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Tyrosine-incorporating amber suppressors in Escherichia coli K12
J. Mol. Biol. (1968) 33,327-329
Tyrosine-incorporating Amber Suppressors in Escherichia cob K12 Earlier, we described an amber (am) suppressor (sum& in Escherichiu coli K12, strain YMel, which is located near the trpt region of the E. coli chromosome (Henning, Dennert, Szolyvay & Deppe, 1965). This suppressor appeared to be identical to the tyrosine-incorporating am suppressors su +-3 of Garen, Garen & Wilhelm (1965) and ST& of Kaplan, Stretton & Brenner (1965). It was possible to obtain defective $80 phages carr.ying both the am suppressor, suGMel, and part of the trp operon (Dennert, Hertel, Deppe & Henning, 1965). Smith, Abelson, Clark, Goodman & Brenner (1966) reported that they could not confirm our latter finding; i.e. in their low-frequency transducing lysates, $80 phages which transduced trp+ never carried the s&r gene. Therefore, since we were confident that our original finding was correct, the question arose again whether or not su&r (su+-3) is identical to suGMel. Here we wish to demonstrate that, in spite of the apparent discrepancy concerning the transducing $80 phages, these suppressors are, in all probability, identical. The amino acid specified by sucMe, was determined using the alkaline phosphatase am mutant H12 (Weigert, Lanka & Garen, 1965; the strain was kindly donated by Dr A. Garen). suGMel was introduced into a trp- derivative of this strain by transduction with phage Pl propagated on YMel (trp+ su&.,). Table 1 shows that tyrosine is incorporated at the H12 site under the influence of SU:~~,.
TABLE
su&,,-directed
tyrosine incorporation Amino
Strain
Wild
type
H12
.su&~
Tryptic
Ll
1
acid
residues
Substitution
peptide LYE
Ala
GUY
Leu
Tyr
Trp
Ll L2
0.98 1.30
2.1
1.42 1.32
1.00 2.00
-
+ -
+ L2
1.98
2.25
2.24
3.00
0.84
-
Trp-t’W
Purification of alkaline phosphatase was performed according to Malamy t Horecker (1984). Fingerprinting of its tryptic peptides, purification of the peptides Ll and L2, and determination of the amino acid composition of the peptides was performed aa described by Weigert & Garen (1965). Because the Tyr-containing peptide Ll does not separate from L2, they were analyzed together (Weigert, Lanka & Garen, 1965). The Leu values were used as reference for the calculation of all other amino acids. Trp was identified by specific staining (Smith, 1953). The wild type was HfrH. The con&u&ion of H12 8ugMer is described in the text. t Abbreviations used: loci on the E. wlichromosome: ace, acetate; Zac, lactose; resistance; trp, tryptophan; HFT, LFT, high and low frequency of transduction, 327
sW,
streptomycin respectively.
tfp + and 8u&
(5 x 10-1)
(5 x IO-‘)
(5 x lo-‘)
(N 1O-1o)
0 +
+
0
+
0
+
0
+
+
0
+
High-frequency transducing capacity of lysates from corresponding heterogenotes for
10
10
6
13
Number of different HFTlysatestested
The origin of 480 and all relevant techniques have been described earlier (Dennert. et al., 1966). The recipient strains for transduction with LFT Zyaates were: YMel-A33 (tryptophan synthetaae negative, Berger & isolated by Dr S. Brenner, kindly donated by Dr J. N. Yanofsky, 1967), for trp+ * CA274 (HfrH lacby ‘selection for both”.%e:‘a%‘trp+ ; ace32 trp- (HfrC oce;& , son-amber tryptophan Abelson) for a~:,,~ synthetase negative; Henning et al., 1965) for simultaneous transduction of trp + and 8u&. The recipient strains for transduction with HFT lysatea were: CA274 for 8~:~ or BU&; M25 (F- 8tTr UDPQ;, Shapiro, 1966, as isolated by Dr S. Brenner, kindly donated by Dr E. Signer), for UDPB+; YMel-A33 and CA274, for trp+ ; ace32 trp- and CA274, for simultaneous transduction of BU&~ and trp+ which was also tested in single infection experiments. Ace32 8u& was obtained by trp+-linked transduction of au&r with phage Pl grown on X7087 (F- ate Lzc,,,,, trp+ 8u&, kindly donated by Dr J. R. Beckwith) using ace32 trp- aa recipient.
YMel
$>re,
trp’)
trp+
tfp+
(UDPG+
trp+)
Ace32.w&
m&
Selective marker for transduction with LFT lysate (frequency of transduction)
YMel
YMel (UDPB+
TABLE 2
of 480 transducing phages for the UDPG + , su,&, SU&~ and trp+ alleles
$80 Lysogen used for production of LFT lysate
Analysis
LETTERS
TO
THE
329
EDITOR
The transducing $80 phages, mentioned above, which carry both the BU&, gene and part of the trp operon, had been obtained by selection for simultaneous transduction of trp f and su&,. Such phages were found in LF’T lysates at frequencies of 10mQ to lo-lo (Dennert et al., 1965). These phages invariably carried the uridine diphosphate glucose pyrophosphorylase locus as well (Table 2, UDPCt+ ; Shapiro, 1966). Repeating these experiments and selecting for either trp+ or .&Mel transducing phages, we found both classes to occur in LFI’ lysates at frequencies around 5 x 10e7. Table 2 demonstrates that transducing phages carrying trp+ do not transduce do carry U D PG + %44el, 41 or UD PG + . Conversely, phages transducing su& but not trp+ (Table 2). We conclude, therefore, that su& (su+-3) and suGMel are most likely the same am suppressor, being located to the left of the 480 attachment site (cf. Signer, Beckwith & Brenncr, 1965). We further conclude that, upon induction, phage $80 can, although rather rarely, become substituted with parts of the bacterial chromosome which originate from both sides of the prophage attachment site. All such phages we isolated are defective; and they may have deletions which do not involve either end (regarding the prophage map) and may thus carry both the phage and the chromosomal attachment sites. Dr C. Yanofsky has kindly informed us that he also found tyrosine incorporation in a tryptophan synthetase amber mutant under the influence of SU&~, (Drapeau, Brammar & Yanofsky, 1968). fiir Biologie
Max-Planok-Institut
G. DENNERT U. HENNINU
Tubingen, Germany Received
17 January
1968
REFERENCES Berger, H. & Yanofsky, C. (1967). Science, 156, 394. Dennert, G., Hertel, R., Deppe, G. & Henning, U. (1965). 2. Vererbuq$ehre, 97, 243. Drapeau, G. R., Br ammar, W. J. & Yanofsky, C. (1968). J. Mol. Biol. in press. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mo2. Biol. 14, 167. Henning, U.. Dennert, G., Szolyvay, K. BE Deppe, G. (1965). J. Mol. BioZ. 13, 592. Kaplan, S., Stretton, A. 0. W. & Brenner, S. (1965). J. Mol. BioZ. 14, 528. Malamy, M. H. & Horecker, B. L. (1964). Biochemistry, 3, 1893. Shapiro, J. A. (1966). J. Bact. 92, 518, Signer, E. R., Beckwith, J. R. & Brenner, S. (1965). J. Mol. BioZ. 14, 153. Smith, I. (1953). Nature, 171, 43. Smith, J. D., Abelson, J. N., Clark, B. F. C., Goodman, H. M. & Brenner, S. (1966). Cold Spr.
Weigert, Weigert,
Harb.
Symp.
Quant.
BioZ.
31, 479.
M. 0. & Garen, A. (1965). J. Mol. BioZ. 12, 448. M. G., Lanka, E. & Garen, A. (1965). J. Mol. BioZ.
14, 522.
Tyrosine-incorporating Amber Suppressors in Escherichia cob K12 Earlier, we described an amber (am) suppressor (sum& in Escherichiu coli K12, strain YMel, which is located near the trpt region of the E. coli chromosome (Henning, Dennert, Szolyvay & Deppe, 1965). This suppressor appeared to be identical to the tyrosine-incorporating am suppressors su +-3 of Garen, Garen & Wilhelm (1965) and ST& of Kaplan, Stretton & Brenner (1965). It was possible to obtain defective $80 phages carr.ying both the am suppressor, suGMel, and part of the trp operon (Dennert, Hertel, Deppe & Henning, 1965). Smith, Abelson, Clark, Goodman & Brenner (1966) reported that they could not confirm our latter finding; i.e. in their low-frequency transducing lysates, $80 phages which transduced trp+ never carried the s&r gene. Therefore, since we were confident that our original finding was correct, the question arose again whether or not su&r (su+-3) is identical to suGMel. Here we wish to demonstrate that, in spite of the apparent discrepancy concerning the transducing $80 phages, these suppressors are, in all probability, identical. The amino acid specified by sucMe, was determined using the alkaline phosphatase am mutant H12 (Weigert, Lanka & Garen, 1965; the strain was kindly donated by Dr A. Garen). suGMel was introduced into a trp- derivative of this strain by transduction with phage Pl propagated on YMel (trp+ su&.,). Table 1 shows that tyrosine is incorporated at the H12 site under the influence of SU:~~,.
TABLE
su&,,-directed
tyrosine incorporation Amino
Strain
Wild
type
H12
.su&~
Tryptic
Ll
1
acid
residues
Substitution
peptide LYE
Ala
GUY
Leu
Tyr
Trp
Ll L2
0.98 1.30
2.1
1.42 1.32
1.00 2.00
-
+ -
+ L2
1.98
2.25
2.24
3.00
0.84
-
Trp-t’W
Purification of alkaline phosphatase was performed according to Malamy t Horecker (1984). Fingerprinting of its tryptic peptides, purification of the peptides Ll and L2, and determination of the amino acid composition of the peptides was performed aa described by Weigert & Garen (1965). Because the Tyr-containing peptide Ll does not separate from L2, they were analyzed together (Weigert, Lanka & Garen, 1965). The Leu values were used as reference for the calculation of all other amino acids. Trp was identified by specific staining (Smith, 1953). The wild type was HfrH. The con&u&ion of H12 8ugMer is described in the text. t Abbreviations used: loci on the E. wlichromosome: ace, acetate; Zac, lactose; resistance; trp, tryptophan; HFT, LFT, high and low frequency of transduction, 327
sW,
streptomycin respectively.
tfp + and 8u&
(5 x 10-1)
(5 x IO-‘)
(5 x lo-‘)
(N 1O-1o)
0 +
+
0
+
0
+
0
+
+
0
+
High-frequency transducing capacity of lysates from corresponding heterogenotes for
10
10
6
13
Number of different HFTlysatestested
The origin of 480 and all relevant techniques have been described earlier (Dennert. et al., 1966). The recipient strains for transduction with LFT Zyaates were: YMel-A33 (tryptophan synthetaae negative, Berger & isolated by Dr S. Brenner, kindly donated by Dr J. N. Yanofsky, 1967), for trp+ * CA274 (HfrH lacby ‘selection for both”.%e:‘a%‘trp+ ; ace32 trp- (HfrC oce;& , son-amber tryptophan Abelson) for a~:,,~ synthetase negative; Henning et al., 1965) for simultaneous transduction of trp + and 8u&. The recipient strains for transduction with HFT lysatea were: CA274 for 8~:~ or BU&; M25 (F- 8tTr UDPQ;, Shapiro, 1966, as isolated by Dr S. Brenner, kindly donated by Dr E. Signer), for UDPB+; YMel-A33 and CA274, for trp+ ; ace32 trp- and CA274, for simultaneous transduction of BU&~ and trp+ which was also tested in single infection experiments. Ace32 8u& was obtained by trp+-linked transduction of au&r with phage Pl grown on X7087 (F- ate Lzc,,,,, trp+ 8u&, kindly donated by Dr J. R. Beckwith) using ace32 trp- aa recipient.
YMel
$>re,
trp’)
trp+
tfp+
(UDPG+
trp+)
Ace32.w&
m&
Selective marker for transduction with LFT lysate (frequency of transduction)
YMel
YMel (UDPB+
TABLE 2
of 480 transducing phages for the UDPG + , su,&, SU&~ and trp+ alleles
$80 Lysogen used for production of LFT lysate
Analysis
LETTERS
TO
THE
329
EDITOR
The transducing $80 phages, mentioned above, which carry both the BU&, gene and part of the trp operon, had been obtained by selection for simultaneous transduction of trp f and su&,. Such phages were found in LF’T lysates at frequencies of 10mQ to lo-lo (Dennert et al., 1965). These phages invariably carried the uridine diphosphate glucose pyrophosphorylase locus as well (Table 2, UDPCt+ ; Shapiro, 1966). Repeating these experiments and selecting for either trp+ or .&Mel transducing phages, we found both classes to occur in LFI’ lysates at frequencies around 5 x 10e7. Table 2 demonstrates that transducing phages carrying trp+ do not transduce do carry U D PG + %44el, 41 or UD PG + . Conversely, phages transducing su& but not trp+ (Table 2). We conclude, therefore, that su& (su+-3) and suGMel are most likely the same am suppressor, being located to the left of the 480 attachment site (cf. Signer, Beckwith & Brenncr, 1965). We further conclude that, upon induction, phage $80 can, although rather rarely, become substituted with parts of the bacterial chromosome which originate from both sides of the prophage attachment site. All such phages we isolated are defective; and they may have deletions which do not involve either end (regarding the prophage map) and may thus carry both the phage and the chromosomal attachment sites. Dr C. Yanofsky has kindly informed us that he also found tyrosine incorporation in a tryptophan synthetase amber mutant under the influence of SU&~, (Drapeau, Brammar & Yanofsky, 1968). fiir Biologie
Max-Planok-Institut
G. DENNERT U. HENNINU
Tubingen, Germany Received
17 January
1968
REFERENCES Berger, H. & Yanofsky, C. (1967). Science, 156, 394. Dennert, G., Hertel, R., Deppe, G. & Henning, U. (1965). 2. Vererbuq$ehre, 97, 243. Drapeau, G. R., Br ammar, W. J. & Yanofsky, C. (1968). J. Mol. Biol. in press. Garen, A., Garen, S. & Wilhelm, R. C. (1965). J. Mo2. Biol. 14, 167. Henning, U.. Dennert, G., Szolyvay, K. BE Deppe, G. (1965). J. Mol. BioZ. 13, 592. Kaplan, S., Stretton, A. 0. W. & Brenner, S. (1965). J. Mol. BioZ. 14, 528. Malamy, M. H. & Horecker, B. L. (1964). Biochemistry, 3, 1893. Shapiro, J. A. (1966). J. Bact. 92, 518, Signer, E. R., Beckwith, J. R. & Brenner, S. (1965). J. Mol. BioZ. 14, 153. Smith, I. (1953). Nature, 171, 43. Smith, J. D., Abelson, J. N., Clark, B. F. C., Goodman, H. M. & Brenner, S. (1966). Cold Spr.
Weigert, Weigert,
Harb.
Symp.
Quant.
BioZ.
31, 479.
M. 0. & Garen, A. (1965). J. Mol. BioZ. 12, 448. M. G., Lanka, E. & Garen, A. (1965). J. Mol. BioZ.
14, 522.