Deletion analysis of two nonessential regions of the T4 genome

VIROLOGY 61,505-523

Deletion

(1974)

Analysis

of Two Nonessential

THEODORE Department

of Molecular

HOMYK,

Biology,

Vanderbilt

Accepted

JR.’

Regions AND

Unioersity,

of the T4 Genome

JON WEIL Nashville,

Tennessee 37235

July 2, 1974

Mutants of T4 which contain a duplication of the rII region also often contain a compensating deletion of a nonessential segment of the genome. This relationship was used to isolate a series of viable deletion mutations. The deletions were mapped genetically, making use of their effect on plaque morphology, and by electron microscopy of DNA heteroduplexes to T2, a T2m4 hybrid, and T4 wild type. They were also tested in several laboratories for various phage functions, enzymes, and proteins. Two different overlapping deletions located between genes 32 and 63 and ten located between genes 39 and 56 were obtained. Analysis of the former class indicates that there are at least 1400 base pairs of DNA between genes 32 and 63 which are unaccounted for by presently known genes. Deletions of the latter class affect the production of exonuclease A, a DNA-dependent ATPase, a 5’.phosphatase, and two proteins revealed by polyacrylamide gel electrophoresis. They also affect modification of host RNA polymerase, suppression of unwinding protein deficiency (sud phenotype), and ability to grow on two different Escherichia coli strains. The effect of different deletions on these properties demonstrates that at least seven genes are involved and allows their approximate locations to be determined. The general applicability of the method used for selecting deletions of nonessential DNA is discussed. INTRODUCTION

An important feature of bacteriophage research is the possibility of isolating and investigating mutations in most or all genes of the organism. For genes which are essential for lytic growth, the isolation of conditional lethal mutants provides a general means of obtaining mutations. However, with complex phages such as T4, exhaustive isolation of conditional lethal mutants has failed to saturate either the genetic map or the calculated coding capacity of the phage (see Wood, 1974). This failure results, at least in part, from the existence of genes which are not essential for lytic growth. A considerable number have been detected mutationally; evidence for others lies in the identification of phage-produced enzymes or proteins which cannot be assigned to known genes. Inspection of the T4 genetic map reveals ‘Present address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada.

several regions, measuring from a few thousand to 20,000 base pairs in length, which are devoid of known essential genes (Wood, 1974). (See Fig. 1, which also shows the location of all genes relevant to the present paper.) Most of these contain some identified nonessential genes. If these regions are entirely dispensable for vegetative growth, one approach to studying the functions they code for is through the isolation of deletion mutants. In addition to providing a means for obtaining mutants of the affected genes, deletions have other useful properties. Since they completely eliminate the gene product, they are useful for physiological experiments and they allow identification of affected proteins by polyacrylamide gel electrophoresis (O’Farrell, et al., 1973) and assays for specific enzymes. They permit DNA-RNA hybridization studies of mRNA synthesis from the affected region (Bautz and Reilly, 1966; Sederoff et al., 1971), and they can be mapped by physical as well as genetic

506

HOMYK

AND WEIL

means (Bujard et al., 1970; Wilson et al., 1972). Previously, deletions of T4 have been isolated as mutations affecting the rI1 genes (Benzer, 1959), the lysozyme gene (Mukai et al., 1967; G. Streisinger, personal communication; Wilson et al., 1972), and the tRNA genes (Wilson et al., 1972). In each case, some of these deletions were shown to extend beyond the gene whose function was used in their selection (Bautz and Bautz, 1967; Dove, 1968; Wilson et al., 1972). However, there has previously been no general method for isolating deletions in T4. Virion morphogenesis results in the packaging of a specific quantity of DNA (Streisinger et al., 1967) so that in contrast to many other phages, deletions cannot be isolated through their physical effect on the virion. An apparently general method for isolating deletions did become available, however, with the demonstration that phage carrying a duplication of the rI1 region (Weil et al., 1965; Parma et al., 1972; Parma and Syder, 1973; Symonds et al., 1972) often contain a deletion as well (Weil and Terzaghi, 1970; Parma et al., 1972). In the present study we have made use of this method to isolate deletion mutants and have obtained deletions which lie in the nonessential regions between genes 32 and 63 and between genes 39 and 56 (see Fig. 1). Phage carrying a duplication of the rI1 region are obtained from a cross between two complementing but overlapping rI1 mutants. One mutant is deletion r1589, which eliminates parts of the A and the B cistrons, yet retains B-cistron activity (Champe and Benzer, 1962). The other mutant lies entirely within the B cistron and overlaps r1589 (e.g., r1236; Barnett et al., 1965). Because the two deletions overlap, a cross between them cannot give rise to wild-type recombinants. Nevertheless, upon plating the progeny of such a cross on Escherichiu coli K(h) (hereafter referred to as K), which is restrictive for rI1 mutants, plaques arise with a frequency of about 2 x lo-‘. These plaques have been shown to contain phage with a tandem duplication of the rI1 region. One duplicate has the rI1 region from the r1589 parent, the other has at

least the A cistron from the r1236 parent, and the phage are thus phenotypically r+ by virtue of complementation between the two rI1 regions. As would be expected, the duplication phage give rise by recombination to haploid segregants which have only one copy of the rI1 region and cannot grow on K. Such segregants may carry either the r1589 deletion or the r1236 deletion. The frequency and ratio with which the two types of segregants arise depends upon the length of the duplicated segment and the location of the rI1 region within it. The observation that rI1 duplication phage often also carry a deletion can be explained by considering the DNA maturation mechanism of the phage. During virion morphogenesis a specific lengt,h of DNA is cut from a concatemer, the length being such as to create a terminal redundancy (Streisinger et al., 1967). A duplication which exceeds the length of the normal terminal redundancy will be lethal because each mature phage particle will be missing a random portion of the genome. Duplications which are shorter than the terminal redundancy will reduce the length of the terminal redundancy. Some of these may also be lethal, because a terminal redundancy sufficiently long to provide a high probability of recombination between the ends of the molecule is apparently required for normal DNA replication (Mosig and Werner, 1969). Thus, in selecting for viable duplication mutations, it appears that some selection is also made for a compensating deletion of nonessential DNA that allows the mature phage to retain sufficient terminal redundancy. We, therefore, isolated duplication mutants and examined them and their haploid segregants for the occurrence of deletions. One deletion isolated in this manner has previously been characterized (Weil and Terzaghi, 1970; Bujard et al., 1970). MATERIALS

Bacteria.

AND

METHODS

E. coli strains G(h), a lysogenic

derivative of B (Arber and Lataste-Dorolle, 1961) referred to as G in this paper, and K-12 112-12-(A,) (Wollman, 1953), referred to as K in this paper, are from our collection. Strains CR63 (Epstein et al.,

DELETION

ANALYSIS

1963), B, BB, and S/6 were obtained from Dr. Gisela Mosig. Strain CTr5x was obtained from Dr. Richard Depew. Strains CR63(Xh), referred to as CR63( X), and B/4 were made during the course of this study. Bactriophage. The rI1 mutants in T4B, described by Benzer (1961) and Champe and Benzer (1962), were those used in a previous study by Weil et al. (1965). The amber mutants, most of which have been described (Epstein et al., 1963; Edgar and Wood, 1966) and the r1 mutant rls (Edgar et al., 1962) are in bacteriophage T4D and were obtained from Dr. Gisela Mosig. T2L and the T2-T4 hybrid phage h44-4 (Beckendorf et al., 1973) were obtained from Dr. Jung-Suh Kim. T4sud mutants (Little, 1973) were obtained from J. Little. T4D derivatives of rI11236 and r111539 were obtained by back crosses as described below. The T4D derivatives used were crossed again to T4D wild-type and progeny plaques examined to verify that they contained none of the plaque morphology markers which segregate in T4D x T4B crosses (Rutberg, 1969). Strains carrying the r1236 mutation and an amber mutation in another gene were isolated from amber x T4D-r1236 crosses. Media. M9, Hershey-Broth (H-Broth), soft agar and plate agar were made as previously described (Foss and Stahl, 1963) with the exception that M9 was made 0.4% in glucose. H-broth used in preparing phage stocks for DNA annealing was adjusted to pH 7.4 with 1 N NaOH. Suspension medium for phage to be used in DNA annealing contained per liter: 2.03 g MgCl,, 5.5 g NaCl, 1.21 g Tris-OH, final pH 8.5 (Kim and Davidson, 1974). Preparation of exponential E. coli. Exponential phase cells were grown to 2 x lOa cells/ml in H-Broth at 37”, chilled on ice under continued aeration, centrifuged, and resuspended in cold H-Broth. Exponential E. coli used for plating was resuspended at 2 x lo9 cells/ml. Plating. Whenever plaque-type discrimination was critical, platings were done with a lo-min preadsorption at 37 ‘. T4B strains were adsorbed in the presence of 20 pg/ml L-tryptophan. B:K mixed plating. Phage were first

OF T4

507

adsorbed to E. coli B at 1 x lo9 cells/ml. One-tenth milliliter of a lo-fold dilution of this adsorption mixture was then plated with 0.1 ml of exponential E. coli K. Platings made in this way allow unambiguous distinction between the rI1 duplications, which produce clear plaques, and the haploid segregants, which produce turbid plaques. Isolation and identification of haploid segregants. Suspensions of rI1 duplication heterozygotes were plated with a mixture of E. coli strains B and K as described above, and haploid segregant plaques picked and suspended in M9. These suspensions were spotted onto plates seeded with 2 x lo8 K cells alone and with K plus 1 x lo7 particles of either T4r1299 or T4r1168. r1236 can recombine with r1168 to allow growth on K, but cannot recombine with r1299. The converse is true for r1589 (see Weil et al., 1965). Phage stocks. Phage stocks were prepared by inoculating a 20-ml exponential E. coli culture (at 2 x IO* cells/ml) growing at 37” with single 5 hr plaques. Stocks of amber mutants, of rI-containing strains, of deletion mutants in the de1(63-32) class, and of the phage mutant in class IV were prepared using E. coli CR63 in M9 with 0.3% Casamino acids (Difco). All other strains were prepared on E. coli BB in M9. Phage stocks for DNA annealing were prepared according to Kim and Davidson (1974). Genetic crosses in liquid culture. Crosses involving amber mutants were performed in E. coli CR63. All other crosses were performed in B. Phage to be crossed were adsorbed to exponential E. coli (1 x lo9 cells/ml) at a multiplicity of infection (m.o.i.) of 5 for each parent. The adsorption mixture was aerated gently at 37’ for 7 min, diluted loo-fold into prewarmed HBroth and aerated vigorously for 1 hr. Lysis was completed with chloroform. Transfer of genetic markers from T4B to T4D. Two backcrosses were performed in which the non-T4D parent received 22 lethal hits from a 15-W General Electric germicidal lamp. Further manipulations were performed under a dim yellow lamp to minimize photoreactivation.

508

HOMYK

Spot crosses. Suspensions of single plaques of each parent were spotted together on plates covered with soft agar seeded with exponential B cells. Samples of the lysis spots formed were suspended in M9 and plated on B for analysis. One-step growth experiments. Exponential B cells were suspended in cold H-Broth at 2 x lO*/ml and used immediately. A phage suspension at 2 x 10’ particles/ml containing 4 x 1O-3 M NaCN was mixed with an equal volume of cells and set on ice for 3 min. Adsorption was then allowed to occur at 37” for 7 min. Unadsorbed phage were removed by incubation with T4 antiserum (Antibodies, Inc.), final K = 6, for 5 min. NaCN and antiserum were removed by two washings, each performed by centrifugation and resuspension of the cells in cold H-Broth. The final suspension, made in 1 vol of H-Broth, was diluted 4 x 104-fold in prewarmed H-Broth and aerated vigorously at 37”. A second growth culture, made by a 20-fold dilution of the first, was prepared for later time samples. Samples taken at regular intervals were either plated directly with B to determine total plaque-forming units or treat,ed with chloroform and plated to determine the total intracellular and extracellular phage. Ultruoiolet inactivation. Four-milliliter samples of each phage at 1 x lo8 particles/ml in M9 were placed in petri plates and exposed to a 15-W General Electric germicidial lamp. Samples were taken at 30-set intervals, diluted, and plated on B. Adsorption, plating, and incubation were performed in the dark to minimize photoreactivation. Test for determining the frequency of rII terminal redundancy heterozygotes (HET test). Terminal redundancy heterozygotes

from a cross between two complementing rI1 mutants such as r1589 and r1236 are able to undergo one round of replication in K (Stahl et al., 1965). However, such heterozygotes are unable to produce plaques on K because the heterozygous condition is not inherited. The HET test is thus designed to measure the proportion of progeny particles which can undergo one round of replication in K: progeny from the cross

AND WEIL

are adsorbed at low multiplicity to K and the infected cells are plated on an rII-permissive host. The test was carried out by the procedure of Weil and Terzaghi (1970), but with the addition of an internal standard to increase the accuracy of the test. The internal standard was a lysate from the cross rIr1589 x rIr1236. The phage from this cross can be distinguished by plaque morphology from those in experimental crosses, which are rI+. Thus, each time a HET test is performed, the frequency of HETs from the rIr1589 x rIr1236 cross is measured and the result obtained for this cross is used to normalize the different HET tests. To the lysate from each r1589 x r1236 cross to be tested, an equal concentration of phage for the cross rIr1589 x rIr1236 was added. The resulting mixture was assayed directly on E. coli BB, on which rI+rII and rIrI1 plaques can be distinguished, and was used in a HET test in which the rII-permissive host was BB. A normalized HET frequency was calculated by the following formula: Titer of rI+ plaques in HET test Titer of r1 plaques in HET test i

Titer of rI+ plaques obtained Titer of rI plaques obtained

by direct assay by direct assay.

The tabulated values (Tables 1 and 2) are reported as a HET ratio, in which the normalized HET frequency for a cross in which one or both parents contained a putative deletion is divided by the normalized HET frequency from a control cross of T4r1589 x T4r1236. The parents in this latter cross do not contain any deletions other than those of the rI1 mutations, and the normalized HET frequency was always close to unity. The HET ratio thus indicates the extent to which the putative deletion enhances the frequency of terminal redundancy HETs. DNA annealing and spreading. The annealing and spreading of phage DNA was done according to Kim and Davidson (1974). Heteroduplex DNA was examined using a Hitachi HU 11 B electron micro-

DELETION

ANALYSIS

OF T4

509

Photographs were made at 5000 fication. These strains were systematically crossed among themselves using the spot and the negatives enlarged 10 or 20 times cross method (see Materials and Methods). on a Nikon profile projector (Model 6C) for Upon examination of at least 1000 plaques tracing. Traced molecules were measured from each cross, the absence of progeny with a Keuffel and Esser map measurer, which produced normal plaques was intermodel 62 0300. Circular single-stranded preted to mean that the plaque morpholand double-stranded @X174 DNA mole- ogy mutations failed to recombine, and cules (a generous gift of Drs. J. S. Kim and they were thus assigned to the same recomA. Forsheit, obtained originally from Dr. R. bination class. To confirm these results, a Sinsheimer) were used as an internal stan- few spot crosses where reconbination had dard of molecular lengths, representing and had not been observed were verified by 5200 bases or base pairs, respectively (Dav- the examination of a thousand progeny idson and Szybalski, 1971). plaques from liquid crosses. On the basis of these crosses, the phage mutants were RESULTS found to comprise four recombination The procedure described by Weil et al. classes, I through IV. The plaque morphol(1965) and outlined in the Introduction was ogy mutant Dj, isolated by Weil et al. used to isolate T4 rI1 duplications. Two (1965) and shown to contain a deletion sets of crosses were performed. In the first between genes 39 and 56 (Bujard et al., set, 48 independent crosses were made 1970; Weil and Terzaghi, 1970), served as a between T4Br1236 and T4Br1589 and the prototype for class I. Of the 50 mutants, 35 progeny were plated on E. coli K to select were in class I, 13 in class II, and one each for rI1 duplications. The resulting plaques in classes III and IV. were picked from the K plates and replated In the second set, 100 crosses were on B for repurification and to allow exami- made using r1236 and r1589 in a T4D genation of plaque morphology. In agreement netic background, and the progeny plated with previous observations (Weil et al., on K. The resulting plaques were then 1965), some of the rI1 duplication mutants plated on E. coli G, which restricts rI1 muproduced normal rII-type plaques while tants yet permits better definition of others had altered plaque morphology. plaque morphology than does K. From each We chose to concentrate on those du- cross a maximum of one duplication strain plication strains which produced abnormal producing normal plaques and one proplaques. On the one hand, we anticipated ducing abnormal plaques was selected. that not all of the duplication strains would Among the abnormal plaque mutants, spot carry a deletion. On the other hand, we crosses placed 19 in class I, one in class II, anticipated that some, although perhaps and one in a new class, V. not all, deletion mutations would affect Terminal redundancy heterozygote test. plaque morphology. Thus, selection of du- Because the T4 DNA maturation mechaplication strains which produce abnormal nism packages a fixed quantity of DNA, plaques appeared to be the simplest means the presence of a deletion mutation infor selecting strains which also carried a creases the length of the terminal redeletion. In addition, the plaque morphol- dundancy, and thus the frequency with ogy phenotype would then allow us to which terminal redundancy heterozygotes follow the deletions in genetic crosses. (HETs) are formed at other loci in a genetic Finally, the fact that plaque morphology cross (Streisinger et al., 1967). Following was altered would ensure that the deleted Weil and Terzaghi (1970), we have meaDNA has some role in the normal phage life sured HET frequency in a preliminary test cycle. for the occurrence of deletions. Five strains From the first set of crosses, 50 duplica- of class I, each having a different plaque tion strains were obtained which produced morphology, and one strain of class II were exclusively abnormal plaques after repuri- chosen for investigation. All strains were

SCOp2.

power on Kodak electron microscope film

510

HOMYK

AND WEIL

deletion strains were transferred into a T4D genetic background by two backcrosses. For each backcross, the strain carrying the morphology marker was irradiated with 22 lethal hits, crossed to T4Dr1589, and the progeny plated on B. Progeny plaques resembling the original mutant were spot crossed to the original mutant (in T4B) to confirm their identity. After the second backcross, crosses were also made of each mutant to T4D-r1589 and the progeny examined for the plaque morphology markers which segregate in T4D x T4B crosses (Rutberg, 1969). No such markers were found in any case. In order to determine whether the presumed deletions had been transferred together with the plaque morphology markers, the T4D-r1589 derivatives of each mutant were crossed to T4D-r1236 and the progeny subjected to a HET test. Table 2 presents the results of these HET tests. The HET ratios are approximately one-half those found in crosses where both parents had contained the presumed deletion (Table 1). This is the result expected, since only one-half of the progeny would contain a deletion and thus have a longer terminal Transfer of the presumed deletions into a redundancy. These results demonstrate that in each T4D genetic baclzground. The plaque morphology markers of the six presumptive case the factor causing enhancement of the HET ratio had been transferred with the TABLE 1 plaque morphology marker. This supports HET TESTthe earlier working hypothesis that the two are identical. HET ratiob Cross between haploid segregants of One-step growth experiments. Table 3 la lb 2 3 4 presents the results of one-step growh experiments at 37” on B for one mutant in 4.0 3.5 3.4 de1(39-5611 class I and one in class II. In each case the 3.0 4.5 3.4 5.4 de1(39-56)2 eclipse and latent periods are increased 3.6 de1(39-56)3 6.4 slightly. The burst size of each mutant is de1(39-5614 -

from the first set of crosses and were thus in T4B. For each strain, an r1589 haploid segregant and an r1236 haploid segregant were isolated, the two segregants were crossed, and the resulting progeny tested to determine the frequency of rI1 HETs. (For the details of this test and a discussion of its rationale, see Materials and Methods.) The results are given in Table 1. For each strain tested, the value listed is the ratio of HET frequency observed with that strain divided by HET frequency observed in a control cross between the original r1589 and r1236 strains. In each case, the ratio is significantly greater than unity, leading to the tentative conclusion that all six strains carry deletions. As will be shown subsequently, class I members contain deletions between genes 39 and 56, and class II members contain deletions between genes 63 and 32. The designations de& 39-56) 1 through del( 39-56)5 and del( 63-32) 1, respectively, have thus been applied to these strains. De1(39-56) 1 was previously called “Dj” (Bujard et al., 1970) and the plaque morphology it produces was referred to as “minute” (Weil and Terzaghi, 1970).

de1(39-5615 de1(63-32)l

5.2

8.5

8.5

-

3.9 -

~Columns la and lb represent two HET tests performed with one set of crosses. The data of columns 2, 3, and 4 are each from a different set of crosses. “The HET ratio represents the normalized frequency of rI1 HETs in experimental crosses divided by the normalized frequency of rI1 HETs in control crosses. The HET ratio is expected to be greater than 1.0 in the case of phage containing deletions and to be 1.0 for phage which do not contain deletions.

TABLE HET

TEST

2

FOR PRESUMED DELETIONS INTO T4D

Cross d&39-56)1-r1589 de1(39-56)2-r1589 de1(39-56)3-r1589 de1(39-56)4-r1589 del(39-56)5-r1589 de1(63-32)1-r1589

TRANSFERRED

HET ratio x x x x x x

r1236 r1236 r1236 r1236 r1236 r1236

2.6 3.0 3.7 2.4 1.9 4.4

DELETION TABLE

ANALYSIS

3

ONE-STEP GROWTH EXPERIMENT Strain

d&39-56)1+1589 &z&63-32)1-r1589 r1589 (control)

Latent period” (min) 28 28 26

Eclipse period” (min) 19 19 17

Burst size (W of control)b 50-60 10-30 100

u In three experiments, the difference between the control and the experimental values was 2 + 0.5 min. b Range of three experiments.

also significantly lower than that of the r1589 control. These parameters suggest that, although dispensable, the deleted functions are beneficial to phage growth.

OF T4

511

ter five strains showed that they also produced plaques with slightly abnormal morphology on B. Since they failed to recombine with class II mutants, they were assigned to this class as members numbered 6-10. When plated on CTr5x, strains 6, 8, and 10 produce small clear plaques while strains 7 and 9 produce barely visible plaques. These observations will be discussed later, when the locations of the deletions in these strains are compared. Electron microscopy of DNA heteroduplexes. Thirteen mutants in class I were

studied by DNA heteroduplex analysis as described by Kim and Davidson (1974). In heteroduplexes of del( 39-56) 1 to T2, a loop of 1100 bases length that is seen in the Examination of phage on E. coli strains standard T2/T4 heteroduplex pattern BB and CTr5x. In order to expand our (Kim and Davidson, 1974) is replaced by a search for deletions which produce ab- loop of 3200 bases. Site Kim and Davidson normal plaques, we screened a large num- place the 1100 base loop between genes 39 ber of m-duplication phage or their hap- and 56 (it lies at the position of 115 loid derivatives on two additional strains kilobases in Fig. 7 of their paper), we infer of E. cob. Because the T4 rl locus lies in that deletion (39-56)l lies between these a large region devoid of known essen- two genes, in agreement with previous tial genes (see Fig. l), the phage were evidence (Weil and Terzaghi, 1970; Bujard examined for the presence of r1 mutations. et al., 1970). This was done on E. coli BB, on which rI1 DNA from r1589 haploid segregants of all mutants produce wild-type plaques but r1 13 mutant strains was annealed to T4D mutants produce r-type plaques. The wild-type DNA and distances measured phage were also tested on E. coli CTr5x. from the r1589 reference loop. The results Depew and Cozzarelli (1974) have shown are presented in Table 4 (see also Fig. 2), that CTr5x restricts the growth of T4 r1 mutants which fail to produce a 3’-phosphatase. The gene for the phosphatase maps between genes 63 and 31 (Depew and Cozzarelli, 1974). Deletions (39-56)l through (39-56)5, de1(63-32)1, 40 other abnormal plaque producers and 72 independently isolated normal plaque producers were streaked on lawns of E. coli B, CTr5x, and BB. None of the phage produced r1 type plaques. However, three abnormal plaque producers in class I, designated I-13, and de1(39-56)11, and 12, failed to produce plaques on 3332 63 CTr5x. In addition, de1(39-56)4 and 5 had FIG. 1. Genetic map of T4 showing essential genes a reduced efficiency of plating (cop) on and genes relevant to this paper. Lines and darkened CTr5x, relative to B. Of the strains classi- areas within the circle indicate the location and, fied as normal plaque producers on B, 67 where known, the extent of essential genes. Symbols produced large clear plaques identical to outside the circle indicate the location of genes which r1589 on CTr5x, while five produced much are relevant to the present paper. The data are from smaller plaques. Reexamination of the lat- Wood (1974).

512

HOMYK TABLE

STRAINS de1(39-56)1-12

4 sudl

AND

HETERODUPLEXED

TO T4” Strain de1 1 2 3 4 5 6 7 8 9 10 11 12 sud 1

Length (A)

Distance to r1589 (B)

Sum of A+B

4,190 6,712 5,696 6,791 6,737 4,583 5,659 4,770 4,519 4,432 8,031 9,841 3,701

6,149 6,068 5,550 6,643 5,343 6,446 6,885

10,339 12,780 11,246 13,434 12,080 11,029 12,544 10,740 11,215 10,736 12,667 14,388 11,343

i 340 * 450 i 361 z+z454 & 472 i 367 l

359

i 294 i 200 * 213 zt 227 i 405 * 158

5,970

6,696 6,304 4,636 4,547 7,642

zt 285 i 355 zt 230 i 325 z+z302 zt 282 + 183 l 319 * 254 +z 291 zt 265 * 165 ziz285

a All measurements are in bases or base pairs. The sum of A + B in column 4 is the distance between r1589 and the rII-distal end point of each deletion. Each measurement represents an average of 7-19 molecules. The standard deviation is given for each set of measurements.

and a representative heteroduplex is shown in Fig. 3. Strains 1 through 5, which were chosen because they produce distinguishable plaque types on B, all appear to contain different deletions. Strains 11 and 12, which were chosen because of their inability to plate on CTr5x, are seen to contain deletions which extend considerably closer to r-11than any of the other strains. Three of the five strains chosen at random from the remaining class I mutants, designated de1(39-56)6-10, appear to contain identical deletions. Strain I-13 contained no deletion in the vicinity of the r1589 reference loop. The data for each deletion in Table 4 are based on the measurement of one long double-strand region (determining the rIIproximal end of the deletion) and one long single-strand region (determining the rIIdistal end and length of the deletion). Thus, the absolute value of the standard deviations is relatively large. Examination of the data in Table 4 revealed, however, that more accurate comparisons between the deletions in strains 1, 6, 8, 9, and 10 could be obtained by annealing them to strain 7. In each such heteroduplex, a

AND WEIL

substitution loop would be formed in which the longer arm comprises the DNA deleted in strain 7 but present in the other strain, while the shorter arm comprises the DNA deleted in the other strain but present in strain 7. Thus, the relative locations of the deletions could be determined by measurement of two short single-stranded regions, with a resulting smaller absolute standard deviation. Figure 4 is an electron micrograph of the substitution loop formed in one such heteroduplex. Table 5 presents the data and calculations from these heteroduplexes. These data agree well with those of Table 4, but the relative locations of these six deletions are determined with considerably more accuracy. The size and location of all 12 deletions is shown in Fig. 2. Little (1973) has isolated T4 mutants which allow phage carrying an amber mutation in gene 32 to grow in ochre-suppressing hosts (sud mutants). These mutants map between genes 39 and 56. Little’s data suggested that some of these mutants, including sudl, are deletions while others, including sud22, are not. In heteroduplexes to r1589, sudl was found to contain a deletion whose size and location are given in Table 4 and Fig. 2. Examination of 11 4Gene sud

Gene 56)

39

I

del(39-5619 del(39-56110 del(39-56)6 del(39-5618 del(39-561

I

del(39-56)7 del(39-5614 del(39-5612 del(39-5613 dal(39-56)5 del(39-56111 del(39-56 0

)I2

4000 DISTANCES

8000 IN BASE

12.000

16,000

PAIRS

FIG. 2. The physical location and extent of deletions in the region between genes 39 and 56. The figures for de&39-56)2, 3, 4, 5, 7, 11, and 12 and sudl are drawn from the data in Table 4. The figures for de1(39-56)1, 6, 8, 9, and 10 are drawn in relation to de1(39-56)7 from the data in Table 5. The scale represents distance from r1589.

DELETION

ANALYSIS

OF T4

513

FIG. 3. Heteroduplex of de1(39-56)3 to T4 wild type. The r1589 loop and the de1(39-56)3 loop are designated r and d, respectively. FIG. 4. Heteroduplex of de1(39-56)10-r1236 to de1(39-5617rl589. The substitution loop D is formed by the nonhomology in the deletions de1(39-56)lO and 7. The substitution loop R is formed as a result of nonhomology between the rII deletions r1236 and r1589.

514

HOMYK AND WEIL TABLE 5

STRAINS de1(39-56)1, 6, 8, 9, AND 10 HETERODUPLEXED TO del(39-56)7

Strain” 1 6 8 9 10

Long arm*

Short arm’

2,588 i 1,551 f 2,105 i 1,495 f 1,590 f

992 zt 123 316 zt 92 863 zt 167 272 f 47 206 * 59

289 201 305 130 210

DWith strains 6 and 10 an r1589/r1236 reference loop was present and used to confirm location on the heteroduplex. bLength of the long arm indicates how much closer the rII-distal end of the deletion is to rI1 than is the rII-distal end of deletion 7. ‘Length of the short arm indicates how much closer the rII-proximal end of the deletion is to rI1 than is the rII-proximal end of deletion 7.

heteroduplex molecules of sud22 revealed no deletion. Heteroduplexes between 12 mutants in class II and the T2-T4 hybrid phage h44-4 (Beckendorf et al., 1973) were examined. These h44-4lT4 heteroduplexes show a specific pattern of single-strand loops with fewer loops than occur in a heteroduplex between T2 wild type and T4 wild type (data not shown). With the exception of one loop, the pattern of loops formed in the h44-4/T4 heteroduplex can be superimposed on the T2/T4 heteroduplex pattern described by Kim and Davidson (1974). h44-4/T4 heteroduplexes, therefore, provide markers with which to identify all parts of the T4 genome. Deletions in strains of class II were located with respect to the nearest marker loop in the h44-4A’4 heteroduplex. This marker loop appears as the left-most member of a pair of small deletion-insertion loops at the position corresponding to 155 kilobases on the T2/T4 heteroduplex map (Fig. 7 of Kim and Davidson, 1974). Figure 5 is an electron micrograph of a representative heteroduplex between de1(63-32)3 and h44-4, with arrows identifying the reference and deletion loops. Eleven of the 12 class II strains examined contain deletions. As seen in Table 6 and Fig. 6, the deletions appear to fall into two

groups, the members within each group being indistinguisable. From the T2/T4 heteroduplex map (Kim and Davidson, 1974), both groups appear to map between genes 63 and 32. Because the deletions within each group appeared identical, they were further examined in heteroduplexes to de1(63-32) 1. Since there was a chance that some of the deletions are, in fact, identical to de1(63-32)1, it was necessary to mark the relevant DNA region so it could be identified in these heteroduplexes. For this purpose, de1(63-32)l was crossed to h44-4 and the progeny plated on E. coli B/4. The recombinant phage de1(63-32)l hZ+, containing the deletion plus the host range of T2, was thus isolated. In heteroduplexes between this phage and T4D wild type, the mutant deletion loop, the host range loop, and several loops in the clockwise direction from rI1 (Kim and Davidson, 1974) were found. The host range and other T2/‘T4 loops provided a convenient means for orienting the heteroduplex molecules. Deletions 2 through 11 of class II were annealed to de1(63-32)l /z2+ and in each case 6-11 molecules containing the region of interest were examined. In heteroduplexes to members 2, 3, 4, 5, 6, 8, 10, and 11 only the T2/T4 loops were found. Thus, the deletions in these phage also appear identical by this method of analysis. It should be noted that Davis and Hyman (1971) found that heteroduplex analysis is suitable for resolving nonhomologous sequences as short as 90 base pairs. Table 7 shows that strains 7 and 9 give very similar substitution loops when annealed to de1(63-32)l h2+. Figure 7 is an example of one such heteroduplex. By analogy with the argument presented for Table 5, these values are more accurate than those of Table 6, and provide stronger evidence that deletions 7 and 9 are identical. Heteroduplexes between the r-1589 haploid segregant of strain I-13 and T4D wild type revealed no deletion loop in the gene 39-56 region. Similarly, heteroduplexes between h44-4 and the twelfth mutant in class II showed no deletion in the 63-32

DELETION

ANALYSIS

OF T4

516

HOMYK TABLE

AND WEIL

6

STRAINS de1(63-32) l-l 1 HETERODUPLEXED TO h44-4” Length (A)

Strain

1 2 3 4b 5 6 7 8 9 10 11

4,978i 4,890 5,040 5,073 4,982 4,619 4,249 4,956 4,203 4,784 4,586

Distance to reference loop (B)

Sum of A+B

3,461* 192 3,465 zt 105 3,612 i 127 3,423 3,525 + 122 3,394 f 102 7,522 +415 3,399* 122 7,185 + 290 3,448i 243 3,431* 121

8,439 8,355 8,652 8,496 8,507 8,013 11,771 8,355 11,388 8,232 8,017

280 i 223 * 174 + 207 zt 269 * 74 + 337 i 192 zt 190 i 139

n Measurements are from 4-12 molecules except for member 4. The standard deviations are included for each measurement. Columns 3 and 4 represent the proximal and distal end points determined relative to the reference loop. b Measurement of one molecule. (Gene

32

Gene

63)

del(63-32

)9

del(63-32)7 del(63-3215 del(63-32) 12,000

I 0,000

BOO0 DISTANCES

6000 IN BASE

4000

2000

I 0

PAIRS

FIG. 6. The physical location and extent of deletions in the de1(63-32) class. The figures for de1(63-32)l and 5 are drawn from the data in Table 6. The figures for de1(63-32)7 and 9 are drawn in relation to de1(63-32)l from the data in Table 7. The scale represents distance from the reference loop.

region. To determine whether these strains might contain deletions elsewhere, their complete genomes were examined in heteroduplexes to h44-4. Examination of each part of each genome in at least five heteroduplex molecules revealed no differences from the T4Dr1589/h44-4 control heteroduplex. Heteroduplexes between h44-4 and each of the single mutations in classes III, IV, and V were constructed. Measurements of each section of each genome in lo-14 heterodunlex molecules revealed no differ-

ences from the control. Since it is possible that some deletion mutations would not affect plaque morphology, segregants of 10 independent rII duplication strains that produce normal plaques on B were studied in heteroduplexes to h44-4. Examination of each section of each genome in at least three molecules revealed no differences compared to the control. Crosses of deletion mutants to amber mutations in linked genes. To confirm the location of the deletions and to determine how close they are to adjacent genetic markers, the r1589 haploid segregants of de1(39-56)12, de1(63-32)1, and de1(63-32)9 were crossed to amber mutants in flanking genes. The data, given in Table 8, confirm the locations of these deletions. They also suggest that, although some deletions are close to amber mutations in flanking genes, they do not extend into the genes. Ultraviolet irradiation inactivation curves. Mutations in several known essential and nonessential genes affect phage survival after UV irradiation. To further characterize our mutants, samples of de1(39-56)12, de1(63-32)1, de1(63-32)9, and the mutants in classes III, IV, and V were subjected to ultraviolet irradiation for varying times and plated on B. Comparison of the inactivation curves of the mutants to that for an r1589 control revealed no differences (data not presented). DISCUSSION

A number of deletions have been isolated which map in two regions of the T4 genome, one between genes 39 and 56, the other between genes 63 and 32. The deletion mutants grow on several of the comTABLE

7

STRAINS de1(63-32)7 AND 9 HETERODUPLEXEDTO de1(63-32)1-h2” Strain 7 9

Long arm

Short arm

3,919 * 173 3,877 zt 178

2,792 4z 113 2,874 + 259

“Measurements are averages of 9 and 11 molecules, respectively. The method for the calculations is similar to that described for Table 5.

DELETION

ANALYSIS

517

OF T4

FIG. 7. Heteroduplex of de1(63-32)7 to t&1(63-32)1 h2+. The loops marked H and r refer to the host range substitution loop and the r1589 loop, respectively. The loop marked D refers to the substitution loop formed as a result of the nonhomology between the deletions de1(63-32)l and 7.

mon laboratory strains of E. coli (B, BB, CR63, S/6) showing that the mutations delete functions dispensable for growth in these hosts. However, all the deletion mutants produce abnormal plaques, and the two mutants examined in a one-step growth experiment have a reduced burst size and increased eclipse and latent periods. This implies that the deleted genes are beneficial for growth in these hosts. The detection and characterization of nonessential genes is important to a total understanding of the bacteriophage, particularly since these genes may have somewhat different physiological roles and evolutionary histories than do essential genes. This existence of nonessential genes can be explained in several ways: (1) their products increase the efficiency of phage production through utilization of available host resources. One example is the T4 nucleases which allow the utilization of nucleotides derived from host DNA (Bose

TABLE

8

GENETIC MAPPING OF THE DELETION MUTATIONS Cross0

Amber in gene

Percent recombination*

de1(39-56112 x amN116 de1(39-56)12 x amE t&1(63-32)1 x amN54 de1(63-32)l x amE t&1(63-32)1 x A453 de1(63-32)l x amN 134 del(63-32)9 x amN54 de1(63-32)9 x amElO72 de1(63-32)9 x A453 de1(63-32)9 x amN134

39 56 31 63 32 33 31 63 32 33

10.2 9.4 18.0 11.5 12.5 15.5 20.8 11.2 5.1 9.7

0 All amber mutant strains except amE in gene 63 also contain the r1236 mutation. hThe cross lysates were plated on B and the percent recombination calculated as the number of large-plaque producers, representing recombinants, divided by the total am+ progeny. The small plaques produced by the leaky mutants in genes 39 and 63 were easily distinguished and were not counted.

518

HOMYKANDWEIL

and Warren, 1969; Hercules et al., 1971; Warner et al., 1972). (2) Their products supplement host functions to give a higher phage yield, as is the case for the phage enzyme thymidylate synthetase (Mathews, 1965). (3) Their products are nonessential in some host strains, but essential for growth on other hosts. An example of this is the tRNA genes of T4 (Wilson, 1973). Another example of the last type is the failure of de1(39-56)ll and 12 to plate on CTr5x. As can be seen in Fig. 2, the deletions in these strains extend approximately 600 base pairs further toward gene 39 than do any of the other deletions. De1(39-56)4 extends further toward gene 56 than does de1(39-56)ll and plates on CTr5x. These results suggest there is a gene between the left ends of de1(39-56)5 and de1(39-56)ll which is required for plaque formation on CTr5x. Angel Rodriguez (personal communication) has obtained a temperature-sensitive mutant, designated AR-S, of an E. coli K12 strain which fails to support the growth of deZ(39-56)3, 5, 11, and 12 at the restrictive temperature. The mutation in AR-8 also affects the growth of the cell at the restrictive temperature. In addition, he has isolated a T4 point mutation, termed cef, which does not recombine with the deletions in strains de1(39-56)3, 5, 11, and 12 and which also prevents growth of T4 on AR-8 at the restrictive temperature. Several of our deletion mutants have been studied in other laboratories to determine the presence or absence of known phage functions. Using enzyme assays, M. Behme and K. Ebisuzaki (personal communication) determined that of all 12 deletion mutants in the de1(39-56) class, only del(39-56) 1 produces a DNA-dependent ATPase (Debreceni et al., 1970). This places the gene for this enzyme between the rII-distal ends of de1(39-56) 1 and 8 (see Fig. 2). Warner et al. (1972) determined that de1(39-56)l through 4, the only ones tested, fail to produce exonuclease A. R. Depew and N. Cozzarelli (personal communication) have determined that del(39-56)2, 3,

and 4 are missing a 5’ phosphatase produced after T4 wild-type infection. J. Little has tested de1(39-56)1, 3, and 10 for the ability to allow growth of gene 32 amber mutants in ochre-suppressing hosts (sud phenotype; Little, 1973) and found that all three have this ability. In a preliminary study, R. Horvitz (personal communication) has determined that all members in the de1(39-56) class except de1(39-56)l and 8 show abnormally low levels of phosphorylation of the (Ysubunit of the host RNA polymerase. Several deletion mutants have also been examined for missing protein bands in polyacrylamide gels of infected cell lysates (O’Farrell et al., 1973 and personal communication; S. Block, personal communication). A 50,000-MW protein is missing from lysates of del(39-56)1, 2, 3, 4, and 5, and a 12,000-MW protein is missing from lysates of de1(39-56)1, 2, 3, and 5, but is present in lysates of de1(39-56)4. (Molecular weights are calculated from the values given by O’Farrell et al., 1973.) Two proteins whose genes are deleted by some member of the de1(39-56) class have been purified. The DNA-dependent ATPase has a molecular weight of 15,000 (Debreceni et al., 1970). Considering its molecular weight and pattern of occurrence, the DNA-dependent ATPase does not appear to be one of the three proteins identified in the polyacrylamide gel studies above. At the present, nothing in this regard can be determined about the T4 exonuclease A, although a MW of 40,000 has been quoted for T2 exonuclease A (Warner et al., 1972). Figure 8 summarizes the location of genes as determined from the studies outlined above. Although the data presented here do not indicate whether the genes for sud and for exonuclease A are identical (intervals 5 and 8 in Fig. S), there is other evidence that they are not (Little, 1973). Thus, at least seven genes are represented by the enzymes, proteins, and phage functions affected by these deletions. Considering the size of the known proteins coded for in this region and assuming an average MW of 30,000 for unidentified gene prod-

DELETION

ANALYSIS

ucts, it is probable that the deleted region includes about 10 genes. It was mentioned earlier that in heteroduplexes of de1(39-56)l to T2, a deletioninsertion loop approximately 3200 bases long replaces a deletion-insertion loop which appears in the normal T2/T4 heteroduplex. The T2/‘T4 deletion-insertion loop is the leftmost of the three loops that occur between positions 115 kb and 120 kb on the standard T2/T4 heteroduplex map (Fig. 7 of Kim and Davidson, 1974) and is approximately 1100 bases long (J. S. Kim, personal communication; unpublished observations). Considering that the deletion loop in de1(39-56)1/T4 wild-type heteroduplexes is 4200 bases long, these data show that this T2/T4 deletion-insertion loop is T4 DNA which is not present in T2. Using enzyme assays (Capco and Mathews, 1973) and plaque formation under special conditions (D. Hall, personal communication), it has been determined that de1(63-32)l is missing thymidylate synthetase but not dihydrofolate reductase, while de1(63-32)9 is deficient in both. Using plaque morphology, Hall has also determined that de1(63-32)l and 9 are probably deficient in ribonucleotide reductase activity. Enzyme assays demonstrate that de1(63-32)l is deficient in endonuclease II activity, while de1(63-32)9 has not been tested (E. M. Kutter, personal communication). Table 9 presents the order and calculated sizes of the known genes between genes 32 and 63. It will be seen that the genes determined to be missing in de&63-32)l or 9 account for at least 4800 base pairs of DNA, while about 8000 base pairs of DNA are deleted by these two mutations (see Fig. 6). Most or all of the DNA deleted by de1(63-32)l can be accou’n1ed for by the genes which it inactivatcp: However, de1(63-32)9 extends approxhately 3000 base pairs beyond de1(63-32)l in the direction of gene 32 (see Fig. 6), and we cannot account for all the DNA in this 3000-base pair region. If de1(63-32)l extends only a short way into the gene for thymidylate synthetase (td),

519

OF T4

I 4

, 2 ,

3 :

4 :

5

67

: fi(39-56)

r-f/. 0 4000

I

,

DISTANCE

,

, 8000

,

FROM

rl589

4 12

,

,

, 12.000

,

,

I

(IN BASE PAIRS)

FIG.8. Positionsof genes determinedfrom enzymes,proteins,and functions missing in de1(39-56)l to 12 and sudl. The lower line represents the extent of de1(39-56)12. The enzymes, proteins, and functions whose gene(s) are located within each interval are as follows: (1) a 5’-phosphatase; (2) a function necessary for plaque formation on CTr5x; (3) a function necessary for growth on AR-S; (4) a protein with MW = 12,000; (5) exonuclease A and a protein with MW = 50,000; (6) a DNA-dependent ATPase; (7) a function affecting the modification of host RNA polymerase and (8) sud. See text for details and references. Note that each interval represents not the interval within which the entire gene must lie, but rather an interval within which at least some part of the gene must lie.

then as much as 1600 of the 3000 base pairs deleted by del(63-32)9 could be accounted for by td and the gene for dihydrofolate reductase (frd). Since de1(63-32)9 does not appear to extend into gene 32, this leaves a minimum of 1400 base pairs of unknown function between genes td and 32. It has been pointed out that while de1(63-32)l and de1(63-32)9 grow equally well on B, as judged by plaque sizes, de1(63-32)9 produces much smaller plaques on CTr5x than does de1(63-32)l. Although inferences concerning the quality of growth made from plaque sizes should be considered with caution, it would be interesting to determine whether this difference is due to the frd gene or to some additional gene missing in del(63-32)9. De1(39-56) 12 and de1(63-32) 1 and 9 have also been examined for the phage RNA ligase reported by Silber et al. (1972). All three strains induced normal levels of this enzyme (J. Cranston and J. Hurwitz, personal communication). The mutations in classes III, IV, and V appear to contain either point mutations or

520

HOMYK AND WEIL TABLE 9 SUMMARY OF FUNCTIONS MISSING IN THE DEL(63-32)

Gene order”:

Gene 32

Dihydrofolate reductase (frd)

Thymidylate syn-

CLASS

Ribonucleotide Endonuclease reductase (nrdA)

(,lFdB)

thetase

Gene 63

11

(denA)

(td)

Molecular weight of gene product

Calculated’ gene length (base pairs) Presence or absence of gene product in:’ de1(63-32) 1 del(63-32)9

29,OOOb

29,ooo’

800

800

+ -

-

85,0006 + 35,0006 3,200 total -g -B

“Yeh and Tessman (1972); b Erickson and Mathews (1971); c Capco et al. (1973); d Berglund (1972). It is not known which gene codes for which products; e Calculated assuming an average molecular weight of 110 per amino acid residue. ‘See text for references: +, presence; - , absence. BIt is not known whether one or both products are missing.

deletions which are too small to be detected by heteroduplex analysis. Further investigation of mutations III and IV has shown that they are low-level suppressors of the exclusion of T4 rI1 mutants by lambda prophage and are in the same gene as the analogous mutations reported by Freedman and Brenner (1972) (Homyk, Rodriguez, and Weil, manuscript in preparation). We had initially hoped that the selection technique we used would yield deletions in many regions devoid of known essential genes. We found, however, that all our deletions lie in one of two regions. This is surprising since Wilson et al. (1972) reported isolating deletions of T4 tRNA genes by screening for suppressor-negative variants of a phage-coded amber suppressor. Their data also demonstrate that several lysozyme deletions extend for considerable distances outside the gene for this enzyme. In addition, Parma et al. (1972) and van de Vate and Symonds (1974) have isolated deletions using methods similar to ours, and some of their deletions appear to affect different regions of the phage genome than do ours (O’Farrell et al., 1973; Symonds and Wilson, personal communication). Several reasons can be suggested to account for our failure to isolate deletions in

more than two regions of the T4 genome: (1) Undetected essential genes exist in some regions. (2) Deletions of some sites have no effect on plaque morphology on B or BB and, therefore, escaped our examination. (3) Deletions of other regions occur, but the mutants suffer a severe growth disadvantage and constitute only a small fraction of the phage in the original selection plaques. (4) Deletions are produced at a much higher frequency in the regions between genes 39 and 56 and genes 63 and 32 than at other locations. Except for our failure to detect deletions in ten unselected rI1 duplication strains which produce normal plaques, the present study provides little information relevant to the first two points above. However, there is some information which favors points (3) and (4). First, in our sample, mutants in the de1(39-56) class were several times more frequent than mutants in the de1(63-32) class. Furthermore, there appear to. be preferential end points for deletions occurring in each class. Of the five independent deletions (numbers 6 through 10) chosen randomly from among mutants in the de1(39-56) class, three appear to be identical. Of 11 independent mutants examined in the de1(63-32) class, nine members form one sublcass containing identical deletions,

DELETION

ANALYSIS

OF T4

521

while the remaining two members form a Mr. Akitsugu Saito for assistance with electron misecond subclass containing identical dele- croscopy, Dr. Gisela Mosig for many helpful discustions. It is interesting to note that T4 rI1 sions and critical reading of the manuscript, and our many collaborators for making available unpublished deletions produced by nitrous acid mutadata. This research was supported by NSF grant genesis also appear to have preferred end- GB27602. points (Tessman, 1962; Bautz and Bautz, 1967), while this is much less the case for REFERENCES spontaneous rI1 deletions (Benzer, 1959; ARBER, W., and LATASTE-DOROLLE, C. (1961). ErDrake, 1970). weiterung des Wirtsbereiches des Bakteriophagen X Previous investigations of T4 rI1 duplicaauf Escherichia coli B. Pathol. Microbial. 24, tion mutants (Weil et al., 1965; Symonds 1012-1018. et al., 1972) have shown that when the BARNETT, L., BRENNER, S., CRICK, F. H. C., SHULMAN, lysate of a cross such as T4r1589 X T4r1236 R. G., and WAWS-TOBIN, R. J. (1967). Phase-shift is plated on K, the plaques which are and other mutants in the first part of the rIIB formed are very heterogeneous with respect cistron of bacteriophage T4. Phil. Trans. Royal Sot. to the rI1 duplications they contain. Many London 252, 487-560. of the plaques contain more than one BAUTZ, F. A., and BAUTZ, E. K. F. (1967). Mapping of deletions in a non-essential region of the phage T4 duplication, as distinguished by the fregenome. J. Mol. Biol. 28, 345-355. quency and ratio with which haploid segregants are produced. Studies by Weil and BAUTZ, E. K. F., and REILLY, E. (1966). Gene-specific messenger RNA: Isolation by the deletion method. Terzaghi (1970) and unpublished observaScience 151, 328-331. tions made in the present study also show BECKENDORF, S. K., KIM, J. S., and LIELAUSIS, I. that the plaques often contain a heteroge(1973). Structure of bacteriophage T4 genes 37 and neous population of deletion mutants. In 38. J. Mol. Biol. 73, 17-35. view of this heterogeneity, selection due to BENZER, S. (1959). On the topology of the genetic fine growth disadvantage may also be imporstructure. Proc. Nut. Acad. Sci. USA 45,1607-1620. tant in determining the frequency with BENZER, S. (1961). On the topography of the genetic fine structure. Proc. Nat. Acad. Sci. USA 47, which different deletion mutations occur in 403-415. these plaques. Therefore, selection could account for the inequality with which the BERGLUND, 0. (1972). Ribonucleoside diphosphate reductase induced by bacteriophage T4. J. Biol. two classes of deletions were obtained in Chem. 247, 7270-7275. this study as well as the absence of deleBOSE, S. K., and WARREN, R. J. (1969). Bacteriotions in other nonessential regions of the phage-induced inhibition of host functions II. Evigenome. dence for multiple, sequential bacteriophageThe analysis of deletion mutations reinduced deoxyribonucleases responsible for degraported here has appreciably increased our dation of cellular deoxyribonucleic acid. J. Viral. 3, knowledge of the regions between genes 39 549-556. and 56 and between genes 63 and 32, and BUJARD, H., MAZAITIS, A. J., and BAUTZ, E. K. F. (1970). The size of the rI1 region of bacteriophage further studies should add even more inforT4. Virology 42, 717-723. mation. This work, plus the results of Parma et al. (1972) and van de Vate and CAPCO, G. R., KRUPP, J. R., and MATHEWS, C. K. (1973). Bacteriophage-coded thymidylate syntheSymonds (1974) discussed above, indicate tase: Characteristics of the T4 and T5 enzymes. that the rII-duplication method of isolating Arch. Biochem. Biophys. 158, 726-735. deletion mutants is of general utility for CAPCO, G. R., and MATHEWS, C. K. (1973). Bacteriothe investigation of nonessential genes of phage-coded thymidylate synthetase. Evidence T4. that the T4 enzyme is a capsid protein. Arch. ACKNOWLEDGMENTS We thank Drs. Norman Davidson and Jung-Suh Kim for their assistance and hospitality in instructing one of us (T.H.) in the technique of electron microscope heteroduplex analysis, Dr. Jane Bibring and

Biochem. Biophys. 158, 736-743. CHAMPE, S. P., and BENZER. S. (1962). An active cistron fragment. J. Mol. Biol. 4, 228-292. DAVIDSON, N., and SZYBALSKI, W. (1971). Physical and chemical characteristics of Lambda DNA. In “The Bacteriophage A” (A. D. Hershey, ed.), pp. 45582.

HOMYK AND WEIL

522

Cold Spring Harbor Laboratory, New York. DAVIS, R. W., and HYMAN, R. W. (1971). A study in evolution: the DNA base sequence homology between coliphages T7 and T3. J. Mol. Bi01. 62, 287-301.

DEBRECENI,N., BEHME, M. T., and EBISUZAKI, K. (1970). A DNA-dependent ATPase from E. coli infected with bacteriophage T4. Biochem. phys. Res. Commun. 41, 115-121.

Bio-

DEPEW,R. E., and COZZARELLI, N. R. (1974). Genetics and physiology of bacteriophage T4 3’-phosphatase: evidence for involvement of the enzyme in T4 DNA metabolism. J. Viral. 13, 888-897. DOVE, W. F. (1968). The extent of rI1 deletions in phage T4. Genet. Res. 11, 215-219. DRAKE,J. W. (1970). “The Molecular Basis of Mutation.” Holden-Day, San Francisco. EDGAR,R. S., FEYNMEN,R. P., KLEIN, S., LIELAUSIS,I., and STEINBERG,C. M. (1962). Mapping experiments with r mutants of bacteriophage T4D. Genetics 47, 179-186. EDGAR,R. S., and WOOD,W. B. (1966). Morphogenesis of bacteriophage T4 in extracts of mutantinfected cells. Proc. Nat. Acad. Sci. USA 55,

90, 648-652.

MOSIG,G., and WERNER,R. (1969). On the replication of incomplete chromosomes of phage T4. Proc. Nat. Acad. Sci. USA 64, 747-754.

MUKAI, F., STREISINGER, G., and MILLER, B. (1967). The mechanism of lysis in phage T4-infected cells. Virology

33, 398-402.

O’FARRELL,P. Z., GOLD, L. M., and HUANG, W. M. (1973). The identification of prereplicative bacteriophage T4 proteins. J. Biol. Chem. 248, 5499-5501. PARMA, D. H., INGRAHAM,L. J., and SNYDER,M. (1972). Tandem duplications of the rI1 region of bacteriophage T4D. Genetics 71, 319-335. PARMA,D. H., and SNYDER,M. (1973). The genetic constitution of tandem duplications of the rI1 of bacteriophage T4D. Genetics 73, 161-183. RUTBERG,B. (1969). A class of hybrids between bacteriophages T4B and T4D. Virology 37,243-251. SEDEROFF, R., BOLLE,A., and EPSTEIN,R. H. (1971). A method for the detection of specific T4 messenger RNAs by hybridization competition. Virology 45, 440-455.

SILBER,R., MALATHI, V. G., and HURWITZ,J. (1972). 498-505. Purification and properties of bacteriophage T4EPSTEIN, R. H., BOLLE, A., STEINBERG,C. M., induced RNA ligase. Proc. Nat. Acad. 5%. USA 69, KELLENBERGER, E., BOY DELA TOUR,E., CHEVAL~EY, 3009-3013. R., EDGAR,R. S., SUSMAN,M., DENHARDT,G. H., STAHL, F. W., MODERSOHN, H., TERZAGHI,B. E., and and LIELAUSIS,I. (1963). Physiological studies of CRASEMANN, J. M. (1965). The genetic struct,ure of conditional lethal mutants of bacteriophage T4. complementation heterozygotes. Proc. Nat. Acad. Cold Spring 375-392.

Harbor

Symp.

Quant.

Biol.

28,

ERICKSON,J. S., and MATHEWS,C. K. (1971). T4 bacteriophage-specific dihydrofolate reductase: Purification to homogeneity by affinity chromatography. Biochem. Biophys. Res. Commun. 43, 1164-1170. Foss, H. M., and STAHL, F. W. (1963). Circularity of the genetic map of bacteriophage T4. Genetics 48, 1659-1672. FREEDMAN,R., and BRENNER,S. (1972). Anomalously revertible rI1 mutants of phage T4. Genet. Res. 19, 1655171. HERCULES,K., MUNRO, J. L., MENDELSOHN,S., and WIBERG,J. S. (1971). Mutants in a nonessential gene of bacteriophage T4 which are defective in the degradation of Escherichia coli deoxyribonucleic acid. J. Viral. 7, 95-105. KIM, J. S., and DAVIDSON,N. (1972). Electron microscope heteroduplex study of sequence relations of T2, T4, and T6 bacteriophage DNA’s Virology 57, 93-111. LI’ITLE, J. W. (1973). Mutants of bacteriophage T4 which allow amber mutants of gene 32 to grow in ochre-suppressing hosts. Virology 53, 47-59. MATHEWS,C. K. (1965). Phage growth and deoxyribonucleic acid synthesis in Escherichia coli infected by a thymine-requiring bacteriophage. J. Bacterial.

Sci. USA 54, 1342-1345.

STREISINGER,G., EMRICH, J., and STAHL, M. M. (1967). Chromosome structure in phage T4. III. Terminal redundancy and length determination. Proc. Nat. Acad. SC. USA 57, 292-295.

SYMONDS,N., VAN DEN ENDE, P., DURSTON,A., and WHITE, P. (1972). The structure of rI1 diploids of Phage T4. Mol. Gen. Genet. 116, 223-238. TESSMAN,I. (1962). The induction of large deletions by nitrous acid. J. Mol. Biol. 5, 442-445. VAN DE VATE, C., and SYMONDS,N. (1974). A stable duplication as an intermediate in the selection of deletion mutants of phage T4. Genet. Res. 23, 87-105.

WARNER,H. R., SNUSTAD,D. P., KOERNER,J. F., and CHILDS, J. D. (1972). Identification and genetic characterization of mutants of bacteriophage T4 defective in the ability to induce exonuclease A. J. Virol. 9, 399-407.

WEIL, J., and TERZAGHI,B. (1970). The correlated occurrence of duplications and deletions in phage T4. Virology 42, 234-237. WEIL, J., TERZAGHI,B., and CRASEMAN, J. (1965). Partial diploidy in phage T4. Genetics 52,683-693. WILSON,J. (1973). Function of the bacteriophage T4 transfer RNA’s J. Mol. Riot. 74, 753-757. WILSON,J. H., KIM, J. S., and ABELSON,J. N. (1972). Bacteriophage T4 transfer RNA. III. Clustering of

DELETION

ANALYSIS

the genes for the T4 transfer RNAs. J. Mol. Biol. 71, 547-556. WOLLMAN, E. L. (1953). Sur le determinisme genetique de la lysogenie. Ann. Inst. Pasteur 84, 281-293. WOOD, W. B. (1974). Genetic map of bacteriophage

OF T4

523

T4. In “Handbook of Genetics” (Robert C. King, ed.), Van Nostrand-Reinhold Co., New York. YEH, Y. C., and TESSMAN, I. (1972). Control of pyrimidine biosynthesis by phage T4. II. In uitro complementation between ribonucleotide reductase mutants. Virology 47, 767-772.