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Nonsense suppressor mutants of bacteriophage BF23
VIROLOGY
90,
133-141
(19%)
Nonsense KIYOTAKA Department
Suppressor OKADA,’ of Biophysics,
Mutants of Bacteriophage
MASAKO Faculty
OHIRA,2
of Science,
Accepted
June
Kyoto
AND HARUO University,
Kyoto
BF23 OZEKI 606, Japan
19, 1978
Nonsense suppressor mutants (bfsu’) of bacteriophge BF23 were isolated as phenotypically wild-type revertants from multiple amber (UAG) mutants. These revertants were found at a frequency expected for a single-base mutation (IO-‘), and backcrosses of them with wild-type phage yielded progeny of amber phenotype carrying the original amber markers. From those bfsu’ strains, suppressor-negative mutants due to either deletion or point mutation were isolated. Some of the deletion mutants lacking a suppressor gene still gave rise to other suppressor mutations. By using those bfsu’ strains and their derivatives, genetic analysis was performed. Three distinctive suppressor genes, bfsul’, bfsu2’, and bfsu3+, were identified, and all of them were mapped together on a dispensable region of the phage genome. These suppressors were general ones capable of suppressing amber mutations in various phage genes. However, amber mutations of the genes on the first-steptransfer (FST) segment of phage DNA appeared not to be suppressed. This phenomenon is discussed in terms of the mode of infection specific to this phage. Inasmuch as it has been known that BF23 carries Wteen or more tRNA genes (Ikemura, T., and Ozeki, H. (i975). Eur. J. B&hem. 57, 117-127), the phage suppressors are most likely to be attributed to these phage tRNAs.
Chen et al., 1976; Hunt et al., 1976). Using two-dimensional polyacrylamide gel electrophoresis, Ikemura and Ozeki (1975) separated more than 15 species of 4 S RNA encoded by BF23. Most of these RNA species contained pseudouridine and ribothymidine in their molecules, and some had similar fingerprinting patterns to those of corresponding tRNA species of T5. This information indicates that BF23 also carries tRNA genes on its genome. A pertinent question is whether these BF23 tRNAs are biologically active. In order to solve this question, attempts have been made to isolate nonsense suppressor mutants. This paper describes the isolation and genetic analysis of BF23 nonsense suppressor mutants (designated bfsu’) active on the amber mutations of this phage. Evidence is also presented showing that the nonsense suppressor genes are dispensable for phage propagation. Preliminary results of this work have been reported (Okada, Ohira and Ozeki, 1973).
INTRODUCTION
Bacteriophge BF23 is a virulent coliphage closely related to T5. Both phages readily recombine with each other and have similar genetic maps in spite of the difference in antigenicity and in receptor site on the host cells (Nishioka and Ozeki, 1968; Mizobuchi et al., 1971; Hendrickson and McCorquodale, 1971). It has been reported that several virulent phages have tRNA genes on their genome. For example, in T4, eight tRNA genes were identified and some of them exhibited nonsense suppressor activity by mutation (McClain, 1970; Wilson and Kells, 1972; McClain et al., 1973; Comer et al., 1974). In the case of T5, at least 14 kinds of tRNA genes were identified and their corresponding genes were mapped on the chromosome of this phage (Scherberg and Weiss, 1970; ’ Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan. *Present address: Research Laboratories of Biotechnology, Mochida Pharmaceutical Co., Ltd., Kamiya l-1-1, Tokyo 115, Japan.
MATERIALS
Media.
Nutrient
AND METHODS
broth
and
nutrient
133 0042~6822/78/0901-0133$02.00/0 Copyright 0 1978 by Academic All righb of reproduction in
Press,
any form
Inc. reserved.
134
OKADA,
OHIRA,
broth containing 1 x lo-” M CaC12 were used for the growth of bacteria and phages, respectively. Phage adsorption buffer was prepared according to Mizobuchi et al. (1971). Bacterial and phage strains. Escherichia coli strains CR63 (sul’) and 594 (sustr’ gal-) were used as permissive and nonpermissive hosts, respectively, for the amber phages. The phage strains used are listed in Table 1. Wild-type BF23 and its amber mutants, amH30, am57, am91, aml59, am177, and am218, were kindly supplied by Dr. Mizobuchi. The order of these markers on the phage DNA is presented at the bottom of Table 1 (Mizobuchi et al., 1971, and Mizobuchi, personal communication). Two markers, amH30 and am57, have been mapped within the 8% segment TABLE BACTERIOPKAGE Strain
STRAINS
Genotype
Wild-type am57 am91 am159 am177 am218 amH30 am9lam159 am91am218 aml77am218 Xl x2 Yl Y2 Zl 22 del-1 del-2 del-3 del-4 del-5 del-6 deL4rl del-4r2 X1-163 X1-214 x1-2110 (amH30 \ 1
1 BF23
am57 am91 am159 am177 am218 amH30 am91 am91 am177 bfsul’ bfsul+ bfsul+ bfsu2+ bfsu3’ bfsul’ bfsulbfsulbfsulbfsu2bfsu2bfsu3bfsul’ bfsul’ bfsulbfsulbfsulam571am91 / The
order
am159 am218 am218 am177 am177 am91 am91 am91 am91 am177 am177 am177 am91 am91 am91 bfsu2bfsu2am177 am177 am177
am218 am218 am218 am218 am159 am159 am218 am218 am218 am218 am218 am159 am91 am218 am91 am218 am218 am218 am218
am218
am177 1
of the markers
used
am159
AND
OZEKI
of BF23 DNA that is transferred to the host cell first. This segment is designated the first-step-transfer (FST) segment by analogy to T5 (Lanni, 1968). Strains bearing multiple amber mutations were constructed by crossing the appropriate amber mutants. Isolation of nonense suppressor mutants. For isolation of the nonsense suppressor mutants of BF23, about 1 x 10’ of appropriate double amber mutants were plated on SU- cells. The rare phenotypically wild-type phages were isolated and purified by single plaque isolation. In order to distinguish the nonsense suppressor mutation from true reversion, the phenotypically wild-type phages were back-crossed with wild-type phage on su+ cells, and then examined to determine whether or not the original amber markers had segregated (see text). The six bfsu’ mutants listed in Table 1 were independently isolated in this manner. Isolation of suppressor defective mutants. Nonsense suppressor-defective mutants, designated bfsu-, were obtained by treatment of nonsense suppressor mutants with ethylmethanesulfonate (EMS) according to the method of Tessman et al. (1964). The mutagenization was stopped by dilution at a survival at 10e4 and the surviving phages were plated on a mixture of indicator cells, one part of SU+ and 20 parts of SK. Several plaques showing the amber phenotype (small turbid plaques) were obtained from about 3000 survivors. These were purified, and their amber markers were examined by complementation tests. Three bfsu- mutants, carrying the amber markers as in the parental phage, were obtained and named X1-163, X1-214, and Xl2110 (Table 1). Complementation test of amber mutants. One drop of amber phage lysate containing about lo8 PFU/ml was spotted on a plate seeded with su- cells, and after it had soaked in thoroughly, one drop of another phage lysate was spotted on the same area. After an overnight incubation at 37”, lysis of the su- cells in the overlayered spot was taken as evidence that complementation had occurred between the two amber mutants tested. Genetic cross of nonsense suppressor
NONSENSE
SUPPRESSORS
mutants. The procedure used for genetic crosses was that of Mizobuchi et al. (1971). The crosses were performed on the SU+ cells of CR63, so that the recombinants, as well as the parental phages, could grow well regardless of their geneotypes. RESULTS
Isolation
of bfsu+ Mutants
As the first step for detecting nonsense suppressor mutations in BF23, we constructed double amber mutants by crossing single amber mutants, and then measured the apparent reversion frequency of these multiple mutants on su- cells. Since the single amber mutants used in the experiment reverted to amber+ at a frequency of 10-5-10-6 (Table 2)) the reversion frequency of the double amber mutants was expected to be 10-10-10-12. Accordingly, if the reversion frequency of a double amber mutant was significantly higher than the expected value, it was thought that the reversion of its phenotype to wild-type was more likely to be due to a nonsense suppressor mutation which had occurred on the phage genome, rather than due to simultaneous reversions of the two amber markers. Table 2 shows the apparent reversion frequency of various multiple amber mutants. The TABLE
2
REVERSIONFREQUENCYOFAMBERSTRAINS Strain
Reversion
frequency”
am91 1.6 x 10 4 am159 9.0 x lo-” am177 3.1 x 10 ‘, am218 4.6 x 10 R am57 2.2 x IO Ii amH30 3.8 x 10 ’ aml77am218 3.4 x 10~’ am91am218 5.8 x 10 ’ am9lam159 4.0 x 10 i am57amH30
IN
PHAGE
135
BF23
three double amber mutants, am177am218, am91am218, and am91am159, reverted at a frequency of approximately 10p7. These values were lC?-104-fold higher than the expected ones. When triple or quadruple amber mutants were examined, the reversion frequency was still the same as that of double amber mutants (Table 2). These results suggested that the phenotypic reversion was due to a single suppressor mutation which was capable of suppressing all those amber mutations. It is worthy of note that the reversion frequency of mutatants carrying an amber mutation in the firststep-transfer (FST) segment (amH30 or am57) was low enough to be characteristic of the expected frequency for a double reversion, indicating that these were not due to suppression (Table 2). It appears, therefore, that the phage suppressor was not active on either am57 or amH30. Possible explanation for this phenomenon are discussed later. In order to confirm that the phenotypitally wild-type revertants from multiple amber mutants were due to nonsense suppressor mutations, the genotype of six independently isolated revertants, Xl, X2, Yl, Y2, Zl, and 22 (see Table l), was analyzed by backcross with wild-type phage. The parental strains of these revertants were am177am218 for Xl and X2, am91am218 for Yl and Y2, and am91am159 for Zl and 22, respectively. Table 3 shows the result of such a cross performed between Xl and wild type. It can be seen that strain Xl segregated recombinants carrying am177 and/or am218. TABLE CROSSBETWEEN _____-Number of progeny amber’ pt%r
-~~.-_--___.~~_
-.-___ 650
3
STRAIN XIANDWILD-TYPE PHAGE" ~.. .__~~ Phenotype -.-___ am177
am218
amli7am218 .__
557 27 20 46 __.__..___~~_..___ ” Strain X1 was crossed with wild-type phage on su+ cells, and progeny phages produced were plated on su+ cells. Plaques formed were stabbed on su and su+ cells using sterilized toothpicks. The genotypes of recombinants showing amber phenotype were checked by complementation tests with the strains carrying either om177 or am218.
136
OKADA,
OHIRA.
This observation indicated that Xl still retained the original amber markers. Similar results were obtained with five other revertants (data not shown). From these observations, it was concluded that, in these six strains, suppressor mutations did take place on the chromosome of BF23 to give rise to phenotypicalIy wild-type phages. The nonsense suppressor gene and its wildtype allele are designated as bfsu’ and bfsu’, respectively. The burst size of these suppressor strains on su- cells was reduced to some extent, compared with that of wild-type phage, about 40% in strain Xl, about 30% in Y2, and about 5% in Zl, respectively.
AND
OZEKI
(7.6%) or between (Table 4).
b&2+
and bfsu3’
Isolation of Deletion Mutants Suppressor Activity
Lacking
(3.5%) the
Hertel et al. (1962) isolated deletion mutants of bacteriophage T5 from survivors after heat treatment at 50” for 60 min in 0.4 M NaCl containing 0.01 M sodium citrate, pH 6.8. We have attempted to search for conditions to efficiently select deletion mutants of BF23, and found that heat treatment at 60” in 0.02 M Tris-HCl, pH 8.5, with 0.02 M EDTA was most effective. This procedure was originally used for the isolation of deletion mutants of bacteriophages A and $30 (Yamagishi and Ozeki, Classification of bfsu+ Suppressors 1972). After heat treatment of phage particles of bfsu’ strains for 20 min, survivors In order to determine whether the inde(10-6) forming plaques on su+ cells were pendently isolated bfsu’ suppressors were picked and tested for their amber phenothe same or not, crosses between the bfsu’ type. Among these survivors, deletion mustrains were carried out on su+ cells, and tants which were associated with the loss the phenotype of the progeny phages was of the suppressor activity were found at a analyzed on su- and su+ cells. The results frequency of 5 X lo-“. Six mutants were are summarized in Table 4. Crosses beindepenently isolated from Xl, Y2, or Zl, tween Xl and Y2 or 21, and between Zl and designated del-1 to del-6 (Table 1). and X2, Y2 or 22 yielded significant numof these bers of amber recombinants; whereas in Table 5 shows some properties deletion mutants. When complementation crosses between Xl and X2, Yl or 22, such tests were performed, the respective delerecombinants could not be detected. On t,he tion mutants were shown to carry two ambasis of these results, the suppressors in the ber markers, which were the same as those six bf;Fu+ strains were tentatively classified into three groups, namely, bfsul+ in strains TABLE 5 Xl, X2, Yl, and 22, bfsu2+ in Y2, and REVEKSION FREQUENCY ANI) BUOYANT DENSITY OF bfiqu3’ in Zl. The gene order of these supDEIXTIOP; MUTANTS pressors is presumably bfsul’ - bfsu2’ ____ ~_--_-~-l’arental hll,). Reverwn frr- Hurwlnt denStrain bfsu3+ since the genetic distance observed pressrx strain q”rncy” (g/ml) -- sit?.” __~ between bfsul’ and bfsu3+ (8.7 - 13.7%) 1.551 Wild-type was further than between bfsul+ and bfsu2’ TABLE REcOMRINATION -Ck-03s _____ Xl x1 x1 x1 Xl Zl Zl Zl
x x x x x x x x
x2 Yl 22 Y2 Zl x2 Y2 Z2
4
BETWEEN SUPPRESSOR ______ STRAINS Number of amber Number of’ progmy phages tested phages (hfsu ) detected F<) -_----204 0 200 0 222 0 368 %a (7.6) 48 (13.7) 350 263 23 (8.7) 286 10 (3.5) 270 29 (10.7)
%.4 x 10 ; 1.54’1 del- 1 Xl del-% Xl “.O x 10 ; 1.537 7.6 x 10 * 1.540 (id9 X1 2.8 x IO ” Y2 1.544 rlel-4 Y8 6.9 x 10 L( 1.540 rleI-5 2.2 x 10; 1.543 clel-6 Zl _ _ ----~~-” Reversion frequency was calculated as described in the footnote of Table 2. “Buoyant density was measured by CsCl equilibrium density gradient centrifugation; 2.5 g of CsCl was dissolved in 2.9 ml of phage suspension, and centrifuged at 35,000 rllm for 20 h at 20°C in SW50-1 rotor (Spinco). As references, bacteriophage h (p = 1.508), hb2b5(0 = 1.483) (Kellenberger-Gujer and Weisberg, 1971) and 1’5(~, = 1.654) (Rubenstein, 1968) were used.
NONSENSE
SUPPRESSORS
of the corresponding parental bfsu’ strains. The buoyant densities of the mutants, which were measured in CsCl equilibrium density gradient centrifugation, were less than that of wild-type phage (Table 5). Furthermore, these mutants were more resistant to heat inactivation than wild-type BF23 (data not shown). The decreasing order of phage density, namely, wild-type > del-4 > del-6 > de&l > del-3 = del-5 > del-2, correlated well with the increasing order of phage stability. Inasmuch as an exceedingly good correlation between DNA content, phage density, and phage stability has been shown in T5 (Hertel et al., 1962) or in X (Parkinson and Huskey, 1971), we concluded that the mutation was due to the deletion of a part of phage genome. Direct measurements of molecular weight of DNA extracted from these deletion mutants by agarose gel electrophoresis later confirmed this conclusion, and showed that del-1,2,3, 4, 5, and 6 deleted 7, 10, 8, 6, 9, and 7% of the whole genome, respectively (Okada, manuscript in preparation). To test whether the bfsu’ gene was deleted in the mutants, they were crossed with the respective bfsu’ strains. As shown in Fig. lA, it is expected that recombinant-s with the suppressor activity would be obtained if a deletion mutant still carried the mutation site for bfsu+, but the expression of suppressor activity was inhibited somehow by the deletion (e.g., lack of the promoter region). Conversely, if the mutation site itself was deleted, no such recombinants would be produced (Fig. 1B). Table 6 presents the results of the crosses. When bfsu”
am1
am2
am1
am2
bfsuo
am1
am2
(bfsu+)
am1
am2
FOG. 1. Diagrammatic representation of a cross between deletion mutants and bfsu’ double amber strains. Two alternative cases are shown: one (A) is that the bfsu+ gene is retained on the chronosome of a deletion mutant, and the other (B) is that the bfsu’ gene is deleted. The wavy region represents the deleted region.
IN
PHAGE
137
BF23 TABLE
6
CROSS BETWEEN DELETION MUTANTS PARENTAL STRAINS Cross del-1
del-2 del-3 de14 del-5 del-6
x x x x x x
am177am218 aml77am218 aml77am218 am91am218 am91am218 am91am159
AND
bfsu”
Phenotypical amber+ progeny phages produced”__~
fl Frequency of phages of amber+ phenotype was calculated as the number of plaque forming units on sum cells as compared with that on su+ cells.
del-1 was crossed with am177am218, the phenotypicahy wild-type phages were detected only at a frequency comparable to the reversion frequencies of both parental phages (see Tables 2 and 5). Accordingly, no positive evidence was obtained indicating that the mutation site of suppressor bfsul+ was retained in the del-1 strain. Similar results were also obtained in the rest of the deletion mutants, except for del-5. In the case of del-5, bfsu‘ recombinants were formed at a frequency of 2.0 x 10e4, indicating that del-5 still retained the bfsu2+ gene, though suppressor activity was not expressed. The frequency of reversion to phenotypically wild-type in each deletion mutant varied from 6.9 x lo-* to 2.8 X 10V6 (Table 5). Considering that these values are in the same magnitude as those of the bfsu strains (see Table 2)) the phenotypic reversion was presumably due to the second suppressor mutation. If this was the case, all of the bfsu’ genes should have been deleted in none of these six strains; some of them should have been retained. In order to confirm this possibility, the following experiments were performed. Second Suppressor Mutation Occurring in a Deletion Mutant (del-4) In order to detect the remaining bfsu’ genes in the suppressor-negative deletion mutants, phenotypic revertants were again isolated by plating the deletion mutants on su- cells and their genotypes were analyzed. Two revertants, designated del-4rl and del4r2, were isolated from del-4. The buoyant
138
OKADA.
OHIRA,
density of these revertants was exactly the same as that of del-4 (data not shown). By crossing with wild-type, amber recombinants carrying am91 and/or am218 were produced among the progeny phages. Thus, it was confirmed that these two revertants still carried the same amber markers as those of del-4. These observations, therefore, indicated that the reversion resulted from the second suppressor mutation which was apparently different from the original bfsu2’. When deldrl or del-4r2 were crossed with either Y2 or Zl, recombinants of amber phenotype were obtained, whereas no such recombinants have so far been detected in the crosses with Xl (Table 7). It appears, therefore, that the sites of the second suppressor mutations occurring in del-4rl or deL4r2 were different from bfsu2’ and bfsu3+, and possibly identical or very close to bfsul+. In strain del-4, therefore, the bfsu2’ gene is presumably deleted, whereas the bfsul” gene appears to be retained. Isolation
of bfsu--defective
Point Mutants
In order to further characterize the nonsense suppressor genes, we have isolated derivatives of a bfsu’ strain which had lost the suppressor activity by point mutation ( bfsu-). After mutagenization of strain Xl (bfsul+) with EMS, phages of amber phenotype were selected, and their genotypes were tested (see Materials and Methods). Three bfsu- mutants, X1-163, X1-214, and X1-2110, were isolated which were confirmed by complementation tests as carrying the same amber markers, am218 and am177, as those in strain Xl. Incidentally, some other strains were found to carry new amber mutations which were not suppressed with bfsul+, but with a host suppressor. They could be attributed to amber mutations in the FST genes, although no further experiments have been done to clarify this point. Table 8 shows the result of crosses made between these bfsu- mutants and suppressor-negative deletion mutants, measuring the frequency of appearance of bfsu+ recombinants. When X1-163 was crossed with de&l, del-2 and del-3, the bfsu’ recombinant was not detected with a frequency
AND
OZEKI TABLE
7
CROSSBETWEEN bfsu’ STRAINSAND PHENOTYPICALLY~ILD-TYPEREVERTANTSFROM del-4 cross
deL4rl deL4rl del-4rl deL4r2 deL4r2 de14r2
x x x x x x
X1 Y2 Zl Xl Y2 Zl
Number of progeny phages tested
Number of amber phages
660 647 648 648 648 648
0 3 2 0 8 5
higher than the reversion frequency of parental phages (see Table 5), although such a recombinant was obtained with a much higher frequency in the cross with del-4. Similar results were obtained with X1-214 and with X1-2110. These data suggested that the mutation sites of these bfsustrains were covered by the deleted regions of the phage genome in strains del-1, del-2, or del-3, but not in del-4. These bfsu- strains were crossed with each other, and also with the original bfsu” strain (am177am218). Bfsu’ recombinants were found at a frequency of lo-” in all the combinations. The results are summarized in Fig. 2. The relative position of the mutation sites, including the original mutation site of bfsul+ in the bfsul gene, was determined on the basis of recombination frequencies, which are indicated as the distances between the sites in Fig. 2. These results indicate that the three bfsu- strains still carry the original mutation site of bfsul+, but are defective by a mutation at other sites. These second mutation sites in the three strains are different from each other, but all of them are located very close to the bfsul+ site, possibly within the bfsul gene. Several suppressor-positive derivatives of these bfsu- strains, obtained by spontaneous reversion, were isolated and crossed with strains Xl (bfsul+), Y2 (bfsu2’), and Zl (bfsu3’). No amber-phenotype recombinant was detected in the cross with Xl, whereas recombinants were obtained with Y2 and Zl to the same extent as in the crosses of Xl x Y2 or Xl X Zl shown in Table 4 (data not shown). These results
NONSENSE
SUPPRESSORS TABLE
CROSSBETWEEN
bfsu- strain
Reversion quency
4.3 x 1o-8 1.9 x 1o-7 7.1 x lo-’
X1-214
PHAGE
139
BF23
8
bfsu--DFNwrIvE MUTANTSANDDELETIONMUTANTS frebfsu’ recombinants per total progeny del-I
X1-163 X1-214 x1-2110
IN
del-2
1.0 x lo-’
X1-163
My/l
del-3
in the cross with
del-4 8.0 x 1o‘4 2.0 x lo-” 1.2 x lo-,’
Xl-~10
FIG. 2. Mutation sites in bfsul- mutants. Three bfsul- strains, X1-163, X1-214, and X1-2110, were crossed with each other, and also with bfsul’ am177 am218. The numeral shows the recombination frequency between the two mutation sites: 2 x bfsu+ recombinants per total progeny phages x 100 (S). bfsul indicates the original mutation site of bfsul+. X1-214, X1-163, and X1-2110 are the mutation sites of these bfsul- strains, by which bfsul’ suppressor became inactive.
indicated that the suppressors in the bfsu+ derivatives of bfsu- were bfsul+, and not bfsu2+ or bfsu3+. Taking all these lines of evidence together, we conclude that the three suppressor-negative strains, del-1, del-2, and del-3, have deleted the bfsul+ gene itself, ,but strain del-4 still carries the bfsul’ gene. DISCUSSION
We have presented genetic evidence indicating that the phenotypic reversion of multiple-amber mutants of BF23 is due to suppressor mutations occurring on the phage genome. Three different suppressors are identified. All of them are not genespecific but general suppressors capable of restoring the amber mutations of various phage genes. They are considered to be amber-suppressors, but the possibility of ochre-suppression still remains because an o&ire-suppressor is known to suppress not only ochre but also amber mutations. In general, suppression of an amber mutation by ochre-suppressors is less efficient than that by amber-suppressors, In this connection, bfsu3’ which shows smaller burst sizes than the other two mutations might be considered as a possible case of an ochresuppressor. This possibility, however, has not yet been examined, because ochre-type
nonsense mutants are unfortunately not available in BF23 at present. Although the nonsense suppressors described in this paper are general ones active on various amber markers of the phage, no suppressor mutant was obtained from the multiple-amber mutants carrying either am57 or amH30. It appears, therefore, that the bfsu+ suppressors are not active on these two markers, which are located on the first-step-transfer (FST) segment of phage DNA. A possible explanation is as follows. Mizobuchi et al. (1971) reported that BF23 DNA is injected into host cells in a two-step manner; namely, the 8% segment (FST) of the DNA molecule in the first step, and the rest in the second step. The second step of DNA transfer was shown to be dependent on synthesis of the pre-early proteins which were encoded by the genes located on the FST segment. Genetic analysis performed by Mizobuchi (unpublished results) revealed that the deleted regions of the suppressor-negative deletion mutants, described in this paper, were located between am91 and am218 on the map shown in Table 1. Thus, the nonsense suppressor genes were shown to be located on the DNA segment transferred in the second step, but urn57 and umH30 were on the FST segment. Accordingly, the fail-
140
OKADA,
OHIRA.
ure of suppression of FST genes by the phage suppressor may be attributed to the failure of transfer of the suppressor genes into the host cells. Moreover, even after complete transfer of the whole DNA molecule, expression of the early (and late) genes, presumably including the suppressors, may also be dependent on expression of a pre-early gene(s) on the FST segment. It has been suggested from the results of transfection experiments that expression of am57 is required for expression of the early and late genes (Benzinger and McCorquodale, 1975). In either case, the failure in selecting suppressor mutants with the amber mutations on the FST segment may be explained by the absence of expression of suppressor characters. In this connection, we are now attempting to construct a mutant carrying the am57 (or amH30) and bfsu+ gene to examine the expression of suppressor activity in the transfection system of BF23 DNA. All the suppressor mutants isolated can grow on su- as well as on su+ bacteria, and the phenotypes of the suppressor genes are always the same, simply “amber suppression,” regardless of the genes. Therefore, it is difficult to distinguish by genetic complementation tests whether two independently isolated suppressors are allelic or not. In the present study, this difficulty was circumvented by an alternative procedure. That is the isolation of second suppressors from strains with deletions which lack the first suppressor gene. The reasoning was that if a second suppressor were detected, it should be different from the first one. Combining this information with the results of genetic recombination tests performed with the suppressor mutants, three distinctive suppressor genes in BF23 were identified. Because BF23 carries a considerable number of tRNA genes (Ikemura and Ozeki, 1975), the suppression may be understood in terms of suppressor tRNAs. In order to test this, analysis of phage tRNAs has been carried out in our laboratory by using the suppressor-negative deletion or point mutants described in this paper. In strain del-5, for instance, the majority of the phage-specified tRNAs were not pro-
AND
OZEKI
duced, indicating that many tRNA genes are clustered together on the phage genome. One of the tRNAs is assigned to the bfsul+ gene product (Ikemura et al., 1978). Inasmuch as the b/K deletion mutants are viable, the BF23 tRNA genes must be dispensable for phage growth at least on E. coli K12. Therefore, the mutation of tRNA genes to suppressor may also be permissive. This in turn suggests that the phage tRNAs are dispensable but still produced in a biologically active form. The observed frequency of a suppressor mutation was roughly 10e7 (Table 2), and this is the value expected for a single-base mutation occurring at a given site. Accordingly, a pertinent explanation for the phage suppression would be as follows. Among the BF23 tRNAs, there are some whose anticodons are convertible by a single-base mutation to amber (or ochre) type. In the present study, at least three such genes (bfsul”, bfsu2’, and bfsu3’) were revealed. Nonsense suppressors have also been isolated in phage T5 (D. Botstein, personal communication). Thus, in all the three phages, T4, T5, and BF23, which are known to code phage-specified tRNA genes, nonsense suppressor mutations have been detected. ACKNOWLEDGMENTS We thank the members of the Laboratory of Molecular Biology, Department of Biophysics, Kyoto University, for useful discussions, and Drs. K. Mizobuchi and T. Nagata for critical reading of the manuscript. This work was supported by a grant from the Ministry of Education of Japan. REFERENCES BENZINGER, R., and MCCORQUODALE, D. J. (1975). Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes. J. Viral. 16, l-4. COMER, M. M., GUTHRIE, C., and MCCLAIN, W. H. (1974). An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA. J. Mol. Biol. 90,665-676. CHEN, M-J., LOCKER, J., and WEISS, S. B. (1976). The physical mapping of bacteriophage T5 transfer RNAs. J, Biol. Chem. 251,536-547. HENDRICKSON, H. E., and MC~ORQUODALE, D. J. (1971). Genetic and physiological studies of bacteriophage T5. I. An expanded genetic map of T5. J. Viral. 7, 612-618.
NONSENSE
SUPPRESSORS
HERTEL, R., MARCHI, L., and MUELLER, K. (1962). Density mutants of phage T5. Virology 18.576-581. HUNT, C., HWANG, L-T., and WEISS, S. B. (1976). Mapping of two isoleucine tRNA isoacceptor genes in bacteriophage T5 DNA. J. Virol. 20,63-69. IKEMURA, T., and OZEKI, H. (1975). Two-dimensional polyacrylamide-gel electrophoresis for purification of small RNAs specified by virulent coliphages T4, T5, T7 and BF23. Eur. J. Biochem. 51, 117-127. IKEMURA, T., OKADA, K., and OZEKI, H. (1978). Clustering of transfer RNA genes in bacteriophage BF23. Virology 90, 142-146. KELLENBERGER-GUJER, G., and WEISBERG, R. A. (1971). Recombination in bacteriophage lambda. I. Exchange of DNA promoted by phage and bacterial recombination mechanisms. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 407-415, Cold Spring Harbor Laboratory, New York. LANNI, Y. T. (1968). First-step-transfer DNA of bacteriophage T5. Bacterial. Rev. 32, 227-242. MCCLAIN, W. H. (1970). UAG suppressor coded by bacteriophage T4. FEES Lett. 6,99-101. MCCLAIN, W. H., GUTHRIE, C., and BARRELL, B. G. (1973). The psu,+ amber suppressor gene of bacteriophage T4: Identification of its amino acid and transfer RNA. J. Mol. Biol. 81, 157-171. MIZOBUCHI, K., ANDERSON, G. C., and McCORQUODALE, D. J. (1971). Abortive infection by bacteriophage BF23 due to the colicin Ib factor. I:
IN PHAGE
141
BF23
Genetic studies of nonrestricted and amber mutants of bacteriophage BF23. Genetics 68.323-340. NISHIOKA, T., and OZEKI, H. (1968). Early abortive lysis by phage BF23 in Escherichia coli K-12 carrying the colicin Ib factor. J. Viral. 2.1249-1254. OKADA, K., OHIRA, M., and OZEKI, H. (1973) Suppressor genes of phage BF23. Jap. J. Genet. 48, 439 (Abstr.). PARKINSON, J. S., and HUSKEY, R. J. (1971). Deletion mutants of bacteriophage lambda. I. Isolation and initial characterization. J. Mol. Biol. 56, 369-384. RUBENSTEIN, I. (1968). Heat-stable mutants of T5 phage. I. The physical properties of the phage and their DNA molecules. Virology 36, 356-376. SCHERBERG, N. H., and WEISS, S. B. (1970). Detection of bacteriophage T4- and T5-coded transfer RNAs. hoc.
Nat.
Acad.
Sci. USA
67.1164-1171.
TESSMAN, T., PODDAR, R. K., and KUMAR, S. (1964). Identification of altered bases in mutated singlestranded DNA. I. In vitro mutagenesis by hydroxylamine, ethylmethanesulfonate, and nitrous acid. J. Mol.
Biol.
9, 352-363.
WILSON, J. H., and KELLS, S. (1972). Bacteriophage T4 transfer RNA. I. Isolation and characterization of two phage-coded nonsense suppressors. J. Mol. Biol.
69, 39-56.
YAMAGISHI, H., and OZEKI, H. (1972). Comparative study of thermal inactivation of phage $80 and lambda. Virology 48, 316-322.
90,
133-141
(19%)
Nonsense KIYOTAKA Department
Suppressor OKADA,’ of Biophysics,
Mutants of Bacteriophage
MASAKO Faculty
OHIRA,2
of Science,
Accepted
June
Kyoto
AND HARUO University,
Kyoto
BF23 OZEKI 606, Japan
19, 1978
Nonsense suppressor mutants (bfsu’) of bacteriophge BF23 were isolated as phenotypically wild-type revertants from multiple amber (UAG) mutants. These revertants were found at a frequency expected for a single-base mutation (IO-‘), and backcrosses of them with wild-type phage yielded progeny of amber phenotype carrying the original amber markers. From those bfsu’ strains, suppressor-negative mutants due to either deletion or point mutation were isolated. Some of the deletion mutants lacking a suppressor gene still gave rise to other suppressor mutations. By using those bfsu’ strains and their derivatives, genetic analysis was performed. Three distinctive suppressor genes, bfsul’, bfsu2’, and bfsu3+, were identified, and all of them were mapped together on a dispensable region of the phage genome. These suppressors were general ones capable of suppressing amber mutations in various phage genes. However, amber mutations of the genes on the first-steptransfer (FST) segment of phage DNA appeared not to be suppressed. This phenomenon is discussed in terms of the mode of infection specific to this phage. Inasmuch as it has been known that BF23 carries Wteen or more tRNA genes (Ikemura, T., and Ozeki, H. (i975). Eur. J. B&hem. 57, 117-127), the phage suppressors are most likely to be attributed to these phage tRNAs.
Chen et al., 1976; Hunt et al., 1976). Using two-dimensional polyacrylamide gel electrophoresis, Ikemura and Ozeki (1975) separated more than 15 species of 4 S RNA encoded by BF23. Most of these RNA species contained pseudouridine and ribothymidine in their molecules, and some had similar fingerprinting patterns to those of corresponding tRNA species of T5. This information indicates that BF23 also carries tRNA genes on its genome. A pertinent question is whether these BF23 tRNAs are biologically active. In order to solve this question, attempts have been made to isolate nonsense suppressor mutants. This paper describes the isolation and genetic analysis of BF23 nonsense suppressor mutants (designated bfsu’) active on the amber mutations of this phage. Evidence is also presented showing that the nonsense suppressor genes are dispensable for phage propagation. Preliminary results of this work have been reported (Okada, Ohira and Ozeki, 1973).
INTRODUCTION
Bacteriophge BF23 is a virulent coliphage closely related to T5. Both phages readily recombine with each other and have similar genetic maps in spite of the difference in antigenicity and in receptor site on the host cells (Nishioka and Ozeki, 1968; Mizobuchi et al., 1971; Hendrickson and McCorquodale, 1971). It has been reported that several virulent phages have tRNA genes on their genome. For example, in T4, eight tRNA genes were identified and some of them exhibited nonsense suppressor activity by mutation (McClain, 1970; Wilson and Kells, 1972; McClain et al., 1973; Comer et al., 1974). In the case of T5, at least 14 kinds of tRNA genes were identified and their corresponding genes were mapped on the chromosome of this phage (Scherberg and Weiss, 1970; ’ Present address: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan. *Present address: Research Laboratories of Biotechnology, Mochida Pharmaceutical Co., Ltd., Kamiya l-1-1, Tokyo 115, Japan.
MATERIALS
Media.
Nutrient
AND METHODS
broth
and
nutrient
133 0042~6822/78/0901-0133$02.00/0 Copyright 0 1978 by Academic All righb of reproduction in
Press,
any form
Inc. reserved.
134
OKADA,
OHIRA,
broth containing 1 x lo-” M CaC12 were used for the growth of bacteria and phages, respectively. Phage adsorption buffer was prepared according to Mizobuchi et al. (1971). Bacterial and phage strains. Escherichia coli strains CR63 (sul’) and 594 (sustr’ gal-) were used as permissive and nonpermissive hosts, respectively, for the amber phages. The phage strains used are listed in Table 1. Wild-type BF23 and its amber mutants, amH30, am57, am91, aml59, am177, and am218, were kindly supplied by Dr. Mizobuchi. The order of these markers on the phage DNA is presented at the bottom of Table 1 (Mizobuchi et al., 1971, and Mizobuchi, personal communication). Two markers, amH30 and am57, have been mapped within the 8% segment TABLE BACTERIOPKAGE Strain
STRAINS
Genotype
Wild-type am57 am91 am159 am177 am218 amH30 am9lam159 am91am218 aml77am218 Xl x2 Yl Y2 Zl 22 del-1 del-2 del-3 del-4 del-5 del-6 deL4rl del-4r2 X1-163 X1-214 x1-2110 (amH30 \ 1
1 BF23
am57 am91 am159 am177 am218 amH30 am91 am91 am177 bfsul’ bfsul+ bfsul+ bfsu2+ bfsu3’ bfsul’ bfsulbfsulbfsulbfsu2bfsu2bfsu3bfsul’ bfsul’ bfsulbfsulbfsulam571am91 / The
order
am159 am218 am218 am177 am177 am91 am91 am91 am91 am177 am177 am177 am91 am91 am91 bfsu2bfsu2am177 am177 am177
am218 am218 am218 am218 am159 am159 am218 am218 am218 am218 am218 am159 am91 am218 am91 am218 am218 am218 am218
am218
am177 1
of the markers
used
am159
AND
OZEKI
of BF23 DNA that is transferred to the host cell first. This segment is designated the first-step-transfer (FST) segment by analogy to T5 (Lanni, 1968). Strains bearing multiple amber mutations were constructed by crossing the appropriate amber mutants. Isolation of nonense suppressor mutants. For isolation of the nonsense suppressor mutants of BF23, about 1 x 10’ of appropriate double amber mutants were plated on SU- cells. The rare phenotypically wild-type phages were isolated and purified by single plaque isolation. In order to distinguish the nonsense suppressor mutation from true reversion, the phenotypically wild-type phages were back-crossed with wild-type phage on su+ cells, and then examined to determine whether or not the original amber markers had segregated (see text). The six bfsu’ mutants listed in Table 1 were independently isolated in this manner. Isolation of suppressor defective mutants. Nonsense suppressor-defective mutants, designated bfsu-, were obtained by treatment of nonsense suppressor mutants with ethylmethanesulfonate (EMS) according to the method of Tessman et al. (1964). The mutagenization was stopped by dilution at a survival at 10e4 and the surviving phages were plated on a mixture of indicator cells, one part of SU+ and 20 parts of SK. Several plaques showing the amber phenotype (small turbid plaques) were obtained from about 3000 survivors. These were purified, and their amber markers were examined by complementation tests. Three bfsu- mutants, carrying the amber markers as in the parental phage, were obtained and named X1-163, X1-214, and Xl2110 (Table 1). Complementation test of amber mutants. One drop of amber phage lysate containing about lo8 PFU/ml was spotted on a plate seeded with su- cells, and after it had soaked in thoroughly, one drop of another phage lysate was spotted on the same area. After an overnight incubation at 37”, lysis of the su- cells in the overlayered spot was taken as evidence that complementation had occurred between the two amber mutants tested. Genetic cross of nonsense suppressor
NONSENSE
SUPPRESSORS
mutants. The procedure used for genetic crosses was that of Mizobuchi et al. (1971). The crosses were performed on the SU+ cells of CR63, so that the recombinants, as well as the parental phages, could grow well regardless of their geneotypes. RESULTS
Isolation
of bfsu+ Mutants
As the first step for detecting nonsense suppressor mutations in BF23, we constructed double amber mutants by crossing single amber mutants, and then measured the apparent reversion frequency of these multiple mutants on su- cells. Since the single amber mutants used in the experiment reverted to amber+ at a frequency of 10-5-10-6 (Table 2)) the reversion frequency of the double amber mutants was expected to be 10-10-10-12. Accordingly, if the reversion frequency of a double amber mutant was significantly higher than the expected value, it was thought that the reversion of its phenotype to wild-type was more likely to be due to a nonsense suppressor mutation which had occurred on the phage genome, rather than due to simultaneous reversions of the two amber markers. Table 2 shows the apparent reversion frequency of various multiple amber mutants. The TABLE
2
REVERSIONFREQUENCYOFAMBERSTRAINS Strain
Reversion
frequency”
am91 1.6 x 10 4 am159 9.0 x lo-” am177 3.1 x 10 ‘, am218 4.6 x 10 R am57 2.2 x IO Ii amH30 3.8 x 10 ’ aml77am218 3.4 x 10~’ am91am218 5.8 x 10 ’ am9lam159 4.0 x 10 i am57amH30
IN
PHAGE
135
BF23
three double amber mutants, am177am218, am91am218, and am91am159, reverted at a frequency of approximately 10p7. These values were lC?-104-fold higher than the expected ones. When triple or quadruple amber mutants were examined, the reversion frequency was still the same as that of double amber mutants (Table 2). These results suggested that the phenotypic reversion was due to a single suppressor mutation which was capable of suppressing all those amber mutations. It is worthy of note that the reversion frequency of mutatants carrying an amber mutation in the firststep-transfer (FST) segment (amH30 or am57) was low enough to be characteristic of the expected frequency for a double reversion, indicating that these were not due to suppression (Table 2). It appears, therefore, that the phage suppressor was not active on either am57 or amH30. Possible explanation for this phenomenon are discussed later. In order to confirm that the phenotypitally wild-type revertants from multiple amber mutants were due to nonsense suppressor mutations, the genotype of six independently isolated revertants, Xl, X2, Yl, Y2, Zl, and 22 (see Table l), was analyzed by backcross with wild-type phage. The parental strains of these revertants were am177am218 for Xl and X2, am91am218 for Yl and Y2, and am91am159 for Zl and 22, respectively. Table 3 shows the result of such a cross performed between Xl and wild type. It can be seen that strain Xl segregated recombinants carrying am177 and/or am218. TABLE CROSSBETWEEN _____-Number of progeny amber’ pt%r
-~~.-_--___.~~_
-.-___ 650
3
STRAIN XIANDWILD-TYPE PHAGE" ~.. .__~~ Phenotype -.-___ am177
am218
amli7am218 .__
557 27 20 46 __.__..___~~_..___ ” Strain X1 was crossed with wild-type phage on su+ cells, and progeny phages produced were plated on su+ cells. Plaques formed were stabbed on su and su+ cells using sterilized toothpicks. The genotypes of recombinants showing amber phenotype were checked by complementation tests with the strains carrying either om177 or am218.
136
OKADA,
OHIRA.
This observation indicated that Xl still retained the original amber markers. Similar results were obtained with five other revertants (data not shown). From these observations, it was concluded that, in these six strains, suppressor mutations did take place on the chromosome of BF23 to give rise to phenotypicalIy wild-type phages. The nonsense suppressor gene and its wildtype allele are designated as bfsu’ and bfsu’, respectively. The burst size of these suppressor strains on su- cells was reduced to some extent, compared with that of wild-type phage, about 40% in strain Xl, about 30% in Y2, and about 5% in Zl, respectively.
AND
OZEKI
(7.6%) or between (Table 4).
b&2+
and bfsu3’
Isolation of Deletion Mutants Suppressor Activity
Lacking
(3.5%) the
Hertel et al. (1962) isolated deletion mutants of bacteriophage T5 from survivors after heat treatment at 50” for 60 min in 0.4 M NaCl containing 0.01 M sodium citrate, pH 6.8. We have attempted to search for conditions to efficiently select deletion mutants of BF23, and found that heat treatment at 60” in 0.02 M Tris-HCl, pH 8.5, with 0.02 M EDTA was most effective. This procedure was originally used for the isolation of deletion mutants of bacteriophages A and $30 (Yamagishi and Ozeki, Classification of bfsu+ Suppressors 1972). After heat treatment of phage particles of bfsu’ strains for 20 min, survivors In order to determine whether the inde(10-6) forming plaques on su+ cells were pendently isolated bfsu’ suppressors were picked and tested for their amber phenothe same or not, crosses between the bfsu’ type. Among these survivors, deletion mustrains were carried out on su+ cells, and tants which were associated with the loss the phenotype of the progeny phages was of the suppressor activity were found at a analyzed on su- and su+ cells. The results frequency of 5 X lo-“. Six mutants were are summarized in Table 4. Crosses beindepenently isolated from Xl, Y2, or Zl, tween Xl and Y2 or 21, and between Zl and designated del-1 to del-6 (Table 1). and X2, Y2 or 22 yielded significant numof these bers of amber recombinants; whereas in Table 5 shows some properties deletion mutants. When complementation crosses between Xl and X2, Yl or 22, such tests were performed, the respective delerecombinants could not be detected. On t,he tion mutants were shown to carry two ambasis of these results, the suppressors in the ber markers, which were the same as those six bf;Fu+ strains were tentatively classified into three groups, namely, bfsul+ in strains TABLE 5 Xl, X2, Yl, and 22, bfsu2+ in Y2, and REVEKSION FREQUENCY ANI) BUOYANT DENSITY OF bfiqu3’ in Zl. The gene order of these supDEIXTIOP; MUTANTS pressors is presumably bfsul’ - bfsu2’ ____ ~_--_-~-l’arental hll,). Reverwn frr- Hurwlnt denStrain bfsu3+ since the genetic distance observed pressrx strain q”rncy” (g/ml) -- sit?.” __~ between bfsul’ and bfsu3+ (8.7 - 13.7%) 1.551 Wild-type was further than between bfsul+ and bfsu2’ TABLE REcOMRINATION -Ck-03s _____ Xl x1 x1 x1 Xl Zl Zl Zl
x x x x x x x x
x2 Yl 22 Y2 Zl x2 Y2 Z2
4
BETWEEN SUPPRESSOR ______ STRAINS Number of amber Number of’ progmy phages tested phages (hfsu ) detected F<) -_----204 0 200 0 222 0 368 %a (7.6) 48 (13.7) 350 263 23 (8.7) 286 10 (3.5) 270 29 (10.7)
%.4 x 10 ; 1.54’1 del- 1 Xl del-% Xl “.O x 10 ; 1.537 7.6 x 10 * 1.540 (id9 X1 2.8 x IO ” Y2 1.544 rlel-4 Y8 6.9 x 10 L( 1.540 rleI-5 2.2 x 10; 1.543 clel-6 Zl _ _ ----~~-” Reversion frequency was calculated as described in the footnote of Table 2. “Buoyant density was measured by CsCl equilibrium density gradient centrifugation; 2.5 g of CsCl was dissolved in 2.9 ml of phage suspension, and centrifuged at 35,000 rllm for 20 h at 20°C in SW50-1 rotor (Spinco). As references, bacteriophage h (p = 1.508), hb2b5(0 = 1.483) (Kellenberger-Gujer and Weisberg, 1971) and 1’5(~, = 1.654) (Rubenstein, 1968) were used.
NONSENSE
SUPPRESSORS
of the corresponding parental bfsu’ strains. The buoyant densities of the mutants, which were measured in CsCl equilibrium density gradient centrifugation, were less than that of wild-type phage (Table 5). Furthermore, these mutants were more resistant to heat inactivation than wild-type BF23 (data not shown). The decreasing order of phage density, namely, wild-type > del-4 > del-6 > de&l > del-3 = del-5 > del-2, correlated well with the increasing order of phage stability. Inasmuch as an exceedingly good correlation between DNA content, phage density, and phage stability has been shown in T5 (Hertel et al., 1962) or in X (Parkinson and Huskey, 1971), we concluded that the mutation was due to the deletion of a part of phage genome. Direct measurements of molecular weight of DNA extracted from these deletion mutants by agarose gel electrophoresis later confirmed this conclusion, and showed that del-1,2,3, 4, 5, and 6 deleted 7, 10, 8, 6, 9, and 7% of the whole genome, respectively (Okada, manuscript in preparation). To test whether the bfsu’ gene was deleted in the mutants, they were crossed with the respective bfsu’ strains. As shown in Fig. lA, it is expected that recombinant-s with the suppressor activity would be obtained if a deletion mutant still carried the mutation site for bfsu+, but the expression of suppressor activity was inhibited somehow by the deletion (e.g., lack of the promoter region). Conversely, if the mutation site itself was deleted, no such recombinants would be produced (Fig. 1B). Table 6 presents the results of the crosses. When bfsu”
am1
am2
am1
am2
bfsuo
am1
am2
(bfsu+)
am1
am2
FOG. 1. Diagrammatic representation of a cross between deletion mutants and bfsu’ double amber strains. Two alternative cases are shown: one (A) is that the bfsu+ gene is retained on the chronosome of a deletion mutant, and the other (B) is that the bfsu’ gene is deleted. The wavy region represents the deleted region.
IN
PHAGE
137
BF23 TABLE
6
CROSS BETWEEN DELETION MUTANTS PARENTAL STRAINS Cross del-1
del-2 del-3 de14 del-5 del-6
x x x x x x
am177am218 aml77am218 aml77am218 am91am218 am91am218 am91am159
AND
bfsu”
Phenotypical amber+ progeny phages produced”__~
fl Frequency of phages of amber+ phenotype was calculated as the number of plaque forming units on sum cells as compared with that on su+ cells.
del-1 was crossed with am177am218, the phenotypicahy wild-type phages were detected only at a frequency comparable to the reversion frequencies of both parental phages (see Tables 2 and 5). Accordingly, no positive evidence was obtained indicating that the mutation site of suppressor bfsul+ was retained in the del-1 strain. Similar results were also obtained in the rest of the deletion mutants, except for del-5. In the case of del-5, bfsu‘ recombinants were formed at a frequency of 2.0 x 10e4, indicating that del-5 still retained the bfsu2+ gene, though suppressor activity was not expressed. The frequency of reversion to phenotypically wild-type in each deletion mutant varied from 6.9 x lo-* to 2.8 X 10V6 (Table 5). Considering that these values are in the same magnitude as those of the bfsu strains (see Table 2)) the phenotypic reversion was presumably due to the second suppressor mutation. If this was the case, all of the bfsu’ genes should have been deleted in none of these six strains; some of them should have been retained. In order to confirm this possibility, the following experiments were performed. Second Suppressor Mutation Occurring in a Deletion Mutant (del-4) In order to detect the remaining bfsu’ genes in the suppressor-negative deletion mutants, phenotypic revertants were again isolated by plating the deletion mutants on su- cells and their genotypes were analyzed. Two revertants, designated del-4rl and del4r2, were isolated from del-4. The buoyant
138
OKADA.
OHIRA,
density of these revertants was exactly the same as that of del-4 (data not shown). By crossing with wild-type, amber recombinants carrying am91 and/or am218 were produced among the progeny phages. Thus, it was confirmed that these two revertants still carried the same amber markers as those of del-4. These observations, therefore, indicated that the reversion resulted from the second suppressor mutation which was apparently different from the original bfsu2’. When deldrl or del-4r2 were crossed with either Y2 or Zl, recombinants of amber phenotype were obtained, whereas no such recombinants have so far been detected in the crosses with Xl (Table 7). It appears, therefore, that the sites of the second suppressor mutations occurring in del-4rl or deL4r2 were different from bfsu2’ and bfsu3+, and possibly identical or very close to bfsul+. In strain del-4, therefore, the bfsu2’ gene is presumably deleted, whereas the bfsul” gene appears to be retained. Isolation
of bfsu--defective
Point Mutants
In order to further characterize the nonsense suppressor genes, we have isolated derivatives of a bfsu’ strain which had lost the suppressor activity by point mutation ( bfsu-). After mutagenization of strain Xl (bfsul+) with EMS, phages of amber phenotype were selected, and their genotypes were tested (see Materials and Methods). Three bfsu- mutants, X1-163, X1-214, and X1-2110, were isolated which were confirmed by complementation tests as carrying the same amber markers, am218 and am177, as those in strain Xl. Incidentally, some other strains were found to carry new amber mutations which were not suppressed with bfsul+, but with a host suppressor. They could be attributed to amber mutations in the FST genes, although no further experiments have been done to clarify this point. Table 8 shows the result of crosses made between these bfsu- mutants and suppressor-negative deletion mutants, measuring the frequency of appearance of bfsu+ recombinants. When X1-163 was crossed with de&l, del-2 and del-3, the bfsu’ recombinant was not detected with a frequency
AND
OZEKI TABLE
7
CROSSBETWEEN bfsu’ STRAINSAND PHENOTYPICALLY~ILD-TYPEREVERTANTSFROM del-4 cross
deL4rl deL4rl del-4rl deL4r2 deL4r2 de14r2
x x x x x x
X1 Y2 Zl Xl Y2 Zl
Number of progeny phages tested
Number of amber phages
660 647 648 648 648 648
0 3 2 0 8 5
higher than the reversion frequency of parental phages (see Table 5), although such a recombinant was obtained with a much higher frequency in the cross with del-4. Similar results were obtained with X1-214 and with X1-2110. These data suggested that the mutation sites of these bfsustrains were covered by the deleted regions of the phage genome in strains del-1, del-2, or del-3, but not in del-4. These bfsu- strains were crossed with each other, and also with the original bfsu” strain (am177am218). Bfsu’ recombinants were found at a frequency of lo-” in all the combinations. The results are summarized in Fig. 2. The relative position of the mutation sites, including the original mutation site of bfsul+ in the bfsul gene, was determined on the basis of recombination frequencies, which are indicated as the distances between the sites in Fig. 2. These results indicate that the three bfsu- strains still carry the original mutation site of bfsul+, but are defective by a mutation at other sites. These second mutation sites in the three strains are different from each other, but all of them are located very close to the bfsul+ site, possibly within the bfsul gene. Several suppressor-positive derivatives of these bfsu- strains, obtained by spontaneous reversion, were isolated and crossed with strains Xl (bfsul+), Y2 (bfsu2’), and Zl (bfsu3’). No amber-phenotype recombinant was detected in the cross with Xl, whereas recombinants were obtained with Y2 and Zl to the same extent as in the crosses of Xl x Y2 or Xl X Zl shown in Table 4 (data not shown). These results
NONSENSE
SUPPRESSORS TABLE
CROSSBETWEEN
bfsu- strain
Reversion quency
4.3 x 1o-8 1.9 x 1o-7 7.1 x lo-’
X1-214
PHAGE
139
BF23
8
bfsu--DFNwrIvE MUTANTSANDDELETIONMUTANTS frebfsu’ recombinants per total progeny del-I
X1-163 X1-214 x1-2110
IN
del-2
1.0 x lo-’
X1-163
My/l
del-3
in the cross with
del-4 8.0 x 1o‘4 2.0 x lo-” 1.2 x lo-,’
Xl-~10
FIG. 2. Mutation sites in bfsul- mutants. Three bfsul- strains, X1-163, X1-214, and X1-2110, were crossed with each other, and also with bfsul’ am177 am218. The numeral shows the recombination frequency between the two mutation sites: 2 x bfsu+ recombinants per total progeny phages x 100 (S). bfsul indicates the original mutation site of bfsul+. X1-214, X1-163, and X1-2110 are the mutation sites of these bfsul- strains, by which bfsul’ suppressor became inactive.
indicated that the suppressors in the bfsu+ derivatives of bfsu- were bfsul+, and not bfsu2+ or bfsu3+. Taking all these lines of evidence together, we conclude that the three suppressor-negative strains, del-1, del-2, and del-3, have deleted the bfsul+ gene itself, ,but strain del-4 still carries the bfsul’ gene. DISCUSSION
We have presented genetic evidence indicating that the phenotypic reversion of multiple-amber mutants of BF23 is due to suppressor mutations occurring on the phage genome. Three different suppressors are identified. All of them are not genespecific but general suppressors capable of restoring the amber mutations of various phage genes. They are considered to be amber-suppressors, but the possibility of ochre-suppression still remains because an o&ire-suppressor is known to suppress not only ochre but also amber mutations. In general, suppression of an amber mutation by ochre-suppressors is less efficient than that by amber-suppressors, In this connection, bfsu3’ which shows smaller burst sizes than the other two mutations might be considered as a possible case of an ochresuppressor. This possibility, however, has not yet been examined, because ochre-type
nonsense mutants are unfortunately not available in BF23 at present. Although the nonsense suppressors described in this paper are general ones active on various amber markers of the phage, no suppressor mutant was obtained from the multiple-amber mutants carrying either am57 or amH30. It appears, therefore, that the bfsu+ suppressors are not active on these two markers, which are located on the first-step-transfer (FST) segment of phage DNA. A possible explanation is as follows. Mizobuchi et al. (1971) reported that BF23 DNA is injected into host cells in a two-step manner; namely, the 8% segment (FST) of the DNA molecule in the first step, and the rest in the second step. The second step of DNA transfer was shown to be dependent on synthesis of the pre-early proteins which were encoded by the genes located on the FST segment. Genetic analysis performed by Mizobuchi (unpublished results) revealed that the deleted regions of the suppressor-negative deletion mutants, described in this paper, were located between am91 and am218 on the map shown in Table 1. Thus, the nonsense suppressor genes were shown to be located on the DNA segment transferred in the second step, but urn57 and umH30 were on the FST segment. Accordingly, the fail-
140
OKADA,
OHIRA.
ure of suppression of FST genes by the phage suppressor may be attributed to the failure of transfer of the suppressor genes into the host cells. Moreover, even after complete transfer of the whole DNA molecule, expression of the early (and late) genes, presumably including the suppressors, may also be dependent on expression of a pre-early gene(s) on the FST segment. It has been suggested from the results of transfection experiments that expression of am57 is required for expression of the early and late genes (Benzinger and McCorquodale, 1975). In either case, the failure in selecting suppressor mutants with the amber mutations on the FST segment may be explained by the absence of expression of suppressor characters. In this connection, we are now attempting to construct a mutant carrying the am57 (or amH30) and bfsu+ gene to examine the expression of suppressor activity in the transfection system of BF23 DNA. All the suppressor mutants isolated can grow on su- as well as on su+ bacteria, and the phenotypes of the suppressor genes are always the same, simply “amber suppression,” regardless of the genes. Therefore, it is difficult to distinguish by genetic complementation tests whether two independently isolated suppressors are allelic or not. In the present study, this difficulty was circumvented by an alternative procedure. That is the isolation of second suppressors from strains with deletions which lack the first suppressor gene. The reasoning was that if a second suppressor were detected, it should be different from the first one. Combining this information with the results of genetic recombination tests performed with the suppressor mutants, three distinctive suppressor genes in BF23 were identified. Because BF23 carries a considerable number of tRNA genes (Ikemura and Ozeki, 1975), the suppression may be understood in terms of suppressor tRNAs. In order to test this, analysis of phage tRNAs has been carried out in our laboratory by using the suppressor-negative deletion or point mutants described in this paper. In strain del-5, for instance, the majority of the phage-specified tRNAs were not pro-
AND
OZEKI
duced, indicating that many tRNA genes are clustered together on the phage genome. One of the tRNAs is assigned to the bfsul+ gene product (Ikemura et al., 1978). Inasmuch as the b/K deletion mutants are viable, the BF23 tRNA genes must be dispensable for phage growth at least on E. coli K12. Therefore, the mutation of tRNA genes to suppressor may also be permissive. This in turn suggests that the phage tRNAs are dispensable but still produced in a biologically active form. The observed frequency of a suppressor mutation was roughly 10e7 (Table 2), and this is the value expected for a single-base mutation occurring at a given site. Accordingly, a pertinent explanation for the phage suppression would be as follows. Among the BF23 tRNAs, there are some whose anticodons are convertible by a single-base mutation to amber (or ochre) type. In the present study, at least three such genes (bfsul”, bfsu2’, and bfsu3’) were revealed. Nonsense suppressors have also been isolated in phage T5 (D. Botstein, personal communication). Thus, in all the three phages, T4, T5, and BF23, which are known to code phage-specified tRNA genes, nonsense suppressor mutations have been detected. ACKNOWLEDGMENTS We thank the members of the Laboratory of Molecular Biology, Department of Biophysics, Kyoto University, for useful discussions, and Drs. K. Mizobuchi and T. Nagata for critical reading of the manuscript. This work was supported by a grant from the Ministry of Education of Japan. REFERENCES BENZINGER, R., and MCCORQUODALE, D. J. (1975). Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes. J. Viral. 16, l-4. COMER, M. M., GUTHRIE, C., and MCCLAIN, W. H. (1974). An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA. J. Mol. Biol. 90,665-676. CHEN, M-J., LOCKER, J., and WEISS, S. B. (1976). The physical mapping of bacteriophage T5 transfer RNAs. J, Biol. Chem. 251,536-547. HENDRICKSON, H. E., and MC~ORQUODALE, D. J. (1971). Genetic and physiological studies of bacteriophage T5. I. An expanded genetic map of T5. J. Viral. 7, 612-618.
NONSENSE
SUPPRESSORS
HERTEL, R., MARCHI, L., and MUELLER, K. (1962). Density mutants of phage T5. Virology 18.576-581. HUNT, C., HWANG, L-T., and WEISS, S. B. (1976). Mapping of two isoleucine tRNA isoacceptor genes in bacteriophage T5 DNA. J. Virol. 20,63-69. IKEMURA, T., and OZEKI, H. (1975). Two-dimensional polyacrylamide-gel electrophoresis for purification of small RNAs specified by virulent coliphages T4, T5, T7 and BF23. Eur. J. Biochem. 51, 117-127. IKEMURA, T., OKADA, K., and OZEKI, H. (1978). Clustering of transfer RNA genes in bacteriophage BF23. Virology 90, 142-146. KELLENBERGER-GUJER, G., and WEISBERG, R. A. (1971). Recombination in bacteriophage lambda. I. Exchange of DNA promoted by phage and bacterial recombination mechanisms. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 407-415, Cold Spring Harbor Laboratory, New York. LANNI, Y. T. (1968). First-step-transfer DNA of bacteriophage T5. Bacterial. Rev. 32, 227-242. MCCLAIN, W. H. (1970). UAG suppressor coded by bacteriophage T4. FEES Lett. 6,99-101. MCCLAIN, W. H., GUTHRIE, C., and BARRELL, B. G. (1973). The psu,+ amber suppressor gene of bacteriophage T4: Identification of its amino acid and transfer RNA. J. Mol. Biol. 81, 157-171. MIZOBUCHI, K., ANDERSON, G. C., and McCORQUODALE, D. J. (1971). Abortive infection by bacteriophage BF23 due to the colicin Ib factor. I:
IN PHAGE
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Genetic studies of nonrestricted and amber mutants of bacteriophage BF23. Genetics 68.323-340. NISHIOKA, T., and OZEKI, H. (1968). Early abortive lysis by phage BF23 in Escherichia coli K-12 carrying the colicin Ib factor. J. Viral. 2.1249-1254. OKADA, K., OHIRA, M., and OZEKI, H. (1973) Suppressor genes of phage BF23. Jap. J. Genet. 48, 439 (Abstr.). PARKINSON, J. S., and HUSKEY, R. J. (1971). Deletion mutants of bacteriophage lambda. I. Isolation and initial characterization. J. Mol. Biol. 56, 369-384. RUBENSTEIN, I. (1968). Heat-stable mutants of T5 phage. I. The physical properties of the phage and their DNA molecules. Virology 36, 356-376. SCHERBERG, N. H., and WEISS, S. B. (1970). Detection of bacteriophage T4- and T5-coded transfer RNAs. hoc.
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TESSMAN, T., PODDAR, R. K., and KUMAR, S. (1964). Identification of altered bases in mutated singlestranded DNA. I. In vitro mutagenesis by hydroxylamine, ethylmethanesulfonate, and nitrous acid. J. Mol.
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WILSON, J. H., and KELLS, S. (1972). Bacteriophage T4 transfer RNA. I. Isolation and characterization of two phage-coded nonsense suppressors. J. Mol. Biol.
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YAMAGISHI, H., and OZEKI, H. (1972). Comparative study of thermal inactivation of phage $80 and lambda. Virology 48, 316-322.