Mutations affecting genetic recombination in bacteriophage T4D

VIROLOGY

88,62-70

Mutations

(1978)

Affecting

Genetic Recombination II. Genetic

RICHARD

in Bacteriophage

T4D

Properties

P. CUNNINGHAM’

AND

HILLARD

BERGER2

Department of Biology, The Johns Hopkins University, Baltimore, Maryland

21218

Accepted February IS,1978 Experiments were performed to assess the effects of mutations in genes 4647,58,59, W, x, and y on the correlation of genetic exchanges, on the production of heterozygotes, and on the extent of high negative interference in bacteriophage T4D. Mutations in these genes of T4 which alter genetic recombination also alter the relative ratio of double to single crossover events and the frequency of heterozygotes for a point mutation in the rIIA cistron. In contrast, the frequency of heterozygotes for a small deletion in the rIIA cistron is essentially constant in wild-type and mutant infections. The reduction in the index of interference caused by substituting a deletion for a point mutation as the central marker in a three-factor cross is eliminated in 46-47-, 59-, W-, and y- infections. INTRODUCTION

High negative interference (HNI) in bacteriophage was noticed by Streisinger and Franklin (1956) in T2 phage and more completely characterized by Chase and Doermann (1958) in T4 phage. HNI is the term for multiple exchanges between closely linked markers that are much more frequent that expected for statistically independent events occuring in two adjacent intervals. Edgar (1961) suggested that HNI and heterozygosis were related phenomena, following Levinthal’s earlier observation of a relationship between heterozygosis and recombination. Two different types of T4 heterozygotes are found among the progeny of mixedly infected cells. Terminal redundancy heterozygotes arise as a consequence of two different alleles of a gene existing on either end of a matured genome. Genomes slightly longer than unit length are a result of the headful packaging mechanism of T4. Internal heterozygotes are the result of two alleles being present in a heteroduplex region of DNA. The heterozygous region, a molecular splice in which DNA strands ’ Present address: Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. ’ Deceased October 28,1975. 0042~6622/78/O&31-0062$02.00/O Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

62

from two genetically distinct molecules are paired, can be flanked by DNA carrying genetic markers which are either in recombinant configuration or in the configuration of one of the parental types, giving rise to recombinant and insertion heterozygotes, respectively. The formation and segregation of insertion heterozygotes can account for HNI in three-factor crosses (Honda and Uchida, 1969). To explain the correlation of a third crossover with the first two in a four-factor cross (Chase and Doermann, 1958), however, one must postulate the reiteration of exchange events. Thus, HNI can be attributed to reiterated recombination events which produce insertion heterozygotes (for reviews, see Mosig, 1970; Broker and Doermann, 1975). Mutations in several different genes have been shown to influence recombination frequencies in phage T4 (Harm, 1964; Bernstein, 1968; Berger et al., 1969; Boyle and Symonds, 1969; Hamlett and Berger, 1975; Shah, 1976). We have previously examined strains mutant in genes 46-47, 58, 59, w, x, and y in an attempt to define pathways of genetic recombination (Cunningham and Berger, 1977). In this paper we have examined the influence of mutations in these genes on the correlation of genetic exchanges in three-factor crosses and on the

RECOMBINATION

IN

frequency of heterozygote formation. Our results allow several generalizations: (1) As recombination frequency between markers increases, the apparent ratio of double crossovers to single crossovers increases while HNI decreases; (2) the frequency of heterozygotes for point mutations increases as recombination frequency increases; and (3) the frequency of heterozygotes for a point mutation is variable while the frequency of heterozygotes for a deletion mutation is essentially constant. These results are consistent with a model for T4 recombination which postulates that singlestranded regions of DNA are necessary for genetic recombination and that enhancement of recombination in short intervals is a consequence of reiterated recombination events, including the production of insertion heterozygotes (Broker and Doermann, 1975). MATERIALS

AND

METHODS

Bacterial strains. The Escherichia coli strains B, B40SuII1, and B40SuIII(h) have been described (Cunningham and Berger, 1977). Bacteriophage. T4D mutants are listed in Table 1. The construction of multiply mutant strains has been described (Cunningham and Berger, 1977). Growth of bacteria and phage. P-broth, H-broth (HB), top-layer agar, and bottomlayer agar were prepared according to Chase and Doermann (1958). Preparation of bacterial cultures and most phage stocks has been described (Krisch et al., 1972). Stocks containing the mutations m22 or xm were prepared as plate lysates (Hamlett and Berger, 1975) using E. coli B40SuIII. Cross procedures. The procedure for phage infection was that of Krisch et al. (1972) for genetic experiments. The host was E. coli B, and all crosses were performed at 30”. The procedure was slightly modified for crosses involving mutants carrying the mutations amN130 and amA which produce low burst sizes. Instead of dilution away from KCN, the cultures were centrifuged at 4’ and resuspended in cold HB. These cultures were diluted lOO-fold into warm HB to start the experiment. We found no concentration effect (Krisch et al., 1972) at the cell densities

BACTERIOPHAGE

T4D TABLE T4D

1

STRAINS Source

r70

A. A. A. A. A. W.

H.Doermann H. Doermann H. Doermann H. Doermann H. Doermann B. Wood

n-20 rdb52 amNl30

: rIIA rIIA rIIA 46

amA

47

W. B. Wood

amE

58 (61)”

W. B. Wood

amC5b

59

D. B. Shah

m22

N. V. Hamlett

Xm

N. V. HamIett

Ym

N. V. Hamiett

1

Reference

Berger Berger Berger Berger Epstein (1963) Epstein (1963) Yegian

(1965) (1965) (1965) (1965)

et al. et al.

et al. (1971) Wu et al. (1972) Hamlett Berger (1975) Hamlett Berger (1975) HamIett Berger (1975)

and

and

and

a Mutations originally assigned to genes 58 and 61 have been found to be in one complement&ion group (Yegim et al., 1971). * This strain contains an additional mutation (Cunningham and Berger, 1977).

which resulted from this protocol. When HNI was measured in prematurely lysed cells, the cultures were centrifuged and resuspended in cold HB. The time when the resuspended cultures were placed in a waterbath at 30’ marked the beginning of the experiment. Premature lysis was achieved by pipetting 0.1 ml of the infected culture into chloroform-saturated P-broth and mixing vigorously. Under these conditions, there was one plaque-forming unit per infected bacterium at 21 min after the experiment began. We observed recombination frequencies from these crosses that were higher than those obtained using standard cross procedures. This effect could be caused either by the high cell densities (Krisch et al., 1972) or residual cyanide (Chase and Doermann, 1958). The 5-min delay in the appearance of intracellular phage suggests that residual cyanide is at least partially responsible for the increase. Cross lysates were plated on E. coli B40SuIII to score for total progeny. E. coli

64

CUNNINGHAM

AND

B40SuIII(h), which supports the growth of r-II+ phage but not rI1 mutants, was used as the selective indicator. The efficiency of plating of T4D+ was the same on the two indicator strains. When the frequencies of heterozygotes were determined, the cross lysates were plated on E. coli B40SuII1, the plates were incubated at 34” for 16 hr, and mottled plaques were scored by inspection. Recombination frequencies were calculated by multiplying by 2 the percentage of wild-type recombinants. Burst sizes were calculated by dividing the total progeny by the number of infected bacteria. The index of interference, i, was calculated using the equation i = R12/(R1 x Rz) where RI and Rz are the recombination frequencies in two adjacent intervals and RIZ is the frequency of simultaneous exchanges in both inter-

BERGER

Boyle and Symonds, 1969; Hamlett and Berger, 1975; Shah, 1976; Cunningham and Berger, 1977). The index of interference, i, also varied in the different mutant infections and was inversely related to the sum of the recombination frequencies between markers (Fig. 1). A similar inverse relationship was found by Chase and Doermann (1958) who examined HNI for a large number of markers in the rI1 region of T4. In their crosses the variation in the frequency of recombination between markers was effected physically by using markers with different map positions. Our results serve to illustrate a problem inherent in the formulation of the index of interference, especially as it is applied to

V&L RESULTS

Correlation of genetic exchanges in mutant infections. We have examined the effect of six mutant genes on the frequency of correlated exchanges in two adjacent intervals in the rIIA cistron of phage T4D (Table 2). The same three rIIA mutations were used in the various two- and threefactor crosses performed in the different genetic backgrounds, the three-factor cross was do-r-20 x r59. The parental strains carried a mutation in gene 58,59, w, x, or y or mutations in genes 46-47. As previously reported, recombination frequencies in the mutant infections were altered (Harm, 1964; Bernstein, 1968; Berger et al., 1969;

FIG. 1. The relationship of the index of interference, i, to the sum of the component recombination frequencies in phage crosses. The crosses r70 x r59, r59 x 12-20, and r70-r2-20 x r59 were performed with strains carrying mutations which alter genetic recombination: T4D+, (0); 46--4T, (0); .W, (0); 59-, (H); w-v (A); x-, (A); and Y-, 0’).

TABLE HNI Mutant gene(s)

T4D+ 4647 58 59 W

x Y

IN THREE-FACTOR RI W

0.999 0.130 8.75 0.177 0.427 0.206 0.132

CROSSES

WITH STRAINS

Rz CW’

Rn CW*

1.04 0.159 7.73 0.219 0.494 0.240 0.132

0.228 0.0132 3.35 0.0277 0.0626 0.0275 0.0162

2

OF T4 ALTERED IN RECOMBINATION i’ Correlation of Burst exchanges’s d

22 f 64 f 5fl 71 f 30 f 56 f 94 zt

6 18 25 2 27 27

a RI and R2 are twice the frequencies of the r’ recombinanta from X i-2-20, respectively. b RI2 is twice the frequency of r+ recombinanta from the three-factor c Unbiased standard deviation of the mean indicated. d Correlation of exchanges is defined CE = (RI* - RIRz)/RI.

0.22 f 0.10 f 0.31 f 0.15 f 0.14 f 0.13 f 0.12 f

0.05 0.01 0.04 0.01 0.06 0.03 0.01

the two-factor cross r70-r2-20

PROFICIENCY size

272 4 125 58 94 35 61 crosses

Number experiments

of

15 2 2 2 2 4 2 r70 x r59 and r59

X r59.

RECOMBINATION

IN

BACTERIOPHAGE

phage genetics. The index of interference is a function which unrealistically overestimates the amount of multiple crossover events with respect to statistically expected recombination in unselected genome intervals within the population. The division of a single term by the product of two numbers, all three of which can be varied by experimental manipulation, leads to large variations in the index of interference. Thus, i can vary 20-fold even though the same selected markers were used in the crosses summarized in Tables 2 and 4. AnTABLE

3

HNI IN THREE-FACTOR CROSSIB WHEN THE CENTRAL MARKER IS EITHER A POINT MUTATION OR A DELETION MUTATIONS Mutant w&d

ipt”

T4D+ 46-47 58 59

22 rfr 6d 78 4.1 58 30 78 117

W

x Y

Burst

idel’

8*2’ 76 2.2 57 27 29 105

size

5 168 79 85 37 78

“The three-factor crosses performed were 170~2-20 x r-59 and r70-12-20 x rdb52. The rdb52 mutation is a short rIIA deletion, and r59 is a point mutation covered by rdb52. The flanking markers sre both rIIA point mutations spanning a distance of approximately 1 map unit. b The index of interference for crosses with r59. ’ The index of interference for crosses with rdb52. dAverage of 15 experiments. Unbiased standard deviation of the mean indicated. ’ Average of eight experiments. Unbiased standard deviation of the mean indicated. TABLE THE EFFECT Cross

r70-r2-20

X r59

r70-r2-20

x rdb52

OF PREMATURE Burst

size

1.9 2.2 82 167 2.9 85 154

LYSIS

65

T4D

other aspect of the difficulty in using the index of interference in T4 genetics is the questionable assumption that each recombination event is independent and that all intervals in all molecules have an equal probability of experiencing a crossover. To circumvent this problem we have formulated an expression for the correlation of exchanges (CE) in a phage cross: CE = (Rlz - R&)/RI. The measure of correlated exchanges expresses the probability that a second, nonrandom genetic exchange will occur once those molecules which have recombined in interval RI have been selected for analysis. Thus, CE deals with subpopulation genetics and, as such, should not be influenced by factors affecting the recombination frequency of the whole population. The relationship of correlated exchanges to recombination frequency (Table 2) and to the frequency of heterozygotes (Table 5) is direct in mutant infections and is insensitive to factors affecting the recombination frequency of the whole population, such as premature lysis (Table 4). As can be seen in Table 2, CE varied threefold in mutant infections, suggesting that T4 phage recombination events occurred with different consequences within the regions studied when specific gene products were inactivated by mutation. HNI in mutant infections when the central marker is a deletion. It has been reported that HNI is reduced when the central marker in a three-factor cross is a deletion, and when wild-type recombinants are scored (Berger and Warren, 1969). When triple mutant progeny are scored, however, 4 ON HIGH

NEGATIVE

INTERFERENCE

RI CW”

Rz W’

Rn (W*

i

0.30 0.38 0.92 1.87 0.16 0.34 0.60

0.32 0.38 1.01 1.87 0.24 0.44 0.84

0.058 0.076 0.16 0.37 0.008 0.010 0.024

60 53 17 11 21 7 5

a RI and Rz are twice the frequencies of the r+ recombinants and n-lb52 x n-20. b RI2 is twice the frequency of the r’ recombinants r70-r2-20 ’ Correlation of exchanges is defined CE = ( RI2 - RIRP)/RI.

from

r70 X r59 and r59 x r2-20

x r59 or r70-r2-20

x rdb52.

Correlation of exchanges’ 0.19 0.20 0.16 0.18 or r70 x rdb52

66

CUNNINGHAM

there is no difference in the index of interference with either a point or a deletion mutation as the central marker (Doermann and Parma, 1967). The difference observed when wild-type progeny are scored has been attributed to the repair of a singlestranded loop of DNA resulting from the formation of a heteroduplex region spanning the deletion (Doermann and Parma, 1967; Berger and Warren, 1969; Broker and Doermann, 1975). We have examined this phenomenon in mutant strains by replacing r-59 with the small deletion rdb52 (Table 3). Since day-to-day variations in recombination frequencies do occur (see Fig. l), we present data for crosses with r59 and with rdb52 which were obtained on the same day and from the same experiment for each mutant strain of T4. The index of interference was reduced for a deletion as compared to a point mutation in wild-type, 58-, and x- infections. In contrast, the index of interference remained approximately equal for the deletion and the point mutation in 48-4T, 59-, w-, and y- infections. These data suggest that either loop repair is deficient in some mutant infections, or that a heterozygous recombinational intermediate containing a loop of single-stranded DNA is not formed in some mutant infections. HNI in prematurely lysed cells. Steinberg and Edgar (1962) reported that the index of interference is higher for progeny from a prematurely lysed culture than for progeny from a normal cross lysate. Since several of the mutants we were studying caused an arrest of DNA synthesis (Warner and Hobbs, 1967; Cunningham and Berger, 1977), we examined HNI in prematurely lysed cells infected with phage carrying rIIA mutations only. Table 4 shows that the index of interference was approximately fivefold higher early in infection than late in infection with a point mutation, as previously reported by Steinberg and Edgar (1962). An inverse relationship between the index of interference and the genetic distance between markers was found over the course of a wild-type infection and was similar to that found in the different mutant infections. These data suggest that the mutations might alter the index of interference and recombination frequency only indirectly by causing an arrest of DNA synthe-

AND

BERGER

sis. The frequency with which a second genetic exchange occurred once an initial exchange had taken place, however, was different in the wild-type and mutant infections. The measure of correlated exchanges, CE, was constant over the entire course of the wild-type infection (Table 4), but varied threefold in the mutant infections (TabIe 2). Thus it appears that the mutations caused variations in the correlation of exchanges in a direct manner. The index of interference also varied over the course of a wild-type infection with the deletion mutation rdb52 as a central marker (Table 4) but was always lower than the index of interference for a point mutation at the same time after infection, suggesting that variations in burst size and recombination frequency do not obscure comparisons of the index of interference for crosses employing either a point or a deletion mutation as the central marker. Heterozygote frequencies in mutant infections. Heterozygotes have been postulated to represent an intermediate in the recombinational process (Levinthal, 1954; Edgar, 1961), and alterations in the frequencies of heterozygotes have been shown to be correlated with alterations in genetic recombination frequencies (Berger et al., 1969). Two types of heterozygotes are formed in T4 infections, terminal redundancy heterozygotes and heteroduplex heterozygotes. Deletions are found only in terminally redundant heterozygous phage particles while point mutations are found in both types of heterozygous phage particles (see Mosig, 1970). We have examined heterozygote frequencies for both point and deletion mutations in am--rIIA+ x am--rIIAcrosses (Table 5). The frequency of heterozygotes formed with a point mutation was altered in 58- and 46+-47 infections as previously reported (Berger et al., 1969) and also was reduced in 59- and y- infections. The frequency of heterozygotes formed with a deletion mutation, however, was relatively constant in all infections. Since the formation of terminal redundancy heterozygotes can be viewed as a recombination event between unlinked markers, genetic equilibrium appears to be fairly constant in most strains. The frequency of heterozygotes in y- infec-

RECOMBINATION

IN

BACTERIOPHAGE

TABLE THE FREQUENCY

OF HETEROZYGOTES

r mutation Mut,r

9 gene T4D 46-47 58 59 Y T4D 46-47 58 59 Y

n The rIIA covering r59.

Point point Point point Point Deletion Deletion Deletion Deletion Deletion and mutations

r59 and rdb52

Mottled plaques

5 FROM Total

MUTANT plaques

40 7 70 13 17 22 15 16 23 11

5002 3308 3282 4389 3623 6252 5208 6316 6732 5005

were employed;

r59 is a point

tions was the lowest observed, and crosses with unlinked point mutations revealed a 30% reduction in recombination frequency (unpublished observations). Our data suggest that there is mixing of parental DNA pools even in mutant infections where DNA synthesis is reduced. DISCUSSION

We have examined the correlation of genetic exchanges and the frequency of heterozygotes in infections by strains of bacteriophage T4D mutationally altered in the ability to produce genetic recombinants. The correlation of genetic exchanges was directly related to both recombination frequency and the frequency of heterozygotes for a point mutation. In contrast, the correlation of genetic exchanges remained constant throughout a wild-type infection while recombination frequencies varied. The frequency of heterozygotes also remained constant throughout a wild-type infection (Hershey and Chase, 1951; Doermann, 1953; Krisch et al., 1972). These findings taken together suggest that alterations caused by mutationally inactivated gene products directly affect the recombinational process and are not merely a reflection of changes in the population of phage chromosomes available for recombination and maturation. We have formulated a function which measures the correlation of genetic exchanges in a subpopulation of phage chromosomes which have recombined at least once in a defined genetic interval. This

67

T4D

INFECTIONS~ Frequency erozygotes

of het(o/o)

Burst

0.80 0.21 2.13 0.30 0.47 0.35 0.29 0.25 0.34 0.22 mutation,

and rdb52

size

247 4 86 30 36 114 3 63 16 26 is a small

deletion

function is relatively insensitive to factors affecting recombination frequency in the whole phage population and is more easily interpreted in molecular terms than the index of interference, which is dependent on recombination frequency. Under the conditions of our experiments, the genetic intervals studied were kept constant while factors altering recombination were experimentally programmed by removing specific gene products by mutational inactivation. In summary, our results show that once a phage chromosome is recombinant in one genetic interval, the likelihood that it will be recombinant in an adjacent genetic interval depends on the presence of products coded for by genes affecting recombination. The fact that the correlation of exchanges varied only threefold while burst sizes varied 30-fold and recombination frequencies varied almost 60-fold probably reflects the requirement for recombinationally generated concatemers for the production of viable phage. Results from heterozygote studies also suggested that packaging requirements can select a certain class of chromosomes. Terminal redundancy heterozygotes can be viewed as arising by recombination between two DNA molecules carrying two unlinked markers. The fact that terminally redundant heterozygotes were present in almost equal frequencies in wild-type and mutant bursts suggested that all viable progeny had an equal chance of being derived from concatemers formed by recombination between nonsibling chromosomes. Thus although

68

CUNNINGHAM

closely linked markers can exhibit great variations in recombination frequency, unlinked markers approach genetic equilibrium in all infections. A simple explanation, which we find appealing, for the reduction in correlated exchanges in mutant infections is that reduced interactions between genomes result in a selection for chromosomes which have experienced a single crossover generating a concatemer suitable for packaging. Insertion heterozygotes, while producing double exchanges, do not produce concatemeric DNA suitable for packaging. Thus in strains producing higher frequencies of recombination in two-factor crosses and proably also undergoing more genomic interactions, the frequency of double crossovers also increases. Other factors which could influence the number of single and double recombination events are the amount of phage DNA synthesis, the length of singlestranded regions of DNA, and the amount of time a molecule is exposed to various enzymes which act on DNA. We have also examined heterozygote frequencies for a point mutation in mutant infections. Heterozygotes are presumed to be intermediates in recombination, and the frequency of heterozygotes in mutant infections varied in a direct fashion with recombination frequency. The lo-fold variation in the frequncy of heterozygotes which we observed can be explained by variations in the number or length of heteroduplex regions of DNA formed in mutant infections, or by variations in the efficiency of a repair system which operates on mismatched base pairs. A reduction of HNI when a deletion replaced a point mutation in a three-factor cross was observed in wild-type, 5&, and xinfections, but not in 46-47-, SF, w-, and y- infections. One explanation for the reduction seen in wild-type crosses is that a loop of excess wild-type DNA formed by pairing of nonsibling chromosomes is preferentially repaired (Berger and Warren, 1969). Two possibilities exist to explain why HNI did not vary for a point or a deletion in some mutant infections. The repair of the loop of DNA could be deficient so that the wild-type allele would not be lost. Alternatively, the length of single-stranded

AND

BERGER

regions of DNA exposed in some mutant infections could be too short to ahow formation of a heteroduplex region spanning the deletion. Thus, a loop would not be formed and loss of the wild-type allele by repair would be eliminated. The existence of clustered exchanges in such crosses would depend upon reiterated insertions of short stretches of DNA into a recipient duplex. The role that the products of genes 46-47 and 58 play in recombination is partially understood. A variety of data (Bernstein, 1968; Berger et al., 1969; Shah and Berger, 1971; Hosoda et al., 1971; Shalitin and Naot, 1971; Prashad and Hosoda, 1972; Broker, 1973; Mickelson, 1974; Hosoda, 1976) suggest that genes 46-47 code for a nuclease which generates regions of single-stranded DNA prerequisite for genetic and molecular recombination. Data from 58 and 46-47--58 infections (Broker, 1973; Hamlett and Berger, 1975) suggest that the gene 58 product acts to control the exonucleolytic erosion of DNA catalyzed by the gene 46-47 nuclease, and that longer than normal single-stranded regions of DNA are exposed in 58 infections. Our findings on the genetic properties of strains mutant for genes 46-47 or 58 are in accord with these proposals. The amount of single-stranded DNA available for pairing could influence the number of interactions between progeny genomes, resulting in the variable ratio of double to single crossovers. The length of the singlestranded regions of DNA interacting during synaptic events could influence the formation of looped-out structures when one parent is a deletion, thus altering the index of interference when a deletion is present in 58 infections but not in 4&-47- infections. Mutants defective in gene w exhibit reduced concatemer formation, reduced genetic recombination, and increased uv-sensitivity (Hamlett and Berger, 1975). Gene 32 protein, a DNA-binding protein (Alberta and Frey, 1970), is overproduced sevenfold in w- infections, and it has been proposed that gene w codes for a late repressor of gene 32 (Russel, 1977). The enzymatic nature of the products of genes X, y, and 59 are not known, nor have their roles in recombination been deter-

RECOMBINATION

IN BACTERIOPHAGE

mined at the molecular level (see Cunningham and Berger, 1977). Further biochemical characterization of the roles of these gene products in replication, recombination, maturation, and packaging of DNA will be necessary to elucidate the precise steps leading to the production of genetic recombinants in bacteriophage T4 infections. Our studies suggest some possible steps at which these gene products might act. ACKNOWLEDGMENTS We thank Nancy HamIett and Peter Gauss for helpful discussions, Marc Rhoades and Philip E. Hartman for their critical reading of the manuscript, and Dr. A. H. Doermann and Dr. John W. Drake for their helpful suggestions and their valuable criticism of this manuscript. Nancy HamIett and D. B. Shah kindly supplied us with phage stocks. We acknowledge the skihful technical assistance of Joan Devine. David Pietrasiuk performed the studies on heterozygotes. One of us (R.P.C.) thanks Robert Dottin for his hospitality while this research was completed in his laboratory. Mike Zaccaria and John TerreII materially aided us. This research was supported by Grants AI08160 from the National Institute of Allergy and Infectious Diseases, GB-43825 from the National Science Foundation, and NP-175 from the American Cancer Society. R.P.C. was a predoctoral trainee on National Institute of General Medical Sciences grant GM-57, and H.B. was a recipient of a Career Development Award, National Institutes of Health. This is contribution 957 from the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218. REFERENCES ALBERTS, B. M., and FREY, L. (1970). T4 bacteriophage gene 32: A structural protein in the replication and recombination of DNA. Nature (London) 227, 1313-1318. BERGER, H. (1965). Genetic analysis of T4D phage heterozygotes produced in the presence of 5&orodeoxyuridine. Genetics 52, 729-746. BERGER, H., and WARREN, A. J. (1969). Effects of deletion mutations on high negative interference in T4D bacteriophage. Genetics 63, l-5. BERGER, H., WARREN, A. J., and FRY, K. E. (1969). Variations io genetic recombination due to amber mutations in T4D bacteriophage. J. Viral, 3, 171-175. BERNSTEIN, H. (1963). Repair and recombination in phage T4. I. Genes affecting recombination. Cold Spring Harbor Symp. Quant. Biol. 33,325-331. BOYLE, J. M., and SYMONDS, N. (1969). Radiation sensitive mutants of T4D. I. T4y: A new radiationsensitive mutant; effect of the mutation on radiation

T4D

69

survival, growth, and recombination. Mutat. Res. 8, 431-439. BROKER, T. R. (1973). An electron microscope analysis of pathways for bacteriophage T4D recombination. J. Mol. Biol. 81, 1-16. BROKER, T. R., and DOERMANN, A. H. (1975). Molecular and genetic recombination of bacteriophage T4. Annu. Rev. Genet. 9,213-244. CHASE, M., and DOERMANN, A. H. (1958). High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43,332-353. CUNNINGHAM, R. P., and BERGER, H. (1977). Mutations affecting genetic recombination in bacteriophage T4D. I. Pathway analysis. Virology 80.67-82. DOERMANN, A. H. (1953). The vegetative state in the life of bacteriophage: Evidence for its occurrence and its genetic characterization. Cold Spring Harbor Symp. Quant. Btil. l&3-11. DOERMANN, A. H., and PARMA, D. H. (1967). Recombination in bacteriophage T4. J. Cell. Physiol. 70 (Suppl. l), 147-164. EDGAR, R. S. (1961). High negative interference and heterozygosis: A study of the mechanism of recombination in bacteriophage T4. Virology 13, I-12. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., SUSMAN, M., DENHARDT, G. H., and LIELAUSIS, A. (1963). Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. HAMLETT, N. V., and BERGEE, H. (1975). Mutations altering genetic recombination and repair of DNA in bacteriophage T4. Virology 63, 539-567. HARM, W. (1964). On the control of uv sensitivity of phage T4 by gene 3~.Mutat. Res. 1,344-354. HERSHEY, A. H., and CHASE, M. (1951). Genetic recombination and heterozygosis in bacteriophage. Cold Spring Harbor Symp. Quant. Biol. 16, 471-479. HONDA, M., and UCHIDA, H. (1969). Genetic recombination between closely linked markers of bacteriophage T4. I. A dual mechanism for recombinant formation. Genetics 63, 743-758. HOSODA, J. (1976). Role of genes 46 and 47 in bacteriophage T4 reproduction. III. Formation of joint molecules in biparental recombination. J. Mol. Biol. 106,277-284. HOSODA, J., MATHEWS, E., and JANSEN, B. (1971). Role of genes 46 and 47 in bacteriophage T4 reproduction. I. In vivo deoxyribonucleic acid replication. J. Viral. 8, 372-387. KRISCH, H. M., HAMLET, N. V., and BERGER, H. (1972). Polynucleotide hgase in bacteriophage T4D recombination. Genetics 72, 187-203. LEVINTHAL, C. (1954). Recombination in phage T2; its relationship to heterozygosis and growth. Genetics 39,169-l&4. MICKELSON, C. (1974). “Identification and Partial Purification of Gene 46 and 47 Protein of Bacterio-

70

CUNNINGHAM

phage T4.” Ph.D. Thesis, University of Rochester, Rochester, New York. MOSIG, G. (1970). Recombination in bacteriophage T4. Aduan. Genet. 15, l-53. PRASHAD, N., and HOSODA, J. (1972). Role of genes 46 and 47 in bacteriophage T4 reproduction. II. Formation of gaps on parental DNA of polynucleotide ligase defective mutants. J. Mol. Biol. 70, 617-635. RUSSEL, M. (1977). “The Regulation of Gene 32 Expression in Bacteriophage T4.” Ph.D. Thesis, University of Colorado, Boulder, Colorado. SHAH, D. B. (1976). Replication and recombination of gene 59 mutant of phage T4D. J. Viral. 17,175-182. SHAH, D. B., and BERGER, H. (1971). Replication of gene 46-47 amber mutants of bacteriophage T4D.

J. Mol. Biol. 57.17-34. SHALITIN, C., and NAOT, Y. (1971). bacteriophage T4 deoxyribonucleic

Role of gene 46 in acid synthesis.

AND

BERGER

J. Viral. 8, 142-153. STEINBERG, C. M., and EDGAR, R. S. (1962). A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47, 187-208. STREISINGER, G., and FRANKLIN, N. C. (1956). Mutation and recombination at the host range region of phage T2. Cold Spring Harbor Symp. Quant. Biol. 21, 103-109. WARNER, H. R., and HOBBS, M. D. (1967). Incorporation of uracil-Cl4 into nucleic acids in Escherichia coli infected with bacteriophage T4 and T4 amber mutants. Virology 33, 376-384. Wu, R., MA, F-J., and YEH, Y-C. (1972). Suppression of DNA-arrested synthesis in mutants defective in gene 59 of bacteriophage T4. Virology 47, 147-156. YEGIAN, C. D., MUELLER, M., SELZER, G., Russo, V., and STAHL, F. W. (1971). Properties of the DNAdelay mutants of bacteriophage T4. Virology 46, 900-919.