infected RNA at 2.9 c.p.m./ng.; gig mutant RNA at 3.4 c.p.m./ng.; and in part B, uninfected early RNA at 1.54 c.p.m./ng.; gigf infected RNA at .9 c.p.m./ng.; and gig mutant infected RNA at 1.15 c.p.m./ng. In both A and B, uninfected RNA is represented by (r—--o), _a_1_l_g+ RNA by (o———————o), and a_l_g mutant infected RNA by (o————o). 77 a o. x .eeazeao u 5 N o. x 25»... .Edd is é C.p.m.input x IO.4 1 l Figure 1 78 circular and some of it becomes supercoiled soon after infection. Lambda DNA which is covalently closed circular and supercoiled can be distinguished from that which is covalently closed, but relaxed, and from linear or nicked circular DNA by ethidium bromide-cesium chloride-gradient centrifugation. To determine if the gig_gene product destroys the super- coiling of circular A DNA, a A lysogen containing radioactive A supercoiled DNA was infected with gigf T4 and gig_mutant T4. The affect of the T4 infections is shown in Fig. 2. Neither the git} T4 (3) nor the gig_mutant T4 (C) alters the supercoiled peak of A DNA. There was also no increase in either the relaxed circular, open circular, or linear DNA peaks. As an aside, the above result indicates that there are no additional T4 nicking activities for cytosine DNA, other than those of the ggg A (Hercules et al., 1971; Warner et al., 1970) and ggg_8_(8runer et al., 1972; Sadowski and Vetter, 1973) gene products, at this time after T4 infection because there are no significant increases in either relaxed open or linear A DNA molecules during either infection. The alc gene product did not inhibit late period A DNA rgpli- ggiigg,--It has been reported that normal late A RNA synthesis depends on viral DNA synthesis (Dove, 1968; Joyner et al., 1968; Skalka et al., 1967). Since the gig gene product might affect late A RNA synthesis by preventing A DNA synthesis, we decided to examine the effect of the gig gene product on A DNA replication by hybridiz- ing pulse labelled DNA synthesized after T4 infection to A DNA 79 Figure 2.--Ethidium bromide cesium chloride gradient centrifugation of supercoiled DNA without 14 infection (A), with §1_C_+ infection, and with T4 gig mutant infection (C). Extracts of a A lysogen which has been infected with labelled A, then superinfected by T4 were lysed, cen- trifuged, and counted as described earlier. Tritiated A DNA is represented by (o~——————o), the position of the marker Col II DNA on the same gradient is repre- sented by the arrow. SCC indicates closed supercoiled circular molecules and 0C indicates open or linear DNA molecules. 80 A SCC 00 BOTTOM IO 20 3'0 4'0 50 com? a s - s'cc OC ‘M‘AAAA‘AA‘.u‘--AAAAA BOTTOM :0 2'0 3'0 4'0 50 soTOP Ci ~5‘ °°IT°M I o 20 30 4'0 50 60 7°? Fraction Number Figure 2 79 Figure 2.--Ethidium bromide cesium chloride gradient centrifugation of supercoiled DNA without T4 infection (A), with gi_T_c_’r infection, and with T4 gig_mutant infection (C). Extracts of a A lysogen which has been infected with labelled A, then superinfected by T4 were lysed, cen- trifuged, and counted as described earlier. Tritiated A DNA is represented by (o~——————o), the position of the marker Col 11 DNA on the same gradient is repre- sented by the arrow. SCC indicates closed supercoiled circular molecules and 0C indicates open or linear DNA molecules. 80 A SCC 00 BOTTOM IO 20 3'0 4'0 50 607°P El 61‘ 5130, (3C WAAAAA.M_AAAAAAAA BOTTOM :0 2'0 3'0 4'0 50 soTOP c N 6‘ sec 00 i i _-------‘-.---——---—— --—‘_-— °°TT°M IO 20 30 4'0 50 60 7°? Fraction Number Figure 2 81 (Table 1). As the data indicates, A DNA replication continues but at a reduced rate during the 219' and gig mutant T4 infections. Somewhat higher rates of A DNA replication are observed after the alc+ infection. We observed this difference both times we performed the experiment and are unsure as to the explanation. The affect of T4 infection on the translation of A late period RNA.--Over the years, a number of indirect experiments have suggested the existence of a T4 mechanism for inhibiting the transla- tion of host mRNA which is independent of the transcriptional shut- off (Kennel, 1970; Svenson and Karlstrom, l976). Because A late RNA synthesis continues during an gig_mutant infection, it is pos- sible directly to determine if T4 has a mechanism to prevent tran- slation of non-T4 RNA molecules. ‘The effects of 219' T4 and alg_mutant T4 on A late protein synthesis were studied by slab gel electrophoresis and autoradio- graphy. The results are shown in Fig. 3. Also shown, for compar- ison, are the labeled proteins of an induced and uninduced A lysogen. Proteins which were labelled in the induced but not the uninduced culture are assumed to be A proteins. It can be seen that no A proteins were synthesized after either an alg' or gig mutant T4 infection. Thus, the late A RNAs made after the gig_mutant T 4 infection are not translated. The size of the A late period RNA synthesized after T, ” infectigg,--One possible explanation for the failure to translate TABLE l.--Lambda DNA synthesis after infection by T 82 . Heat induced cells (06 75Acl857susS7/DG75) in M95 suppiemented with 5 ug./ml. thymidine and 10 ug./ml. tryptophan were infected with T4 and pulse labelled with l0 uCi./ml. methyl-H thymi— dine as described. Pulse labelled DNA from 5 ml. aliquots was extracted and hybridized to 20 ug. of A DNA per filter. Filter Filter Time After Input + A DNA - A DNA % Input T4 Induction c.p.m. c.p.m. c.p.m. Hybridized A None Uninduced l092 23 22 .09 B None Late 8958 2085 86 22.3 c Alc' Late 3042 551 45 l6.6 D Alc- Late 4373 436 31 9.3 83 Figure 3.--Slab gel electropherograms of proteins synthesized in an uninduced culture of a A lysogen and in heat induced lysogen infected by T The columns contain, A: induced 4. late period proteins, B: Uninduced host proteins, C: alg' T4 infected proteins, and D: alc mutant T4 infected pro- teins. Tentative identification of some late A protein bands and T bands was by comparison to published data 4 (Murialdo and Siminovitch, 1972; O'Farrell and Gold, 1973). 85 the A late mRNAs made after a T4 gig_mutant superinfection could be that they are non-translatable fragments. Accordingly, the jn_vjvg_ size of late period A RNA synthesized during an gig_mutant infection was examined. Fig. 4A shows the size of the A RNA made late in the A cycle without T4 infection. Lambda RNA is detected throughout the gradient, but particularly in the high molecular weight region and most sedimented faster than the 165 ribosomal marker. This was expected, because A late mRNA has been shown to be polycistronic (Garilglio and Green, 1973). Like the A late RNA synthesized with- out T4 infection, the bulk of the A RNA synthesized during an 319 mutant infection sediments faster than the 165 ribosomal marker (Fig. 48). Therefore, the failure to translate the A late RNAs made after an alg_mutant T4 infection is not due to the size of the A RNA. In the same experiments we examined the size of T4 RNA by hybridizing the fractions of the same gradients to T4 DNA. As expected, a considerable amount of RNA synthesized during the alg_ mutant infection hybridized to T4 DNA (Fig. 4B). The sedimentation profile of the T4 RNA corresponds with previous profiles of T4 RNA (Ricard and Salser, 1975). Thus, there did not appear to be exten- sive degradation during the extraction of RNA. Without T4 super- infection, the RNA in the gradient fractions hybridized at back- ground levels to T4 DNA (Fig. 43), as expected, since there is little sequence homology between A RNA and T4 DNA (Kennel, l968; Skalka et al., l967). 86 Figure 4.--Sucrose gradient centrifugation of heat induced late period RNA synthesized without and with T4 infection. Late induced A RNA (A) and late induced RNA synthesized during an gig_mutant superinfection (B) were isolated as in Fig. lA and heat denatured for 3 min. before layering 0n 5 ml. 5-20% sucrose gradients. The methods for disaggregation and sucrose gradient centrifugation were described by Ricard and Salser (1975). Centrifugation was performed in an SN 50.l rotor at 45,000 r.p.m. for 5 h. at 4°C. The gradient in part A contained 33 ug. of RNA. At the end of centrifugation, 0.2 ml. fractions were collected from the top with an 1500 fractionator. The positions of the ribosomal subunits were determined with a u.v. monitor. Each fraction was subjected to hybridization as described earlier to one of the following: l0 ug. of A DNA (H), 10 pg. of T4 DNA (o-———————o), no DNA (V). The yield of hybridized counts in A and B were 98% and 95% of the input hybrid radioactivity. 87 Au no.3 .13. 3“. 023 x .0» la 95 nu b p b) .A . 233 i :03 i 45 i - d 2 I re. 14205.2...53 33.13.5023 x am. no Ru ‘Q a; p n p b 1.5 9. a nu c. was nwnlv VS a s 6" .m n. w I' '5 d d d u 4w so a; .u «o. x 220: £1.53 Fraction Number £3--9- Fi ure 4 DISCUSSION Like the shutoff of T4 true late transcription from cytosine T4 DNA and some host transcription, the shutoff of A transcription by T4 during the late period of the A lytic cycle requires the T4 gig_gene function. Since alg_mutants are defective in preventing at least some transcription from T4, §;_ggli, and A DNA templates, there must be something similar about the three types of transcrip- tion which is affected by the gig_gene product. It is clear from the data that the 210 gene product neither blocks A DNA synthesis nor directly removes supercoils from closed circular supercoiled A DNA molecules. Therefore, the molecular basis for the alg;mediated shutoff of some types of transcription remains undiscovered. Sev- eral plausible mechanisms for gig_function remain. To give two possible mechanisms, the gig_gene product could act directly on some promoters, provided they contain cytosine, and prevent their utilization. Alternatively, the gig function could act on some other DNA structure such as the "cores" of nucleoids and thereby indirectly remove supercoiling which, in turn, may be required for the utilization of some types of promoters. Both of these explana- tions have the gig_function blocking the initiation, but not the elongation of A late in RNA synthesis which may be untenable because of the following considerations. All A late transcription is 88 89 thought to begin at a promoter to the right of g_and proceed through the late region requiring around l0 minutes to transcribe the entire region (Green et al., l970). However, the gig_function has shut off A late transcription within 4.5 min. of T4 superinfection and, since a finite interval is required for the transcription and tran- slation of the gig_gene, the time for alg to function is actually less than this. Therefore, A late transcription may be shut off more quickly than might be expected if only the initiation of tran- scription is blocked suggesting that the 319 function can block the elongation and not just the initiation of the synthesis of mRNA. Because T4 inhibits early period A transcription indepen- dently of gig_function, it was not possible to determine if the gig_ product also effects A early period transcription. We suspect that .319 does not effect A early period transcription because the shutoff of early period A transcription was less complete than the £12? mediated shut off of T4 and A late transcription. Regardless, the results indicate that another T4 mechanism can inhibit A early transcription. This mechanism may be merely a manifestation of an inherent property of T4 DNA. For example, T4 early promoter sequences may have a greater affinity for the RNA polymerase of the host than A early promoter sequences. Experiments jn_vjt§9_demon- strating that T4 is a more active template for transcription than A DNA, even with RNA polymerase from uninfected bacteria (Mailhammer et al., 1975), provide some support for this mechanism. Alterna- tively, a T4 induced function may be responsible for the 90 non-al§:mediated inhibition of A early RNA synthesis. For example, the bacteriophage T4 induces several changes in the host RNA poly- merase which include both adenylation (Horvitz, l974) and addition of polypeptides to the polymerase (Stevens, l972; Horvitz, 1973). Either the adenylations or the added polypeptides could be respon- sible for the non-alg inhibition of early A transcription. Preli- minary evidence indicates that the adenylations of the polymerase are not responsible because A early RNA synthesis is blocked after infection by an altf, mpg? mutant infection (R.E.P. unpublished observations). But, since an algemediated shutoff could be super- imposed on a shutoff due to adenylation of the RNA polymerase, it may be necessary to construct a T4 containing mutations which make it altf mggf and gig_mutant to investigate the effects of the adenylations on A transcription. With respect to the addition of T4 induced polypeptides to the host RNA polymerase, it has been reported that there are four, of which two are the products of known T4 genes 33 and 55 (Stevens, 1972; Horvitz, l973). The other two are good candidates for the shutoff of A early transcrip- tion. In this connection, we have reported that an gig_mutant affects the binding of one of the T4 induced polypeptides to the host RNA polymerase (Sirotkin et al., l978), probably the l5,000 M.N. polypeptide of Stevens (l972). This is not a general property of gig_mutants and many of them show normal amounts of this poly- peptide 0n the RNA polymerase. Because all of our attempts to isolate deletion mutants or nonsense mutants of the gig gene have 91 failed, we think alg_may be an essential (i.e. indispensible) gene for T4 development. If alg_is an essential gene, then all 319 mutations may be only partially inactivating. If this is the case, then all gig_mutants may have the RNA polymerase binding polypeptide, but it may be lost in some alg mutants from the RNA polymerase dur- ing purification. We also think that T4 gig_mutants may accumulate second site mutations perhaps because they are selected with cytosine in their DNA and without the phage induced dCTPase. Perhaps a second site mutation, in an gig_mutant, was in the gene for the l5,000 dalton polypeptide of Stevens (1972). More experiments are needed to clarify this issue. Out data suggest that a T4 mechanism inhibits A late period DNA synthesis and, even more dramatically, A early period DNA syn- thesis (REP, unpublished observation). It has been reported that the T4 Egg gene product inhibits host DNA synthesis (Snustad et al., l976a) and may have a similar effect on A DNA synthesis. However, the parent of the alg_mutant we used has a deletion which includes .ggd, so this gene product could not have been responsible in our case. It should be pointed out that the shutoff we saw could have been an indirect effect of shutting off A translation. For example, the Q_gene product of A is an unstable protein (Wyatt and Inokuchi, l974) and stopping its synthesis will result in a cessation of A DNA synthesis (Klinekert and Klein, 1978). We have presented direct evidence for a T4 mechanism that prevents translation of A late RNA molecules. We think this is the 92 most dramatic demonstration to date of a T4 induced mechanism which prevents translation of non-T4 mRNA molecules. The failure to tran- slate the A late RNA was not due to its size because high molecular weight A RNA was synthesized, but still not translated. Other mech- anisms can be proposed. For example, the shutoff of host and A protein synthesis could be due to one of the several T4 induced ribo- some binding proteins which are synthesized during a T4 infection (Dube and Rudland, 1972; Smith and Haselkorn, l969). The gggA_ protein, which is involved in translation regulation of early T4 proteins (Karam et al., 1974; Trimble et al., l976; Niberg et al., 1973), might also affect the shutoff of translation. However, the rggA_gene product may not be synthesized early enough to be respon- sible for the translation shutoff we observe (J. Wiberg, personal communication). Whatever the mechanism, we predict that T4 mRNA has some property which distinguishes it from host and A mRNA. A potential candidate would be an unusual sequence at the 5' end of T4 mRNA which is involved in the initiation of protein synthesis. We have begun to try to isolate mutations which prevent the shutoff A late protein synthesis after a T4 31g mutant superinfection. LITERATURE CITED Blair, 0. G., B. D. Clewell, D. J. Sheratt, and D. R. Helinski. l97l. Strand specific supercoiled DNA-protein relaxation complexes: Comparison of the complex of bacterial plasmids 2T0 544and Col E2. Pro. Natl. Acad. Sci. U.S.A. 68: Bolle, A., R. H. Epstein, N. Salser, and E. P. Geiduschek. 1968. Transcription during bacteriophage T development: Syn- thesis and relative stability of early and late RNA. J. Mol. Biol. 31: 325—348. Brunner, R., A. Souther, and S. Suggs. 1972. Stability of cyto- sine-containing deoxyribonucleic acid after infection by certain T4 rII-D deletion mutants. J. Virol. 10: 88-92. De Wyngaert, M. A., and D. C. Hinkle. l979. Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA. J. Virol. 29: 529-535. Dove, w. F. 1966. Action of the Lambda chromosome. J. Mol. Biol. l9: 187-201. Dube, S. K. and P. S. Rudland. I970. Controls of translation by T phage: Altered binding of disfavored messengers. Nature 236: 820-823. Eisen, H. A., C. R. Fuerst, L. Siminovitch, R. Thomas, L. Lambert, L. P. Da Silva, and F. Jacob. 1966. Genetics and physio- logy 0f defective lysogeny in K12: Studies of early mutants. Virol. 30: 222-24l. Frederick, R. J. and L. Snyder. l977. Regulation of anti-late RNA synthesis in bacteriophage T4. J. Mol. Biol. ll4: 461-476. Garilglio, P. and M. H. Green. 1973. Characterization of poly- cistronic late Lambda messenger RNA. Virol. 53: 392-404. Green, M. H., Hayward, N. S., and P. Gariglio. l970. A method for localization of active promoters. Cold Spring Harb. Symp. Quant. Biol. 35: 295-303. 93 94 Hayward, N. 5., and M. H. Green. 1965. Inhibition of Escherichia oli and bacteriophage Lambda messenger RNA synthesis by c T4 Proc. Nat. Acad. Sci. U.S.A. 54: l675-l678. Hercules, K., J. L. Munro, S. Mendelsohn, and J. S. Wiberg. l97l. Mutants in a nonessential gene of bacteriophage T which are defective in the degradation of Escherichia cgli deoxyribonucleic acid. J. Virol. 7: 95-l05. Herskowitz, I., and E. Signer. l970. Control from the r_strand of bacteriophage Lambda. Cold Spring Harb. Symp. Quant. Biol. 35: 355-368. Horvitz, H. R. l974. Bacteriophage T4 mutants deficient in altera- tion and modification of the Escherichia coli RNA polymerase. J. Mol. Biol. 90: 739-750. Horvitz, H. R. l973. Polypeptide bound to the host RNA polymerase is specified by T4 control gene 33. Nature New Biol. 244: l37-l40. Joyner, A., L. H. Isasacs, H. Echols, and N. S. Szybalski. 1966. DNA replication and messenger RNA production after induc- tion of wild type and mutant Lambda. J. Mol. Biol. l9: l74-l86. Karam, J. D. and M. G. Bowles. l974. Mutation to over roduction of bacteriophage T4 gene products. J. Virol. 3: 428-438. Kennel, D. l970. Inhibition of host protein synthesis during infec- tion of Escherichia coli by bacteriophage T4. J. Virol. 6: 208-2l7. Klinkert, J. and A. Klein. 1978. Roles of A gene products 0 and P during early and late phases of infection cycle. J. Virol. 25: 730-737. Kutter, E., A. Beug, R. Sluos, L. Jensen, and 0. Bradley. I975. The production of undegraded cytosine-containing DNA by bacteriophage T4 in the absence of dCTPase and enconucleases II, IV and its effect on T directed protein synthesis. J. Mol. Biol. 99: 591-509. Mailhammer, R., H-L. Yang, G. Reiness, and G. Zubay. 1975. Effects of bacteriophage T4 induced modification of Escherichia coli RNA polymerase on gene expression jn_vitro. Proc. Natl. Acad. Sci. U.S.A. 72: 4928-4932. 95 Murialdo, H., and L. Siminovitch. 1972. The morphogenesis of bacteriophage Lambda: Identification of gene products and control of the expression of the morphogenetic information. Virol. 48: 785-823. Nijkamp, H. J. J., K. Bdure, and N. Szybalski. l970. Controls of rightward transcription in coliphage A. J. Mol. Biol. 54: 599-604. O'Farrell, P. Z.-and L. M. Gold. l973. Bacteriophage T4 gene expression: Evidence for two classes of prereplicative cistrons. J. Biol. Chem. 248: 5502-55ll. 0da, K., Y. Sakakibara, and J. Tomizawa. l969. Regulation of transcription of the A bacteriophage genome. Virol. 39: 901-918. Ogawa, T., J. Tomizawa, and M. Fuke. T968. Replication of bacterio- phage DNA. Structure of replicating DNA of phage Lambda. Proc. Natl. Acad. Sci. U.S.A. 60: 86l-866. Pettijohn, D. E. and T. Hecht. l973.. RNA molecules bound to the folded bacterial genome stabilize DNA folds and segregate domains of supercoiling. Cold Spring Harb. Symp. Quant. Biol. 38: 3l-4l. Puga, A. and I. Tessman. 1973. Mechanism of transcription of bacteri0phage Sl3: Dependence of messenger RNA synthesis on amount and configuration of DNA. J. Mol. Biol. 75: 86-98. Ricard, B. and w. Salser. l975. The structure of bacteriophage T4 specific messenger RNAs. J. Mol. Biol. 94: l63-l72. Ross, P. and P. Howard-Flanders. l977. Initiation of rec A+-depen- dent recombination in Escherichia coli (A). J. Mol. Biol. ll7: l37-l58. Runnels, J. and L. Snyder. l978. Isolation .of a bacterial host selective for bacteriophage T4 containing cytosine in its DNA. J. Virol. 27: 815- 8l8. Ryan, M. J. l976. Coumermycin A]: A preferential inhibitor of replicative DNA synthesis in Escherichia coli: 13 vivo characterization. Biochem. TS: 3769- 3777. Sadowski, P. D. and D. Vetter. 1973. Control of T4 end0nuclease IV by the 02a region of bacteri0phage T4. irology. ' 544-546. 96 Schnos, M. and R..Inman. l970. Position of branch points in replicating A DNA. J. Mol. Biol. 51: 6l-74. Skalka, A., B. Butler, and H. Echols. l967. Genetic control of transcription during development of phage A. Proc. Natl. Acad. Sci. U.S.A. 58: 576-583. Sirotkin, K., J. Nei, and L. Snyder. l977. T bacteriophage-coded subunit blocks host transcription and flnfolds the host chromosome. Nature 2l5: 28-32. Smith, F. L. and R. Haselkorn. l969. Proteins associated with ribosomes in T infected §;_coli. Cold Spring Harb. Symp. Quant. Biol. 5: 9l-94. Snustad, D. P., C. J. H. Bursch, K. A. Parson, and S. H. Hefeneider. l976a. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: Shutoff of host DNA and protein synthesis, gene dosage experiments, identification of a restrictive host and possible biological significance. J. Virol. l8: 268-288. Snustad, D. P., M. A. Tigges, K. A. Parson, C. J. H. Bursch, F. M. Caron, J. F. Koener, and D. J. Tutas. l976b. Identifica- tion and preliminary characterization of a mutant defec- tive in bacteriophage T4 induced unfolding of the Escheri- chia coli nucleoid. J. Virol. 17: 622-64l. Snyder, L. R. and D. L. Montgomery. l974. Inhibition of T4 growth by an RNA polymerase mutation of Escherichia coli. Virol. 62: 184-l96. Snyder, L., L. Gold, and E. Kutter. l976. A gene of bacteriophage T whose product prevents true late transcription on cytosine-containing T4 DNA. Proc. Natl. Acad. Sci. U.S.A. 73: 3098-3l02. Stevens, A. 1972. New small polypeptides associated with DNA- dependent RNA polymerase of Escherichia coli of the infec- tion with bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 69: 603-607. Stonington, 0. G. and D. E. Pettijohn. 197]. The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc. Natl. Acad. Sci. U.S.A. 68: 6-9. Studier, N. F. l973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79: 237-248. 97 Svenson, S. B. and 0. H. Karlstrom. l976. Bacteriophage T induced shutoff of host specific translation. J. Virol. l : 326- 334. Takahashi, H., M. Shimizu, H. Soito, and Y. Ikeda. 1979. Studies of viable T bacteriophage containing cytosine substituted DNA (T4dC Phage). Mol. Gen. Genet. l68: 49-53. Takahashi, S. 1974. The rolling circle replicative structure of bacteriophage A DNA. Biochem. Biophy. Res. Comm. 61: 657-633. Takahashi, S. l975. The starting point and direction of rolling circle replicative intermediates of coliphage A DNA. Mol. Gen. Genet. l42: l37-l53. Taylor, K., 2. Hradenca, and W. Szybalski. l967. Asymetric dis- tribution of the transcribing regions on the complementary strands of coliphage A DNA. Proc. Natl. Acad. Sci. U.S.A. 57: l6l8-1625. Tigges, M., C. J. H. Bursch, and D. P. Snustad. l977. Slow switch- over from host RNA synthesis to bacteri0phage RNA synthesis after infection of Escherichia coli with a T4 mutant defec- tive in the bacteriophage T induced unfolding of the host nucleoid. J. Virol. 24: 75-785. Trimble, R. B. and F. Maley. l976. The level of specific prerepli- cative mRNAs during bacteriophage T4 reg A“, 43' T 43‘ infection of Escherichia coli B. J. Virol. l7: 38-549. Tutas, D. J., Wehner, J. M., and J. F. Koerner. l974. Unfolding of the host genome after infection of Escherichia coli with bacteriophage T4. J. Virol. l3: 548-550. Warnaar, S. 0. and J. A. Cohen. l966. A quantitative assay for DNA-DNA hybrids using membrane filters. Biochem. Bi0pny. Res. Comm. 24: 554-558. Warner, H. R. and S. E. Barnes. l966. Evidence for a dual role for the bacteriophage T -induced deoxycytidine triphosphate nucleotidehydrolase. P 0c. Nat. Acad. Sci. U.S.A. 56: lZ33—l240. Warner, H. R., D. P. Snustad, S. Jorgensen, and J. F. Koerner. 1970. Isolation of bacteriophage T mutants defective in the ability to degrade host deoxyribonucleic acid. J. Virol. 5: 700-708. 98 Wiberg, J. S., S. Mendelsohn, W. Warner, K. Hercules, C. Aldrich, an J. Munro. l973. SP62, a viable mutant of bacter10- phage T defective in regulation of phage enzyme synthesis. J. Viro . l2: 775-792. Wilson, G. G., V. I. Tonyashin, and N. Murray. 1977. Molecular cloning of fragments of bacteriophage T4 DNA. Mol. Gen. Genet. l56: 203-214. Worcel. A. and E. Burgi. l972. 0n the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 7l: 127-147. Wu, A. M., Ghosh, S., Echols, H., and W. G. Spiegelman. l972. Repression by the cI protein of phage A: in vitro inhibi- tion of RNA synthesis. J. Mol. Biol. 67: 407-42l. Wu, R., and E. P. Geiduschek. l975. The role of replicative pro- teins in the regulation of bacteriophage T4 transcription. J. Mol. Biol. 96: 5l3-538. Wyatt, W. M. and H. Inokuchi. 1974. Stability of A 0 and P replication functions. Virol. 58: 313-3l5. Young, E. T., and R. L. Sinsheimer. 1968. Vegetative A DNA: III: Pulse labelled components. J. Mol. Biol. 33: 49-59. ACKNOWLEDGMENT This work was supported by NSF grant PCM 77-24422. This is Article No. of the Michigan Agricultural Experiment Station. 99 APPENDIX 100 APPENDIX The Shutoff of Lambda Monomeric Circle Replication Lambda DNA replicates as monomeric circles during the first ten minutes of development at 37°C and then replicates as concatamers. Because monomeric circle replication differs from concatameric DNA replication, T4 may effect the two forms of Lambda DNA replication differently. To examine the affects of T4 on monomeric circle replication, an experiment analogous to that used to investigate the affect of T4 on Lambda concatameric replication was performed. The results of the experiments are presented in Table 2. Also presented, for comparative purposes, are the results of the experi- ment showing that the alg_gene product did not block A concatameric DNA replication. It was clear from the data that both monomeric circle DNA replication and some concatameric DNA replication were shut off during T4 superinfection. But, monomeric circle replica— tion was more sensitive to a T4 shutoff mechanism than concatameric DNA replication. There are several plausible explanations for the different sensitivities of Lambda monomeric circle DNA and Lambda concatameric DNA replication to T4 superinfection. One explanation, is that the difference may be simply due to the difference in the number of lOl 102 TABLE 1.--Lambda synthesis after infection by T3. Heat induced UPP cells (DG 75Ac1857susS7/DG75) in M95 lemented with 5 ug./ml. thymidine and 10 ug./m1. tryptophan were infected with T and pulse labelled with 10 uCi./ml. methyl-H3 thymi ine as described. Pulse labelled DNA from 5 ml. aliquots was extracted and hybridized to 20 ug. of A DNA per filter. Filter Filter Time After Input + A DNA - A DNA % Input T4 Induction c.p.m. c.p.m. c.p.m. Hybridized A None Ininduced 1092 23 22 .09 B None Early 8238 262 38 2.7 c A1c+ Early 1164 30 24 .5 D Alc- Early 1586 33 23 .6 B None Late 8958 2085 86 22.3 c A1c+ Late 3042 551 45 16.6 D Alc- Late 4373 436 31 9.3 103 replicating genomes at the time of T4 superinfection. Alternatively, the difference could be due to the shutoff of Lambda protein syn- thesis. Because both monomeric circle replication and concatameric DNA replication require different unstable Lambda gene products, the shutoff of Lambda protein synthesis may affect Lambda monomeric circle replication more severely than Lambda concatameric replica- tion. Another possibility is that T4 induces several functions that prevent DNA synthesis. Perhaps, monomeric circle replication was more sensitive to a shutoff function than concatameric DNA replica- tion. The Shutoff of Lambda Late Transcription After alt’ mod- MDtantT4 Infection T4 blocks Lambda late transcription during superinfection. At least one T4 gene involved in the shutoff of late transcription has been identified as the T4 gig_gene. Recently, the T4 induced adenylation of the host RNA polymerase has been linked to the shut- off host RNA synthesis and may be involved in the shutoff of Lambda late transcription. This can be studied with altf mggf mutant T4 because this strain of T4 fails to adenylate the host RNA polymerase during infection (see above). An experiment analogous to those investigating the affect of gig_gene on Lambda late period transcription was performed with gltf mggf mutant T4. The results of this experiment are presented in Figure 5 and, for comparative purposes, the experiment on the 212? mediated shutoff of A late period transcription is also presented. 104 Figure l.--Hybridizati0n of A RNA synthesized after T4 infection to A DNA. A heat induced lysogen (S655/594) in M95 supplemented with 10 ug./ml. tryptophan was infected with T4 as described earlier. The RNA was pulse labelled with 10 uCi./ml. 5-H3 uridine, extracted, and hybridized to 25 ug./ml. of Lambda DNA. Each point represents the amount of RNA hybridized to DNA minus the amount of RNA hybridized without DNA. The specific activities of the RNAs were Uninfected late RNA at 6.6 c.p.m./ng., alg' infected RNA 2.9 c.p.m./ng., gig mutant RNA at 3.4 c.p.m./ng., and altf mggf RNA at 1.5 c.p.m./ng. Uninfected RNA is represented by (0—__———_O)s 31C+ RNA by (0-—-————0), gig_mutant RNA by ( ________ ), and alt- mod' RNA by ( _______ ). 105 gm 0. x 39: .Edd em _ r. 1 N 0| x 'ngq/(H 'uJ'd'o 9 106 It is clear that the alt‘ mod" mutant T shut off late RNA synthe- 4 sis. Therefore, the T4 induced adenylation of the host RNA poly- merase is not involved in the shutoff of Lambda late period transcription. There are a number of explanations for the failure to detect an effect of the T4 induced adenylations on the shutoff of late period transcription. First, the T4 induced adenylation of the RNA polymerase may not be related to the shutoff of Lambda late transcription. 0n the other hand, adenylation of the RNA polymerase may be involved in the shutoff of Lambda late period transcription, but the algfmediated transcriptional shutoff may be superimposed on it. To study this alternative, a triple mutant containing gltf @997 and gig_mutants may be necessary to determine if the adenylation of the RNA polymerase is responsible for some of the shutoff of Lambda transcription. ._./ “I “at - T 1111111111 1006 RR 12 MICHIGAN STA 11111111111111“) 31293 RIES ”HI 1