VIROLOGY
55, 285-288 (1973)
Location of the DNA-Adenine Methylase Gene on the Genetic Map of Phage T2 JOAN BROOKS
STANLEY HATTMAN
AND
Department of Biology, University of Rochester, Rochester, New York 14627 Accepted June
4, 1973
The location of the gene controlling DNA-adenine methylase activity is reported to be in the region between genes 49 and 1'1 on the T2 genetic map. A new nomenclature is proposed to describe the various phage types affected by mutations in the methylase gene.
Mutants of phages T2, T4, and T6 defective in the synthesis of glucosyl transferase(s) (yt-) are generally restricted by strains of Escherichia coli (1, 2); certain strains, designated rql:', are permissive for the nonglucosylated gt- and T*-forms (3; see 4- for an excellent recent review). A class of the gt- mutants (designated 70['1) are also restricted, but not modified, by PI-lysogenic bacteria (5-7). However, both T2 gt- l'P1 and T4 at- l'P1 can mutate to a form which, though still gt-, is resistant to PI-restriction (6, 7). These mutants, designated ef'J, have been shown to direct synthesis of an altered DNA-adenine methylase (8, 9) and to hypermethylate qt: phage DNA (10) j the additional methylation presumably protects the DNA against the PI-restriction nuclease, These and other results (8-10) have led to the conclusion that the 1.tPl mutation occurs in the structural gene controlling synthesis of the phage specific DNAadenine methylase (11-13). DNA methylase activity appears shortly after phage infection (11-13) as does the activity of other so-called "early-enzymes." Since a nonrandom clustering of genes according to function has been observed on the phage genetic map, it was of interest to determine where the methylase gene is located. In the present communication we report the location of this gene (designated clam, see Table 1) and propose a new nomenclature for this system.
The previously used symbols (vis., rPl, uP1, u RP1, ete.), though appropriate when
the system was first investigated, are no longer adequate or precisely descriptive. Since the mutations all appear to affect the DNA-adenine methylase gene of T2, we propose to replace the old notations with clam, for DNA-adenine-methylase. lVIarinus and Morris (14) have already adopted this symbol for the B'. COl1: DNA-adenine methylase genes, Listed in Table 1 are some of the various T2 phage forms in existence, and their phenotypic characters relevant to methylation and restriction. For the remainder of the discussion, we will use the new nomenclature. Previous mapping studies with various T2 and T4 mutants indicated that the darn gene is located 26 map units (in a clockwise direction on the map) from the structural gene for o-glucosyl transferase (7, 15). In a preliminary experiment, we also observed that 13% of the progeny in a two-factor cross between T2 gt1- clamh X T2 wildtype (dam+) were recombinant T2 gtc clam+ (data not shown) j this result was obtained even though the host and temperature used for the cross were different from those of Revel and Georgopoulos (7). In order to more precisely localize the dam. gene with respect to known markers, it was necessary to carry out three-factor crosses; the bacterial and phage strains used in these studies
285 Copyright © 1973 by Academic Press, Inc, All rights of reproduction in any form reserved.
28G
SHORT COMMUNICATIONS TABLE I PROPOSED NOMENCLATURE FOR DNA-ADENINE METHYLASE MUTANTS
Phage-
Methylase genotype
Pi-directed restriction
DNA-adenine methylation
Relevant references
normal slightly higher" than T2 wild-type slightly higher" than T2 wild-type hypermethylated little or no methylation
('1, to) ('1, to)
T2 wild-type T2 gt- r,Pt
darn+ dam»
resistant resistant
T2 gt- rPt
dam+
sensitive
T2 gt- uPt T2 gt uRPJ
dam b ' dam b dam-Id
resistant sensitive
('1, to) ('1, to) (9)
• The gt- mutant lacks et-glucosyl transferase. The r.Pt and rPt types differ in their sensitivity to PI-restriction. The r,Pt phage is conditionally sensitive to PI-restriction, i.e., it becomes susceptible to restriction after growth in UDPG-Iess hosts ('7). o The higher content of N°-methyl adenine in gt- DNA, as compared to T2 wild-type has been attributed to the absence of glucose, and not to a mutational alteration in the methylase (10). , It is not known how many sites can mutate to create the PI-resistant phenotype; crosses between independent dam b mutants to produce PI-sensitive dam+ recombinants have been obscured by the high background of second site dam mutants (9). d Several revertants from PI-resistance to PI-sensitivity have been studied and appeal' to map on either side of the original dam h mutation (9). Mutants lacking NG-methyl adenine or defective in DNAadenine methylase activity have been described (to, .15); these are presumably dam mutants, but they have not been mapped yet.
are listed in Table 2. The gt- are derived from T2H; the T2 1'1 and amber mutants are from T2L. Although the availability of markers is scant in the region we believed to be of interest, the data summarized in Table 3 show that it was possible to assign a location for the clam locus. In these experiments, it was not possible to identify all the possible genotypes. Consequently, the various genotypes are grouped according to distinguishable phenotypic classes; e.g., in class II of cross A, gt+ am clam+ (parental type) and gt+ am clamh (recombinant) possessidentical plating properties. Despite this drawback, the relative linear order of markers could be determined in each cross, and a composite yields the relative location of the clam locus. The known relative linear order of the markers used here is a gt - am - rI (16). The data in Table 3 can be analyzed in several ways. First, for each cross, the frequency of 2-factor recombinants involving the gt- marker can be compared. In cross A, the fraction of gtdam+ recombinants (0.114) was more frequent than the fraction of gt- am recombinants (0.058); this suggests the relative order of gt - am - dam. Thus, one would anticipate that the least frequent classes of
recombinants would be gt- am dam h and gt+ am» dam», The latter genotype cannot be scored (Table 3, Class III); however, the former is observed to be present in the smallest fraction among recombinant classes (Class VI). A similar analysis can be made in Cross B, although the additivity is not as good as in Cross A. The frequency of recombinants between gt- and domr was reduced about four-fold in Cross B compared to Cross A; this discrepancy is not yet understood. The 2-factor data show that the gt- dam" recombinants occur less frequently than the gt- rI recombinants (0.028 versus 0.068). Analysis of the 3-factor recombinants is also consistent with a relative linear order of gt - dam - 1'1; viz. qt: dam+ rI+ is the smallest recombinant class (the reciprocal recombinant cannot be unambiguously scored). It should be pointed out, however, that only a small number of Class V and VI recombinants were scored in Cross B (10 and 25, respectively, out of 1132 total progeny) ; thus, there may still be some question as to which class really constitutes the least frequent one. However, the agreement with the 2-factor data strengthens the assignment of gene order. From the relative order determined in
287
SHOHT COMMUNICATIONS TABLE 2 BAC'J'JmIAL "\NV PI-lAW, H'l'RAINS
Escllerichia eolia strains
Ability to restrict phage gt- dam+
gt- damb
+
+
K12 1'6+ 1';',4 K12 1'6- 1';',4 = K rgl1100 1'6- 1'2".4 = 1100 rqt: 1100 rgl- (PI) B
+
Amber suppressor
+ + +
+
+
Growth on
Phage!' strains Rrgl-
1100 rgl-
11001'gl- (PI)
K r6'!- 1".4
B
+ +
+ +
+ +
+
+
+
+
+
+ +
+ +
+ +
+
'1'2 wild-type '1'2 gt l - 1lPl (='1'2 gt l - clam h ) '1'2 giJ- rPl (='1'2 gt l - dam+)
'1'21'1 '1'2 am 2 (gene 49)
The K12 and 1100 strains (2,4) were obtained from Helen Hevel. The '1'2 gh- clam+, '1'2 gt l - dam h (7) and 1'2 am 2 were obtained from Helen Revel; '1'21'1 was provided by Martha Baylor. a
I,
TABLE 3 ANALYSIS OF PROfJlCNY DERIvm) FROM THREE-FACTOR CROSSES INVOLVING 1'2 gte dam ha Cross B: 1'2 gr 1'1+ damb X
Cross A: 1'2 gr a11l+ damh
X
1'2 gt+ atn dam"
Class
Genotype
1'2 gt+1'1 dam+
Fraction of
Class
Genotype
Fraction of progeny!'
0.30'1 0.455
I II
0.435 0.435
0.114
III
0.046
IV V VI
gt- rl" dam» at+ r1 dam h at+ r1 dam+ gt+ rl+ dam h gt+ rI" dam+ gt- rI dam b gt- rI" dam+ at- r1 dam+ Order: at - dam - rl
progeny"
I II
III IV V VI
am+ clamh arn darn h am clarn+ am+ darn h am+ clam+ am dant" am+ clam+ am dam h Order: gt - am - clam gtgt+ gt+ gt+ gt+ gtgtgt-
0.068 0.012
0.054 0.049 0.009 0.019
a E. coli 1100 rgl- bacteria were grown to 1 X lOB Iml at 30°0 in broth and infected with approx, 5 phage of both parental genotypes. After 5 min adsorption, anti-T2 serum was added (K. = 20 final) j after 5 min inactivation of free phage, the infected cells were serially diluted 10- 4 in broth and incubated for 90 min at 30°C with vigorous aeration. Lysis was completed by adding CHCl a ,and the progeny phage were plated for individual plaques on 1100 rgl-. Plaques were randomly picked with toothpicks into 0.2 ml phage buffer (10) saturated with CHC1 3 contained in separate wells (disposable U-plates, Scientific Products). Spot tests were made with a brass stamper onto agar plates seeded with appropriate host, cells. The various genotypes which could be unambiguously identified by their phenotypic behavior are grouped together in distinct classes. In Cross B, the rI phages were recognized by virtue of a halo they produced around the confluently lysed area of the spot test. • 585 and 1,132 randomly picked plaques were analyzed in Cross A and B, respectively.
288
~HOHT
COMMUNICATIONS
each of the two crosses (Table 8) we conclude that the dam locus is located between genes 49 and rI on the 1'2 genetic map. The close agreement observed between the 1'2 and 1'4 genetic maps (16) 'would allow a tentative assignment of the 1'4 clam gene to a corresponding region. It is interesting to note that the nonessential gene for thioredoxin (nnZC) in 1'4 has been recently assigned to the regionbetween genes 49 and rlalso (1'l). ACKNOWLEDGMENTS This work was supported by a Public Service Grant No. AI-08738 and a grant from the National Science Foundation No. GB-32125. S. H. is the recipient of a Public Health Service Research Career Development Award (K04 AI-28022); J. B. is a pre-doctoral trainee supported in part by !1 Public Health Service Training Grant No. 5 TOl-GM 06658-12. REFERENCES 1.
RI~VJ~L,
H. R., HATTMAN, S., and LURIA, S. E., Biochem, Biophys. Res. Commun. l8, 545-550 (1965). 2. REVJ~L, H. R., Virology 31, 688-701 (1967). 3. HATTMAN, S., and FUKAsAwA, T., Proc. Nat. Accul. Sci. USA 50,297-300 (1963).
4. REVEl" H. R., ancI LUl\Ii\., 8. Eo, Amm. Rev. Genet. 4, 177-192 (1970). 5. KLEIN, A., Z. Vererbungsl. 96, 346-363 (1965). 6. MOLHOLT, B., Ph.D. Thesis, University of Indiana, Bloomington, Indiana (1967). 7. HEVEL, H. H., ancI GEORGOPOULOS, C. P., Virology 39, 1-17 (1969). 8. HEHLMA.NN, R., and HATTMAN, S., J. Mol. Biol. 67, 351-360 (1972). 9. Rl,:VEL, H. R' I and HATTMAN, S. M., Virology 45,484-495 (1971). 10. HATTMAN, S., Virology 42, 359-3!17 (1970). 11. FUJIMOTO, D., SRINIVASAN, P. R., and Bomcx, E., Biochemistry ,j" 2849-2855 (1965). 12. GOLD, ~I., HAUSMANN, R., ~I.UTHA, U., and HURWITZ, J. Prot'. Nat. Acad. Sci. USA 52, 292-297 (1964). 13. HAUSMANN, H", and GOLD, M., J. Biol, Chem, 2'U, 1985-1994 (1966). 14. MARINUS, M. G., and MOHRIs, R. N., (1973) J. Bacleriol. in press. 15. GEORGOPOULOS, C. P., Ph.D. Thesis, Massachusetts Institute of Technology, Cambridge, Mass. (1969). 16. RUSSI'JLL, R. L., Ph.D. Thesis, California Institute of Technology, Pasadena, California (1967). 17. TESSMAN,!', and GREENDEHG, D. B., Virology 49,337-338 (1972).