4""? 1" 3'37». ' 23*; I > W... ‘ PM r. ' 3'f. ., 'IIIr' 2-4:" '3 I.' Hfid'flh 36* Ihag‘d 3. :I'II'i‘r 'IIH . . P O I III III" I I .'4 4 I42.- ”I; 224222.234 I. WHII fl: 3313:3’47’ .- MY: ill M 3; \ ." I . ”3:? VIII'W “13?. “WC“, II I 2“ t€$3k Iak any“ *é‘J'. z'J‘I I‘D-333933;.) 14"“ ' 2'“' fl '4‘; 4' 4' "22" 2""42523 43'3"" 2312'." «25".! sf. 47143 -'II"~I:. 4 24 242" 4'.I' 3’3 -.2*4I2“24"4:':I’4.:‘-‘134121443.742 4‘" N22. ‘21?- '.4_'3.- I - "4442". .3624. ""23" 2“ ' ' ""324 "431%? ‘ . ~22.. ...3. .II2~I 1.. M42 2 23'1'2' '2 )I IIIIIIII '.""' 3"“.{237 3 mIvf"'"3 l ' 4. . I3 ‘0' 1% .24. 4 4 ’ 7'4" . 242-41: 44 3"; "I '2' . 4 . ..42 4,4 «2.222422 ' "Pun“ "I'"'33:I"¥ )3": "3'&:"."il .123} '("‘2;'.III:A 3} “ix "3'43'm4 fin“ -' *‘l‘l IIII"'W’!'I:I.'.'“I”;' ' ffi'id3 ILL 244 4 I... 4 4.44.... 422442 2 '4 4 '.I.'.,I,I_ 242. 2223.3 .2 "434:4 ."3I4"""‘MT' 33331333 .333 I*1 4. :3!" ""94' ':.':?"."34'4;-"I 522" I‘III7;"".‘."‘ 1‘ ' . " ‘ f.‘"". .' ... 2322'- :“93'M2fi'4-i35323' "T ' '7': 1' 4' $13-34": WE 2.2!... "332%322&II 4‘44' ' 'Pt "'1'. o ttfqu WISE" '\l I‘II 3'}! :'("""'I II723‘ {OIL-IVE? 3v $.12 ‘0‘“. I" 9+"4'" {IE 3'3‘4'"4'"3" ,, I33I' $3“qu .'2 "'33,.2' .2‘“ .3' g "3' '3' "3-3,". Ifl'“ "‘ )‘2‘ 3'1'g'wij'3ufl'"'"""J‘~""$""3'I"‘i' l '2' ”1‘9”:de '3'.21:° . "ti-'3 ' Y 7322.": :42; ' "44.4"i-'2;'.4I . . " '22" 'JL"'@EES~;3": g" ' ' .2722 33‘”I‘6"'".'I3'P»:3' 4.. PU" I“? ‘12” QI'flwm. 1313 '2 23'333 'D'... "13313111! P". 433F1'4t2gw..m\133;’32\“4 *4 521423“ ".41“. ' '.'~ , '3 " ‘ ‘ .3 ' 442243.23 4 4.:.: 22 4 .'2'2'22“ 2" 41332124444 -4 $2224. .. "”4322" ”I212?" IE"... ‘4 "'II'3'I " ”(N-34121232.” ’3 52'2"" . ._4 .. 2.222. 2 .Iw..m w "':33I.:1"":'3374'\:$':t"' 2125953.; .. ,I4.II'III. II'I- In; , ‘2' \x . ""'“.“th ..'3" :3 IIIIIII ' 442. 2.2.4 "-'4‘*44 =4 '4" 44*" 324 4 42. ""2,“ "'}3 . ol"'q'|'I"|"."" "Vi—"2' 2 2 , 2"33' 33:32. 3""""" .‘4- 44- . 2’. 5“ 3 'l' I"" ""'"-2' . .2222. 4 4.211.234. ‘2 44: ;'-33" III-'IIU3'I'I .3 3 I' 3. 2!.IIYI I ' 2 .. ‘|II.I. I I .II 2..) .I ’3‘ . ‘ ., .242. .3944.“ 71.1.1. 1‘ . 1 422‘ '.I,4 [2-'," 3"..."3 3"“ " '3'4'I3 'I‘I'l c ' 3 "I" I.I..4..I.II- ,. "13....“ '4' :4 2:24:44- ".2 "742.222 .- . . -. - - 4.4- 2224 a~22222242~24 ' "4 " ‘ 4 4. ' ..'3 'L 4 ' 2;2"| ' .12': I... ‘ '. I2 ‘ '2 'II ' I ' 1!. '12,. . "ply " "" .".',' .I I"?"'IT..." :I ' 7 I ' .' ‘3 -2 .2. I II '2 . 2 "".' I“: I‘ 2 "4; 2 - . N 222." ' " I" "'*4"""2i"" 2"" 4.2" -. ..24:4..I.4 II, 44.24.114 .43.: IIJIMt'II'I 'II"]". 'IIIvI :IIIiI .2 I .ZI'I I :323II,I I! I "'.:',"'JI-'31H3 . .31.. 3'". '. '.4 422?} I6 4I' 4."'i:' '4-2' .7“ " I-;- 3.. ".42 'Y. . 2 " 1" ‘ I...» .. I ""."" ' ' '. ' 3' I)" “2'“ B ".3" 2"“ 4 v" "'I " ' ,'|'|I3:l|-' I’!"":'. '42 .""I ,3'II 3": ..'3'3III 3 l',“ rIII:,|I II, II yII3'3 .‘ I :‘I I. '..I I.” I 2 .3"! I '..3 ":l V ' r'.| " ' ' "33' " ' “1'93“ 3 _ '3'P'f')"" "'53 '2': 432:2 ' .I-:""'24’.i*";24'.' ”""2I4' '.I4.'. . ‘~.'-4 " 2 It" 44» 2444-42. .I44...:;444 4II,I,4' 422.22.‘ 4‘ "73'2“” ,. 3.4.4-4212II“" . . "... ..2 4-; 22f ..H ' 2 4 *I- 1 ~. 44 [,3 .3 I2.3; I'... 2 ' 2U: . .. ,. ""’" " l '3 "I' ' ""' "' ' ' ' 244" ""' "‘ lI ' IIIIw'fII.’ I '5'2'2 2“ I f. '2'“ .' ‘4' 'I". IIIu '32 31 I" I. ' '3" ".' . 23'1” III... , ' 5 ""'I'.""'""I" v ‘,""‘ ‘., 3. .4 .' ..3'4 ' '4‘ 342 I'IJ " ”.‘II... "2 2'33. '3'. .4'2 . ,"4 'I'24'2 .I3I:'4 'Il 3I2' 4 . M4132 "'2:.' .' '."I"'.'.. ""'4 ' "" I'I'HHJ ;.'. 3"20 I 2 “2244 l '03'2 'fim'll'nfi ‘23:", , "‘33 W'..:' """",' I422. 1 . .| 3 l' ._ 4 2 , 4m "3 'l ' . 245'4.4.4.I,,...1.,.4 "ll 2 4““ .- -'4'f *J-o-*.I"'.4:*w2h. 2122.922 ' £3 '1 . THESIS This is to certify that the thesis entitled STUDIES ON THE BIOLOGICAL ROLE AND GENETICS OF THE T4 BACTERIOPHAGE RNA LIGASE presented by Judith Marie Runnels has been accepted towards fulfillment of the requirements for PhoDo degree in Genet'iCS Major professor Date %ovember 14, 1980 0-7639 LIBRARY A Michigan Sum W: 25¢ per day per item REFUNDS LIBRARY MATERIALS: Place in book return to remove charge from circulation records // |\\ J (line-{\\ is 5 ' any}: , STUDIES ON THE BIOLOGICAL ROLE AND GENETICS OF THE T4 BACTERIOPHAGE RNA LIGASE By Judith Marie Runnels A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY The Genetics Program 198l Copyright by JUDITH MARIE RUNNELS 198l ABSTRACT STUDIES ON THE BIOLOGICAL ROLE AND GENETICS OF THE T4 BACTERIOPHAGE RNA LIGASE By Judith Marie Runnels The psgl_gene of bacteriophage T4 encodes a protein which has 5' polynucleotide kinase 3' phosphatase activity. Mutants in this gene fail to grow on an g, gglj_host called CTrSX. CTrSX is a hybrid strain between laboratory g, gglj-and a clinical isolate. The phenotype associated with psgl_mutant infections of CTrSX is: (1) T4 DNA synthesis is reduced to between 30% to 50% of its normal level; (2) T4 DNA is approximately half the normal size; (3) the DNA which is made is not packaged efficiently; and (4) depressed levels of T4 late proteins are made. In addition, the pseT phenotype can be suppressed by: (1) growth at 42°C; (2) a T4 extracistronic suppressor mutation called sip; and (3) introduc- tion of an efficient amber suppressor into the CTr5X host genome. CTrSX am su+ will allow growth of psgl_point and deletion mutants, indicating that the amber suppressor is suppressing a host rather than the phage mutation. While pursuing a search for mutants in the psgl_gene, I discovered a second cistron in T4 which expressed the phenotype of Judith Marie Runnels p§g1_mutants. I called this cistron "125? fer interacts with phosphatase kjnase. The jpk_cistron mapped close to pseT. Further genetic study utilizing three and four factor crosses mapped ipk_to gene 63. T4 gene 63 has previously been shown to encode a protein with two activities. It enhances the rate of attachment of the six tail fibers to the baseplate (TFA), and it ligates single- stranded RNAs and DNAs (RNA ligase). RNA ligase will also cyclize RNA and DNA oligomers. The biological role of the RNA ligase activity is unknown, despite the fact that the enzyme's versatility makes it immensely useful for nucleic acid biochemistry. No pheno- type had previously been described for phage deficient in RNA ligase activity. Gene 63 mutants deficient in TFA, on the other hand, are incapable of plaque formation at temperatures at or below 19°C. Their low infectivity is the result of a reduced number of tail fibers on the virion.‘ Tail fiber attachment will proceed in the absence of the gene 63 protein, although it is only at 1% to 5% the rate observed with the gene 63 product. Further genetic analysis of the 125 mutations demonstrated that they were interspersed among previously described gene 63 amber mutations. The jpk_mutants appeared to be normal in TFA ability as determined by their plating characteristics. Hence, I set about to determine if the phenotype associated with jpk.muta- tions could be due to an RNA ligase deficiency. I measured the amount of cyclization activity in crude extracts of T4-infected cells using 5' P-terminus labeled oligo-poly A as the substrate. Judith Marie Runnels The conversion of linear to circular molecules was measured by the conversion of radioactivity from terminal to internal positions on the substrate molecules. None of the 125 mutants possessed any RNA ligase activity. Thus, I have shown that an RNA ligase deficiency leads to the inability of T4 to grow on g, gglj_CTr5X because of deficiencies in DNA metabolism and late gene expression. In addition, I have crossed an stp_mutation into a gene 63 amber mutant background. The gene 63 amber mutant was deficient in both TFA and RNA ligase. Introduction of the stp_mutation restored growth of the gig, am63 double mutant on CTrSX; however, the TFA deficiency was not corrected as the double mutant was still incapable of growth at 19°C on a non-amber suppressing host. These results confirm genetically what had previously been suspected from biochemical data: the TFA and RNA ligase activities associated with gene 63 protein are functionally unrelated. ACKNOWLEDGMENTS I wish to thank my committee members for their guidance during my education at Michigan State University. My gratitude is extended to Albert H. Ellingboe, Paul T. Magee, Leonard G. Robbins, and Robert R. Brubaker who served in this capacity. Special appreciation is merited by two people who also served on my committee. I thank Michele M. Fluck whose fosterage, both psychological and scientific, were invaluable to my maturation as a scientist during this period. Likewise, I extend my gratitude to my mentor, Loren R. Snyder, whose curiosity and devotion to science and whose perceptive analyses of data and adroitness in formulating questions could only serve to develop my skills as a competent researcher. I wish also to express appreciation to Olke C. Uhlenbeck and Dan Soltis at the University of Illinois. I thank them for their biochemical expertise and assistance in performing the RNA ligase assays as well as the hospitality they extended to me while those experiments were in progress. Lastly, I am grateful for the financial support proffered me while I was a graduate student at Michigan State University. This support was given in the form of teaching, administrative, and research assistantships. ii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES INTRODUCTION LITERATURE SURVEY . Scope of the Literature Survey Requirements for T4 Late Transcription T4 DNA Synthesis . . . . Gene Products 33 and 55 Gene Product 45 True- Late Transcription with Cytosine- Containing DNA: The PseT Gene . Gene 63. . Tail Fiber Attachment Activity. T4 RNA Ligase . The Relationship Between RNA Ligase and Tail Fiber Attachment Activity . . . The Phenotype of RNA Ligase Deficient T4 . The Role of RNA Ligase and 5' Polynucleotide Kinase 3' Phosphatase in T4 Infection . . . . References . . ARTICLE I. GENETIC AND PHYSIOLOGICAL STUDIES OF THE ROLE OF THE T4 BACTERIOPHAGE RNA LIGASE . . Summary . . . . . . . Introduction Results . . . Genetic Mapping of jpkf Mutations . . Assays of RNA Ligase 1n _pk_ Mutants . Phenotypes of _pkf Mutations . . Discussion . . . iii Page vi Experimental Procedures Phage Strains Crosses . Physiological Experiments . Preparation of 5' Labeled Poly A RNA Ligase Activity . Acknowledgments References . APPENDICES A. The Map Order of 125,.319, and pseT Mutations . B. The Order of Some alg_Mutations C. Isolation of a Bacterial Host Selective For Bacteriophage T4 Containing Cytosine in Its DNA iv LIST OF TABLES Table Page 1. The Genetic Mapping Data for jpk_and Gene 63 Markers . 55 2. The Position of ipk_Mutations with Respect to pseT . . 84 3. Recombination Between alg_Markers . . . . . . . 89 LIST OF FIGURES The Assembly and Attachment of T4 Tail Fibers Map of Gene 63 Region of T4 Assays of RNA Ligase Activity in Extracts of Cells Infected by 125. Mutants of T4 . . . . . The Rate of T4 DNA Synthesis after Infection of E, coli CTr5X by an 1pkf Mutant . . . . Late Protein Synthesis on E. coli CTr5X with T4 Gene 63 Mutants . . . . . . . . The Effect of Growth Temperature on T4 Late Protein Synthesis after Infection of E. coli CTr5X by pseT' and11p_.' Mutants . Map Order of Genes ipk, alc, pseT, and 3l Map Order of Some glg_Mutations . vi Page 20 56 58 61 64 66 85 90 INTRODUCTION The laboratory within which this work was done is interested in the control of gene expression in the minor early region of T4 genes consisting of genes 63, 112, and psgl, The alg_gene permits transcription of T4 late genes when the viral DNA contains cytosine rather than the normally present modified base, hydroxymethylcytosine. In addition, alg_is responsible for "unfolding" the E, 2911_nucleoid after T4 infection. This laboratory began its study of psgl_mutants because of their relationship to alg_mutants. .Esg1_mutants are often isolated as psgl, alg_double mutants, and the two genes may be contiguous. The p§g1_gene codes for the enzyme 5' polynucleotide kinase 3' phosphatase. Under some conditions pggl_mutants are also deficient in T4 late protein synthesis. The research described herewith began as an offshoot of the study of the p§§I_gene, and evolved into a study of gene 63. Many gene 63 mutants express the same phenotype as pggl_mutants, although the two genes code for separate genetically complementable enzymes. This research describes the phenotype associated with mutations in either gene, links the gene 63 mutant phenotype to the absence of T4 RNA ligase activity, and allows speculation regarding the biological role of the 5' polynucleotide kinase 3' phosphatase and RNA ligase in T4 infection. The data derived from this research is presented in Article I which has been submitted for publication. The appendices contain genetic data and fine structure maps of the region. Some of these data were used to order genes 63, gig, and psgl, but have not been presented in Article I. Appendix B is a map of some markers in the §1§_gene. This laboratory had not previously been able to obtain recombinants in the al§_gene due to the close proximity of the alg_mutations. The capacity to force recombination between alg_markers is a direct consequence of the work done on the psgl_and 63 genes presented in Article I. Appendix C describes the isolation of a bacterial host which selects for T4 312 phage containing cytosine rather than hydroxymethylcytosine in the viral DNA. This article has been published in the Journal of Virology, g1; 815-8l8 (T978). LITERATURE SURVEY Scope of the Literature Survey This dissertation is concerned with the genetics and biological role of the T4 gene 63 product (gp63). As presented in Article I of this work, gp63 functions in DNA and late protein synthesis, at least under some conditions. To date, the action of the RNA ligase activity of gp63 jg_111g_is not known; however, much information is available on the chemical mechanism of enzymatic action jn_11159, The opposite situation exists with the tail fiber attachment (TFA) activity associated with gp63: the jn_1119_action of the protein is known, but the mechanism of the reaction is not well understood. This literature survey, therefore, includes sections devoted to the state of knowledge regarding the biology and chemistry of the RNA ligase and tail fiber attachment activities to facilitate proposing a possible 13_1119 action of the RNA ligase activity. The genetics presented in this work inextricably link the RNA ligase activity of gene 63 and the 5' polynucleotide kinase 3' phosphatase activity of the psgl_gene. Phenotypes of mutants in both genes are identical with the exception of their respective enzymatic activities. Therefore, this literature survey embodies a section on the chemistry and physiology of the defect in pseT mutants. The phenotype of either gene 63 or psgl mutants is a depressed rate of DNA and late protein synthesis on a restrictive host. What is known regarding the mechanism of T4 DNA synthesis and the requirements for T4 late protein synthesis is also reviewed here. The pggl_and 63 genes are separated by a short distance on the T4 genetic map. The interval between the two genes includes the alg/gnj_gene (which may be contiguous with 63 and psgl). The genome of T4 is constructed so that genes with related functions are clustered. Indeed, 115/311 has been shown to control late gene expression under special circumstances. Three of the four 63 muta- tions isolated in this laboratory and analyzed in this work were isolated as gig/uni, 63 double mutants. Likewise, many psgl_mutants have been isolated as alc/unf, pseT double mutants. Because of the special relationship between these three genes a section on alg/unj_ has been incorporated. Finally, a discussion of the possible action of RNA ligase and 5' polynucleotide kinase 3' phosphatase in the T4 infection process is included as well as recommendations for future experi- ments to test the hypotheses. Requirements for T4 Late Transcription Within one minute after infection T4 specific transcripts are made from the ”l" strand of the phage chromosome (Rabussay and Geiduschek, l977b). Synthesis of many early transcripts ceases at approximately the time DNA synthesis begins. Those early transcripts which continue to be made throughout infection are referred to as "quasi-lates" (Salser et al., l970). Early in infection, quasi-late transcripts are made at a low frequency from an unreplicated template. Late in infection, their transcription occurs at a high frequency from the newly replicated DNA (Bruner and Cape, 1970). True-late transcription in T4 is temporally correlated with the onset of DNA replication at approximately nine minutes after infection at 30°C. The ultimate products of true-late transcription are the structural components of the virion. True-late transcripts are read from the "r" strand of the DNA. The prerequisites for this transcription are that DNA synthesis occurs; gp33, 55, and 45 are functional; and the DNA contains the modified base, hydroxymethyl- cytosine rather than cytosine (Bolle et al., 1968). T4 DO mutants, a class of mutants which make no DNA, do not synthesize late pro- teins; late protein synthesis is thymidine dependent in host and T4 combinations which are thymidine dependent for DNA synthesis; and UV irradiated T4 which does not synthesize DNA, does not make late proteins (Bolle et al., l968). Furthermore, in marker rescue exper- iments with phage defective in a late gene and DNA synthesis, the late marker could not be rescued by superinfecting phage defective in DNA synthesis, but with a wild type allele for the late_gene. Some marker rescue was seen, however, when the late function was encoded by a quasi-late rather than a true-late gene (Bruner and Cape, 1970). Several mutations which severely limit the amount of T4 DNA synthesized, however, do allow substantial synthesis of true-late gene products. These include mutations in genes 30 (DNA ligase) (Bolle et al., T968), 4l (DNA replication), 46 and 47 (endonucleases) (Riva et al., l970b). All of these mutations cause a stabilization of interruptions in the DNA (Rabussay and Geiduschek, l977a). These data have led to the theory that true-late transcription requires a "competent" template; i.e., a specific structural conformation of the DNA (Bolle et al., T968; Riva et al., l970a, Riva et al., l970b). This template contains interruptions which are necessary for the binding-initiation of RNA polymerase at late regions, and the inter- ruptions can be generated by DNA replication or very controlled nuclease action (Riva et al., l970b). Replication-independent true-late transcription, however, occurs after a delay and at a much reduced rate compared to replication coupled true-late trans- cription. It is multiplicity and temperature-dependent (Rabussay and Geiduschek, l977a; Wu and Geiduschek, 1975). T4 DNA Synthesis Replication of the T4 genome is a two step process. Initially, approximately 20 genomes are synthesized during the first 6-l2 minutes after infection at 30°C. Afterward, covalent joining of the genomes occurs to create linear concatemers, and a second wave of replication begins (c.f. Broker and Doermann, I975). The progeny genomes are synthesized in the form of concatemers and later cleaved and packaged. T4 DNA synthesis is a membrane associated phenomenon (Earhart et al., 1973). Soon after infection the parental DNA associates with the cell envelope presumably at the origin of T4 DNA replication (c.f. Siegel and Schachter, 1973). This association requires no phage-induced enzyme (Earhart, 1970) or DNA (Earhart et al., 1973) synthesis. However, it does require RNA synthesis as rifampicin added prior to infection will prevent the association. Energy poisons also inhibit membrane attachment (Earhart et al., 1973). Attachment of the parental T4 genomes to the host cell membrane does not involve detachment of the cellular DNA, an observation which implies that there are multiple DNA attachment sites on the cell membrane. In support of this hypothesis, it has been observed that when as much as 75% of the cellular DNA has been degraded by T4, the remaining DNA is all found in association with the membrane (Earhart, 1970). Both the host and parasite DNA remain associated with the membrane until the end of the eclipse period. Unlike attachment, detachment requires phage specific late protein synthesis. It will not occur in the presence of chloramphenical or in DNA negative mutants (Earhart, 1970). An RNA-DNA copolymer has been isolated from T4-infected cells soon after infection (Buckley, Kosturko, and Kozinski, 1972; Speyer et al., 1972). The RNA segment is covalently joined to the DNA segment, and the RNA segment constitutes approximately 95% of the copolymer. The DNA segment hybridizes specifically to the "1" strand of T4 DNA (Buckley et al., 1972). The 1 strand is the template for early mRNAs. The RNA-DNA copolymer is believed to be an essential intermediate in DNA synthesis (Buckely et al., 1972). Likewise, early in infection enough 3H-thymidine is joined to parental T4 genomes to increase their size by 6% (Murray and Mathews, l969a). The amount of incorporation is proportional to the multiplicity of infection. Although the size of the parental DNA is altered, the density of the DNA is not changed. Since less than 1% of the injected DNA becomes acid-soluble intracellularly, the incorporation of label appears not to be the result of a DNA repair mechanism (Murray and Mathews, l969a). It occurs in DNA synthesis negative mutants (Murray and Mathews, l969a,b). The addition of nucleotides to this intermediate is complete by five minutes post infection at 37°C. Curiously, the newly added nucleo- tide sequence will hybridize to rapidly sedimenting replicating T4 DNA, but not to mature T4 DNA (Murray and Mathews, l969a). These data could not be explained by these authors. It has been proposed that this early DNA structure is a necessary intermediate involved in concatemer formation. Both formation of the RNA-DNA copolymer and the slightly extended parental DNA molecules depends on host enzymes and occurs in the absence of phage protein synthesis. It is not until 4-5 minutes after infection that viral proteins become associated with the DNA (Miller and Kozinski, 1970). No concatemers have been seen early in infection. There is no evidence in T4 for a rolling circle mechanism operating in DNA concatemer synthesis. Instead, it appears that approximately 10 minutes after initiation of infection at 30°C concatemers are formed through a recombination mechanism (Tomizawa, 1967). Murray and Mathews (1969b) found that 5 minutes after infection at 37°C the DNA of a gene 44 mutant (DNA negative) shifted to a position on sucrose gradients indicating it was a larger molecule than the parental DNA. This corresponds with the finding of Tomizawa (1967) and Kozinski and Felgenhauer (1967) that gene 44 mutants can form concatemers consisting of linearly joined parental T4 DNA molecules. Thus, in the absence of DNA synthesis, concatemers can be formed through the joining of parental genomes. Electron micrographs of isolated complexes of replicating T4 DNA indicate that the complex is one compact mass associated with cellular membrane components (Huberman, l968). Dissociation of the DNA-membrane complex occurs as the DNA is packaged into T4 heads. Gene Products 33 and 55 Genes 33 and 55 are the muturation defective genes of T4, and they code for two proteins which associate with the E, 9911_RNA polymerase (Stevens, 1972). The addition of gp33, 55, 45, and possibly two other small peptides permits T4 to utilize the host RNA polymerase for its late transcription. Unlike true-late transcripts, quasi-late mRNAs are made at low levels in the absence of gp55 early in infection, but later they are transcribed along with true-lates from replicated templates 10 in the presence of gp55 (Bruner and Cape, 1970). Their rate of synthesis, however, never approaches that of true-lates (Salser et al., 1970). Gene Product 45 Like gp33 and 55, gp45 is also a protein associated with the RNA polymerase (Ratner, 1974). Gp45 is less tightly bound to the polymerase than either gp33 or 55, hence, it does not copurify with the RNA polymerase. This gene product is required for both DNA replication and late protein synthesis. The requirement for gp45 is more stringent than that for gp33, 55 or other 00 genes. In the absence of gp45 neither DNA replication-dependent nor replication-independent transcription of lates occurs (Wu and Geiduschek, 1975). True-Late Transcription with QytOsine-Containing_DNA The enzymes required for the conversion of cytosine to HMC residues are T4-induced. Introduction of the appropriate mutations into the T4 genome forces the incorporation of cytosine into the DNA. The T4 triple mutant, gene 56' (deoxycytidine- triphosphatase), den A' (endo II, a nuclease specific for double- stranded DNA and active on the host chromosome), den 8' (endo IV, a nuclease specific for single-stranded DNA and active on progeny T4 molecules with cytosine) synthesizes full sized T4 genomes containing cytosine (Kutter et al., 1975). It does not, however, make late proteins, although Wu and Geiduschek (1975) have reported 11 that substantial amounts of RNAs from the r strand are synthesized late in 56- infections. However, the requirement of true-late transcription for T4 hydroxymethylcytosine (HMC)-containing DNA can be circumvented by another mutation in the glg/ggf_gene (Snyder et al., 1976). The glg/ggf gene maps between genes 63 and p§g1_on the T4 chromosome (Snustad et al., 1976; Sirotkin et al., 1977). It has been shown to possess two activities: it 211ows late transcription from a gytosine-containing template (212; Snyder et al., 1976), and it "ggfolds" the host chromosome (£51; Snustad et al., 1976). In T4, genes involved in the same function are often clustered, and it is intriguing to speculate the alc/unf, pseT, and RNA ligase genes may have related functions. The effect of the glg/ggf_gene on transcription has been demonstrated in more than just T4 late transcription. E. Kutter (personal communication) reports that glg_also functions in T4 early gene expression. The 119/131 gene product has been shown to prevent late gene expression in the bacteriophage lambda when lambda lysogens were induced and then superinfected with T4. N0 glgjgnf_effect was seen on lambda early gene expression (Pearson and Snyder, 1980). The glg/gflf_protein has been shown to unfold the host nucleoid after T4 infection (Snustad et al., 1976; Tutas et al., 1974). The host nucleoid is a compact chromosomal structure consisting of between 12 and 80 individual supercoiled domains (Worcel and Burgi, 1972). T4 infection causes movement of the 12 host nucleoid so that it is dispersed along the cell envelOpe. This is accomplished through the action of the T4 gene ngg_(guclear gjsruption deficient) (Snustad and Conroy, 1974). The g1g/ggj_gene acts to open the chromosome into a linear rather than nucleoid conformation, but has no cytological effect. Ultimately, the host chromosome is degraded, and the nucleotides are re-utilized in T4 DNA. RNA transcription is vital to the integrity of the nucleoid. Exposure of cells to rifampicin prior to lysis precludes the iso- lation of intact nucleoids (Pettijohn and Hecht, 1973). It is reasonable to hypothesize that the glgjggf gene's true function is in the cessation of host transcription. Evidence to support this theory is proffered by experiments which indicate that an increased amount of RNA hybridizes to E, £911_DNA after 212: infections compared to glgf infections (Sirotkin et al., 1977; Tigges et al., 1977). The effect of the glg/ggf gene on cytosine- containing T4 DNA may be viewed as a result of the inability of T4 to distinguish its own genome from that of the cell and subse- quent inactivation of the phage genome by glg/ggf_($irotkin et al., 1977). Given the behavior of the 119/ggf_gene, it is not clear which effect, if either, is primary, and which is secondary. The glg/ggf_gene could code for an activity which unfolds the nucleoid and, consequently, disrupts transcription, or it could prevent transcription causing the nucleoid structure to disintegrate. Pearson and Snyder (1980) have shown that glg/ggf_does not l3 exert its effect on lambda transcription through a nickase or topoisomerase activity. Presumably, the two activities of this gene are related; however, it is not currently clear how they are related. The ngT Gene The product of the p§g1_gene possesses two activities: 5' polynucleotide kinase and 3' phoSphatase (Cameron and Uhlenbeck, 1977; Sirotkin et al., 1978). The enzyme's active form is a tetramer consisting of monomers of approximately 33,000 molecular weight (Panet et al., 1973; Lillehaug, 1977). The 5' polynucleotide kinase transfers terminal phosphates from ATP to 5' hydroxyl termi- nated DNAs and RNAs. The pH optimum for this reaction has been reported to be a broad range from 6.5-8.5 (Cameron and Uhlenbeck, 1977). The kinase can phosphorylate single-stranded molecules and molecules with protruding 5' ends. It will also work on double- stranded nucleic acids and at the sites of gaps, nicks, and protruding 3' ends; however, the ATP concentration for these reactions must be greater than 20 times higher than for reactions with single-stranded molecules (c.f. Kleppe and Lillehaug, 1979; Lillehaug and Kleppe, 1977; Lillehaug et al., 1976). The 3' phosphatase prefers deoxyribonucleotides and DNAs to ribonucleotides and RNAs (Becker and Hurwitz, 1967), and operates maximally around pH 6.0 (Cameron and Uhlenbeck, 1977). It removes terminal 3' phosphates from nucleic acids. There is no ATP requirement for phosphatase activity. Even though these l4 differences exist between the kinase and phosphatase, the two activities may act coordinately 1g_1119, A mutant which was described as deficient in the kinase activity by its enzymatic behavior in crude extracts was incapable of complementing a mutant deficient in the phosphatase activity when both mutants co-infected a restrictive host (Sirotkin et al., 1978). Soltis (personal com- munication), however, has found that the kinase deficient mutant has less than 3% of the wild type phosphatase activity 1g_111§g, Although the kinase activity was discovered in 1965 (Richardson, 1965; Novogrodsky and Hurwitz, 1966), and the phos- phatase was discovered in 1967 (Becker and Hurwitz, 1967), the biological role of the p§§1_gene product is still not clearly understood. It is not clear why the infected cell needs a nucleic acid 3' phosphatase activity as no E, £911_or T4 enzymes have been discovered which yield DNA with 3' phosphate termini (c.f. Koerner, 1970). Isolated cellular DNA with 3' phosphate termini have been reported (Richardson and Kornberg, 1964), but the occurrence of these ends has been attributed to the isolation procedure (Richardson, 1966). Damage to DNA incurred through gama irradia- tion does include 3' phosphate termini formation, however (Bopp et al., 1973). If DNA 3' phosphate termini are the lethal inter- mediates accumulated in restrictive pgglf infections, the number of these termini capable of exerting such an effect must be small since pgglf DNA isolated from restrictive infections and analyzed on alkaline sucrose gradients is approximately one-half the size of normal DNA (Depew and Cozzarelli, 1974). 15 Chan and Ebisuzaki (1970) reported that T4 kinase deficient mutants were not deficient in DNA recombination, replication, or repair on common laboratory strains of E, £911_K and B. Depew and Cozzarelli (1974), however, showed that lack of the 3' phosphatase activity prevented growth of these 14 mutants on E, £911_CTr5X. E, 9911_CTr5X is a hybrid strain between E, £911 K and a clinical isolate, E, £911_CT196. Depew and Cozzarelli (1974) used a EEEI. mutant which lacked the phosphatase, but not the kinase activity (Sirotkin et al., 1978; Cameron et al., 1978). In CTr5X infections the p§31_mutant (1) made DNA on CTr5X at one-half the rate of wild type T4, (2) made DNA of approximately one-half the normal size, and (3) packaged the DNA to a lesser extent than wild type T4 does (Depew and Cozzarelli, 1974). In addition, they found that the E§g1_defect on CTr5X could be overcome by a suppressor mutation in a second T4 gene called §1p_(§uppressor of Ehree prime phos- phatase mutants). The 112_gene maps near the rII region, a region which is rich in genes coding for membrane proteins and nucleases. An §12_mutation restored DNA synthesis, size, and packaging to normal in pggl, §12_double mutant infections of CTr5X. The authors hypothesized that the §12_gene probably coded for a nuclease. The first evidence that the kinase and phosphatase activi- ties were associated with the same protein came from biochemical data. Uhlenbeck and Cameron (1977) fOund a persistent contamination of phosphatase activity when they purified the kinase protein. Later, the phosphatase activity was shown to be associated with the same molecule as the kinase by several criteria: the 16 activities co-purified following DEAE cellulose, phosphocellulose, hydroxylapatite, and Sephadex 6200 chromatography; heat inactivation curves for the two activities were identical; and kinase (but not phosphatase) substrates protected both activities from heat inactivation (Cameron and Uhlenbeck, 1977). This relationship was confirmed genetically by Sirotkin et a1. (1978) who showed that mutants isolated for lacking one activity usually lacked the other. The lack of either enzymatic activity caused loss of viability on CTr5X, and 1n_1119_a mutant which appeared to lack the kinase activity only did not complement a mutant lacking the phosphatase (Sirotkin et al., 1978; Soltis, personal communication). Sirotkin et a1. (1978) also showed that p§g1_mutants on CTr5X made less than normal amounts of late proteins. Early proteins appeared to be unaffected. The depressed level of late protein synthesis may account for the DNA packaging defect observed by Depew and Cozzarelli (1974). An important observation reported by Sirotkin et a1. (1978) bears upon the relationship of the host cell to p§§1_mutants. In addition to suppression by T4 §12_mutations, p§21_mutations can also be suppressed by introduction of an efficient amber suppressing mutation into the CTr5X host. The efficient amber suppressing CTr5X will permit growth of Egg: point mutants and deletions. Hence, growth is not due to suppression of the p§g1_mutation directly, but may be due to suppression of a host mutation in a gene whose product can substitute fer the pggl_gene product. These results also explain the inconclusive results of Chan and 17 Ebisuzaki (l970). E§g1_mutants would be expected to show no pheno- type on laboratory strains if those strains possessed an activity which could substitute for the pggl_activity. No 5' polynucleotide kinase activity has been observed in E, £911_cells, but 3' phosphatase activities have been reported. Recently, Cooley et a1. (1979) have been able to isolate E, ggl1_mutants using common laboratory strains which behave towards pggl_mutants similarly to E, £911 CTr5X. These strains carry a double mutation called 111 A and 8 (late 1nhibitors of 14). E11_B maps at 25 minutes on the E, 9911_chromosome and describes a new gene. The position of’111LA is less certain. Although the 111_defects are not characterized, these mutants are not deficient in 3' phosphatase activity. The evidence to date indicates that the pgel_gene acts at the level of the DNA and ultimately functions in the control of late protein synthesis. This action may consist of shuttling phosphates from 3' to 5' termini (Sirotkin et al., 1978). Recent evidence (Soltis and Uhlenbeck, unpublished) indicates that the kinase-phosphatase both act at nicks, in effect converting a 3' phosphate to a 5' phosphate. However, the phosphate fixed to the 5' terminus is derived from ATP rather than the 3' terminus of the molecule at the site of the nick, and the phosphate which was originally at the 3' terminus is released. Eggl_control of late protein synthesis is probably attained through the regulation of late transcription as: (1) the EEEI. product interacts with the DNA since the effect of p§g1_mutations can be seen as early as DNA synthesis begins (Depew and Cozzarelli, 18 1974); (2) other gene products which affect true-late gene expres- sion do so by affecting transcription (Bolle et al., 1968); (3) other mutations which affect DNA synthesis to a far greater extent do not inhibit late gene expression (Sirotkin et al., 1978); and (4) pre- liminary results on "mixed competitor" hybridization experiments with RNA from pgel 2 and wild type T4 indicated that p§g1_mutants make less late mRNA than wild type (Sirotkin et al., 1978; Snyder, unpublished). Gene 63 Gene 63 codes for a 42,000 molecular weight protein product (Vanderslice and Yegian, 1974). As early as 1969, gene 63 had been implicated in attachment of T4 tail fibers to the baseplate (Wood and Henninger, 1969). However, it was not until 1977 that it became apparent that the product of gene 63 was also responsible for the RNA ligase activity in extracts from T4 infected cells (Snopek et al., 1977). Thus, the mapping of the RNA ligase protein to gene 63 came five years later than the discovery and purification of this enzy- matic activity (Silber et al., 1972). Tail Fiber Attachment Activity The protein product of gene 63 has long been known to function in the attachment of tail fibers to the baseplate of 14 (Wood and Henninger, 1969). Despite the fact that gp63 was impli- cated in this function much earlier than in its role in nucleic acid metabolism (Snopek et al., 1977), the mechanism by which gp63 acts in tail fiber attachment still remains elusive. Curiously, 19 in this reaction gp63 may be a member of an odd class of proteins which appear to act catalytically, yet neither break nor form covalent bonds in their substrate molecules (Wood and King, 1979; Wood, 1979). Four non-structural proteins participate in the process of T4 tail fiber assembly and subsequent attachment. All of these exhibit the curious behavior of non-perturbation of covalent bonds (Wood and King, 1979; Wood, 1979; Bishop et al., 1974). These feur proteins are gp38, 57, 63, and wgg, The structural elements of tail fibers consist of proteins encoded by genes 34, 35, 36, and 37 (Wood and King, 1979; Wood, 1979; Bishop et al., 1974; Dickson, 1973; Wood and Bishop, 1973; King and Wood, 1969). A diagram of T4 tail fiber assembly is presented in Figure l. The assembly of tail fibers initiates with the dimerization of gp34 by gp57. The gene 34 protein dimer constitutes the proximal half fiber of the tail fiber. The gene 57 product is then used along with gp38 to dimerize the gene 37 protein. Gp36 is then added to the gp37 dimer. The last step in the formation of the distal half fiber is the addition of gp35 to the gp36-37 complex. Whole tail fibers are formed by linkage between the proteins encoded by genes 34 and 35. T4 tail fibers are approximately 1400 A long and 45 A wide with a kink at the midpoint which forms an angle of 160° (Wood and Bishop, 1973; Ward et al., 1970). Tail fibers are joined to the phage at the baseplate through the action of gp63 (Wood and Henninger, 1969) and apparently the product of the wgg_gene. Six tail fibers normally attach to the baseplate which has a six-fold symmetry. The baseplate consists of six identical 20 gp34 gp37 9P57 9P57 gp38 34 d' gp37 dimer gp. 1mer gp36 (prox1mal half fiber) gp37-36 complex 9935 gp37- -36- 35 complex (distal half fiber) whole tail fiber - gp wac completed head ,/”\\ %and sheath 4» gp wac and gp 63 tail fiber completed interaction virion Figure l.--The Assembly and Attachment of T4 Tail Fibers. 21 protein wedges which form a hexagon (c.f. Eiserling, 1979; Wood and King, 1979). T4 particles can infect with as few as three tail fibers (Wood and Henninger, 1969) although infectivity is greatly reduced under these conditions. Gp wgg_(whisker gntigen gpntrol) constitutes the collar and whisker protein (Follansbee et al., 1974; Yanagida and Ahmad-Zadeh, 1970) located at the head-tail junction of the virion. It has been fOund that tail fibers attach to Egg? tail fiberless phage ten times more rapidly than they do to wggf tail fiberless phage (Wood and Conley, 1979). Upon further dissection, the wag? tail fiber inter- action appears to involve whiskers and distal half fibers. Although 1n_1119 TFA appears to require the formation of completed tail fibers (King and Wood, 1969; King, 1968) it is possible to cause half fibers to interact with the completed head—tail structure of the virion 1n_11159, This phenomenon has permitted the use of half fiber reactions to analyze the contributions of gp wgg_and gp63 in TFA. 1nH11159, phage lacking proximal half fibers and having distal half fibers attach proximal half fibers significantly more rapidly than phage lacking distal half fibers but having proximal half fibers attach distal half fibers (Terzaghi, 1971). Wood and Conley (1979) have fbund that Egg: phage can attach proximal half fibers as well as wggf phage, but wag? phage are significantly deficient in activating proximal half fibers into whole fibers by the addition of distal half fibers. The interpretation of these data is that the rate limiting step in tail fiber attachment is 22 the interaction between whiskers and the distal half fiber. Once this occurs the proximal half fiber is readily attached to the baseplate. Furthermore, whole tail fibers attach more slowly to 312?, 34' phage than to Eggf, 37' phage, suggesting that the presence of gp37 in wggf, 34' viroids competitively inhibits the interaction of the added whole tail fibers and whiskers (Terzaghi, 1971). The data indicate that T4 whiskers recognize and bind to a site on the distal half fiber to assist in aligning the whole tail fiber, although Terzaghi et a1. (1979), and Singh and Terzaghi (1974). have evidence that whiskers also interact with a site on the proximal half fibers. It appears that the wgg_structure serves as a "jig" site for the attachment of the tail fibers (Terzaghi et al., 1979; Terzaghi, 1971): its presence on the phage particle increases the rate at which tail fiber attachment occurs probably because it serves to increase the “target size" for interaction between free tail fibers and the assembled head and tail components (Wood, 1979; Terzaghi et al., 1979). The fact that the collar-whisker complex is added after head-tail attachment (Wood and King, 1979; Coombs and Eiserling, 1977) explains early results that attachment of free tail fibers required joined head-tail structures (Terzaghi, 1971). The collar-whisker complex interacts with tail fibers in the process of tail fiber retraction also. The evidence is that the interactions are much the same for TFA as well as tail fiber retraction (Wood and Conley, 1979) since a defective 36 mutant attaches poorly to whiskers under some conditions and is also impaired in tail fiber retraction. 23 The role of gp63 in TFA has been deduced from experiments carried out by Wood and colleagues. When purified tail half fibers were added to assembled heads and tails in the presence of gp63 the rate of the slow and probably non-physiological attachment of proximal half fibers was stimulated five times compared with the less than 1.5 fold stimulation for attachment of distal half fibers to attached proximal half fibers (Wood et al., 1978). The process of tail fiber attachment is therefbre believed to involve an interaction of the whiskers probably with gp36 of the distal half fiber of the tail fiber (Wood and Conley, 1979; Wood, 1979) followed by attachment of gp34 of the proximal half fiber to the baseplate mediated by gp63. It is not known to which protein of the baseplate gp34 attaches; however, a strong candidate is gp9 (c.f. Eiserling, 1979). In electron micrographs of baseplates with attached tail fibers the fibers end close to, if not at, gp9 (Crowther et al., 1977), and phage missing gp9 do not attach tail fibers (King, 1968). The TFA reaction mediated by gp63 is an intriguing one. Evidence that gp63 acts catalytically in TFA is: (1) TFA proceeds in the presence of gp63 at a much faster rate than in its absence; (2) the rate of TFA varies linearly with the amount of gp63 present; and (3) the final yield of infectious particles is dependent upon the concentrations of purified fibers and fiberless particles and not on gp63 concentration (Wood, 1979; Wood et al., 1978; Bishop, et al., 1974). As previously mentioned, no covalent bonds appear to be involved in this reaction. The presence of gp63 is not 24 absolutely required fer TFA (Wood et al., 1978) or phage viability. In the absence of the TFA protein, tail fiber attachment occurs, albeit at between 1% and 5% of the rate which occurs in the presence of gp63 (Wood, 1979; Wood et al., 1978; Snopek et al., 1977). Burst sizes under these conditions range between 10-15 phage per cell (Snopek et al., 1977). The attachment of tail fibers can be stimu- lated in the absence of functional gp63 by increasing the growth temperature or prolonging the latent period (Snopek et al., 1977). Unlike other catalytic proteins enormous quantities of gp63 are required for the TFA reaction. 1n.11119, the molar quantity of gp63 required for an adequate reaction rate is ten times the number of free tail fibers present and several orders of magnitude greater than the number of attachment events which occur (Wood et al., 1978). 1n_1119, the number of gp63 molecules synthesized by the end of a single viral life cycle is approximately equal to the number of tail fibers synthesized (Ward et al., 1970; Wood et al., 1978). Gp63 constitutes 1% of the total soluble cellular protein at this point. T4 RNA Ligase T4 RNA ligase was discovered and purified by Hurwitz and coworkers in 1972. The enzyme exhibited the unusual ability of cyclizing oligomers of poly A (Silber, Malathi, and Hurwitz, 1972). Since its discovery T4 RNA ligase has proven itself versatile and immensely useful in nucleic acid synthesis. In addition to its activity in cyclizing oligoribonucleotides of chain length greater 25 than eight (Kaufman, Klein, and Littauer, 1974), the enzyme can catalytically join oligoribonucleotides to other oligoribonucleotides (Walker et al., 1975; Uhlenbeck and Cameron, 1977; Sugino et al., 1978), oligodeoxyribonucleotides to oligoribonucleotides (Sugino et al., 1976; Sugino et al., 1978) and oligodeoxyribonucleotides to oligodeoxyribonucleotides (Sugino et al., 1976; Snopek et al., 1976; Sugino et al., 1978; Hinton and Gumport, 1978; McCoy and Gumport, 1980). Circularization of oligodeoxyribonucleotides has also been reported (Sugino et al., 1976; Sugino et al., 1978) as well as the single addition of 2'deoxyribonucleoside, 3',5'-bisphosphates to deoxyribonucleotides (Hinton et al., 1978). T4 RNA ligase catalyzes phosphodiester bond formation between a 3'OH-terminated "acceptor" molecule and a 5'P-terminated "donor" molecule. The reaction mechanism may be diagrammed as follows: +2 M9 E + ATP # E-AMP + PP]. (Cranston et al., 1974) Acceptor E-AMP + 5'P—donor e======é E + A(5')P-P(5')Donor E A(5')P-P(5')Donor + Acceptor =FB Acceptor-P-Donor + AMP (Sugino et al., 1977b) The first step of the reaction is the adenylation of the enzyme where ATP reacts with the enzyme to form an enzyme-AMP complex with the release of pyrophosphate. The presence of polynucleotides is unnecessary for this step to occur (Cranston 26 et al., 1974). In the second step of the reaction the adenylated enzyme complex transfers the adenyl group to the donor molecule to form an adenylated donor molecule. AMP is covalently bound to the donor in a 5'-5' pyrophosphate linkage (Sugino, et al., 1976; Ohtsuka et al., 1976; Sugino, et al., l977b). This step requires the presence of an hydroxyl terminated acceptor molecule (Sugino et al., 1976; Sugino et al., 1977b). The donor molecule may be an oligonucleotide or a nucleoside 3',5' bisphosphate, but cannot be a single nucleoside monophosphate (Hinton et al., 1978; England and Uhlenbeck, 1978). After the activated donor molecule is formed the reaction proceeds in the absence of ATP (Ohtsuka et al., 1976; Sugino et al., 1977b; England and Uhlenbeck, 1978) or a 3' P04 group on a nucleotide donor (England and Uhlenbeck, 1978; Hinton et al., 1978). The final step of the reaction joins the 3' phosphate of the acceptor molecule to the 5' phosphate of the donor molecule with the concomitant release of AMP. Donor activation and the final ligation step have not been shown to be reversible (Sugino et al., l977b). Generally, the preferred reaction for T4 RNA ligase is the Circularization reaction of single-stranded nucleic acid (Sugino et al., l977b; Kaufmann and Kallenbach, 1975; Sugino et al., 1976). In this situation the 5' end of one molecule acts as the donor and the 3' end acts as the acceptor. Intermolecular joining reactions can be effected through the use of small oligomers which sterically inhibit the production of a circular product (Sugino et al., 1976) or substrates with appropriately altered termini 27 which are incapable of engaging in unimolecular reactions (Uhlenbeck and Cameron, 1977). As mentioned above, RNA ligase has a rather broad substrate specificity, especially with respect to donor molecules (England and Uhlenbeck, 1978). Oligoribonucleotides and oligodeoxyribonucleotides can both act efficiently as donors (Snopek et al., 1976; Uhlenbeck and Cameron, 1977). In fact, ligation of a DNA donor molecule to an RNA acceptor molecule can proceed as efficiently as a cyclization reaction (Sugino, et al., 1977b). Requirements for acceptors, on the other hand, are more stringent. At the minimum, an acceptor molecule must be a 3'OH- terminated trinucleotide (Kaufman and Kallenbach, 1975; Sugino et al., 1976; England and Uhlenbeck, 1978). Apparently, the enzyme "sees" only the 3' terminal trinucleotides of a potential acceptor molecule as dinucleotides are inactive as acceptors, and acceptor oligomers longer than three nucleotides do not significantly alter the reaction rate, yields, or Km values compared to values achieved with trinucleotides (Kaufman et al., 1974; England and Uhlenbeck, 1978). Placement of an rU residue in the 3' terminal trinucleotide block of an oligomer greatly reduces its efficacy as an acceptor molecule (England and Uhlenbeck, 1978). Oligo- deoxyribonucleotides act as acceptor molecules approximately an order of magnitude more poorly than oligoribonucleotides (Sugino, et al., 1978). When optimum conditions for RNA acceptor reactions are employed in reactions using DNA acceptors, the yields have generally been reported as poor. Sugino et a1. (1976) reported 28 obtaining intermolecular DNA ligation by using oligomers too small to cyclize or missing an hydroxyl group at the 3' end. To improve yields with DNA acceptors new reaction conditions have been established. High yields may be obtained by using larger amounts of enzyme, incubation periods of between two and seven days (as compared to a few hours with RNA acceptors) at 17°C rather than 37°C, an ATP regenerating system to keep the ATP concentration low, 2 for the normal requirement of "9+2 and the substitution of Mn+ (Hinton et al., 1978; Hinton and Gumport, 1979; McCoy and Gumport, 1980). For long incubation periods it is necessary to incubate the reaction mixture at lower temperatures because RNA ligase becomes inactivated at 37°C, and initial reaction rates are not maintained throughout the reaction (Uhlenbeck and Cameron, 1977; England and Uhlenbeck, 1978). Also, lower temperatures may stabilize the Michaelis complex between reactants in the final step of the reaction (Hinton et al., 1978). Adequate levels of ATP are required to ensure activation of the donor molecule; however, high levels of ATP inhibit the final ligation step of the reaction which requires free enzyme rather than the adenylated form (Hinton et al., 1978; McCoy and Gumport, 1980). The use of an ATP regeneration system for long term reactions rather than high concentrations of ATP circumvents this problem. Mn+2 in the incubation mixture may act by relaxing the enzyme's nucleotide specificity requirements as the ion does in DNA and RNA polymerase reactions, thus, making a DNA acceptor more acceptable to RNA ligase (Hinton et al., 1978). 29 2 requiring It is not clear, however, how this may occur if theMg+ step is the adenylation of the enzyme. The importance of the acceptor molecule in determining reaction rate may be attributed to its bifunctional role in the reaction. The acceptor molecule first is an obligatory participant in the donor activation step, and secondly, it is itself a substrate in the terminal ligation reaction. These two steps of the reaction are separable, as the same acceptor need not function in both steps. Indeed, RNA acceptors have been used to effect donor activation followed by ligation of the donor molecules to DNA acceptors. This phenomenon has been designated "acceptor exchange" (Sugino et al., 1977b). The RNA ligase' preference for the cyclization reaction may be interpreted then as a consequence of the proximity of donor and acceptor ends in cyclizable molecules. Indeed, the reaction rate is maximum between 10 and 20 nucleotides but decreases as ends become more distant in longer molecules (Kaufmann et al., 1974). The mechanism of T4 RNA ligase reaction is similar to that of T4 and E, £911_DNA ligases (Lehman, 1974). All these enzymes first form an adenylated enzyme complex, which transfers the adenyl group to the donor molecule to form the activated donor intermediate. The T4 enzymes cleave the pyrophosphate bonds of ATP molecules, whereas the E, g911_ligase cleaves the pyrophosphate bond of an NAD molecule. An important difference between T4 RNA ligase and these DNA ligases is that the DNA ligases covalently join DNA ends which are held in juxtaposition by hydrogen bonding with complementary strands. 30 T4 RNA ligase has no such requirement for the positioning of substrate molecules (Sugino et al., 1976), and, in fact, duplex molecules inhibit the reaction. Curiously, however, addition of T4 RNA ligase to T4 DNA ligase reaction mixes greatly stimulates the joining of blunt-end double-stranded DNA molecules (Sugino et al., 1978; Sugino et al., l977a). Using a synthetic duplex decamer Sugino et a1. (l977a) found that especially at low concen- trations of T4 DNA ligase, the addition of T4 RNA ligase could encourage blunt-end joining to the point where the turnover number for the reaction approximated the turnover number achieved in reactions joining DNA cohesive ends. Essentially, then, addition of RNA ligase enabled the DNA ligase to behave in blunt-end joining as though it had been given the advantage of prealignment of the DNA strands by complementary base pairing. The products of the blunt-end joining were largely linear, but some circular duplexes were formed. In contrast to the high efficiency conferred upon blunt-end joining by the RNA ligase, the effect on cohesive end joining was only moderate, and T4 RNA ligase conferred no advantage to E, £911_DNA ligase in any reaction. The efficiency of the duplex joining reaction lead these authors to suggest that this enzymatic activity has a physiological role in T4 infection. The Relationship Between RNA Li ase and TailiFiber AttaChment Activ ty The discovery that gene 63 coded for the T4 protein responsible fbr RNA ligase activity came when mutants deficient in RNA ligase activity were mapped to gene 63 (Snopek et al., 1977). 31 It was shown that neither RNA ligase nor TFA activity is present in gene 63 amber mutants when they infected non-amber suppressing hosts; both activities appear simultaneously when the T4 mutants infect an amber-suppressing host; both activities are more heat labile than normal when infection is of an amber suppressor F strain, and a revertant of a gene 63 amber mutation has simul- taneously lost both phenotypes. When gp63 is purified, it functions 1n vitro in both reactions. The roles of gp63 appear to be functionally unrelated. Reaction conditions and inhibitors are different for the activities. The RNA ligase reaction requires the presence of ATP and Mg+2 (Snopek et al., 1977); TFA requires only divalent or monovalent cations (Wood and Henninger, 1969). TFA occurs in the presence of pyrophosphate, EDTA (Wood and Henninger, 1969), and ammonium ions (Snopek et al., 1977). RNA ligase activity, however, is inhibited under these conditions. Both activities are sensitive to exposure to sulfhydril blocking agents such as parachloromercurobenzenesulfonate or N-ethylmaleimide, and each is stabilized by ATP or DTT (Wood et al., 1978; Snopek et al., 1977). Stabilization by reducing agents and inhibition by sulfhydril blocking reagents indicates that a sulfhydril group is essential to both activities of this protein (Snopek et al., 1977). RNA ligase activity is associated with the monomer form of the protein. Early reports on TFA activity describe the dimer fOrm as the active form (Wood and Henninger, 1969); however, 32 Snopek et a1. (1977) have found activity in monomers and that the protein tends to behave as a dimer under conditions similar to those used by Wood and Henninger (1969) to purify the enzyme. The Phenotype of RNA Ligase Deficient T4 At the time I began my analysis very little was known about the role of RNA ligase in the infection process. No phenotypes had been attributed to a deficiency in this enzymatic activity. Wood and Henninger (1969) isolated a double mutant which was deficient in TFA as well as DNA synthesis. Upon backcrossing, the mutant segre- gated two progeny types; one which gave minute plaques (1g), and another which behaved as an amber mutant. Analysis of the mutants demonstrated that the amber mutant was deficient in TFA and became known as the gene 63 amber mutant, M69. In later work (Snapek et al., 1977), M69 was demonstrated to be defective in RNA ligase activity in jn_vitro assays. Recombination between M69 and the minute mutant yielded 3% recombination frequency and mapped the mfl_marker to the region between genes 32 and 63. Minute mutants were normal in TFA ability. Neither of the mutants was tested for DNA synthesis, but m1_was assumed to be responsible for this defect since it mapped in a region coding for DNA synthesis enzymes. I have fbund that gene 63 mutants are defective in DNA synthesis, but only when they infect certain restrictive hosts (Article I). The laboratory strains used by Wood and Henninger (1969) do not belong to this class of 33 restrictive hosts, and hence, the DNA deficiency they saw was not due to the M69 mutation. ' Article I of this work describes my contribution to the current state of knowledge regarding the mutant phenotype associated with an RNA ligase deficiency. I have found that the E, £911_CTr5X strain, which does not support growth of T4 p§g1_mutants, does not support growth of many gene 63 mutants. Furthermore, the defect on this strain is independent of the TFA phenotype. Gene 63 mutants which I have isolated because of their inability to grow on CTr5X were not deficient in TFA activity. When they were tested 1n_11119_ for their ability to cyclize poly A oligomers, however, none possessed any RNA ligase activity. As with 2§EI_mutants, an infection of CTr5X by RNA ligase deficient phage results in a defect in DNA and late protein synthesis. In addition, conditions which suppress the phenotype of p§g1_mutants on CTr5X also suppress the RNA ligase mutant phenotype. Hence, RNA ligase mutant phage can overcome their late protein synthesis defect if grown at 42°C; RNA ligase mutants grown on altered CTr5X cells containing an efficient amber suppressor will produce normal amounts of DNA and late proteins; and lastly, introduction of an §12_mutation into the phage genome allows the double mutant to grow on CTr5X. These data implicate the products of the pseT and 63 genes in the same T4 metabolic pathway. 34 The Role of RNA Ligase and 5' Polynucleotide Kinase 3' Phosphatase in 14 Infection It is easy to envision how the 5' polynucleotide kinase 3' phosphatase and RNA ligase could work in concert 1n_1119, The activity of the 5' polynucleotide kinase 3' phosphatase can produce a nucleic acid which is a substrate for the RNA ligase in either a donor or acceptor capacity. Beyond this hypothesis, however, their roles in the infection process are much less clear. In organisms where RNA splicing occurs, the need for an RNA ligase activity is obvious. In yeast mitochondria, the existence of a circular RNA byproduct of splicing has even been reported (Halbreich et al., 1980). However, no circular RNAs have been reported in T4-infected cellular extracts, or, for that matter, have any instances of mRNA splicing been reported in prokaryotic systems (c.f. Abelson, 1979). Whatever the role of RNA ligase in the infection process, the fact that an absence of this enzyme has ramifications for both DNA and late protein synthesis must be taken into account. The importance of a "competent" form of DNA for late transcription has long been known. RNA ligase may function in forming a nucleic acid structure which is competent for DNA and late protein synthesis. A model must also be consistent with the fact that T4 DNA synthesis is depressed from the beginning of the synthesis period in an RNA ligase mutant. I think that the effect on late transcription of an RNA ligase deficiency is not the result of its effect on DNA synthesis. 35 Other T4 mutants which do not make DNA do not synthesize late mRNAs, but continue to synthesize early transcripts past the time when early mRNA synthesis normally ceases. In CTr5X infections RNA ligase and p§g1_mutants, on the other hand, cease making early transcripts normally even though late gene expression is prevented. This observation is consistent with the hypothesis that these enzymes function in formation of a competent template. Either nucleoid formation or DNA-membrane attachment could be essential to the formation of a competent DNA structure. The §1g_gene, which maps between genes 63 and Eggl, is responsible for destruction of the integrity of the host nucleoid. Since genes with related functions are clustered in T4, it is tempting to propose that the gene 63 and p§21_products are involved in the formation or maintenance of a T4 nucleoid-type structure. The integrity of cellular nucleoids is destroyed when transcription is terminated by the addition of rifampicin. Unfolding of the host chromosome by the T4 glg_gene product is accompanied by the loss of the DNA's ability to be used as a transcriptional template (Sirotkin et al., 1977). Hence, late transcription in T4 could depend on the proper fblding of the T4 chromosome into a nucleoid structure. It is easy to imagine how RNA ligase could function in nucleoid formation if core formation required the covalent linkage of DNA to RNA. The fact that DNA donors can be ligated to RNA acceptors at a high efficiency lends support for a possible physiological role for this reaction. 36 Sugino et al. (1977a) suggested that the amount of RNA ligase available in the cell especially at late times in infection would permit the DNA ligase-catalyzed, RNA ligase-stimulated duplex DNA blunt-end joining reaction to occur at a high efficiency, and thus, the reaction is probably physiologically significant. Perhaps the products of genes 30 (DNA ligase) and 63 form a complex which behaves as a topoisomerase. The topoisomerase could be involved in preparing a competent template for T4 DNA and late mRNA synthesis. Type II DNA topoisomerases have been demonstrated to reversibly break double—stranded DNA (Liu et al., 1980). The T4 DNA ligase, RNA ligase complex possesses the joining ability, but has not been shown to affect DNA superhelicity. That activity could reside in another peptide or peptides. In this model, the CTr5X host may be missing a topoisomerase activity which can substitute for this T4 complex, or alternatively, it may be missing a subunit which E, ggl1_normally contributes to the T4 complex. Sugino et a1. (l977a) suggested the DNA ligase-catalyzed, RNA ligase-stimulated blunt-end duplex DNA joining reaction was involved in T4 illegitimate recombination. If so, RNA ligase or DNA ligase mutants would be expected to accumulate duplications and deletions to a lesser degree than wild type T4. This hypothesis could be tested by comparing the spontaneous frequency of deletions occurring in an unessential region such as the 0 region (Depew et al., 1975) in an RNA or DNA ligase mutant and wild type T4. We now know that the T4 gene 63 product functions in DNA and late mRNA synthesis. The mechanism by which it accomplishes 37 this result is unknown; however, whatever that mechanism may prove to be, its elucidation will surely contribute substantially to the understanding of molecular biological phenomena. 38 References Abelson, J. 1979. RNA Processing and the Intervening Sequence Problem. pp. 1035-1069 in Annual Reviews of Biochemistry, vol. 48, eds. E. E. Snell, P. D. Boyer, A. Meister, and C. C. Richardson (Annual Reviews, Inc.). Becker, A. and J. Hurwitz. 1967. The Enzymatic Cleavage of Phosphate Termini from Polynucleotides. J. Biol. Chem., 112; 936-950. Bishop, R. J., M. P. Conley, and W. 8. Wood. 1974. Assembly and Attachment of Bacteriophage T4 Tail Fibers. J. Supramol. Struct., E; 196-201. Bolle, A., R. H. Epstein, W. Salser, and E. P. Geiduschek. 1968. Transcription During Bacteriophage T4 Development: Requirements for Late Messenger Synthesis. J. Mol. Biol., 33: 339—362. BOpp. A., S. Carpy, B. Burkart, and U. Hagen. 1973. Action of Exonuclease III on y-Irradiated DNA. Biochem. Biophys. Acta., 223; 47-56. Broker, T. R., and A. H. Doermann. 1975. Molecular and Genetic Recombination of Bacteriophage T4. pp. 213-244 in Annual Reviews of Genetics, vol. 9, eds. H. L. Roman, A. Campbell, and L. M. Sandler (Annual Reviews, Inc.). Bruner, R. and R. E. Cape. 1970. The Expression of Two Classes of Late Genes of Bacteriophage T4. J. Mol. Biol., §§; 69-89. Buckley, P. J., L. D. Kosturko, and A. W. Kozinski. 1972. ln_Vivo Production of an RNA-DNA Copolymer after Infection of Escherichia coli by Bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A., 62, 3165-3169. Cameron, V., D. Soltis, and O. C. Uhlenbeck. 1978. Polynucleotide Kinase from a T4 Mutant which Lacks the 3' Phosphatase Activity. Nucl. Acids Res., E; 825-833. Cameron, V. and O. C. Uhlenbeck. 1977. 3' Phosphatase Activity in T4 Polynucleotide Kinase. Biochem., 1E, 5120-5126. Chan, V. L. and K. Ebusazaki. 1970. Polynucleotide Kinase Mutant of Bacteriophage T4. Mol. Gen. Genet., 199: 162-168. 39 Cooley, W., K. Sirotkin, R. Green, and L. Snyder. 1979. A New Gene of Escherichia coli K-12 whose Product Participates in T4 Bacteribphage Late Gene Expression: Interaction of 111_with the T4-Induced Polynucleotide 5'Kinase 3' Phosphatase. J. Bact., 159: 83-91. Coombs, D. H., and F. A. Eiserling. 1977. Studies on the Structure, Protein Composition, and Assembly of the Neck of Bacterio- phage T4. J. Mol. Biol., 11E, 375-405. Cranston, J. W., R. Silber, V. G. Malathi, and J. Hurwitz. 1974. Studies on Ribonucleic Acid Ligase. Characterization of an Adenosine Triphosphate-Inorganic Pyrophosphate Exchange Reaction and Demonstration of an Enzyme-Adenylate Complex with T4 Bacteriophage-Induced Enzyme. J. Biol. Chem., ggg, 7447-7456. Crowther, R. A., E. V. Lenk, Y. Kikuchi, and J. King. 1977. Molecular Reorganization in the Hexagon to Star Transition of the Baseplate of Bacteriophage T4. J. Mol. Biol., 1_1_6_: 489-523. Depew, R. E. and N. R. Cozzarelli. 1974. Genetics and Physiology of Bacteriophage T4 3' Phosphatase: Evidence for Involve- ment of the Enzyme in T4 DNA Metabolism. J. Virol., 115 888-897. Depew, R. E., T. J. Snopek, and N. R. Cozzarelli, 1975. Char- acterization of a New Class of Deletions of the 0 Region of the Bacteriophage T4 Genome. Virol., E4, 144-152. Dickson, R. C. 1973. Assembly of Bacteriophage T4 Tail Fibers. IV. Subunit Composition of Tail Fibers and Fiber Precursors. J. Mol. Biol., 12, 633-647. Earhart, C. F. 1970. Association of Host and Phage DNA with Membrane of Escherichia coli. Virol., 42, 429-436. Earhart, C. F., C. J. Sauri, G. Fletcher, and J. L. Wulff. 1973. Effect of Inhibition of Macromolecule Synthesis on the Association of Bacteriophage T4 DNA with Membrane. J. Virol., 11; 527-534. Eiserling, F. A. 1979. Bacteriophage Structure. pp. 543-580 in Comprehensive Virology, vol. 13, eds. H. Fraenkel-Conrat and R. R. Wagner (Plenum Press). England, E., and O. C. Uhlenbeck. 1978. Enzymatic Oligoribo- nucleotide Synthesis with T4 RNA Ligase. Biochem. 11, 2069-2076. 4O Follansbee, S. E., R. W. Vanderslice, L. H. Chavez, and C. D. Yegian. 1974. A New Set of Adsorption Mutants of Bacteriophage T40: Identification of a New Gene. Virol., EE: 180-199. Halbreich, A., P. Pajet, M. Foucher, C. Grandchamp, and P. Slonimski. 1980. A Pathway of Cytochrome b mRNA Processing in Yeast Mitochondria: Specific Splicing and an Intron-Derived Circular RNA. Cell, 12, 321-329. Hinton, D. M., J. A. Baez, and R. I. Gumport. 1978. T4 RNA Ligase Joins 2'-Deoxyribonuc1eoside 3'5'-Bisphosphates to 01igodeoxyribonucleotides. Biochem., 11, 5091-5097. Hinton, D. M. and R. I. Gumport. 1979. The Synthesis of Oligo- deoxyribonucleotides Using RNA Ligase. Nucl. Acids Res., 1; 453-464. Huberman, J. A. 1968. Visualization of Replicating Mammalian and T4 Bacteriophage DNA. Cold Spr. Harb. Sym. Quant. Biol., EE: 509-524. Kaufmann, G. and N. R. Kallenbach. 1975. Determination of Recognition Sites of T4 RNA Ligase on the 3'-0H and 5'-P Termini of Polynucleotide Chains. Nature, EEQ: 452-454. Kaufmann, G., T. Klein, and U. Z. Littauer. 1974. T4 RNA Ligase: Substrate Chain Length Requirements. F.E.B.S. Lett., 4E, 271-275. King, J. 1968. Assembly of the Tail of Bacteriophage T4. J. Mol. Biol., 1g; 231-262. King, J. and W. 8. Wood. 1969. Assembly of Bacteriophage T4 Tail Fibers: The Sequence of Gene Product Interaction. J. Mol. Biol., 12; 583-601. Kleppe, K. and J. R. Lillehaug. 1979. Polynucleotide Kinase. pp. 245-275 in Advances in Enzymology and Related Areas of Molecular Biology, vol. 48, ed. A. Meister (John Wiley and Sons, Inc.). Koerner, J. F. 1970. Enzymes of Nucleic Acid Metabolism. pp. 291- 322 in Annual Reviews of Biochemistry, vol. 39, eds: E. E. Snell, P. D. 80 er, A. Meister, and C. C. Richardson (Annual Reviews, Inc. . Kozinski, A. W. and Z. Z. Felgenhauer. 1967. Molecular Recombina- tion in T4 Bacteriophage Deoxyribonucleic Acid. 11. Single- Strand Breaks and Exposure of Uncomplemented Areas as a Prerequisite for Recombination. J. Virol., 1: 1193-1202. 41 Kutter, E. M., A. Beug, R. Sluss, L. Jensen, and 0. Bradley. 1975. The Production of Undegraded Cytosine-Containing DNA by Bacteriophage T4 in the Absence of dCTPase and Endonucleases II and IV, and its Effects on T4-Directed Protein Synthesis. J. Mol. Biol., 92; 591-607. Lehman, I. R. 1974. DNA Ligase: Structure, Mechanism, and Function. Science, 1§E5 790-797. Lillehaug, J. R. 1977. Physicochemical Properties of T4 Poly- nucleotide Kinase. Eur. J. Biochem., 1;, 499-506. Lillehaug, J. R. and K. Kleppe. 1977. Phosphorylation of tRNA by T4 Polynucleotide Kinase. Nucl. Acids Res., 4, 373-380. Lillehaug, J. R., R. K. Kleppe, and K. Kleppe. 1976. Phosphoryla- tion of Double-Stranded DNAs by T4 Polynucleotide Kinase. Biochem., 1E, 1858-1865. Liu, L. F., C. C. Liu, and B. M. Alberts. 1980. Type II DNA Topoisomerases: Enzymes that Can Unknot a Topologically Knotted DNA Molecule via a Reversible Double-Strand Break. Cell, 12, 697-707. McCoy, M. I. and R. I. Gumport. 1980. T4 Ribonucleic Acid Ligase Joins Single-Strand 01igo(deoxyribonuc1eotides). Biochem., 1_9: 635-642. Miller, R. C., Jr. and A. W. Kozinski. 1970. Early Intracellular Events in Replication of Bacteriophage T4 Deoxyribonucleic Acid. V. Further Studies on T4 Protein Deoxyribonucleic Acid Complex. J. Virol., E, 490-501. Murray, R. E. and C. K. Mathews. l969a. Addition of Nucleotides to Parental DNA Early in Infection by Bacteriophage T4. J. M01. Biol., 45: 233-248. Murray, R. E. and C. K. Mathews. l969b. Biochemistry of DNA- Defective Amber Mutants of Bacteriophage T4. II. Intra- cellular DNA Forms in Infection by Gene 44 Mutants. J. Mol. Biol., 44, 249-262. Novodgrodsky, A. and J. Hurwitz. 1966. The Enzymatic Phosphoryla- tion of Ribonucleic Acid and Deoxyribonucleic Acid. 1. Phosphorylation at 5' Hydroxyl Termini. J. Biol. Chem., 241, 2923-2932. Ohtsuka, E., S. Nishikawa, M. Suguira, and M. Ikehara. 1976. Joining of Ribonucleotides with T4 RNA Ligase and Identi- fication of the 01igonucleotide-Adenylate Intermediate. Nucl. Acids Res., 3: 1613-1623. 42 Panet, A., J. H. van de Sande, P. C. Loewen, H. G. Khorana, A. J. Raae, J. R. Lillehaug, and K. Kleppe. 1973. Physical Characterization and Simultaneous Purification of Bacteriophage T4 Induced Polynucleotide Kinase, Polynucleotide Ligase, and Deoxyribonucleic Acid Polymerase. Biochem., 1E: 5045-5050. Pearson, R. E. and L. Snyder. 1980. The Shutoff of Lambda Gene Expression by Bacteriophage T4: Role of the A19 Gene. J. Virol., §§, 194-202. Pettijohn, D. E. and R. Hecht. 1973. RNA Molecules Bound to the Folded Bacterial Genome Stabilize DNA Folds and Segregate Domains of Supercoiling. Cold Spr. Harb. Sym. Quant. Biol., EE, 31-41. Rabussay, D. and E. P. Geiduschek. l977a. Regulation of Gene Action in the Development of Lytic Bacteriophages. pp. 1-196 in Comprehensive Virology, vol. 8, eds. H. Fraenkel-Conrat and R. R. Wagner (Plenum Press). Rabussay, D. and E. P. Geiduschek. 1977b. Phage T4-Modified RNA Polymerase Transcribes T4 Late Genes jn_Vitro. Proc. Natl. Acad. Sci., U.S.A., 14; 5305-5309. Ratner, D. 1974. The Interaction of Bacterial and Phage Proteins with Immobilized Escherichia coli RNA Polymerase. J. Mol. Biol., §§; 373-383. Richardson, C. C. 1965. Phosphorylation of Nucleic Acid by an Enzyme from T4 Bacteriophage-Infected Escherichia coli. Proc. Natl. Acad. Sci., E4: 158-165. Richardson, C. C. 1966. The 5'-Terminal Nucleotides of T7 Bacteriophage Deoxyribonucleic Acid. J. Mol. Biol., 1E: 49-61. Richardson, C. C. and A. Kornberg. 1964. A Deoxyribonucleic Acid Phosphatase-Exonuclease from Escherichia coli. I. Purifi- cation of the Enzyme and Characterization othhe Phosphatase Activity. J. Biol. Chem., 112; 242-250. Riva, S., A. Cascino, and E. P. Geiduschek. 1970a. Coupling of Late Transcription to Viral Replication in Bacteriophage T4 Development. J. Mol. Biol., E5: 85-102. Riva, S., A. Cascino, and E. P. Geiduschek. l970b. Uncoupling of Late Transcription from DNA Replication in Bacteriophage T4 Development. J. Mol. Biol., E5; 103-119. 43 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 TgatZSomg are "Turned Off" at Late Times. J. Mol. Biol., 4 : 7 - 95. Siegel, P. S. and M. Schaechter. 1973. The Role of the Host Cell Membrane in the Replication and Morphogenesis of Bacterio- phages. pp. 261-282 in Annual Review of Microbiology, vol. 27, eds. M. P. Starr, J. L. Ingraham, and S. Raffel (Annual Reviews, Inc.). Silber, R., V. G. Malathi, and J. Hurwitz. 1972. Purification and Properties of Bacteriophage T4-Induced RNA Ligase. Proc. Natl. Acad. Sci., U.S.A., E2, 3009-3013. Singh, M. K. and E. Terzaghi. 1974. Selective Blockage of Bacteriophage T4 Assembly by Chemical Modification. Nature, EEE, 321-323. Sirotkin, K., W. Cooley, J. Runnels, and L. R. Snyder. 1978. A Role in True-Late Gene Expression for the T4 Bacteriophage 5' Polynucleotide Kinase 3' Phosphatase. J. Mol. Biol., 12;: 221-233. Sirotkin, K., J. Wei, and L. Snyder. 1977. T4 Bacteriophage-Coded RNA Polymerase Subunit Blocks Host Transcription and Unfolds the Host Chromosome. Nature, EEE, 28-31. Snopek, T. J., A. Sugino, K. L. Agarwal, and N. R. Cozzarelli. 1976. Catalysis of DNA Joining by Bacteriophage T4 RNA Ligase. Biochem. Biophys. Res. Commun., EE; 417-424. Snopek, T. J., W. 8. Wood, M. P. Conley, P. Chen, and N. R. Cozzarelli. 1977. Bacteriophage T4 RNA Ligase is Gene 63 Product, the Protein That Promotes Tail Fiber Attachment to the Baseplate. Proc. Natl. Acad. Sci., U.S.A., 14: 3355-3359. Snustad, D. P. and L. M. Conroy. 1974. Mutants of Bacteriophage T4 Deficient in the Ability to Induce Nuclear Disruption. I. Isolation and Genetic Characterization. J. Mol. Biol., E9: 663-673. Snustad, D. P., M. A. Tigges, K. A. Parson, C. J. H. Bursch, F. M. Caron, J. Koerner, and D. J. Tutas. 1976. Identification and Preliminary Characterization of a Mutant Defective in the Bacteriophage T4-Induced Unfolding of the Escherichia coli Nucleoid. J. Virol., 11: 622-641. 44 Snyder, L., L. Gold, and E. Kutter. 1976. A Gene of Bacteriophage T4 Whose Product Prevents True-Late Transcription on Cytosine-Containing T4 DNA. Proc. Natl. Acad. Sci., U.S.A., 1;: 3098-3102. Speyer, J. F., J. Chao, and L. Chao. 1972. Ribonucleotides Covalently Linked to Deoxyribonucleic Acid in T4 Bacterio- phage. J. Virol., 19: 902-908. Stevens, A. 1972. New Small Polypeptides Associated with DNA- Dependent RNA Polymerase of Escherichia coli after Infection with Bacteriophage T4. Proc. Natl. Acad. Sci., U.S.A., E2: 603-607. Sugino, A., H. M. Goodman, H. L. Heyneker, J. Shine, H. W. Boyer, and N. R. Cozzarelli. l977a. Interaction of Bacteriophage T4 RNA and DNA Ligases in Joining of Duplex DNA at Base Paired Ends. J. Biol. Chem., Egg, 3987-3994. Sugino, A., N. P. Higgins, T. J. Snopek, A. P. Geballe, and N. R. Cozzarelli, 1978. Ligation of DNA by Bacteriophage T4 RNA Ligase. pp. 10-12 in Microbiology, 1978, ed. D. Schlessinger (American Society for Microbiology). Sugino, A., T. J. Snopek, and N. R. Cozzarelli. 1976. Catalysis of DNA Joining by Bacteriophage T4 RNA Ligase. Fed. Proc., EE, 1377 (Abs.). Sugino, A., T. J. Snopek, and N. R. Cozzarelli. l977b. Bacterio- phage T4 RNA Ligase. Reaction Intermediates and Interaction of Substrates. J. Biol. Chem., Egg; 1732-1738. Terzaghi, B., E. Terzaghi, and D. Coombs. 1979. The Role of the Collar/Whisker Complex in Bacteriophage T4D Tail Fiber Attachment. J. Mol. Biol., 1E1; 1-14. Terzaghi, E. 1971. Alternative Pathways of Tail Fiber Assembly in Bacteriophage T4? J. Mol. Biol., E9, 319-327. Tigges, M., C. J. H. Bursch, and D. P. Sunstad. 1977. Slow Switchover from Host RNA Synthesis to Bacteriophage RNA Synthesis after Infection of Escherichia coli with a T4 Mutant Defective in Bacteriophage T4-InducedSUnfolding of the Host Nucleoid. J. Virol., EA, 775-785. Tomizawa, J. 1967. Molecular Mechanisms of Genetic Recombination in Bacteriophage: Joint Molecules and Their Conversion to Recombinant Molecules. J. Cell. Physiol., 29, Suppl. 1: 201-214. 45 Tutas, D. J., J. M. Wehner, and J. F. Koerner. 1974. Unfolding of the Host Genome after Infection of Escherichia coli with Bacteriophage T4. J. Virol., 11; 548-550. Uhlenbeck, 0. C. and V. Cameron. 1977. Equimolar Addition of Oligoribonucleotides with T4 RNA Ligase. Nucl. Acids Res., 1; 85-98. Vanderslice, R. W. and C. D. Yegian. 1974. The Identification of Late Bacteriophage T4 Proteins on Sodium Dodecyl Sulfate Polyacrylamide Gels. Virol., E9, 265-275. Walker, G. C., 0. C. Uhlenbeck, E. Bedows, and R. I. Gumport. 1975. T4-Induced RNA Ligase Joins Single-Stranded Oligoribonucleotides. Proc. Natl. Acad. Sci., U.S.A., 2E; 122-126. Ward, 5., R. B. Luftig, J. H. Wilson, H. Eddleman, H. Lyle, and W. 8. Wood. 1970. Assembly of Bacteriophage T4 Tail Fibers. II. Isolation and Characterization of Tail Fiber Precursors. J. Mol. Biol., E15 15-31. Wood, W. B. 1979. Bacteriophage T4 Assembly and the Morphogenesis of Subcellular Structure. pp. 203-223 in The Harvey Lectures, vol. 73 (Academic Press). Wood, W. B. and R. J. Bishop. 1973. Bacteriophage T4 Tail Fibers: Structure and Assembly of Viral Organelle. pp. 303-324 in Virus)Research, eds. C. F. Fox and W. F. Robinson (Academic Press . Wood, W. B. and M. P. Conley. 1979. Attachment of Tail Fibers in Bacteriophage T4 Assembly: Role of the Phage Whiskers. J. Mol. Biol., 111, 15-29. Wood, W. B., M. P. Conley, H. L. Lyle, and R. C. Dickson. 1978. Attachment of Tail Fibers in Bacteriophage T4 Assembly. Purification, Properties, and Site of Action of the Accessory Protein Coded by Gene 63. J. Biol. Chem., Z§§9 2437-2445. Wood, W. B. and M. Henninger. 1969. Attachment of Tail Fibers in Bacteriophage T4 Assembly: Some Properties of the Reaction ‘1¥_Vitro and Its Genetic Control. J. Mol. Biol., 12; 603- 8. Wood, W. B. and J. King. 1979. Genetic Control of Complex Bacterio- phage Assembly. pp. 581-633 in Comprehensive Virology, vol. 13, eds. H. Fraenkel-Conrat and R. R. Wagner (Plenum Press . 46 Worcel, A. and E. Burgi. 1972. On the Structure of the Folded Chromosome of Escherichia coli. J. Mol. Biol. 11: 127-147. Wu, R. and E. P. Geiduschek. 1975. The Role of Replication Proteins in the Regulation of Bacteriophage T4 Transcription. I. Gene 45 and Hydroxylmethyl-C-Containing DNA. J. Mol. Biol., 9E; 513-538. Yanagida, M. and C. Ahmad-Zadeh. 1970. Determination of Gene Product Positions in Bacteriophage T4 by Specific Antibody Association. J. Mol. Biol., E1, 411-421. ARTICLE I GENETIC AND PHYSIOLOGICAL STUDIES OF THE ROLE OF THE T4 BACTERIOPHAGE RNA LIGASE Judith M. Runnels and Larry R. Snyder Manuscript submitted for publication to Cell 47 Summary The product of the T4 pseT gene, which is both a 5' poly- nucleotide kinase and a 3' phosphatase (Depew and Cozzarelli, 1974; Cameron and Uhlenbeck, 1977; Sirotkin et al., 1978) is nonessential for T4 development on most laboratory strains of Escherichia coli. However, 14 pgglf mutants fail to multiply on E, £911.CTr5X, a hybrid between E, £911 K-12 and a clinical isolate, E, 5911 C1196 (Depew and Cozzarelli, 1974), because of defects in late gene expres- sion and T4 DNA replication. While isolating T4 pgglf mutants, we discovered another class of closely linked mutations which caused phenotypes similar to those caused by pgglf mutations but which defined another complementation group. These mutations, which we have named 1pE_mutations because the gene product may 1nteract with polynucleotide Ejnase, map within T4 gene 63 and inactivate the RNA ligase activity, but not the tail fiber attachment activity asso- ciated with the product of this gene. We conclude that, at least in E, £911.C1r5x, the T4 polynucleotide 5' kinase 3' phosphatase and RNA ligase interact 1n.1119 in some reaction required for normal T4 DNA replication and 14 late gene expression. Introduction The product of the T4 bacteriophage gene, pggl, a tetramer of identical subunits of about 30,000 daltons (Lillehaug, 1977; Panet et al., 1973), is both a 5' polynucleotide kinase (Richardson, 1965; Novogrodsky and Hurwitz, 1966; Sirotkin et al., 1978) and a 3' phosphatase (Becker and Hurwitz, 1967; Depew and Cozzarelli, 1974; 48 49 Cameron and Uhlenbeck, 1977). The 5' kinase activity can transfer the v-phosphate of ATP to a 5' hydroxyl terminus of either RNA or DNA and is used extensively in nucleic acid biochemistry. The 3' phosphatase can remove 3' terminal phosphates from either DNA (Becker and Hurwitz, 1967) or RNA (Cameron and Uhlenbeck, 1977). It is not known whether the kinase and phosphatase work together 1n,1119, or are just two enzymes "tacked together." A clue comes from the T4 pgglj mutant. This mutant is phenotypically similar to other pgglf mutants even though it only lacks the phosphatase activity and has normal kinase activity (Sirotkin et al., 1978; Cameron et al., 1978). Either all the phenotypes are due to the 3' phosphatase or the two activities work together in a composite reaction. The two activities can work together jn_111[9, There is indirect evidence that poly- nucleotide kinase can convert a 3' phosphate terminated "nick" in DNA into a substrate fer DNA ligase (Champoux, 1978). However, nicks in DNA are a poor substrate fer the polynucleotide kinase 1n_11119_(Li11ehaug et al., 1976) and they may not be the real substrate 13 111g . The product of the T4 p§e1_gene is nonessential to phage development in most strains of E, 9911, However, when E, £911_ CTr5X, a hybrid of E, £911_K-12 and a clinical isolate, E, 9911 C1196 (Depew and Cozzarelli, 1974), is infected with péglf mutants no phage are produced because of defects in T4 DNA replication (Depew and Cozzarelli, 1974) and T4 late gene expression (Sirotkin et al., 1978). The defects in late gene expression, DNA replica- tion, and phage production only occur when the cells are grown and 50 infected at lower temperatures. At 42°C both late gene expression and phage production are normal (Cooley et al., 1979; Cooley and Snyder, unpublished results). The inability of T4 pgglf mutants to multiply on E, £911_ CTr5X can be suppressed either by extracistronic phage mutations or by host mutations. T4 mutations which inactivate a gene close to rII, the §12_gene, allow T4 pgglf mutants to multiply in E, 9911 CTr5X (Depew and Cozzarelli, 1974). Also, mutations which convert E, £911_CTr5x to an efficient amber-suppressing strain permit growth of p§g1_mutants (Sirotkin et al., 1978). In the latter case the effect is not due to suppression of the pgglf mutations themselves, but probably to the suppression of an amber mutation in a host gene whose product is required for a productive infection in the absence of a functional T4 p§e1.9ene product (Sirotkin et al., 1978). The 14 gene 63 is closely linked to the p§g1_gene. The product of gene 63 also has two activities, one which, at low temperatures, is required for the attachment of T4 tail fibers to T4 tails (TFA) (Wood and Henninger, 1969; Wood and Bishop, 1973), and another which can catalyze the joining of single-stranded polynucleotides jg 11159_(RNA ligase) (Silber et al., 1972; Snopek et al., 1977). On most strains of E, 9911, the only phenotype of amber mutations in gene 63 is the failure to attach tail fibers at low temperatures leading to the production of non-infectious, fiber-deficient particles (Wood and Henninger, 1969; Wood and Bishop, 1973). It seems unlikely that the RNA ligase activity is involved in tail fiber attachment because 1n_vitro its requirements 51 are different from those of the TFA activity (Snopek et al., 1977; Wood et al., 1978). In the process of isolating T4 pgglf mutants, we discovered another class of T4 mutants which fail to multiply on E, EQ11_CTr5X. Although the responsible mutations define a different complementation group they cause phenotypes similar to péglf mutations. Thus, we named them 1pEf mutations since the product of the gene may 1nteract with polynucleotide Ejnase. T4 125? mutations map within T4 gene 63 and inactivate the RNA ligase, but not the TFA activity associated with the product of this gene. Results T4 mutants which cannot form plaques on E, ggl1_CTr5X are quite common and map in many places in the T4 genome. Included among these are many amber mutants with mutations in essential genes since E, £911 CTr5X has a very inefficient amber suppressor gene. In order to distinguish pgglf mutants from amber mutants and from a myriad of other types, we eliminated those which were "leaky" or which gave high recombination frequencies with p§§I_mutants. In this way, we isolated many more candidates fer EEEI: mutants including four strains which proved to have mutations closely linked to, but in a different complementation group from, E§g1_mutations. We named the feur mutants ngf mutants, and the responsible mutations 121:11, ipk-15, ipk-25 and 125:11, Because the product of the ipE_gene could be involved in the same nucleic acid pathway as the poly- nucleotide kinase we decided to further characterize ipk' mutations. 52 Genetic Mapping of ipk' Mutations We obtained recombination frequencies of about 4% between 125: and pgpl: mutations suggesting they are closely linked. The p191 gene of T4 maps between genes 31 and 63 (Depew and Cozzarelli, 1974). Another gene in this region is the ppj]glp_gene (Snustad et al., 1976; Sirotkin et al., 1977) which maps between p§g1_and gene 63 (Sirotkin et al., 1978). To order 1pEf mutations with respect to these other genes, we performed three and four factor genetic crosses. The following crosses were used to establish the order of unflalc, pseT and 1pE_mutations. An 1pEf, 115: double mutant was crossed with a pgplf single mutant and the recombinants which formed plaques on E, pplj_CTr5X, and were therefore both 1pEf and pgplf, were tested for their plp_phenotype as described in Experimental Procedures. In both this cross and a cross between a pgplf,‘glpf double mutant and an 1pEf single mutant, about 50% of the 1pET p531? recombinants were 215:. The simplest interpreta- tion of these results is that 319: mutations lie between 1pEf and ppplf mutations. To confirm that the ppjyplp gene lies between the p§§1_and 1pE_genes, we tested a specific prediction of this map order. The frequency of 319? recombinants should greatly increase among the ipk+, pseT+ progeny of crosses between 1pEf, 219:, and pggIE, 31p: double mutants when the plpf mutation coupled with the 1pEf mutation is clockwise of the 212: mutation coupled with the ppplf mutation. In this orientation an glpf recombinant is the product of a single crossover while the reciprocal orientation requires a double crossover to yield plpf recombinants. As 53 predicted, for any two plp_mutations the frequency of 319? recombi- nants was greatly increased among pgplf, 1pEf recombinants compared to the frequency among the total progeny of such crosses, particu- larly when the four parental markers were in one orientation. Because of the short genetic distances between 915: mutations we had not previously been able to obtain 115? recombinants in two factor crosses. These results allowed us to order some 219: mutations (data not shown). We also determined the order of the four 1pEf mutations with respect to each other and with respect to the p15 gene and gene 31 using three factor crosses. We crossed 1pEf, glpf double mutants with 1pEf single mutants and tested the 1pEf recombinants for their plp_phenotype. We obtained the unambiguous order 1pE-25-13-15-77-glp, The same order was obtained using a ts mutation in gene 31, rather than 215: mutations, as the outside marker confirming that plp_and gene 31 are on the same side of 1pE mutants. From all our data we conclude the map order is ipk-alc-pseT-gene 31. It remained to order 1pE_mutations with respect to gene 63 mutations. Some gene 63 amber mutations are very leaky on most strains of E, ppl1_which has made them difficult to use as genetic markers. However, we observed that all the known amber mutations in gene 63 do not form plaques, or fbrm very tiny ones, on E, p911 CTr5X making possible the detection of ipk+ am+ recombinants. This allowed us to perform three factor crosses using a ts mutation in gene 31 as the outside marker. We selected ipk+ am+ recombinants 54 and tested these for the ts phenotype. Also, where possible, we used an 1pEf mutation as the outside marker in crosses between two amber mutants in gene 63. The result of all these crosses are shown in Table l. The data revealed that 1pEf mutations lie within the coding region of gene 63 intermingled with the known amber mutations. A composite map of the gene 63 region of T4 is shown in Figure 2. It is of interest that 1pEf mutations are scattered throughout gene 63 rather than clustered in one region suggesting that the 1pE_activity is not associated solely with one region of the polypeptide product. Assays of RNA Ligase in jpk‘ Mutants As mentioned above, the product of gene 63 has two known activities, one which promotes the attachment of T4 tail fibers to the baseplate at low temperatures (TFA) and another which joins single-stranded polynucleotides (RNA ligase). Because 1pEf mutants form normal sized plaques at or below 23°C on most laboratory strains of E, 9911, we concluded that they were not deficient in TFA. We therefore tested them for their ability to induce RNA ligase activity. Because RNA ligase can join two single-stranded poly- nucleotides, it will cyclize short oligonucleotides of poly A. If the poly A has been labeled at its 5' terminus with phosphorous- 32 one can detect RNA ligase activity as the conversion, with time, from bacterial alkaline phosphatase (BALP) sensitive to BALP TABLE l.--The Genetic Mapping Data for 1pE and Gene 63 Markers. 55 Recomb. _ 1 Cross Tested 1;, or 1pE_ Order Oeduced ipk-ZS, tsA56 x amm15.6 an." 1p1+ 25; 1111411155 - ipk-ZS - tsA56 1111111155. tsA56 x ipk-ZS 55: 1211-25. tsA56 x 111111469 am” ipk” 67% ipk-25 - anM69 - tsA56 amM69, tsA56 x 1911-25 44% ipk-25, tsA56 x anmza am+ 1px+ 57: ilk-25 - 1111141423 - tsA56 anmza, tsA56 x ipk-ZS m ipk-l3, tsA56 x anm15.5 m" 1le 21: mamas - 1pk-l3 - tsA56 amMN15.6, tsA56 X ipk-l3 62% ipk-l3, tsA56 x anme9 am“ 1ka 13: anfl69 - ipk-13 - tsA56 amM69, tsA56 X ipk-l3 57% ipk-13, tsA56 X amMN23 am+ ipk+ 60% ipk-l3 - amMN23 - tsA56 amMN23. tsA56 X ipk-l3 40% 1911-15. tsA56 x mmsa am“ ipk” 17: ”12115.5 - ipk-lS - tsA56 anflNlS.6, tsA56 x ipk-lS 64% ipk-l . tsA56 x was am+ ipk” 13: amM69 - ipk-15 - tsA56 mass. tsA56 x ipk-lS 54: 12k-15. tsA56 x anmza am+ 1pk” 66% ipk-lS - arrMN23 - tsA56 anmza. tsA56 x ipk-ls 40: aum15.6. ipk-77 11 mag am” 72: amuse - amM69 - ipk-77 amM69, ipk-77 X anrm15.6 21% 1911-77, tsA56 x anmza 11m+ um” 29: ammza - ipk-77- tsA56 anmza. tsA56 x ipk-77 53: 56 .xgaapwnee use mmucaumpc FF< .mwsu sow mucmuw>m.wwm+ww1oc m>mg m3 zmaogpFa magma mzosmwucou mm execm mew pmmm new upa\m:= .mo meow .ep co cowmwm mm meow mo aa:uu.~ wezmwm _m hmma u_a\+c= mm 1+... ... 1411.111? m m A Ne-¥ae mmzz m_-¥aw m_-xa_ me: mm-xaw e.mpzz 57 resistant radioactivity as the poly A becomes cyclized and the 5' phosphate becomes internalized. To determine if 1pEf mutants induce RNA ligase activity, E, pplj_cells were infected with various mutants of T4, the cells were lysed, and the lysates were assayed for poly A cyclizing activity. The results are shown in Figure 3. Shown fer comparison are assays with extracts of cells infected with wild type T4 and a p§p1_mutant, pgglg, both of which induce RNA ligase activity. The amber mutants in gene 63 induced no RNA ligase, in agreement with the results of others (Snopek et al., 1977). Also, all four 1pEf mutants induced no RNA ligase. In the experiment shown in Figure 3A,the mutant 1pE;1E_seemed to induce some activity but this result did not repeat in a subsequent experiment. To be certain that the 1pEf mutations were inactivating the RNA ligase directly rather than inhibiting it or causing an abnormally rapid degradation of the poly A substrate, we performed the controls shown in Figure 3B and C. If 1pEf mutants were inducing an inhibi- tor, then purified RNA ligase added to the extracts should also be inhibited. The activities measured when purified RNA ligase was added to extracts of either wild type T4- or 1pE;1E-infected cells were the sums of the activities seen with the purified ligase alone and the wild type or 1pE-l5 extracts alone (Figure 3C). Thus, the absence of RNA ligase activity in 1pEf mutant extracts was not due to the presence of an RNA ligase inhibitor. Also, there was very little, if any, degradation of the poly A substrate during the assays either in the extracts of cells infected with 1pEf mutants 58 .cwmuogn Fe\m: mm op mm Saga 0 use m cw ”seepage _E\m: “mm on NPN sage umcrmucou < cw waxes ammma ask .ucmewgmaxm mama as» sage wen u one m cw upon ash .uuaepxo umuumecw +¢P op woven mmmmwp <2”. 8:23 71115 38.56 8885 g 3 828 38.: <5. 8:22. AI): 85$ 33: <21 3:23 A-IIII-V “+3. 74111.4; a; 312.19 .3833 33.8 o“ mmamFP wuua :oPpMNFFuaU co uummmm use we czogm .u .uumhxm flaw. A01. .13 mommmwp eeu< macaw; 2,ooo Ci/mmole). The reactions were incubated at 37°C before 25 uI aliquots were shifted to 65°C, an equal volume of 2.5 mg/ml bacterial alkaline phosphatase in 0.2 flTris, pH 8.2, 10 mM MgC12, 0.4 11 NaCl was added, and the incubation continued for an additional 30 min. The reactions were spotted onto squares of Whatman DE-81 paper which were washed once with H20, four times with 0.3 M_ammonium formate, and once with 95% ethanol for 5 min each before drying and counting. Acknowledgments We wish to thank Olke Uhlenbeck in whose laboratory the RNA ligase assays were performed. We also wish to thank Vicky Cameron for technical assistance. This work was supported by grant PCM7724422 from the NSF. 78 References Becker, A. and Hurwitz, J. (1967). J. Biol. Chem. 112, 936-950. Cameron, V., Soltis, D., and Uhlenbeck, 0. C. (1978). Nucl. Acids Res. E, 825-834. Champoux, .1. J. (1978). .1. Mol. Biol. 11p, 441-445. Cameron, V. and Uhlenbeck, 0. C. (1977). Biochemistry 1E, 5120-5126. Cooley, W., Sirotkin, K., Green, R., and Snyder, L. (1979). J. Bacteriol. 139, 83-91. David, M., Vekstein, R., and Kaufmann, G. (1979). Proc. Natl. Acad. Sci. USA 15, 5430-5434. Depew, R. E. and Cozzarelli, N. R. (1974). J. Virol. 11, 888-897. Depew, R. E., Snopek, T. J., and Cozzarelli, N. R. (1975). Virology fl. 144-152. Duckworth, D. H. (1970). Bacteriol. Rev. 11, 344-363. England, T. E. and Uhlenbeck, 0. C. (1978). Biochemistry 11, 2069-2076. Johnson, R. A. and Walseth, T. F. (1979). Advances in Cyclic Nucleotide Research 19, 135-167. Kaufmann, G. and Littauer, U. Z. (1974). Proc. Natl. Acad. Sci. U.S.A. _7_1, 3741-3745. Knapp. G., Ogden, R. C., Peebles, C. L., and Abelson, J. (1979). Cell 1_8_. 37-45. Lillehaug, J. R. (1977). Eur. J. Biochem. 11, 499-506. Lillehaug, J. R., Kleppe, R. K., and Kleppe, K. (1976). Biochem. 1_5. 1858-1865. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). J. Biol. Chem. 121, 265-275. McCoy, M. I. M. and Gumport, R. I. (1980). Biochemistry 19, 635- 642. Novogrodsky, A. and Hurwitz, J. (1966). J. Biol. Chem. E41, 2923- 2932. 79 Panet, A., van de Sande, J. H., Loewen, P. C., Khorana, H. G., Raae, A. J., Lillehaug, J. R., and Kleppe, K. (1973). Biochemistry 1E, 5045-5050. Pearson, R. and Snyder, L. (1980). J. Virol. §§, 194-202. Richardson, C. C. (1965). Proc. Natl. Acad. Sci. USA E4, 158-165. Riva, S., Cascino, A., and Geiduschek, E. P. (l970a). J. Mol. Biol. 55. 85-102. Riva, S., Cascino, A., and Geiduschek, E. P. (l970b). J. Mol. Biol. .5_4. 103-119. Silber, R., Malathi, V. G., and Hurwitz, J. (1972). Proc. Natl. Acad. Sci. USA g2, 3009-3013. Silver, S. (1965). Proc. Natl. Acad. Sci. USA §§, 24-30. Sirotkin, K., Cooley, W., Runnels, J., and Snyder, L. R. (1978). .1. Mol. Biol. 1_23_, 221-233. Sirotkin, K., Wei, J., and Snyder, L. (1977). Nature EEE, 28-31. Snopek, T. J., Sugino, A., Agawal, K. L., and Cozzarelli, N. R. (1976). Biochem. Biophys. Res. Commun. EE, 417-424. Snopek, T. J., Wood, W. B., Conley, M. P., Chen, P., and Cozzarelli, N. R. (1977). Proc. Natl. Acad. Sci. USA 14, 3355-3359. Snustad, D. P., Tigges, M. A., Parson, K. A., Bursch, C. J. H., Caron, F. M., Koerner, J. F., and Tutas, D. J. (1976). J. Virol. 11, 622-641. Snyder, L., Gold, L., and Kutter, E. (1976). Proc. Natl. Acad. Sci. USA 131, 3098-3102. Sugino, A., Goodman, H. N., Heyneker, H. L., Shine, J., Boyer, H. W., and Cozzarelli, N. R. (l977a). J. Biol. Chem. EEE, 3987- 3994. Sugino, A., Snopek, T. J., and Cozzarelli, N. R. (l977b). J. Biol. Chem. 252, 1732-1738. Tessman, I. (1968). Viral. EE, 330-333. Walker, G. C., Uhlenbeck, 0. C., Bedows, E., and Gumport, R. I. (1975). Proc. Natl. Acad. Sci. USA 12, 122-126. 80 Wood, W. B. and Bishop, R. J. (1973) in Virus Research, C. F. Fox and W. F. Robinson, eds. (N.Y.: Academic Press), pp. 303- 324 . Wood, W. 8., Conley, M. P., Lyle, H. L., and Dickson, R. C. (1978). .1. Biol. Chem. g3, 2437-2445. Wood, W. B. and Henninger, M. (1969). J. Mol. Biol. 12, 603-618. APPENDICES 81 APPENDIX A THE MAP ORDER DE 1’1, 9.1g- and pseT MUTATIONS 82 APPENDIX A THE MAP ORDER or 1 k, _a_1_e, and pseT MUTATIONS The fellowing data were used to order initially the muta- tions responsible for 1pEf, p1pf, and peel? phenotypes. The map position of the p§e1_gene with respect to genes 31 and 30 had pre- viously been determined (Depew and Cozzarelli, 1974; Sirotkin et al., 1978; Sirotkin, Ph.D. dissertation). To order 1pE, p19, and pgel, three-factor crosses were performed. In the first set of crosses, recombination was forced between gene 30 and 31 markers (tsA80 and tsA56, respectively) by plating the cross progeny at 42°C. The position of the 1pE_marker was determined by the frequencies of occurrence of 1pEf and 1pEf types in the temperature sensitive+ progeny of reciprocal crosses. Likewise, reciprocal crosses were set up using 1pE, p15, and p§e1_markers. Recombination was forced between the 1pE_and p§e1_markers by plating progeny on CTr5X. To determine the g1p_genotype these crosses were done in an amE51(56'), 002(denB'), nd28(denA') background. Approximately one half of the progeny of these crosses were 315:, and the other half were 3191, indicating that 312 maps between the 1pE and pseT markers. 83 84 -o_e me one .11. + mm me home epeomm x m-mmfl nope o_e om x8e - + co oe home m-mmm x mahomm nmflm 12m em ow $3 11 + we ea? epeomm x mpope m-mmm -u? S we 33 11 + mm ea? e-Pomm ePoPe x m-mmm -xee we om om