The S and U genes of bacteriophage Mu are located in the invertible G segment of Mu DNA

VIROLOGY 92, 108-l%

(1979)

The S and U Genes of Bacteriophage Mu Are Located Invertible G Segment of Mu DNA

in the

MARTHA M. HOWE,*’ JAMES W. SCHUMM,” AND AUSTIN L. TAYLOR? *Department of Bacteriology, College of Agriculture and Life Sciences, University Madison, Wisconsin 53706, and TDepartment of Microbiology and Immunology, Medical Center, Denver, Colorado 80262

of Wisconsin-Madison, University of Colorado

Accepted September 18, 1978

An F’ plasmid containing only the /3 and G segments of bacteriophage Mu DNA in a p-G-p structure was isolated as a LacZ- segregant of an F’loc plasmid containing Mu prophages inserted in the 1oxZ and 1acY genes. The segregant arose by homologous recombination between the similarly oriented G regions located in oppositely oriented Mu prophages whose a segments were directed toward the 1acZ gene. Electron microscopic observation of singlestranded plasmid DNA from the segregant revealed the expected p-G-p stem and loop structure in which the double-stranded stem was the same length asp and the single-stranded loop was the same length as G. Marker rescue experiments with Mu amber mutants defective in each of the known essential Mu genes showed that strains carrying this F’P-G-/3 plasmid contained the wild-type alleles for S and U mutations but not for mutations in other genes. More detailed mapping of the R S U region by deletion mapping in Mu prophages and ApMu transducing phages produced two gene orders, R S U and R U S, thus indicating that the S and U genes were located in the invertible G segment rather than in p. Confirmation of this conclusion was obtained from results of DNA heteroduplexing experiments which showed that deletions ending within gene U end physically within G segment DNA. The location of essential genes S and U within G and the location and extent of G segment DNA which is nonessential for growth (L. T. Chow, R. Kahmann, and D. Kamp, 1977, J. Mol. Biol. 113, 591-669), taken together, reveal that the order of genes in viable Mu phage with G in the G(+) orientation is R S U. INTRODUCTION

plaques (Ramp et al., 19’78). Not all of G is essential, however, because phage with deNear the right or variable end of Mu DNA letions in the rightmost 1.3 kb of G in a G( +> is a 3kilobase (kb) pair segment, the G seg- orientation are viable plaque-forming phage ment, which has the ability to invert via (Chow et al., 1977). The G orientation effect intramolecular recombination between small would be easily understood if there was an regions of homology located at its ends essential gene located within G, yet mapping (Daniel1 et al., 1973a; Hsu and Davidson, of the original set of essential genes A 1974). During the last two years it has become through S (Abelson et al., 1973) located clear that this invertible G segment plays an them all in the cysegment to the left of the G important role in Mu development (see segment (Daniel1 et al., 1973b). FurtherHowe, 19’78,for review). Phage which con- more, recent analysis of 300 new conditional tain G in one orientation, the G(+) orienta- lethal amber mutations has not revealed the tion, are viable plaque-forming phage, while existence of any new genes located to the phage containing G in the opposite orienta- right of mutations originally assigned to tion, the G(-) orientation, cannot form gene S and now recognized to comprise two genes S and U (O’Day, Schultz, Ericsen, * To whom requests for reprints should be ad- Rawluk, and Howe, manuscript in preparation). dressed. 9942~6822/79/010198-17$02.99/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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MU GENES

S AND U MAP IN THE G SEGMENT

109

Recent electron microscopic evidence Bacterial Strains has demonstrated that phage Pl DNA also The bacterial strains used are listed in contains an invertible segment and that this segment is homologous to the G segment of Table 1. The derivations of several strains Mu (Chow and Bukhari, 1976). There is also are given below. AT3325. A dilysogen containing two genetic evidence for partial homology between Pl and Mu since Mu phage defective copies of wild-type Mu prophage in the lac in genes S and U can recombine with Pl to operon was obtained as a genetic recomproduce nondefective Mu phage (Toussaint binant from a cross between Hfr strain AT3124 (ZacZ88::+Mu, Pro+, SW, transfer et al., 1978). These results led us to reconsider the pos- origin of Hfr Cavalli) and F- strain AT827 sibility that part of Mu genes S or U might (lacYlL7:: -Mu, proBl7, StrR). (See legend be located within the G segment of Mu to Table 1 for meaning of ::, +, and - noDNA. In reexamining the results of the pre- tation.) Pro+ StF recombinants were selected vious correlation of the genetic and physical on minimal plates containing succinate as sole maps of Mu by analysis of XpMu phage carbon and energy source, plus streptomycin. (Magazin et al., 1977), we realized that the Since recombination between Zuc1and lacy sensitivity of resolution of the DNA hetero- was expected to be infrequent in this cross, duplex and restriction enzyme analyses per- Xgal (5bromo-4-chloro&indolyl-P-Dgalactoformed was not sufficient to prove or dis- side, Sigma) and tONPG (O-nitrophenyl-Pprove this hypothesis. Therefore we have D-thiogalactoside, Vega-Fox Biochemicals) combined a somewhat different approach were included in the selective medium to for generating partial Mu prophages with help discriminate between the parental and the use of a large number of new amber recombinant lac phenotypes. At a concenmutations for mapping these prophages. tration of 320 mg/liter tONPG partially inThe results obtained have demonstrated hibits the growth of constitutive lacy+ cells that part, if not all, of Mu genes S and U are while permitting normal growth of lacy- or inducible lacy+ bacteria. At 20 mg/liter, located within the invertible G segment. Xgal permits direct scoring of ZacI- constitutive clones as blue pigmented colonies. MATERIALS AND METHODS Pro+ recombinants which were both lac constitutive (Zac188::Mu) and permease negaMedia tive (ZacYl2?‘::Mu) were thus detected as LB broth, LB agar, soft agar, SB broth, fast growing blue colonies on the medium. and tryptone broth have been described The frequency of appearance of these dilysopreviously (Howe, 1973a). LBM and SBM genie recombinants ranged from 0.2 to 0.8% contain 2 x 10e3M MgS04 in addition to LB in three separate experiments. One of these and SB. LBMC is LB containing2 x 10e3M recombinants was mated with strain ATMgS04 and 2 x 10e3M CaCl,. SBPM is SB 3315, a donor of the plasmid F’42 which containing 1 x lop3 M Pb(C,H,O,), and 2 carries a wild-type lac operon. The heterox low3 M MgSO+ SM contains per liter 5 g genetic transconjugant (F’42 lac~ilacI88:: NaCl, 2.7 g Tris-HCl, and 0.32 g Trizma Mu ZacY127::Mu styA) produced from this base and is supplemented with MgSO, to a cross was screened for spontaneous homogfmal concentration of 2 X low3 M after auto- enotes (F’42 Zac188::Mu lacY127::Mu/lucclaving. SMC is SM containing 2 x 1O-3M Z88::Mu lacYl27’::Mu &A) which contained CaCI,. TCM contains 0.01 M Tris, pH 7.5, the Mu-induced Zac- alleles in the plasmid. 0.01 M MgS04, and 0.01 M CaCl,. Minimal Although the presence of the Mu prophage medium is OM of Ozeki (1959) supplemented in the ZacY gene makes the cell Lac-, the after autoclaving with vitamin B, at 1 pg/ml, presence of the ZacZ::Mu mutation makes sugars at 0.2%, and desired amino acids at synthesis of P-galactosidase constitutive 20 pg/ml. MacConkey lactose medium was and therefore results in a partially Lac+ purchased from Difco. phenotype on both minimal lactose and Mac-

HOWE, SCHUMM, AND TAYLOR

110

TABLE 1 BACTERIALSTRAINS Strain

Genotype”

AT827 AT3124 AT3315 AT3321 AT3325 BUS082 BU8093

F-lacy 127::-Mu thr-1 proB17 thi-1 strA (A+) HfrC lacZ&?::+Mu metB1 thi-l F’42 lac+ilac+ F-Apro-lac recA56 spc F’42 locZ88::Mu lacZ+ lacY127::MuJApro-lac spc recA F-Apro-lac trp strA (hcZ857S7 plac917::Mu c+) F’pro+lacZ8305::(Mu cts62)lApro-lac trp strA (AcZ857S7

CR63 Ml07 MH112 MH118 MH119 MH130 MH131 MH165 MH394 MH594 MH690

SuI+ hh+s hhs F-Aloe SuI+ strA araD ph (leu::MuAc-A--am) araD pA(leu::MuAc-R--am) araD pA(leu::MuAc-R--ara) araD pA(leu::MuAS--ara) araD pA(leu::MuAL-U S--ara) araD thyA pA(lys::MuAcb) F-Apro-lot his met tyr strAc F-Apro-lot his met tyr strA NalR c HfrHApro-lac T6s thyA

MH2500 MH2501 MH2856 MH2907

F- gal strA recA F- gal strA (A+) F-Apro-lac spc pA(leu::MuAL-U S--am) Nals F’42 locZ88::Mu lacZ+ lacy 127::MulApro-lac spc ph (leu::MuAL-U S--aru) F’42Alac::pGp/Apro-lacspcpA(leu::MuAL-US--am)NaIs F’421aeZ&?::MullacZ+ lacY127::MuiApro-lac his met tyrstrA NaIs F’42Alac:$GZ3/Apro-lac his met tyr strA Nala F- SuIII+ me1 pro (P2) F- SuIII+ me1 pro (A) F- SuIII+ me1 pro (Ai434)

plac8305::Mu

MH2908 MH2909 MH2911 MH30’72 MH3073 MH3074 MH3077 MH3078 MH3080 NlOO $5003 WA5047 WD5021

cts62)

galE groN785 F- gal strA (Ai434) F- gal strA (P2) (Adbio24-5ASA439[AAO-chlA])” W3102 galK recA F- SuII+ thr leu tonA lac F- SuIII+ me1 pro F- trpASS AsSpcs recA F- gal strA

Source/reference/derivation A.L.Taylor (in prep.) A.L.Taylor (in prep.) A.L.Taylor (in prep.) A.L.Taylor (in prep.) This work Bukhari and AIlet (1975) Bukhari and Allet (1975) Appleyard et al. (1956) Howe (19’73a) Howe (1973a) Howe (1973a) Howe (1973a) Howe (1973a) Howe (1973a) Howe (1973a) 533 of Zeldis et al. (1973) MH394 NaIs thyA derivative of strain 462 of D. Zipser WD5021 recA of F. van Vhet WD5021 (A+) This work This work This work This work This work QD5003 (P2) QD5003 (A) QD5003 (A?) Georgopoulos (1971) WD5021 (ArU) Kayajanian (1970) Little and Gottesman (1971) Howe (1973a) Howe (1973a) Howe (1973b) Howe (1973a)

a The ::Mu notation indicates that the Mu prophage is integrated in the gene immediately preceding the ::. The + Mu and -Mu notation designates the orientation of the prophage; + orientation means that if one moves around the E. coli map in a clockwise direction, the immunity end of the Mu prophage will be reached first. (See Howe and Bade [1975] for a more complete description of notation.) The pA(leu::MuAc-A--am) notation indicates a partial deletion of the region within the parentheses; in this case the deletion removes DNA from the c and A genes of a Mu prophage inserted in leu through part of ara. b This strain was originally thought to contain a deletion extending from thyA into the Mu prophage (Howe, 1973a)but reversion of the thyA mutation indicatesthat the prophage deletion and thyA mutation are separate. c The tyr mutation in this strain is extremely leaky but addition of tyrosine to minimal medium does stimulate the growth rate. d The only ADNA remaining in this strain is the c1 region from the right end of the bio substitution within gene rez to the left end of the SA439 deletion within gene 0.

MUGENESSANDUMAPINTHEGSEGMENT

Conkey lactose plates. Potential homogenotes were detected on the basis of their pink colony color on MacConkey lactose plates and were distinguished from cells cured of the F’42 plasmid by their continued sensitivity to the male-specific phage MS2. One of these homogenotic derivatives was then mated with the recipient strain AT3321 to generate strain AT3325. MH2856. The starting strain MH131 (Howe, 1973a), a partial Mu prophage deletion strain, was originally derived from RS54 HfrH araD (Schleif, 1969), which had lost its high-frequency transfer ability. The strain was presumably F+ since it was still sensitive to male-specific phage R17 and was a poor recipient in matings. Strain MH645 was isolated as an R17 resistant derivative of strain MH131 arising after treatment of the culture with 50 pg/ml acridine orange to cure the F+ factor (Hirota, 1960). A SpcRGal- derivative of strain MH645 was then isolated by plating an unmutagenized culture first on LB medium containing 200 pg/ml spectinomycin (the generous gift of G. B. Whitfield, The Upjohn Company) and then plating that derivative on medium containing 2deoxygalactose (Alper and Ames, 1975) to select a Gal- mutant. The resulting strain was mated with strain MH690 (HfrH thy Apro-lac T6R) at 34”for 3 hat 2-5 x lo8 cells/ml, and the mixture was plated on minimal plates containing galactose, proline, and leucine to select Gal+ Thy+ recombinants. These were then screened for their Leu, Pro, Lac, and Spc phenotypes by streaking on appropriate media. A recombinant retaining the partial Mu prophage ara-leu::Mu cts A-K, the pro-lac deletion, and spectinomycin resistance was isolated and designated MH2855. Strain MH2856 was selected as a spontaneously occurring mutant of strain MH2855 resistant to 40 pg/ml of nalidixic acid. MHZgOY, MH.2908, MH2909, and MH2911. Strains MH594, AT3325, and MH2856 were grown in LB to 2 x lo8 cells/ml. Matings between strain AT3325 and each of the other strains were performed by mixing the cultures in a lF’:3F- ratio and incubating the mixtures for 3 hr at 34”. Transconjugant

111

strains receiving the F’ZacZ::Mu ZacY::Mu plasmid from strain AT3325 were selected by virtue of their partially Lac+ phenotype by plating the mixtures on selective media: minimal medium containing lactose, proline, leucine, and nalidixic acid for the MH2856 mating and minimal medium containing lactose, proline, histidine, methionine, tyrosine, and nalidixic acid for the MH594 mating. The transconjugants were purified and detected as pink colonies on MacConkey lactose and designated MH2907 for the F’I MH2856 strain and MH2909 for the F’/ MH594 strain. Strains MH2908 and MH2911 were derived from strains MH2907 and MH2909, respectively, as spontaneous Lacsegregants (l-20% of population) making white colonies on MacConkey lactose plates but still retaining the F’ factor as indicated by their sensitivity to phage R17. Bacteriophage Strains Mu c+ and its heat-inducible derivative, Mu cts62, have been described previously (Taylor, 1963; Howe, 1973a). Mu ~25 is a phage containing a spontaneous clear mutation. Mu phage with amber mutations 1001 to 1999 and 7001 to 7367 were isolated and characterized by Howe (1973a) and O’Day, Schultz, Rawluk, Ericsen, and Howe (manuscript in preparation). R17 was obtained from J. F. Atkins. The XpMu phage 201725 were isolated by heat induction of dilysogens containing a Mu prophage integrated in a kI857S7 plac5 prophage using the method of Bukhari and Allet (1975) slightly modified as described (Magazin et al., 1977; O’Day, Schultz, Rawluk, Ericsen, and Howe, manuscript in preparation). hpMu 251-299 and 400-449 were obtained from strain BUS082 while ApMu 201-249 and 600-625 were obtained from strain BU8093. The hpMu strains 4M133 and 4M134 were constructed in vitro by Moore et al. (1977) by cloning the rightmost EcoRI endonuclease fragment of Mu DNA into a hCharon 4 cloning vector. All work with these phage was carried out under Pl and EKl conditions as specified by the “NIH Guidelines for Recombinant DNA.” These

112

HOWE.

SCHUMM,

phage contain the XKH54 immunity deletion (Blattner et al., 1974), the nin5 deletion (Court and Sato, 1969), and the QSR region from phage $80 (Franklin et al., 1965). In addition, they contain a contiguous block of Mu genes 1Mthrough U and DNA from the G, f3, and variable end regions. The host DNA segments in the variable end differ in the two phage. Since these phage contain less DNA than A, deleted derivatives could not be isolated by selecting chelating agent resistance (Parkinson and Huskey, 1971) using our standard plate procedure. Therefore, a derivative of 4M134, h134imm434, containing more DNA was isolated by crossing 4M134 with hh43%mm434(obtained from W. Szybalski). The cross was performed by mixing the phage in a spot on a lawn of strain QD5003 on an LB plate, incubating the plate overnight at 37”, resuspending the phage spot in SM, and plating the mixture on strain MH3080. On strain MH3080 only Mu+ (i.e., Red-Gam-) imm434 recombinants can grow: immX phage are repressed by the immh partial prophage and Red+Gam+ phage are unable to grow due to the P2 prophage (Zissler et al., 1971). The recombinants were tested for their genetic markers by assaying their growth on various strains: - on strain CR63 indicated they were h”; + on strain MH3072 and - on strain NlOO indicated they were missing at least the red region of A and therefore probably contained the Mu DNA substitution; + on strain MH3073 and - on strain MH3074 indicated they were imm434; and - on strain MH3077 indicated they were nin+. The presence of Mu DNA was confirmed by amber mutant marker rescue assays. The deleted phage were then selected from the Ah”pMu imm434 phage by adsorbing varying amounts of lysate to strain QD5003 in 1 x low3M MgS04 and plating 0.2 ml of the mixture in 2.5 ml soft agar on OM minimal plates containing glucose, proline, and 1.9 x 10e3 M sodium citrate on which deleted phage grow well. Phage from plaques, which arose at a frequency of approximately 10m5, were purified first on lawns on the same medium and then on LB. Lysates derived

AND TAYLOR

from these phage were then tested for Mu markers by amber mutant marker rescue. Bacteriophage

Techniques

(a) Mu lysate preparation by lytic infection [modiified from Howe (19Z?a)]. Cells

were grown in LBM to 2 x lo8 cells/ml at 37”. One fresh plaque was picked with a Pasteur pipet and added to 0.3 ml of cells, vortexed, incubated at 37” for 15 min, and diluted with 6.0 ml of LBM at 37”. The culture was grown at 37” until lysis (3-5 hr). If the cells became more concentrated than 10s/ml, they were diluted threefold with fresh LBM. After lysis, the lysates were chilled, chloroform was added, and debris was removed by centrifugation (Sorvall SS34 rotor for 30 min at 12,000 rpm). This procedure generally yielded titers of lo*1O’OPFU/ml. (b) Mu lysate preparation

by induction

of a lysogen. Lysogens were isolated as described previously (Howe, 1973a, b). A heat inducible lysogen was grown in SBPM to 2-4 x lo8 cells/ml at 32”. Phage development was induced by adding an equal volume of medium at 55” and incubating the culture at 42”. After 20 min the culture was shifted to 37” until lysis. Chloroform was added, and debris was removed by centrifugation. (Titers: 1 x 109 to 2 x 10’0 PFU/ml.) (c) A Lysatepreparation. Five- to twentymilliliter lysates on the various ApMu phage were grown using the same procedure used to grow Mu by infection, except that twice as many cells were used. (d) Titrations. Titrations of Mu and A were performed using lysates diluted in SM or SMC, adsorbed for 20 min to 10s cells freshly grown in LBM , and plated in 2.5 ml soft agar on LB plates. Plates were incubated overnight at 37” unless noted otherwise. (e) R17. Phage R17 lysates were grown by infection as for Mu, except that an Hfr strain was used as the host, and the medium contained 1 x lop3 M CaCl,. Tests for R17 sensitivity were performed by spotting the lysate on lawns of cells suspended in 2.5 ml

113

MU GENES S AND U MAP IN THE G SEGMENT

soft agar containing 0.1 ml of 1 M CaCl, over an LB plate and incubating overnight at 32 or 3’7”. Isolation and Electron Microscopic sis of F’ Plasmid DNA

Analy-

Exponentially growing bacteria at 4 X lo8 cells/ml were harvested from 25-ml LB broth cultures and disrupted by the lysis procedure of Sharp et al. (1972). The viscous lysates were sheared by repeated passage through a 5-ml Falcon plastic pipet and then incubated for 30 min at 37” with Proteinase K (200 pg/ml, EM Laboratories, Inc.). Lysates were adjusted to a density of 1.58 g/ml with CsCl, and ethidium bromide was added to 0.5 mg/ml. The final volume was brought to 10 ml with TES (0.05 M Tris-HCl, pH 8.0,5 mM Na,EDTA, 0.05 M NaCl), and the DNA was centrifuged to equilibrium at 40,000 rpm for 40 hr at 15” in a Be&man Type 65 rotor. The resulting bands were visualized in long-wave ultraviolet light and the thick upper band, containing sheared chromosomal DNA, was drawn off from the top of the gradient and discarded. The remaining lower band of super-coiled plasmid DNA was collected, adjusted to 1.55 g/ml with CsCl in TES with ethidium bromide at 0.5 mg/ml, and immediately centrifuged at 35,000 rpm for 24 hr at 15” in an SW 50.1 rotor. The lower bands were collected by side puncture and stored at 4” in the dark. Approximately 50% of the supercoiled DNA was converted into relaxed circles by X- irradiation at an energy level of 100 kVp and 5 mA to deliver a total dose of 125 R at a rate of 9.36 R/min. After nicking, the DNA was rebanded in a CsCl-ethidium bromide gradient as above and the upper band, which contained the relaxed form, was collected for examination in an RCA Model EMU4 electron microscope. The methods used for spreading native and renatured homoduplex molecules by the formamide method were essentially those described by Sharp et al. (1972). Contour lengths of DNA molecules were measured from tracings of photographic negatives enlarged

with a Scherr-Tumico optical comparator. Samples of phage PM2 DNA (9.7 kb) (Pettersson et al., 1973) and phage 4X174 DNA (5.375 kb) (Sanger et al., 1977) were included in the spreads as calibration standards for double- and single-stranded DNA segments. Purijkation UM133

and

Heteroduplexing

of

and N3hA22imm434

Approximately 1.5 x lo9 stationary phase cells of strain QD5003 were mixed with 1.4 x lo6 phage, incubated for 20 min at 37”, and diluted into 2 liters tryptone broth containing 1 x 10e2M MgS04. After 8.5 hr of growth at 37” the lysed cultures were treated with chloroform, chilled, and centrifuged to remove debris (Sorvall GSA, 30 min at 7500 rpm). Four percent NaCl (w/v) and 14% (w/v) polyethylene glycol (Fisher Carbowax 6000) were dissolved in the lysate and the mixture was allowed to stand at 4” overnight. The mixture was then centrifuged at 7500 rpm for 30 min (Sorvall GSA rotor). The pellet was resuspended in SM containing 1 x low2 M MgSO., and recentrifuged three times, saving each supernatant. The second and third supernatants containing the majority of phage were pooled and layered over CsCl (0.02 M Tris-HCl, pH 7.4,0.1 M MgCl,) step gradients containing steps of 6 ml p 1.413 g/ml, 6 ml p 1.50 g/ml, and 3 ml p 1.70 g/ml and centrifuged for 3 hr at 20,000 rpm at 4” in a Beckman SW 25.2 rotor. The resulting phage bands were removed, combined, adjusted to a density of 1.495 g/ml, and centrifuged to equilibrium at 28,000 rpm in an SW 40 rotor at 4” for 42 hr. The phage bands were removed with a syringe inserted through the side of the,centrifuge tube. Heteroduplexes of these phage DNAs were made and spread using the formamide procedure of Davis et al. (1971). After shadowing with platinum in a Kinney vacuum Model KDTG-3P they were viewed with a Carl Zeiss electron microscope Model 9A and photographed. The negatives were projected onto a screen and measured using a Numonics Corporation electronic graph-

114

HOWE,

SCHUMM,

its calculator. ColEl plasmid DNA was added before spreading to serve as a length calibration standard. The value of 6750 base pairs used for the length of ColEl was derived from the relative lengths of ColEl and P4 (Tanaka et al., 1975), the relative lengths of P4 and 4x174 RF (Younghusband et al., 1975), and the absolute length of 4X174, 5375 bases @anger et al., 1977).

AND TAYLOR

gave a significant number of plaques (revertants) on the control plates, marker rescue was judged positive if the ApMuinfected plates had greater than twice the number of plaques on the control plates. This assay was 50- to lOO-fold more sensitive than the spot test.

Marker Rescue from Partial Prophage Deletion Strains (a) Spot tests. For Mu-sensitive strains Aplkfw Marker Rescue approximately lo8 cells of each strain were Marker rescue was tested by infecting suspended in 2.5 ml of soft agar and plated an Su- cell containing a himmh prophage on LB plates. Lysates of amber mutant (MH2501) or a Aimm434 prophage (MH3078) phage at lo* to lo9 phage/ml were spotted with ApMu immA or ApMu imm434, respec- on the lawns, and the plates were incubated tively, and with a Mu amber mutant. The overnight at 37”. For immune strains a only phage able to grow under these condi- mixed lawn containing lo8 cells of the deletions were Mu which had rescued the am+ tion strain and lo8 cells of the nonlysogenic allele from the ApMu phage. Su- parent strain RS54 was used, and incu(a) Spot tests. Strains MH2501 or MH3078 bation was at 42” to inactivate the heatwere grown to lo9 cells/ml in LBM contain- sensitive immunity of the prophage. Control ing 0.02% maltose. Two-tenths milliliter of plates containing strain MH165, which a ApMu lysate at a concentration of 109- lOlo allows rescue of all the amber mutations, PFU/ml was added to 0.1 ml of cells, incu- and strain RS54 or MH594, which allow bated for 15 min at 37”, and plated in soft rescue of none of the mutations, were done agar on an LB plate. Mu amber mutant ly- in parallel. Experimental plates were scored sates at concentrations of 108-lo9 PFU/ml by comparison to the control plates such were spotted onto the plate and onto a con- that rescue was scored as positive if spots trol plate containing only strain MH2501 on both the experimental plate and the or strain MH3078. Both plates were incu- strain MH165 plate were lysed while those bated overnight at 37”. The presence of on strain RS54 or MH594 were not lysed. confluent lysis or individual plaques in a spot Rescue was scored as negative if spots on on the ApMu-infected plate and none on the the experimental and strain RS54 or MH594 control plate indicated rescue of the urn+ plates were not lysed but those on strain marker from the ApMu. MH165 were lysed. (b) Whole plate assay. For more sensitive (b) Whole plate assay. For final analysis of mutants mapping near a deletion end mapping of mutations near a deletion end point, marker rescue was measured by point, the spot test procedure was modified incubating a mixture of 0.2 ml of ApMu by incubating the cells for 20 min directly lysate, 0.2 ml of amber mutant lysate, and with 0.1 ml of a single Mu amber lysate, 0.1 ml of strain MH2501 (or strain MH3078) plating that mixture in soft agar on an LB for 15 min at 37”, plating in soft agar on plate, and incubating the plate overnight an LB plate, and incubating the plate over- at 37 or 42” as described above. The rescue night at 37”. The resulting plaques were was scored as positive if the experimental counted. Control infections lacked ApMu strain gave more than 10 plaques and more lysate. For amber mutant lysates which than twice as many plaques as the parent gave no plaques on the control plates, marker strain RS54 or MH594. The rescue was rescue was interpreted as positive if the scored as negative if the experimental ApMu-infected plates had more than 10 strain gave the same number of plaques plaques. For amber mutant lysates which as strain RS54 or MH594 and if strain MH165

MU GENES S AND U MAP IN THE G SEGMENT F’lac

I:: Mu

loo Y ::Mu

F’lao

A:: PGp

FIG. 1. Intramolecular recombination between prophages in the lac operon of F’42 plasmid DNA. The structure of the plasmid F’locZ::Mu lacY::Mu is depicted in the upper left corner. The two prophages contained in this plasmid are inserted in opposite orientations with respect to the la& gene. The mean contour length of this DNA is 216 ? 3.8 kb, which is roughly equivalent to the length of parental F’42 DNA at 14’7* 1.5 kb plus two Mu genome lengths. Denaturation of F’lucZ::Mu lacY::Mu DNA, followed by renaturation, results in intrastrand annealing of Mu sequences to generate the structure diagrammed in the lower left corner. Further documentation for these intramolecular reactions will be reported in a separate publication. Genetic recombination between the G regions of the two prophages, as described in the text, results in the structure shown in the upper right corner. Denaturation and reannealing of this plasmid DNA produces the stem and loop configuration illustrated in the lower right corner.

gave at least 500 plaques or 50-fold more than the number on strain RS54 or MH594. This plate assay was 50- to lOO-fold more sensitive than the spot test.

115

measurements, the availability of ApMu phage containing desired regions of the DNA, and the lack of knowledge concerning the specific location of amber mutations within the DNA sequence comprising each gene. Although this same approach could have been applied to determine whether any of the essential genes of Mu were located in the G region, it would have suffered from the same limitations as those above and from the further limitation that it might be difficult to prove that a specific ApMu phage rescuing certain markers did not contain any of the (Ysegment DNA. Therefore, a different approach was chosen in which a strain containing only intact p and G segments of Mu and none of the (Ysegment would be constructed and then tested for Mu gene content by marker rescue analysis. The basic rationale of the strain construction was to obtain a lysogen containing two Mu prophages in opposite orientations with their (Y segments close to each other and then allow for deletion of the (Y segments and intervening DNA by homologous recombination between similarly oriented G regions. A brief description of the strain construction will be presented here; specific technical details are given under Materials and Methods section. Isolation and Characterization

of F’PGP

The first step was to construct an F’lac plasmid containing two Mu prophages inRESULTS serted into it, one in the lacl gene, the Previous attempts to define the locations second in the lacy gene. These prophages of specific Mu genes on the physical map were oriented in opposite directions, as of Mu DNA have relied on correlating (1) shown in Fig. 1, such that both the (Ysegthe results of restriction enzyme and DNA ments were adjacent to the ZacZ gene. Due heteroduplex analyses of XpMu (Magazin to constitutive expression of the 1acZ gene, et ccl., 1977) and Xdpgl Mu (Daniel1 et al., this plasmid conferred a partially Lac+ 19’73b)phage carrying varying amounts of phenotype on the cells. Since the G segments Mu DNA with (2) the Mu gene content of in the Mu prophages could invert, some those phage as determined by marker res- cells in the population contained the G segcue, i.e., the ability of Mu amber mutant ments in the same orientation relative to phage to rescue the corresponding wild-type the plasmid DNA. In these cells recombiallele from the XpMu or Adpgl Mu phage. nation between the homologous G segments The precision of these correlations was would result in deletion of the intervening limited by the size of the restriction frag- DNA, i.e., the (L segments, the lucZ+ gene, ments, the accuracv of the heterodunlex and a total of one G segment. with con-

116

HOWE,SCHUMM,ANDTAYLOR

con&ant production of a Lac- F’ plasmid containing only the /3 and G segments of Mu in a p-G/3 structure. As expected, this recombination event was completely dependent on the homologous recombination functions of the host: No Lac- segregants were observed in the recA strain AT3325 In Ret+ strains such as MH2907 and MH2909, Lac- segregants arose at a high frequency and comprised l-20% of a saturated population grown from a single Lac+ colony. That the Lacphenotype was due to deletion of lad and not to curing of the plasmid was indicated by the continued sensitivity of the Lacstrains to the male-specific phage R17. The Lac- segregants of MH2909 also lacked Mu immunity, as would be expected if the plasmid were deleted for both copies of the (Y segment which contains the Mu c gene necessary for immunity. To confirm that the plasmid was still present in the Lac- strains and that the expected /3-G-j?structure was in the plasmid, plasmid DNA was prepared from a freshly isolated Lac- segregant of the Reef strain MH2909. The supercoiled F’ plasmid DNA was isolated by CsCl-ethidium bromide centrifugation and converted into relaxed circular molecules as described under Materials and Methods. The relaxed plasmid DNA fraction was recovered from a second CsCl-ethidium bromide density gradient and then examined by electron microscopy. A parallel sample of wild-type F’42 Zac+plasmid DNA was similarly prepared for comparison with the Lac- segregant. Samples of both preparations were spread, photographed, and measured to determine the contour lengths of intact native circular molecules. The mean lengths obtained for six molecules of Lac-F’ DNA and for five molecules of F’42 Zac+DNA were 148 f 2.1 and 147 + 1.5 kb, respectively. The close similarity in molecular size of the two plasmids is consistent with the genetic prediction that (Ysegments of both Mu prophages and most of the lac operon are absent in the segregant LacF’ plasmids (Fig. 1). Positive identification of the P-G-P structure in the Lac-F’ plasmid was obtained - -_-. from electron photomicrographs of UNA

preparations which were denatured and self-annealed prior to spreading. As illustrated in Fig. 1, the nucleotide sequences of the two p segments which flank G are complementary to each other in the same DNA strand and should therefore reanneal to form a characteristic stem and loop structure. Several examples of this p-G-/3 stem and loop were seen in the self-annealed Lac-F’ DNA (Figs. 2B and C); whereas no such structures could be found in similar preparations of the parental F’42 la& plasmid DNA. The contour lengths of stem and loop structures in plasmid DNA and of /? and G segments in Mu DNA were measured from mixtures containing self-annealed plasmid DNA and self-annealed Mu DNA extracted from mature virions. (Fig. 2A shows an example of a self-annealed Mu DNA molecule that contains a single-stranded G bubble and single-stranded variable ends connected by a duplex p segment). The data recorded in Table 2 show first that the duplex stems in Lac-F’ plasmid DNA are equal in length to the p segment of Mu and, second, that the terminal loops in the plasmid molecules are also equal, within experimental error, to the molecular length of the G region of Mu DNA. The close agreement between these parallel measurements strongly supports the conclusion that Lacsegregants of strain MH2909 harbor F’ plasmids which contain the G and /3segments of phage Mu. Strains MH2908 and MH2911 containing the F’ p-G-p plasmids were then tested for the presence of Mu genes by assaying the ability of Mu phage containing amber mutations to rescue the wild-type allele of the amber mutation from the plasmidcontaining strains. This was done first using spot tests and then using a more sensitive and quantitative whole plate assay. Strains MH2908 and MH2911 allowed rescue of all 10 U mutations and all 26s mutations (shown in Fig. 3) which were tested. They did not allow rescue of any of the 13 R mutations proximal to the S and U genes. The strains were also tested for rescue of 2 or 3 mutations in each of the other essential genes located to the left of gene R , i.e., A through Q, T, V, W, Y, and lys. As expected, strain

117

MU GENES S AND U MAP IN THE G SEGMENT

FIG. 2. Electron photomicrographs of phage Mu DNA and F’Alac:$-G-P DNA. (A) Right end portion of a Mu homoduplex molecule showing a G inversion bubble at the top, a duplex p segment, and single-stranded variable ends at the bottom. (B, Cl Stem and loop structures in self-annealed F’AZac:$-G/3 DNA showing duplex p segments and single-stranded terminal G loops. All three micrographs are shown at the same magnification. Bar = 1.0 kb. Junctions between double- and single-stranded portions of the molecule are indicated by short lines.

MH2911 did not allow rescue of any of these mutations, while strain MH2908 allowed rescue for mutations in only genesA through K which were present in the chromosome of this partial prophage deletion strain. Therefore, these results demonstrate that the 5’ and U mutations are located in either the G or /3 segments of Mu and confirm that

mutations in the remaining essential genes are located in the cr segment. The identical behavior observed in strains with and without an immune chromosomal partial prophage deletion demonstrates that the presence or absence of Mu immunity during plasmid transfer and G-G recombination had no effect on the marker rescue results.

TABLE 2 MOLECULAR LENGTH OF STRUCTURES OBSERVED IN SELF-ANNEALED

Lac- F’ plasmid DNA

PLASMID AND PHAGE DNA”

Phage Mu DNA

Double-stranded stems

Single-stranded loops

Double-stranded p segments

Single-stranded G bubbles

1.6 2 0.2 (15)

3.0 -t 0.3 (15)

1.7 2 0.1 (10)

3.1 + 0.1 (16)

a Mean contour lengths ? SE are given in kilobases or kilobase pairs for single- and double-stranded DNA segments, respectively. The total number of structures measured is indicated in parentheses.

118

HOWE, SCHUMM, AND TAYLOR

R

---

s I6W

U

P

IOU 1517 IO50 I520 7135 #I2 1001 lwo 7152 1004 Iwo low 1646 1910 7161 I063 I637 7046 1204 IO66 IO23 72l2 7ou we 1010 7662 1066 7044 lo49 law 7555 7ow Is66 7011 706l 7ow 7157 I609 7057 75.55 7505~725ll7036( 7uo~7045(70l2 756417220 b

722Oj7504(7OE

(70431

7140~7056(726l

--_--_--__ --------_ --------_ -------__

Xp.MU WA161454 AppMu220 MHIIO Ml+14 hpMu6W.

--_--_--_ --_----_ --------_ --------------_

hiHI Mlill8, b+4u 600,6l7,624, \pMu134A22i454 XpM”219 hpMu295 ApMu215,401,409,416

617,624,

MH29ll

J751Xj

---_

MH 291 I

FIG. 3. Deletion mapping of the R S U region. The four-digit numbers across the top of the map represent specific amber mutations in genes R, S, and U. The bars represent the extent of Mu markers present in the ApMu or partial prophage deletion strains indicated on the right. The arrows indicate the relative orientations of the map of S and U markers with respect to R markers and are inverted with respect to each other for the two sets of strains.

Deletion Mapping of S and U Mutations

that the deletions arose by loss of a single contiguous segment of DNA. The map demonstrates, as predicted, that the deletion strains derived both from Mu prophages

To distinguish whether genes S and U are located in the invertible G segment or in the noninvertible /3 segment we assayed for inversion of S and U markers using the TABLE 3 following rationale. Mu prophages and ApMu phage with intact G and /3 segments invert WHOLE PLATE MARKER RESCUE RESULTS” their G segments normally; however, when No. am+ plaques produced one end of the G segment is removed by by infection with deletion, inversion of G no longer occurs Bacterial or phage Mu S am phage (Allet and Bukhari, 1975; Chow et al., 1977). strains being tested Since such deletions might occur when G for rescue S7306 s7140 s7008 is in either orientation, a deletion should freeze the G segment in either the G(+) A. MHZ911 (%-/3-G/3) >lO,OOO 4,000 >lO,OOO or the G(-) orientation. If genes S and U 0 MH594 (Su-) 0 20 were located in the G segment, deletion 19 >lO,OOO >10,006 mapping of S and U mutations should re- B. MH119 (Su-PA) 39 15 40 RS54 (Su-) sult in two different orders with respect to gene R: one order for the G(+) orientation 22 >10,006 >lO,OOO C. hpMu 220 10,000 10,000 ApMu 219 58 and the opposite order for the G( -) orienta1,000 7 93 ApMu 416 tion. Confirmation of this prediction was ApMu 82-111 17 16 62 obtained by mapping the S and U mutations by marker rescue from partial prophage (1Marker rescue was performed as described under deletion strains and from hpMu transducing Materials and Methods. Numbers represent the numphage which carry portions of the S U re- ber of am+ plaques produced per plate after infection gion of the Mu genome. An example of this with lysates of the designated S amber mutant phage. marker rescue data is given in Table 3. The Sections B and C were performed with the same S am resulting map of this region, presented in lysates on the same day. Plaques produced with negaFig. 3, was derived by ordering the muta- tive control strains MH594, RS54, and ApMu 82-111are tions by making the standard assumption due to am+ revertants present in the S am lysates.

119

MU GENES S AND U MAP IN THE G SEGMENT

and from ApMu phage fall into two classes: one which gives a map order R S U and a second which gives the inverted map order R U S. Therefore, these mapping results are consistent with the interpretation that the S and U genes are located in the G segment and not the p segment of Mu. Heteroduplex

Mapping

of Gene U

Further confirmation of the location of the U gene within G was obtained by physical anal-

ysis of the deletion strain h134A22imm434, in which one end of the deletion is located within the U gene and can, therefore, be used to identify the position of the U gene in the physical structure of the DNA. The analysis was performed by observing DNA heteroduplexes between h4M133 and X134A22imm434. As shown in Fig. 4, these two phage are homologous except for the host sequences in the Mu variable ends, the different immunity regions, the nin5 deletion, the QSR-80 substitution, and the deletion in A134A22imm434 which removes Mu genes Q through R and part of U but leaves theS gene intact (see Fig. 3). Heteroduplexes of these phage are particularly useful for this analysis because they contain two characteristic substitution bubbles which flank and therefore serve to locate the Mu DNA segment; one is due to the different host DNA sequences in the Mu variable end DNA while the other is due to the nonhomologous imm434 and immAAKH54 immunity regions. Many different heteroduplex structures in the Mu DNA region could be predicted depending on whether the gene order was R S U or R U S, whether the S and/or U genes were in (Y, G, or p, and whether the G regions in the two annealed DNA strands were in the same or VE

ul

opposite orientations. The two types of heteroduplex structures observed allowed a unique resolution of these possibilities. First, let us consider the question of gene order. Since A22 removes genes Q through R and part of gene U without removing gene S, heteroduplexes of DNA strands containing G segments in the same orientation should show a single deletion loop if the gene order is R U S and two deletion loops if the order is R S U. No structures with two deletion loops were found but structures with a single deletion loop, such as that shown in Fig. 5a, were observed. This indicates that the deletion arose by removal of a single contiguous DNA segment with the genes in the order R U S. The length of the duplex DNA segment between the deletion loop and the variable end substitution bubble was 3 kb (Fig. 5b), a length equivalent to fi plus approximately 1.3 kb of the G segment, thus indicating that the deletion ends within the G segment. Heteroduplex structures observed for molecules containing G segments in opposite orientations confirmed that the deletion ends within G. Let us consider the heteroduplex structures expected if U were located in CY,in G, or in p. If gene U were located in the (Y segment, then deletion of part of U would not remove G; normal G bubbles in addition to the deletion loop should be visible in heteroduplexes of DNA strands containing G in opposite orientations. If the region between G and the deletion were too small to hybridize stably, the structures might run together to form a substitution bubble, but in this case both strands of the bubble would be longer than the normal G length. If gene U were located within G, normal G bubbles should not be

AZ2

m

)---(

l-H-1

X~MWJ

FIG. 4. Physical maps of h134A22imm434 and X4M133 drawn approximately to scale. The thin lines represent ADNA while the thick lines represent Mu DNA. Boxes indicate substitutions; parentheses indicate deletions. The leftmost box represents the small amount of loc5 DNA remaining after cloning. The box adjoining the lac5 DNA represents the cloned variable end DNA (VE) from Mu which differs in sequence and length in the two phage. The BIO substitution is the small amount of bio256 DNA remaining after cloning.

120

HOWE, SCHUMM, AND TAYLOR

b

FIG. 5. DNA heteroduplex structures observed in mixtures of DNA from h134A22imm434 and h4M133. All electron photomicrographs are shown at the same magnification. Circular ColEl DNA was included as an internal length standard of 6.75 kb. (a) Electron photomicrograph of relevant portion of heteroduplex between DNA strands from h134A22imm434 and h4M133 whose G segments are in the same orientation. (b) Schematic diagram of heteroduplex structure shown in (a) indicating the average lengths of the relevant DNA segments in kb as determined from measurements of four molecules. The 6.9* value represents the measurement from a single molecule. VE and Pa4 denote the substitution bubble structures arising due to different variable end sequences and different immunity region sequences, respectively, in the two phage. (c) Electron photomicrograph of relevant portion of heteroduplex between DNA strands from A134A22imm434 and A4M133 whose G segments are in opposite orientations. (d) Schematic diagram of heteroduplex structure shown in (c) indicating the average lengths of the DNA segments in kb as determined from measurements of at least seven molecules. (e) Electron photomicrograph of G bubble formed by annealing of DNA strands from A4M133 phage whose G segments are in opposite orientations. It is included here to allow comparison of a normal G segment length (2.9 2 0.5 kb) with that observed in (c). Independent measurements of Mu heteroduplex structures indicate that the p region is 1.7 ? 0.1 kb in length.

present, but rather a substitution bubble containing one strand shorter than G should be found. If gene U were located completely within ,B,the G region would be completely deleted resulting in a heteroduplex structure containing a single deletion loop located at a distance less than /3 length from the variable end nonhomology. The heteroduplexes observed were of the type shown in Fig. 5~. They contain a single substitution bubble with one side whose length is shorter

than G by approximately 1.7 kb (Fig. 5d). Thus, these structures demonstrate that the deletion ending in gene U ends physically within the G segment DNA and proves that at least part of the U gene is located within the G region. DISCUSSION

A bacterial strain containing only the /3 and G segments of Mu was constructed

MUGENESSANDUMAPINTHEGSEGMENT

and found to contain the wild-type DNA sequences corresponding to the S and U genes of Mu. The more precise location of those genes to the invertible G segment was demonstrated genetically by the discovery of two map orders of U and S mutations with respect to gene R, i.e., R U S and R S U, and physically by demonstration that deletions which end within the U gene end within the G segment DNA. In this work no heteroduplexing was done to confirm that gene S is located in the G segment; however the results of the previous analysis of ApMu 220 performed by Magazin et al. (1977) support this conclusion. As shown in Fig. 3, ApMu 220 arose by deletion of Mu genes A through R and part of gene S but not gene U. Restriction analysis of ApMu 220 DNA showed that Hind11 restriction nuclease fragments 7 and 8 from the Mu G region were still present in this phage. Heteroduplexes of XpMu 220 DNA with Mu DNA had a single duplex region (no deletion loops) with a length of 3.7 + 0.4 kb which would be equivalent to the 1.7 kb of p plus 2.0 kb of G. The simplest interpretation of these data is that the ApMu 220 deletion end point located within gene S ends within G segment DNA and, therefore, that gene S is located within the G segment. The conclusion that genes S and U are located within G conflicts with the conclusion of Daniel1 et al. (1973a,b) that the S gene is located within the (Ysegment DNA to the left of G. That conclusion was based on marker rescue and electron microscopic heteroduplex analysis of Adpgl Mu phages carrying varying amounts of the S G /3 end of a Mu prophage. The critical observation was that one phage (Adpgl Mu 6b) contained an intact G region whose inversion was detectable by observation of heteroduplexes, yet this phage did not allow rescue of a mutation in the S gene. There are two possible explanations for this conflict with the results reported here. The first is that the entire S gene is not located within the G segment and that the particular S mutation tested by Daniel1 et al. (1973a,b) is located outside of G in the (Ysegment. While this possibility seems unlikely since all the

121

other S and U mutations are located within G, it cannot be ruled out on the basis of existing data. The second is that the spot test procedure used by Daniell et al. (1973a,b) for rescue of the S mutation may not have been sensitive enough to detect the presence of the marker. Since this S amber mutant strain has been lost, we have been unable to determine which of these explanations is correct. Kamp et al. (1978) have found that phage containing G in one orientation, the G(+) orientation, are viable plaque-forming phage; while phage containing G in the opposite orientation, the G(-) orientation, cannot form plaques. The discovery that mutations in essential genes S and U are located within the G segment suggests a simple explanation for the inviability of phage containing G in the G(-) orientation-namely, that genes S and/or U are not properly expressed when the G segment is in that orientation. This might result if one of the genes spans the CYGboundary or if the promoter needed for transcription of one or both genes is located outside the G segment. (The location of the promoter(s) is not yet known.) In either case inversion of G to the G(-) orientation would result in the inability of the normal gene products to be made and would therefore render the phage defective. A critical test of the validity of this explanation will be a comparison of the properties of inviable G( -) phage and S and U defective phage. As yet, very little is known about these phage. Recent work of Bukhari and Ambrosio (1978) indicates that G(-) phage are defective in adsorption or penetration of the phage DNA into the cell. While similar studies for S and U defective phage have not yet been completed, there are several findings which suggest that at least the S gene product may be involved in phage adsorption or penetration. (1) When lysates made by induction of Mu S- lysogens are examined with the electron microscope, normal numbers of phage particles are observed (Howe and Pate, unpublished observations). These particles differ slightly from particles observed in Mu S+ lysates but further experiments are needed to define the differences. (2) Serum blocking experi-

122

HOWE, SCHUMM, AND TAYLOR

ments indicate that it is the interaction of antiserum with the S gene product that inactivates the plaque-forming ability of the wild-type phage particle (Howe, unpublished observations). Since in the case of phage A (Mount et al., 1968; Buchwald and Siminovitch, 1969) and T4 (Edgar and Lielausis, 1965) neutralizing antibody is directed against the tail fibers, this result suggests that the S gene product may be a tail fiber component. (3) Toussaint et al. (1978) have found that Mu S- and Mu U- phage can recombine with phage Pl in a region including the invertible C DNA segment of Pl which is homologous to the Mu G segment. The Mu S+ and Mu lJ+ recombinants obtained fall into two classes, those which retain the host range of Mu, and those which acquire the broader host range of Pl. Some of these hybrid phage are less sensitive to inactivation by antiserum directed against Mu, but the rate of inactivation does not correlate with the presence of Pl host range properties. Thus, the G region of Mu and C region of Pl appear to encode products involved in host range, adsorption, and penetration; but the precise contribution of Mu S and U gene products to these processes is not yet clear. Further experiments to determine the functions of theS and U gene products are in progress. The genetic mapping data presented in Fig. 3 do not demonstrate whether the gene order present in viable phage particles is R S U orR U S because there was no selection for functional S and U gene expression during the isolation of the deleted strains. Attempts to determine the gene order in viable phage by three-factor crosses of Mu c+ Sam with Mu cts Uam and Mu cts Sam with Mu c+ Uam were unsuccessful since the S+ U+ recombinants contained equal proportions of c+ and cts markers (Howe, unpublished observations). However, a strong argument can be made from the heteroduplex data that the orientation in viable phage is R S U. The orientation of genes in h134A22imm434 was shown by heteroduplexing to be R U S. In this phage, in which part of gene U is deleted, 1.2 + 0.2 kb of the G segment is retained and must encode the remainder of gene U and gene S.

Chow et al. (1977) have shown that the rightmost 1.3 kb of the G segment in the G(+) viable orientation is not essential for phage development. Since this nonessential segment is too large to fit in the G DNA segment remaining in h134A22imm434 it must be located in the G region deleted in that phage. This means that the order in A134 imm434 just prior to deletion was R-nonessential-U-S. Since in the viable phage the nonessential DNA is at the rightmost end of G (Chow et al., 1977), the order in the viable phage must be the inverse of that in A134622imm434 or R-S-U-non-essential. ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by NSF Grant PCM75-02465 A01 and NIH Grant AI 12731 to M.M.H., NIH Predoctoral Training Grant GM 07133 to J. W.S., and NIH Grants AI 14025 and General Research Support RR-05357 to A.L.T. The authors are grateful to D. W. Schultz, R. Heinzen, V. A. Chapman, and S. S. DeLong for technical assistance. REFERENCES ABELSON, J., BORAM, W., BUKHARI, A. I., FAELEN, M., HOWE, M., METLAY, M., TAYLOR, A. L., TOUSSAINT, A., VAN DE PUTTE, P., WESTMAAS, G. C., and WIJFFELMAN, C. A. (1973). Summary of the genetic mapping of prophage Mu. Virology 54, 90-92. ALLET, B., and BUKHARI, A. I. (1975). Analysis of bacteriophage Mu and A-Mu hybrid DNAs by specific endonucleases. J. Mol. Biol. 92, 529-540. ALPER, M. D., and AMES, B. N. (1975). Positive selection of mutants with deletions of the gal-chl region of theSalmonella chromosome as a screening procedure for mutagens that cause deletions. J. Bacterial. 121, 259-266. APPLEYARD, R. K., MCGREGOR,J. F., and BAIRD, K. M. (1956). Mutation to extended host range and the occurrence of phenotypic mixing in the coliphage lambda. Virology 2, 565-574. BLA~NER, F. R., FIANDT, M., HAAS, K. K., TWOOSE, P. A., and SZYBALSKI, W. (1974). Deletions and insertions in the immunity region of coliphage A: Revised measurements of the promoter-startpoint distance. Virology 62, 456-471. BORAM, W., and ABELSON, J. (1973). Bacteriophage Mu integration: On the orientation of the prophage. Virology 54, 102-108.

MU GENES S AND U MAP IN THE G SEGMENT BUCHWALD, M., and SIMINOVITCH, L. (1969). Pro-

duction of serum-blocking material by mutants of the left arm of the A chromosome. Virology 38, l-7. BUKKARI, A. I., and AMBROSIO, L. (1978). The invertible segment of bacteriophage Mu DNA determines the adsorption properties of Mu particles.

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KAMP, D., KAHMANN, R., ZIPSER, D., BROKER,T. R., and CHOW, L. T. (1978). Inversion of the G DNA segment of phage Mu controls phage infectivity. Nature (London) 271, 577-580. KAYAJANIAN, G. (1970). Deletion mapping of the Nature (London) 271, 575-577. CIII-CII region of bacteriophage lambda. Virology BUKHARI, A. I., and ALLET, B. (1975). Plaque-forming 41, 170-174. A-Mu hybrids. Virology 63, 30-39. LISLE, J. W., and GOITESMAN, M. (1971). Defective CHOW,L. T., and BUKHARI, A. I. (1976).The invertible lambda particles whose DNA carries only a single DNA segments of coliphages Mu and Pl are identicohesive end. In “The Bacteriophage Lambda” cal. Virology 75, 242-248. (A. D. Hershey, ed.), pp. 371-394. Cold Spring CHOW, L. T., KAHMANN, R., and KAMP, D. (1977). Harbor Laboratory, Cold Spring Harbor, N. Y. Electron microscopic characterization of DNAs MAGAZIN, M., HOWE, M., and ALLET, B. (1977). of non-defective deletion mutants of bacteriophage Partial correlation of the genetic and physical maps Mu. J. Mol. Biol. 113, 591-609. of bacteriophage Mu. Virology 77, 677-688. COURT, D., and SATO, K. (1969). Studies of novel transducing variants of lambda. Dispensability of MOORE, D. D., SCHUMM,J. W., HOWE, M. M., and genes N and Q. Virology 39,348-352. BLATTNER, F. R. (1977). Insertion of Mu DNA DANIELL, E., ABELSON,J., KIM, J. S., and DAVIDSON, fragmentsinto phage h invitro. In “DNA Insertions, N. (1973a). Heteroduplex structures of bacterioPlasmids and Episomes” (A. I. Bukhari, J. Shapiro, phage Mu DNA. Virology 51, 237-239. and S. Adhya, eds.), pp. 567-574. Cold Spring DANIELL, E., BORAM, W., and ABELSON, J. (1973b). Harbor Laboratory, Cold Spring Harbor, N. Y. Genetic mapping of the inversion loop in bacterio- MOUNT, D. W. A., HARRIS, A. W., FUERST, C. R., phage Mu DNA. Proc. Nat. Acad. Sci. USA 70, and SIMINOVITCH,L. (1968). Mutations in bacterio2153-2156. phage lambda affecting particle morphogenesis. DAVIS, R. W., SIMON, M., and DAVIDSON, N. (1971). Virology 35, 134-149. Electron microscope heteroduplex methods for OZEKI, H. (1959). Chromosome fragments participating mapping regions of base sequence homology in in transduction in Salmonella typhimurium. Genetnucleic acids. In “Methods in Enzymology,” Vol. 21: ics 44, 457-470. “Nucleic Acids,” Part D (L. Grossman and K. Mol- PARKINSON, J., and HUSKEY, R. (1971). Deletion dave, eds.), pp. 413-428. Academic Press, New York. mutants of bacteriophage lambda. I. Isolation and EDGAR, R. S., and LIELAUSIS, I. (1965). Serological initial characterization. J. Mol. Biol. 56, 369-384. studies with mutants of phage T4D defective in PETTERSSON, U., MULDER, C., DELIUS, H., and genes determining tail fiber structure. Genetics SHARP, P. A. (1973). Cleavage of adenovirus type 2 52, 1187-1200. DNA into six unique fragments by endonuclease FRANKLIN, N., DOVE, W., and YANOFSKY, C. (1965). R.Rl. Proc. Nat. Acad. Sci. USA 70, 200-204. The linear insertion of a prophage into the chromoSANGER, F., AIR, G. M., BARRELL, B. G., BROWN, some of E. coli shown by deletion mapping. Biochem. N. L., COULSON, A. R., FIDDES, J. C., HUTCHISON Biophys. Res. Commun. 18, 910-923. III, C. A., SLOCOMBE, P. M., and SMITH, M. (1977). GEORGOPOUL~S,C. (1971). A bacterial mutation Nucleotide sequence of bacteriophage $X174 DNA. affecting N function. In “The Bacteriophage Lambda”

(A. D. Hershey, ed.), pp. 639-646. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HIROTA, Y. (1960). The effect of acridine dyes on mating type factors in Escherichia coli. Proc. Nat. Acad. Sci. USA 46, 57-64. HOWE, M. M. (1973a). Prophage deletion mapping of bacteriophage Mu-l. Virology 54, 93-101. HOWE, M. M. (197313). Transduction by bacteriophage Mu. Virology 55, 103-117. HOWE, M. M. (1978). Invertible DNA in phage Mu. Nature

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HOWE, M. M., and BADE, E. G. (1975). Molecular biology of bacteriophage Mu. Science 190, 624-632. HSU, M. T., and DAVIDSON, N. (1974). Electron

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