VIROLOGY 63, 591-595 (1975)
Amber
Red-
JANET E. MERTZ,’ Department
of Biology,
Mutants
of Phage Lambda
ETHAN R. SIGNER, AND FRED SCHAEFER
Massachusetts
Institute
Accepted
of Technology,
Cambridge,
Massachusetts
02139
October 15, 1974
A positive selection procedure for obtaining recombination-deficient (red-) mutants of bacteriophage lambda has been used to isolate and partially characterize a series of amber red- mutants.
Recombination-deficient (red-) mutants of bacteriophage lambda recombine poorly in ret- bacteria (1-3). Analysis of such mutants (4) shows that two red genes (Fig. 1) exist, each coding for a protein that has been purified. Reda codes for an exonuclease (5), and red/3 codes for the beta protein (6; see 7 for review). Red- mutations also affect lambda growth under certain conditions, particularly when combined in various ways with mutations in y and 6, two other genes located in the same region of the lambda genome and probably also involved in recombination (8-11). Compared with wildtype, growth of 6-red-yis restricted under some conditions and enhanced in others. The experiments described here rely on three such growth-altering phenomena, summarized in Table 1. Though none of them are yet understood in molecular terms, they are nevertheless very useful. Using these phenomena, we first selected new red- mutations appearing in a a-y5 stock by growing &-red+75 in bacterial lysogenic for the unrelated phage P2 in which 6-red-75 has a large growth advantage over Cred+r5. Then, red+ recombinants, or suppressed red- amber mutants, of these new a-red+75 phages were selected from or identified in a mixture with unsuppressed a-red-75 phages by plating on bacteria where a-red+75 recA-sup+ ’ Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA.
phages efficiently form plaques. Third, red- mutants were mapped by crossing them with a set of b-red- delet,ion mutants and scoring for the red+ recombinants which can grow in polymerase I-deficient bacteria (PO&, refs. 3, 12). In this manner, we have isolated and partially characterized a series of amber red- mutants. The mutants were obtained in the 6-ystock bio7-20-y5, in which gene 6 is deleted (Fig. l), by isolating phage from the rare plaques that appear when this stock is plated on a lawn of the P2 lysogen W311O(P2) (9). To insure the independence of separate isolates, plaques of bio72075 were suspended in 0.5 ml buffer each and individually plated on W311O(P2); 0.3 ml of suspension gave between 0 and 50 plaques. A background of several thousand minute plaques, a result of the low but finite plating efficiency of bio7-2075 on this host (Table l), was also seen but ignored. The larger plaques were presumed to represent new spontaneous and independent red- mutants. These new red- mutants (of presumed genotype b-red-y-) were screened for amber suppressibility by plating on P2lysogenic hosts that either did (sup’) or did not (sup-) carry an amber suppressor. Since neither bio7-20 nor y5 is amber, only suppression of the new mutation would restore inability to grow on a P2 lysogen. The new mutants that appeared to be amber were picked directly into buffer and further tested for their amber red- phenotype; each was streaked with an inocula-
,591 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
592
---
SHORT
COMMUNICATIONS
d I
I blo II,22
%iFxm3+227 L--J-
blo
-
7-20
FIG. 1. Map of the inner right quarter of lambda showing genes and deletions used (8,9). Transcription is from right to left on the map as drawn. TABLE 1 PLATING EFFICIENCY OF X MUTANTS ON VARIOUS E. coli HOST@ Mutant Wild-type 6red8-red6-75 d-y210 6-red-yF &red-y210
P2 lysogen
recA -b
-c 2.5 x 10-Sd 0.6” +’ +
+ + + + +
+ + -
3 ,+lop
I
Amber
Red-
JANET E. MERTZ,’ Department
of Biology,
Mutants
of Phage Lambda
ETHAN R. SIGNER, AND FRED SCHAEFER
Massachusetts
Institute
Accepted
of Technology,
Cambridge,
Massachusetts
02139
October 15, 1974
A positive selection procedure for obtaining recombination-deficient (red-) mutants of bacteriophage lambda has been used to isolate and partially characterize a series of amber red- mutants.
Recombination-deficient (red-) mutants of bacteriophage lambda recombine poorly in ret- bacteria (1-3). Analysis of such mutants (4) shows that two red genes (Fig. 1) exist, each coding for a protein that has been purified. Reda codes for an exonuclease (5), and red/3 codes for the beta protein (6; see 7 for review). Red- mutations also affect lambda growth under certain conditions, particularly when combined in various ways with mutations in y and 6, two other genes located in the same region of the lambda genome and probably also involved in recombination (8-11). Compared with wildtype, growth of 6-red-yis restricted under some conditions and enhanced in others. The experiments described here rely on three such growth-altering phenomena, summarized in Table 1. Though none of them are yet understood in molecular terms, they are nevertheless very useful. Using these phenomena, we first selected new red- mutations appearing in a a-y5 stock by growing &-red+75 in bacterial lysogenic for the unrelated phage P2 in which 6-red-75 has a large growth advantage over Cred+r5. Then, red+ recombinants, or suppressed red- amber mutants, of these new a-red+75 phages were selected from or identified in a mixture with unsuppressed a-red-75 phages by plating on bacteria where a-red+75 recA-sup+ ’ Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA.
phages efficiently form plaques. Third, red- mutants were mapped by crossing them with a set of b-red- delet,ion mutants and scoring for the red+ recombinants which can grow in polymerase I-deficient bacteria (PO&, refs. 3, 12). In this manner, we have isolated and partially characterized a series of amber red- mutants. The mutants were obtained in the 6-ystock bio7-20-y5, in which gene 6 is deleted (Fig. l), by isolating phage from the rare plaques that appear when this stock is plated on a lawn of the P2 lysogen W311O(P2) (9). To insure the independence of separate isolates, plaques of bio72075 were suspended in 0.5 ml buffer each and individually plated on W311O(P2); 0.3 ml of suspension gave between 0 and 50 plaques. A background of several thousand minute plaques, a result of the low but finite plating efficiency of bio7-2075 on this host (Table l), was also seen but ignored. The larger plaques were presumed to represent new spontaneous and independent red- mutants. These new red- mutants (of presumed genotype b-red-y-) were screened for amber suppressibility by plating on P2lysogenic hosts that either did (sup’) or did not (sup-) carry an amber suppressor. Since neither bio7-20 nor y5 is amber, only suppression of the new mutation would restore inability to grow on a P2 lysogen. The new mutants that appeared to be amber were picked directly into buffer and further tested for their amber red- phenotype; each was streaked with an inocula-
,591 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
592
---
SHORT
COMMUNICATIONS
d I
I blo II,22
%iFxm3+227 L--J-
blo
-
7-20
FIG. 1. Map of the inner right quarter of lambda showing genes and deletions used (8,9). Transcription is from right to left on the map as drawn. TABLE 1 PLATING EFFICIENCY OF X MUTANTS ON VARIOUS E. coli HOST@ Mutant Wild-type 6red8-red6-75 d-y210 6-red-yF &red-y210
P2 lysogen
recA -b
-c 2.5 x 10-Sd 0.6” +’ +
+ + + + +
+ + -
3 ,+lop
I
