Genetic Map of Bacteriophage Lambda

Home , Bacteriophage P2, Harrison Echols, Lambda phage

MICROBIOLOGICAL REVIEWS, Sept. 1978, p. 577-591 Vol.42, No. 3 0146-0749/78/0042-0577$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Genetic Map of Bacteriophage Lambda HARRISON ECHOLS* AND HELIOS MURIALDO Department ofMolecular Biology, University of California, Berkeley, California 94720, * and Department of Medical Genetics, University of Toronto, Toronto M5S 1A8, Canada INTRODUCTION .5..577 PHYSICAL STATES AND ACTIVITY OF THE A GENOME 577 GENETIC MAP OF THE A GENOME .577 ACTIITrY MAP OF THE A GENOME .583 COMMENTS ON GENETIC ORGANIZATION ...... 5*583 LITERATURE CITED .584

INTRODUCTION that are the obligatory precursors for the The study of bacteriophage A has been a cen- cleaved, linear molecules packaged into a phage tral endeavor of molecular biology for a number head (Fig. 1). The lysogenic pathway involves a of years. Phage A has been the creature of choice repression of transcription and a site-specific for many investigators interested in deoxyribo- recombination event that inserts A DNA into nucleic acid (DNA) transcription, replication, the host Escherichia coli genome. This integra- and recombination, in nucleoprotein assembly, tive recombination between the phage and host and in the organization of these processes into attachment sites (att) generates a genetic struc- temporally regulated pathways. As a result of ture that is permuted from the linear order this intensive study, a great deal of information found in the phage particle because the phage is now available about A genes and how they attachment site (a a' or P P) is approximately work; however, the communication of this in the center of the mature DNA molecule (Fig. knowledge is sometimes hindered by a "culture 1). The prophage has structurally distinct at- gap" between "lambdologists" and those lacking tachment sites: a left attL site (b a' or B P') an understanding ofthe array ofgene names and and a right attR site (a b' or P B'); these in sites for protein activity. In an effort to remedy turn can recombine to detach the prophage this difficulty, we present an annotated genetic DNA when the virus is induced to lytic devel- map of phage A. We begin with a brief summary opment, regenerating the phage attP site (a a' of the A life cycle; present two maps, one of gene or P P') and the original host attB site (b b' or order and one of gene activity; and conclude B B'). with a short on the More detailed descriptions of the diverse life- commentary genetic orga- styles of phage A can be found in the general nization. review articles by Echols (47; in J. R. Sokatch PHYSICAL STATES AND ACTIVITY OF and L. N. Ornston, ed., The Bacteria, in press) THE X GENOME and Herskowitz (88). The lysogenic pathway has been recently reviewed by Weisberg et al. (233); The genome of phage A is a double-stranded the lytic pathway has not been selectively re- DNA molecule about 47,000 base pairs in length. viewed for some time, but the general features In the phage particle, A DNA has single- are covered in the review Echols stranded, complementary ends 12 bases in by (46). length, termed mature or cohesive ends m and m'. Within an infected cell, A DNA forms a circle GENETIC MAP OF THE X GENOME through pairing ofthe single-stranded DNA, and The order of genes along the linear A DNA is replicated and transcribed as a circular mole- molecule is shown in the upper part of Fig. 2; cule during the replication-oriented early phase the function of certain gene clusters is indicated of A development. below the map. The lower part of Fig. 2 gives a After this early stage, A development may blowup of the right half of the A map, designed proceed along the productive (or lytic) pathway to focus on the activity sites for the develop- or along the alternative lysogenic pathway. The mental events noted in the preceding section. encapsulation-oriented late stage of the produc- To construct Fig. 2, we used three principal tive pathway involves a transcription switch to types of information: (i) "traditional" genetic synthesis of head, tail, and lysis proteins and a mapping; (ii) physical mapping by heteroduplex replication switch to a rolling-circle mode that analysis of genetically characterized deletion generates multimeric A genomes (concatemers) mutations; and (iii) molecular weights of gene 577 578 ECHOLS AND MURIALDO MICROBIOL. REV.

DNA encapsulation Phage Late proteins Monomer DNA on Injected DNA replicotion mm' Eor1yEorly~~~~~~~mm' f" proteinsEoff K> proteins o mmmiil ° C ) Lytic DNA integration Prophoge proteins mm o,f fI _110. bo' mm' oab' Integrotion on b bI FIG. 1. Developmental pathways for bacteriophage A. The injected DNA forms a covalently closed circle through pairing of the mature ends, m and mi', followed by ligation. After an early phase common to both pathways, viral development may follow the productive or lysogenic pathways. In the productive pathway, synthesis of encapsulation and lysis proteins is turned on, synthesis of early proteins is turned off, and replication switches to a rolling-circle mode that generates the multimeric DNA used as a substrate for encapsulation of linear DNA with free mature ends. In the lysogenic pathway, synthesis of lytic proteins is turned off, and the circular viral DNA is inserted into the host genome by a specific recombination event.

GENETIC MAP Nu! P.u3 Ef Fl Fl z U V G rt L K i uooint xis redX redB gem i! ciiicralrox cro cl OP S R mAmtXW W-t;tX'lCO]D 4g4HS2 /aD?J 4+ 1~~~~'V ClII o$g lm 0 10 20 30 40 50 60 70 80 90 100 Phage heod Phoge tail Integration, Replication sis excision, and ReglatonRegulation Late recombination regulationlateo GENE ACTIVITY MAP Through heod and toil

00 PL CI PR CrO fg1 Cl 'np 0 or P fe2 P's int xis cmIII ILAN9 0 m 50 60 70 2 80 90 too Pt SOL! PM Pt

FIG. 2. Genetic map of bacteriophage A. The genes that code forproteins of defined function are shown in the upper part of the figure; the vertical line marks the approximate center of the genes (see Table 1). The b region is silent in terms of defined viral functions, although it does code for several proteins (see text and Table 1). The regulatory sites and their function are indicated in the lower part of the figure; these are described in the text and in Table 1. The different stages of transcriptional activity and the DNA regions involved are indicated by the arrows; the actual length of DNA transcribed can be determined from the intersection of the promoter (p) and terminator (t) lines with the horizontal calibrated line representing the A genome. products. The principles (and examples) are examples of this type of overestimate exist (e.g., given in the articles by Campbell (20), Davidson the V protein has an electrophoretic estimate of and Szybalski (38), and Szybalski and Szybalski 32,000 daltons, but a calculated value from (211). The functions of the genes and sites are amino acid composition of 26,000 daltons). summarized with references in Table 1. Based on this assessment, we have reduced the Based on the physical mapping data of Par- size of some genes (3 to 10%) to conform to the kinson and Davis (156) and the estimated mo- physical map; these reduced values are used for lecular weights of the gene products (Table 1), the "Coordinates" column of Table 1. The mo- the head and tail region is "oversaturated" lecular weight values originally reported for the (codes for more amino acids than available nu- proteins are given in the "Gene function and/or cleotides). We believe that the discrepancy most protein activity" column. likely results from overestimates of protein size Most readers of this article are aware already by the standard method of polyacrylamide gel that A nomenclature has not evolved in a logi- electrophoresis in sodium dodecyl sulfate, rather cally consistent way. Genes for proteins essential than from overlapping genes, because several to productive growth were originally given up- VOL. 42, 1978 GENETIC MAP OF PHAGE LAMBDA 579 TABLE 1. Genes ofphage A and their function co- Gene symbol Approxordinatesamap Gene function and/or protein activity Referencesb m 0 Left cohesive end of the mature DNA mol- 13,31,38,57,60,61,69,87,146, ecule; the first 12 nucleotides of the 5' 147, 151, 206, 218, 228, 229, end of the I (transcribed leftward) strand 237,238 protrude as a single-stranded chain, complementary to m'. Nul 0/0.5 Involved in DNA packaging and cohesive 8, 145, D end formation; may activate A protein. A 0.5/4.7 DNA packaging into proheads and forma- 5-7, 19, 67,90, 96, 103, 109-111, tion of cohesive ends; protein is 79,000 132, 139, 144, 145, 155, 197, daltons (79K). 204, 206, 210, 226, 229, 231 w 4.7/5.0 Modifies DNA-filled heads in an unknown 28, 93, 132, 155, 205, 226 way to allow FII action; protein is 5-10K. B 5.0/8.3 Structural component of the capsid; B pro- 7, 19, 67, 85, 86, 91, 120, 139, tein and a cleaved derivative B form 140, 141, 142, 145, 155, 169, the head-tail connector-, B is 59-62K; B" 204, 223, 231, 242 is 53-56K. C 8.3/11.3 Structural component of capsid; the 7, 19, 67, 84-86, 91, 120, 139, 56-61K C protein is present in the capsid 140, 141, 142, 144, 145, 155, as two cleaved derivatives, fused to a 169, 204, 223, 231, 242 cleaved derivative of E; the two cleavage-fusion products, termed Xl and X2, are 29 and 27K, respectively. Nu3 11.3/12.3 Transient morphopoietic core for capsid 7, 86, 91, 103, 140, 141, 144, assembly; protein is 19K. 145, 169, 242 D 12.3/12.8 Major component of the phage head; the 4, 7, 19, 29, 91, 92, 95, 98, 111, 11-12K protein is incorporated into the 129, 132, 139, 143-145, 204, capsid during or after DNA packaging. 207, 226, 231, 234 E 12.8/14.8 Major component of the capsid; 10 to 12 of 4, 7, 16, 19, 27, 29, 85, 86, 91, 94, the 420 molecules of the 37K E protein 98, 101, 103, 120, 132, 139, are present in the capsid fused to C, 140, 141, 144, 145, 155, 169, forming Xl and X2 (see C). 204, 223, 226, 231, 234 FI 14.8/15.7 Packaging and maturation of DNA; the 7, 8, 14, 19, 142, 144, 155 17K protein may confer specificity on A protein. FII 15.7/16.3 Structural component of DNA-filled 12, 14, 15, 19, 24, 26, 28, 132, heads; the 11.5K protein mediates tail 144, 145, 155, 226 attachment. z 16.3/17.4 Structural component of the proximal end 113-116, 155, 218, 219 of the tail; the 20K protein is involved in proper positioning ofthe right end of the DNA molecule in the tail. U 17.4/18.4 Structural component of the tail; the 113-117, 120, 126, 139, 144, 14-16K protein is the tail length deter- 155 mination factor. V 18.4/19.9 Major protein of the tail tube; protein is 6, 113-117, 126, 143, 144, 155 25-32K. G 19.9/21.7 Involved in the assembly of the tail initia- 67, 116, 120, 126, 139, 144, 155, tor-, the 33K protein is possibly a struc- 231 tural component. . T 21.7/22.6 Structural component of the tail; the 16K 67, 139, 144 protein is involved in the assembly of the tail initiator. H 22.6/27.3 Cleaved form of the 87-90K H protein, the 19, 83, 103, 113, 116, 120, 126, 78-79K H*, is a structural component of 139, 143, 144, 155, 183, 231 the tail, involved in DNA injection. M 27.3/27.9 Structural component of the tail; the 10K 19, 113, 114, 116, 117, 120, 139, protein is involved in tail initiator assem- 155, 231 bly. L 27.9/29.6 Structural component of the tail initiator; 19,103, 114, 116,120, 126,139, protein is 29K. 144, 155, 231 K 29.6/31.2 Temporarily associated with the tail initi- 19, 103, 114, 116, 120, 126, 139, ator, function unknown; protein is 27K. 144, 155, 231 I 31.2/31.8 Involved in assembly of the tail initiator. 19, 103, 113, 116, 155, 223, 231 31.8/39.4 Structural component of the tail; the 17, 19, 42, 67, 82, 113, 114, 116, 130-140K protein forms the tail fiber 120, 126, 139, 143, 144, 155, and determines host range ofadsorption. 188, 231 580 ECHOLS AND MURIALDO MICROBIOL. REV. TABLE 1.-Continued co- Gene symbol Approxordinatesamap Gene function and/or protein activity Referencesb b 39.4/57.3 Although this region of the chromosome is 32,40,81,82, 119, 128, 134, 144, called the silent region, it codes for sev- 157, 173, 177, B eral proteins of unknown function. a a'/(P P' or attP) 57.3 Determines location and specificity of site- 39, 40, 50, 99, 127, 192, 194 specific recombination; 15-base homol- ogy with host site b b'. int 57.4/59.8 Integrative and excisive recombination; 3, 45, 50, 59, 70, 71, 73, 74, 121, the 40K protein provides sequence rec- 122, 138, 148, 194, 243 ognition for a* a'. xis 59.8/60.2 Excisive recombination. 45, 59, 76, 108 Pi (60.2) Promoter for transcription from the int 59, 190, 191 gene under positive regulation by cII and cIII proteins. redX (64.7/66.1) General recombination; the 24K protein 23, 49, 131, 165, 167, 193, 194 ("A-exonuclease") is a 5'-exonuclease active on double-stranded DNA. redB 66.1/67.8 General recombination; the 28K protein 23, 49, 165, 193, 194 ("C'-protein") associates with A-exonuclease. gam 67.8/68.7 Regulation of DNA replication; the 16K 58, 112, 180, 195, 225, 244 ("gamma") protein inhibits the RecBC DNase, an antagonist of the rolling-circle mode of replication. kil (68.7/69.2) Loss of host cell viability, associated with 75 an inhibition of cell division. CIII (68.8/70.7) Establishment of lysogeny (together with 33, 34, 36, 51, 55, 106, 118, 122, cII); the cII and cHII proteins activate 133, 170, 201 transcription from the cI and int genes and repress transcription from the lysis, head, and tail (and probably replication) genes (see cHI, PE, and pi). ral (70.7/71.6) Partial alleviation of restriction of X DNA A by K-12 restriction endonuclease. tL (71.8) Termination for the earliest (immediate- 123, 126, 174 early) stage of RNA synthesis initiated at pi., a p-mediated event. N 72.5/73.3 Positive regulation of early development; 1, 19, 66, 80, 124, 126, 149, 166, the 13K protein activates delayed-early 174, 189, 196, 221, 222 transcription from recombination, replication, and regulation genes; N protein prevents termination events at tL, tRi, and tR2. PL 73.5 Promoter for transcription of the N gene 10, 11, 124, 136, 174, 217 during the immediate-early stage of development and for the N through recombination region during the N-activated delayed-early stage of development (see N); repressed by Cro during the late stage of lytic development and by cI during the maintenance stage of lysogeny (see cI and cro). OL 73.5/73.6 Operator for regulation of transcription 52, 96, 102, 107, 135, 136, 159, from PL; the OL sequence defines three 163, 172, 199, 203, 213, 214, 17-base binding sites, OLI, oL2, oL3; the cI 235, 236 and Cro proteins bind at OL to prevent binding by RNA polymerase and thus transcription from pL. tM (74.2) Terminator for transcription of the cI and 79, 102 rex genes (initiated at PM) during the maintenance stage of the lysogenic pathway. rex (74.2/75.9) Restricts growth of T4rII mutants and 77, 97, 130, B helps cell growth in limiting carbon sources; protein is 29K. VOL. 42, 1978 GENETIC MAP OF PHAGE LAMBDA 581 TABLE 1.-Continued co- Gene symbol Approxordinates'map Gene function and/or protein activity Referencesb cI 76.9/78.4 Maintenance oflysogeny through a repres- 30, 53, 55, 102, 106, 107, 137, sion of RNA from early genes; the 26K 161, 162, 171, 172, 182, 203, protein binds as a dimer (or tetramer) to 236 OL and OR, repressing transcription from pL andpR and regulating transcription of the cI and rex genes, positively at low cI levels and negatively at high cI levels (see OL and OR). PM (P.) 78.4 Promoter for transcription of the cI and 55, 79, 102, 135, 160, 170, 171, rex genes during the maintenance stage 241 of lysogeny; the cI and Cro proteins bind at OR to regulate this transcription (see OR, cI, and cro). OR 78.4/78.5 Operator for regulation of transcription 52, 64, 96, 102, 104, 107, 135, fromPM andpR; the OR seqeuence defines 137, 160, 163, 171, 203, 213, three 17-base binding sites, ORI, OR2, OM; 214, 235, 236 the cI and Cro proteins bind at ORI and oR2 to prevent binding of RNA polymerase and thus transcription from pi,; cI binding at ORI (and/or oR2) enhances transcription from PM, whereas cI and Cro binding atoR3 prevents transcription frompM. PR 78.5 Promoter for transcription of the cro gene 10, 11, 124, 135, 174, 217, 227 (and limited cIIOP transcription) during the immediate-early stage of development and for the cro gene onwards during the N-activated delayed-early stage of development (see N); repressed by Cro during the late stage of lytic development and by cI during the maintenance stage of lysogeny (see cI and cro). cro (tof, fed) 78.6/79.0 Regulation of late stage of lytic develop- 18, 35, 52, 55, 63, 64, 65, 1(00, ment; Cro represses early transcription 104, 123, 154, 158, 159, 176, and is also required directly or indirectly 186, 199, 200, 213, 214, 215 for normal late replication; the 7K protein binds as a dimer to OL and OR, repressing transciption form p,, PR, and pM (see oL and OR). tRI 79.2 Terminator for most of the earliest ("im- 80, 149, 174, 178, 186 mediate-early") stage of RNA synthesis initiated at PR, a p-mediated event. Pa (Pre) 79.2 Promoter for transcription of the cI and 51, 170, 201, C rex genes during the establishment stage of lysogeny; positively regulated by cII and cm proteins. cH 79.2/79.8 Establishment of lysogeny (together with 33, 34, 36, 51, 55, 106, 118, 122, cIII); the 11K cII protein (aided by cIII) 133, 170, 186, 201, 240, B activates transcription from the cI and int genes and represses transcription from the lysis, head, and tail (and probably replication) genes (see cIII, pE and px). Po 79.9 Promoter for transcription of the 4S or 37, 79, 184 "OOP" RNA, an 81-base RNA, termi- nating in the absence of p, that so far lacks a clearly defined function (it has been postulated to be involved in initiation of replication or the establishment of repression). 0 79.9/81.9 Initiation of the early (simple-circle) mode 19, 56, 68, 105, 152, 164, 186, of DNA replication (with P); the 34K 212, 224, 240, B protein probably interacts with the origin sequence and P protein, and the complex directs the host DNA propagation enzymes to replicate A DNA; 0 and P proteins may also be required for the propagation stage of replication (see P and ori). 582 ECHOLS AND MURIALDO MICROBIOL. REV. TABLE 1.-Continued Approx map co- GeneGenesymbol ordinates' Gene function and/or protein activity References' ori 80.9 Origin (initiation site) for the bidirectional 41, 44, 168, 185, 208 early (simple-circle) mode of DNA replication; activation of the o-rigin requires O and P proteins (and probably RNA synthesis through or near the site) (see O and P). p 81.9/83.3 Initiation of the early (simple-circle) mode 56, 105, 152, 153, 212, 224, B of DNA replication (with 0); the 24K protein probably interacts with 0 protein, and the resultant O/P/ori complex directs the host propagation enzymes to replicate A DNA; 0 and P proteins may also be required for the propagation stage of replication (see 0 and ori). tR2 (83.3) Terminator for the residual fraction (after 80, 149, 178 tRi) of the earliest (immediate-early) stage of RNA synthesis initiated at PR, a p-mediated event. Q 90.8/92.1 Positive regulation of the late stage of lytic 19, 42, 89, 105, 149, 150, 175, development; the 23K protein activates 196, 198 late transcription (initiated at P'R) from the lysis, head, and tail genes; Q might provide for new initiation events or prevent termination of a small 198-base 6S RNA synthesized (at least in vitro) from the P'R region (see P'R) P R (93.1) Promoter used for transcription of the 89, 175, 198 head, tail, and lysis genes, subject to positive regulation by Q protein (see Q). S (93.1/93.9) Cell lysis (together with R); the S protein 2, 72, 78 is probably involved in a turnoff of certain host functions as a necessary pre- requisite for cell lysis, perhaps through a direct effect on the cell membrane; S- mutants accumulate large numbers of intracellular phage. R (93.9/95.0) Cell lysis (together with S); the 18K (en- 9, 22, 54, 62, 216 dolysin) protein is an endopeptidase that produces lysis through hydrolysis of the cross-linking bond in murein. 100 Right cohesive end of the mature DNA 13, 31, 38, 57, 60, 61, 69,87, 146, molecule; the first 12 nucleotides of the 147, 151, 206, 218, 228, 230, 5' end of the r strand protrude as a 237,238 single-stranded chain, complementary to m. 'Approximate map coordinates were determined mainly from electron microscopic data on heteroduplex structures of genetically characterized deletions and from molecular weights of proteins; the extensive set of heteroduplex data by Szybalski and co-workers has been particularly helpful in this effort (11, 38, 99, 211). Coordinates likely to be off by more than ±0.2 units are in parentheses. b Reference numbers in roman type denote studies of gene function; boldface numbers denote studies of activity in vitro with purified proteins; italic numbers denote nucleotide or amino acid sequences. Letters refer to manuscripts in preparation or submitted for publication but not yet in press: (A) L. De Brouwere, M. Zabeau, M. Van Montagu, and J. Schell; (B) C. Epp and M. L. Pearson; (C) M. Jones, R. Fischer, I. Herskowitz, and H. Echols; (D) R. A. Weisberg and N. Stemnberg. percase designations (19); genes for regulatory explain it (Table 1). In order not to have the proteins needed for lysogeny were given lower- same name for two things (e.g., gene P and case designations (106); and "other" genes non- attachment site P), we have used lowercase italic essential for productive growth (or thought to letters throughout for sites of protein activity be) were given three-letter designations (e.g., int, and retained uppercase italic letters and italic red). We consider this situation unfortunate, but three-leter designations for genes that code for suspect that an effort for complete consistency proteins. The proteins themselves are desig- at this stage will cause more confusion than it nated by nonitalic letters, generally followed (in will remedy. Thus we have adopted most of the the text) by protein; in the field of bacteriophage current nomenclature and have only sought to morphogenesis, gene products are more usually VOL. 42, 1978 GENETIC MAP OF PHAGE LAMBDA 583 designated by the symbol of the gene preceded grative recombination involves recognition of by the lowercase letters gp (for gene product) the attachment site (a-a') by the Int protein, (e.g., gpA = A protein). leading to the breaking and joining event (a* a' x b b') that inserts the viral DNA into that of ACTIVITY MAP OF THE X GENOME the host. The reverse excisive recombination The activity map of A DNA shown in the requires recognition ofthe prophage attachment lower part of Fig. 2 indicates the known DNA sites (b a' and a- b') in a reaction involving the sites at which the proteins essential for A devel- Int and Xis proteins. The generation of the free opment act. Promoter (initiation) sites for RNA mature ends (m and m') most likely involves synthesis are designated p and termination sites specific nucleolytic cleavage by the A protein in t; the transcription pattern that characterizes a reaction that also requires at least the presence productive growth is shown above the line rep- of phage head precursors (proheads) and possi- resenting A DNA, and the transcripts specific for bly host factors. lysogenic development are shown below the line. During the initial "immediate-early" phase of COMMENTS ON GENETIC ORGANIZA- productive growth, ribonucleic acid (RNA) TION chains initiated at the early promoter sites pL Some remarkable features of genetic organi- and pRzare mainly terminated at the end of the zation in phage A are evident from a considera- N and cro genes (termination sites tL and tRi, tion of Fig. 2 and the accompanying discussion. respectively); some rightward RNA chains con- We note the following points: (i) the clustering tinue through the replication genes (to tR2) of genes with similar function (e.g., head, tail, (-). During the next "delayed-early" phase, the DNA); (ii) the proximity of genes for proteins N protein provides for extension of the imme- acting on X DNA to their site of action (e.g., cI diate-early transcripts into the remainder of the and cro to OL and oR, int and xis to att, 0 and P early-gene region, providing for synthesis ofrep- to ori); (iii) the remarkable economy in the use lication, recombination, and regulatory proteins of most of X DNA; and (iv) the puzzling "silent" (--). During the late phase, the Cro protein acts b region. More extensive discussions than the at the operator sites OL and OR to reduce initia- following one may be found in the articles by tion of the early RNA; Q protein provides for Thomas (220), Stahl and Murray (202), Dove transcription of the lysis, head, and tail regions (43), Casjens and Hendrix (26), Echols et al. (48), by a transcript initiated at the late promoter site Campbell (21), and Echols (in press). P'R and continuing through the entire late-gene The clustering of genes with similar functions region (- - -*) (the lysis genes are joined to the is ofpotential value in three ways: the regulatory head and tail genes in the simple-circle or roll- needs of viral development are simplified be- ing-circle molecule). cause a limited number of regulatory proteins During the establishment phase of lysogenic and sites are required to control a large number development, the cII and cIII proteins activate of genes whose products act together (at least in leftward transcription of the cI and int genes, time); genes for proteins that must recognize initiated at the promoter sites PE and pi (oo, each other are rarely separated by recombina- and inhibit rightward transcription of lytic tion; and the evolution of new phage species is genes. During the maintenance phase, the cI facilitated by recombinational transfer of func- protein acts at OL and OR to repress nearly all A tional "modules" (e.g., a phage can acquire a transcription by preventing synthesis of early- new tail with different adsorption properties). gene RNA from the early promoter sites pL and The proximity of genes to target sites on the PR; the cI protein also regulates its own further DNA also serves these three potential purposes; synthesis by controlling cI gene transcription in addition, a protein is synthesized close to its from the maintenance promoter PM (**e+, ex- activity site, thus conferring a potential kinetic erting positive regulation at low cI concentration increment on protein activity because the pro- and negative regulation at high cI concentration. tein does not need to diffuse through a vast The operator sites OLand OR define three binding number of nonspecific interactions to reach its sites for cI protein, and the different binding target site. constants probably account for the functional Although A has not been demonstrated to diversity of the repressor-operator interaction. have the "gene-within-a-gene" economy of The symbol ori represents the initiation site 4OX174 and G4 (181, 187), the intensive use of A for the early mode of DNA replication specified DNA is nevertheless impressive. With the ex- by the 0 and P proteins; the initiation site for ception of the b region and a stretch between P the late, continuous rolling-circle mode is not and Q, there appears to be very little DNA not known with any certainty. Specificity for inte- used to code for proteins of known utility, and 584 ECHOLS AND MURIALDO MICROBIOL. REV. some genes probably remain to be identified Natl. Acad. Sci. U.S.A. 72:581-585. (e.g., control of the switch to rolling-circle rep- 6. Becker, A., M. Marko, and M. Gold. 1977. lication). In fact the recombination region may Early events in the in vitro packaging of bac- and teriophage A DNA. Virology 78:291-305. be oversaturated (211), suggesting that gam 7. Becker, A., H. Murialdo, and M. Gold. 1977. kil may be analogous to the 4X174 overlap. As Studies on an in vitro system for the packaging might be expected for an economic regulatory and maturation of phage A DNA. Virology system involving a small number of proteins, the 78:277-290. amount of A DNA devoted to regulatory sites is 8. Benchimol, S., A. Becker, H. Murialdo, and very small. M. Gold. 1978. The role of the bacteriophage With all of the evolutionary development ev- lambda FI-gene product during phage head ident in the highly organized, intensively used, assembly. Virology, in press. and tightly regulated X genome, the "silent" (or 9. Black, L. W., and D. S. Hogness. 1969. The lysozyme of bacteriophage A. I. Purification "useless") b region remains an embarrassment. and molecular weight. J. Biol. Chem. 244: Three possible reasons for the existence of such 1968-1975. a silent region are: (i) the proteins coded by this 10. Blattner, F. R., and J. E. Dahlberg. 1972. RNA region serve a valuable viral function under some synthesis startpoints in bacteriophage A: are set of conditions in which X is not normally the promoter and operator transcribed? Nature studied in the laboratory (e.g., different hosts, (London) New Biol. 237:227-232. growth medium, etc.); (ii) the DNA itself serves 11. Blattner, F. R., M. Fiandt, K. K. Hass, P. A. a function (e.g., size or structural aspects of Twose, and W. Szybalski. 1974. Deletions packaging); (iii) the silent DNA is useless to the and insertions in the immunity region of coli- vestige of a recombina- phage lambda: revised measurement of the virus, an evolutionary promoter-startpoint distance. Virology 62: tional event with another phage or with host 458-471. DNA, preserved by the kindness of the friendly 12. Bode, V. C. 1971. Incomplete lambda bacterio- molecular biologist, who has reduced further phage heads produced by a gene F mutant. J. evolutionary pressure to a minimum. Since Virol. 8:349-351. phage deleted for most of this silent region can 13. Bode, V. C., and A. D. Kaiser. 1965. Changes grow and make phage particles normally, the in the structure and activity of A DNA in a second possibility does not seem tenable, except superinfected immune bacterium. J. Mol. Biol. as a special case of the first. We think that the 14:399-417. third possibility has some conceptual merit, in 14. Boklage, C. E., E. Chun-te Wong, and V. C. Bode. 1973. The lambda F mutants belong to the sense that molecular biologists often assume two cistrons. Genetics 75:221-230. that whatever is, has function. 15. Boklage, C. E., E. Chun-te Wong, and V. C. ACKNOWLEDGMENTS Bode. 1974. Functional abnormality of lambda phage particles from complemented F11-mutant We thank the community of lambdologists for pro- lysates. Virology 61:22-28. viding the information used in constructing the map 16. Buchwald, M., H. Murialdo, and L. Siminov- and for making lambda research a pleasant as well as itch. 1970. The morphogenesis of bacterio- fascinating field of endeavor. 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