Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4623-4627, December 1976 Genetics
Genetic analysis of a transposable suppressor gene in Saccharomyces cerevisiae (gene mapping/nonsense suppression/transfer RNA)
HOWARD M. LATEN, JOHN GORMAN, FRANCES WEBB, AND ROBERT M. BOCK Department of Biochemistry and Laboratory of Molecular Biology, University of Wisconsin,
Madison, Wisc. 53706
Communicated by Oliver E. Nelson, Jr., October 8, 1976 ABSTRACT We have demonstrated in Saccharomyces cerevisiae the transposition of a gene coding for an efficient ochre (UAA) suppressor from a centromere-linked site on chromosome III to two new sites in the yeast genome. One site is on chromosome VI, very close to, if not allelic with, SUP)), one of eight genes coding for a tyrosine-inserting suppressor. The second site is on chromosome III, unlinked to the centromere and distal to the mating type locus. This site is very close to those mapped for the recessive lethal amber suppressors, SUP-RL1 and SUP61.
Gene transposition is a term that has been used to describe the phenomenon of gene movement as observed in the genomes of Zea mays (1-3), Drosophila melanogaster (4, 5), bacteria and their plasmids (6-12, 16-20), and temperate bacteriophages (13-16). For the purpose of this paper, we will define transposition as the movement of a gene from one locus on the yeast chromosomal complement to another, without regard to mechanism. Transposable elements in maize were first described by McClintock (1) and Brink and Nilan (2). These elements appear to modulate the expression of specific genetic information by repressing the action of adjacent genes (1-3). In D. melanogaster, spontaneous transposition of a controlling element from a location on the X chromosome to a new site on an autosome was reported by Green (4). The controlling element is integrated at the site of the white-crimson eye color gene, Wc (5), and when transposed, carries along a portion of the WC gene
(4).
In prokaryotic systems, the term transposition has been applied to both specific and nonspecific insertions of DNA from one genome into another or from one genetic locus into another. The transposition of the lac region of the Escherichia coli chromosome to other sites on the chromosome via an Flac episome intermediate has been reported by -several authors (6-9). Analogous transpositions involving the arabinose operon have also been reported (10). Insertion of DNA segments of R factors (antibiotic resistant plasmids) into other plasmids (11, 12) and into bacteriophage X (13) has also been demonstrated. Other insertions include those of the mutator phage, Mu, into the E. coli K12 chromosome (14), those of an altered phage P22 into the Salmonella typhimurium chromosome (15), and those of the insertion sequences (IS) into E. coli (16), plasmid (17-19), and bacteriophage X (16) genomes. Saedler et al. (20) have shown that, like the eukaryotic controlling elements, IS2 can control gene expression, specifically that of the galactose operon in E. coli. Our observations of gene transposition in Saccharomyces cerevisiae occurred during the characterization of several classes of yeast ochre suppressors isolated in our laboratory. This paper describes the genetic examination of one of the suppressor Abbreviations: SUP, suppressor; cM, centimorgan; PD, parental ditype; NPD, non-parental ditype; T, tetratype.
genes as it is transposed to new sites in the S. cerevisiae genome.
EXPERIMENTAL PROCEDURES Strains. GB700:2 1-16 was isolated in our laboratory by the selection for simultaneous reversion of the ochre (UAA)
mutations ade2-1, canl-100, his5-2, lysl-l, and trp5-48. The 2:2 segregation of this mutation at meiosis establishes it as a suppressor, designated SUPI16. SUPI 16 is linked to the mating type locus on chromosome III, and its frequency of second division segregation, 0.10, indicates it is proximal to the mating type locus, 5 centimorgans (cM) from the centromere. In addition to suppressing the above alleles, SUP 16 suppresses the ochre alleles arg4-17 and cycl -9. It has no effect on the amber
(UAG) alleles tyr7-1 and trpl-1. This pattern of suppression strongly suggests that SUP 16 may code for a class I, set I sup-
pressor. Class I, set I suppressors are a collection of efficient yeast ochre supersuppressors (21, 22) that insert tyrosine in response to the UAA codon (23). Eight such loci have been identified (21, 22) but none map on chromosome III. Another ochre suppressor, SUQ.5, a serine inserter, is an efficient suppressor only in the presence of the cytoplasmically inherited factor, *+ (24). Because all strains used in this study are '', it is unlikely that S UPI 16 is related to SUQ5. HL2038B was one of 20 independently derived strains selected after ethyl methanesulfonate treatment as a canavanine resistant revertant of strain GB700:2 1-16. GB700:2 1-16 is sensitive to the arginine analogue, canavanine, by virtue of the suppression of canl-100. The strains were shown to carry additional canavanine resistance mutations, presumably in the canl locus (Laten, unpublished results). The pattern of suppression of SUPI 16 in these strains was not altered. A complete list of strains used in this study is given in Table 1. Gene Designations. SUPI 16 refers to the suppressor gene in general, or as found in strain GB700:2 1-16. SUPr¶16 refers to the suppressor gene in all strains where transpositions of the gene are suspected or demonstrated. Genetic Methods. Strains were constructed by utilizing standard techniques of crossing, sporulation, and ascus dissection. Nutritional requirements were determined by growth on synthetic medium containing 0.67% Bacto-yeast nitrogen base (without amino acids), 2% glucose, 2% Bacto-agar, and appropriate supplements. Segregation of the suppressor was monitored by the suppression of two or more ochre nutritional markers. The ochre alleles used in this study include ade2-1, his5-2, lysl-l, trp5-48, and arg4-17. Mating type was determined by complementation of segregants with the multiplymarked aa strain, WY91, on unsupplemented medium. Gene Mapping. Distances between genes were calculated from the following equation (25): 100(6NPD + T) d 2(PD + NPD + T)
4623
4624
Genetics: Laten et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Strain designations and genotypes
Strain no.
Genotype*
Source or parentage
GB700:2 I-16 GB701-14 HL2038B
a SUPI 16 (ade2-1 his5-2 lysl-l trp5-48 canl-100) leul-12 a ade2-1 his5-2 lysl-l trp5-48 canl-100 urab a SUP Jr6 (ade2-1 his5-2 lysl-l trp5-48) leul-12 canl-100-Rt
HL2038B-3D X2928-3D-1A X3424-6C X1049-9C JK373-21C JK421-9B GB502-4B
a
SUPtr16 (ade2-1 his5-2 lysl-l trp5-48) leul-12
Gorman and Bock Gorman and Bock See experimental procedures
HL2038B X GB701-14 Yeast Gen. Stock Ctr., Berkeley, Calif. Yeast Gen. Stock Ctr., Berkeley, Calif. Yeast Gen. Stock Ctr., Berkeley, Calif. Gorman
GB502-25B GB502-30A HL103-2B
adel gall leul his2 ura3 trpl metl4 ilv3 leu2 his6 ade2 arg4 lysl gal2 asp5 his2 trpl ura3 lys7 his8 ade2 arg8 trp5-48 ura3-0 ce trp5-48 a ura3-0 arg4-1 7 lysl-1 trpl-1 tyr7-1 as lysl-I arg4-17 a ade2-1 lysl-1 tyr7-1 a ura3-0 ade2-1 lysl-1 trpl-1 a (ade2-1 his5-2 lysl-l trp5-48)
HLZlA-11C
a
SUPit%6 (ade2-1
HLZlA-23C
a
SUPI 16 (ade2-1 arg4-1 7 lysl -1)
HL103-2B x GB502-11C
HLZ1C-7C HLZ1A1-2C HLZ1A3-1D HL301-2A HL302-9
a
SUPtr6 (ade2-1 trp5-48)
HL103-2B x JK421-9B
GB502-11C
a cs a a
Gorman Gorman and Bock Gorman and Bock Gorman and Bock Gorman and Bock HL2038B-3D X GB701-14
SUPi-6
lysl-l)
HL103-2B X GB502-11C
SUPit16 (lysl-l arg4-17) a SUPir16 (ade2-1 lysl-l) tyr7-1 a adel his2 ura3 trpl metl4 tyr7-1 arg4-1 7 lysl-1 a ade2 his6 leu2 ura3 ilv3 trp5-48 a his8 arg8 asp5 trpl ura3 ade2-1 lysl-l
HLZlA-23C x GB502-11C HLZlA-11C x GB502-25B X2928-3D-1A X GB502-4B X3424-6C X JK373-21C X1049-9C x GB502-30A
a
HL303-1B
Parentheses indicate suppressed alleles. t R denotes reversion of this allele.
*
where d is the distance in cM, and PD, NPD, and T are the number of parental ditype, nonparental ditype, and tetratype asci, respectively, for the marker pair.
RESULTS Several of the strains derived by ethyl methanesulfonate mutagenesis from GB700:2 I-16 were crossed with GB701-14. After sporulation of the diploids and dissection of the asci, the ascus types, PD, NPD, and T with respect to S UP, 16 and mating type were noted. Segregation of the suppressor gene was monitored by the suppression of the ochre alleles ade2-1, his5-2, lysl-l, and trp5-48. Although the strains carry the additional ochre
allele, canl-100, the presence or absence of this marker was not scored. Tetrads from 20 crosses were analyzed. In 14 of the crosses, SUP-16 was still centromere-linked, proximal to the mating type locus on chromosome III, as in GB700:2 I-16. However, tetrad analysis of the segregants from six of the crosses indicated that the SUP 1.6 gene was no longer linked to the mating type locus, a/la, as evidenced by a significant number of T and NPD asci. A list of the ascus types derived from one of the six crosses appears in Table 2A. Two genes are considered unlinked when the ratio of PD to NPD asci approaches 1:1. Perkins (25) has derived an equation that predicts the frequency of crossing-over between gene x and its centromere [X] when the frequency of crossing-over between gene y and its
Table 2. (A) Linkage relationships of SUPi% 6 from tetrad analyses of the segregants from the cross: HL2038B-3D x GB701-14, and (B) second division segregation frequency of SUPi-16
B*
Expected second
Calculated second
division division segregation segregation Frequency of frequency of frequency of tetratype asci known marker suuptr
A*
Number of asci PD
NPD
T
PD:NPD
(T)
(Y)
(X6
SUPIr16-a/a
15
10
14
1.5:1
0.36
0.38
0.00
SUPr,6-leult
16
18
5
0.9:1
0.12
0.02
0.10
Marker pair
(A) Segregant genotypes were determined (see Experimental Procedures), and the ascus types for the appropriate marker pairs noted. (B) The second division segregation frequencies for SUP I6 were calculated from the equation X = (T - Y)/[1 - (3/2) 1]. t The tight linkage of leu I to the centromere of chromosome VII, Y = 0.02, minimizes the absolute error of the calculated value of X.
*
Proc. Natl. Acad. Sci. USA 73 (1976)
Genetics: Laten et al.
4625
GB700:2 1-16
HL2038B X GB701-14 GB701 -14 X HL2038B-3D
HL103-2B X JK421-9B | X3424-6C X JK373-21C
GB502-1 1 C X H L1 03-2B
GB502-1 1C X HLZ1A-23C
HLZ1A-11C X GB502-25B HLZ1C-7C X HL302-9
X2928-3D-1A X GB502-4B
I
GB502-30A X X1049-9C
H L312
HL301-2A X HLZ1A 1-2C
HL303-1B X HLZ1A3-1D
I
HL31 1
HL313
FIG. 1. Lineage of HL311, HL312, and HL313. Solid lines connect segregants with matings from which they derive. Dotted line denotes a mutagenesis. The genotypes of all strains are listed in Table 1. centromere
[Y] and the frequency of tetratype asci [T]
are
known: X
=
(T
-
Y)/(1
-
3Y
[1]
The results of the application of this equation to the data in Table 2A are given in Table 2B. They indicate that the new SUPr_16 locus, although no longer on chromosome III, is still centromere-linked. To map the location of the transposed SUPs'16 gene in HL2038B, we constructed strains that carried several centromere-linked markers in addition to appropriate suppressible mutations. Strains carrying SUPI'16 along with ochre markers thereby ensuring that its segregation could be monitored by the suppression of two such alleles were also constructed. Fig. 1 outlines the lineage of the strains used to map the transposed suppressor gene. The genotypes and sources or parentage of all strains are given in Table 1. Three crosses involving 14 genetic loci, 12 of which were centromere-linked, were made. In cross HL313, SUP [16 showed no linkage to any of the markers in the cross: hWs8, asp5, trpl, ura3, arg8, or a/a (data not shown). In cross HL311 (Table 3), however, SUPr,31 shows linkage to the his2 locus on chromosome VI and is calculated to be 26 cM from this locus. An analysis of the asci in HL311 (data not shown) indicates that there were no crossovers between trpl and its centromere and metl4 and its centromere. This facilitates an ordering of these asci and reveals that the frequency of crossing-over between the suppressor gene and its centromere is 0.06. This is consistent with the findings presented in Table 2B, and confirms the transposition of SUP 116 to a site proximal to his2, 3 cM from the centromere. Although the mapping of the SUPI,31' locus in HL311 was straightforward, the data reveal one feature that is not consistent with results expected from a conventional genetic cross. This feature is the presence of two NPD asci with respect to the his2-SUP/!,361" marker pair, The frequency of T asci, from Table 3, roughly reflecting the frequency of single crossovers between the two genes, is 0.17. Because the formation of an NPD ascus requires a four-strand double crossover, and such crossovers constitute only 25% of the total number of double crossovers, the frequency of NPD asci in the absence of interference is expected to be [0.17]2[0.25] or 0.007 [only positive chiasma interference has been observed in yeast (26), and such interference would result in the decrease of this frequency; chromatid interference has been observed in yeast only rarely (26)]. From the binomial expression, the probability of two NPD asci
resulting from chromosomal recombination in this cross is 0.03. If one of the NPD asci were disregarded, the distance between his2 and SUP3' would shorten to 18 cM. One possible explanation for this finding is that one of these asci resulted from the transposition of SUP16 to a new site unlinked to hs2. To test this hypothesis, suppressor positive segregants from both NPD asci were crossed with appropriate nonsuppressor strains. The results demonstrated that in both cases SUPI-16 was still linked to his2 (data not shown). Finally, from the data from cross HL312 (Table 4), the 6 gene shows linkage to a/a. The map distance between the two genes is 37 cM. The data also indicate that SUP-2 is loosely linked to leu2, a centromere-linked gene on chromosome III separated from a/a by the centromere. If SUPI2 were proximal to a/ca it would exhibit much closer linkage (fewer NPD and T asci) to leu2. The suppressor gene is therefore distal to a/a and constitutes another SUPI16 site, the second on chromosome III. Table 3. Tetrad mapping of SUPfJ6 from the segregants of cross HL311
Linkage relationship or intergenic distance
Number of asci
Marker pair
PD
NPD
T
(cM)
ade1-SUP~r311
17
14
4
Unlinked
his2-SUPIJr3l1
27
2
6
ura3-SUPtr31l 1-16 trpl1-SUP~r3j metl 4-SUPtr3ll
14
12
9
Unlinked
17
16
2
Unlinked
18
15
2
9
6
20
11 6
6 7
17 22
Unlinked Unlinked Unlinked Unlinked
tyr7-SUPfjr311 a/10-SUPJr361 a/a-his2
26*
Segregant genotypes were determined (see Experimental Procedures), and the ascus types for the appropriate marker pairs noted. Map distances were calculated from the equation d = 100 (6NPD + T)/2(PD + NPD + 7). The a/a-his2 marker pair is a control that demonstrates non-linkage of a/a to his2. * This figure reduces to 18 cM with the statistical elimination of one NPD ascus (see Results).
4626
Genetics: Laten et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 4. Tetrad mapping of" I 6 from the segregants of cross HL312
Marker pair
PD
NPD
T
Linkage relationship or intergenic distance (cM)
his6-SUpIr312 I-16
13
12
11
Unlinked
leu2-SUPfr3j2
19
6
11
Linked
ura3-SUPtr3f2 ilv3-SUPtr3l2 'I"1-16 a/a-SUPtr312
12
15
9
Unlinked
8
15
13
Unlinked
19 15
2 0
15 17
Number of asci
a/ca-leu2*
37 27
See Table 3 for description of methods. The a/a-leu2 marker pair is a control that demonstrates the expected linkage of a/a to leu2. * Because the leu2 marker was suppressible, the genotypes of the segregants with respect to leu2 could not be determined in four out of the 36 asci. Two of these asci were either PD or T with respect to the a/a-leu2 marker pair and the other two were either T or NPD. Backcrosses necessary to determine the ascus types of these four asci were not made.
This finding, however, is not consistent with the data presented in Table 2B that demonstrate SUPr116 to be centromere-linked in HL2038B, nor is it consistent with the observation that SUPr16 is not linked to the mating type locus in any cross previous to HL312 (results not shown). The best explanation for these observations is that the SUP"2 site on chromosome III is the result of the transposition of the suppressor gene from chromosome VI (the site mapped in HL311) during the sporulation of the ZIC-7 ascus.
DISCUSSION The data presented demonstrate the transposition of the SUPI 16 gene from a site on chromosome III to a centromere-linked site on chromosome VI. In addition, the transposition of the SUP"I16 gene presumably from this new site to a site 37 cM distal to a/la on chromosome III is also demonstrated. We have no evidence to suggest that our data reflect the transposition of a controlling element analogous to those reported in maize (1-3), Drosophila (4), and E. coli (20); however, the nature of those factors responsible for the observed high frequency of transposition in both the presence and absence of ethyl methanesulfonate is not known. The alternative explanation that additional suppressor mutations have been generated is unlikely in view of the observation that the suppressor phenotype segregates as a single genetic marker in all crosses reported. That the observed transpositions are due to double mutations whereby one mutation results in the reversion of the suppressor SUPI 16 while the second mutation creates a new suppressor, SUPX, is also very unlikely. The method of screening for the canavanine-resistant revertants of GB700:2 I-16, one of those revertants being HL2038B, selected those strains which retained the function of an efficient ochre suppressor. The strains selected contained either the original suppressor, SUPI 16, or a new suppressor, SUPX,, but not both. The presence of two efficient ochre suppressors greatly retards growth in haploids (21). That the frequency of mutation under the conditions of this mutagenesis was not abnormally high is evident from the observation that,
among 97 canavanine-resistant revertants of GB700:2 1-16, none of the four point mutations, ade2-1, his5-2, lysi-1, and trp5-48 had reverted, there were no additional mutations (other than suppressible ochre mutations) in these loci, nor were there any additional nonsuppressible conditional lethal mutations in any of the several enzymes involved in the synthesis of adenine, histidine, lysine, tryptophan, or leucine (unpublished results). These observations together with independent observations of the stability of the SUPI 16 gene, make it clear that an unreasonably high frequency of mutation for both the reversion of SUPI 16 and the creation of SUPX would be required to generate the observed six out of 20 strains in which the suppressor is at a new locus. Thus, the preferred interpretation is that of gene transposition at a high frequency, reminiscent of the observations of high frequencies of transposition in maize. In this study, we have not attempted to answer the question of whether or not the observed transpositions are site specific. However, the site mapped for SUPir311 places it very close to the SUPlJ locus, one of eight genes that code for a tyrosineinserting suppressor in S. cerevlsiae (21-23, 27, 28). In addition, our finding of a second division segregation frequency of 0.06 for SUP ?.31 is identical to that reported for SUPI I by Hawthorne and Mortimer (22). Whether SUPII and SUP'r36 are at the same locus remains unclear. SUP-62 is on chromosome III distal to the mating type locus. A gene coding for the recessive lethal amber suppressor, SUP-RL1 (29), has been found to map between the thr4 and MAL2 loci, 30 cM distal to a/a (30). SUP61, also a recessive lethal amber suppressor, maps 39 cM distal to a/a (31), and may be identical to SUP-RL1. SUPuJ361 maps 37 cM distal to a/a (see Table 4) and could be at the same locus as either SUP-RL1 or SUP61. The contrasting characteristics of the mutations make it unlikely that S UPI 16 and SUP-RLI or SUP61 are lesions in identical genes. However, both these genes probably encode tRNAs and could exhibit a significant amount of sequence homology. Gesteland et al. (32) have demonstrated that two of the eight genes coding for the tyrosine-inserting suppressors SUP4 and SUP6, and the gene coding for SUP-RL1 code for tRNAs. In addition, Capecchi et al. (33) have shown that the tyrosine-inserting suppressor SUP7 and the serine inserter, SUQ5, also code for tRNAs. The sequence homologies that exist between tRNA species and the apparent redundancy of tRNATYr in S. cerevtsiae may be significant in the mechanism of the observed transpositions. This possibility is currently being
investigated. Note Added in Proof. By using the Poisson distribution, the expected frequency of NPD asci for the his2-SUPr3l marker pair is 0.005. The probability given as 0.03 in the text should be 0.01. This research was supported by National Science Foundation Grant GB-41275 and by National Institutes of Health Grant GM12395. 1. McClintock, B. (1951) "Chromosome organization and genic expression," Cold Spring Harbor Symp. Quant. Biol. 16, 1347. 2. Brink, R. A. & Nilan, R. (1952) "The relation between light variegated and medium variegated pericarp in maize," Genetics
37,519-544. 3. Fincham, J. R. S. & Sastry, G. R. K. (1974) "Controlling elements in maize," Annu. Rev. Genet. 8, 15-50. 4. Green, M. (1969) "Controlling element mediated transpositions
of the white gene in Drosophila melanogaster," Genetics 61, 429-441.
Genetics: Laten et al. 5. Green, M. (1969) "Mapping a Drosophila melanogaster 'controlling element' by interallelic crossing over," Genetics 61, 423-428. 6. Cuzin, F. & Jacob, F. (1964) "Deletions chromosomiques et int6grations d'un episome sexuel F-lac+ chez Escherichia coli K12," C.R. Hebd. Seances Acad. Sci. 258,1350-1352. 7. Beckwith, J. & Signer, E. (1966) "Transposition of the lac region of E. coli. I. Inversion of the lac operon and transduction of lac by ,80," J. Mol. Biol. 19, 254-265. 8. Beckwith, J., Signer, E. & Epstein, W. (1966) "Transposition of the lac region of E. coll," Cold Spring Harbor Symp. Quant. Biol. 31,393-401. 9. Ippen, K., Shapiro, J. & Beckwith, J. (1971) "Transposition of the lac region to the gal region of the Escherichia coli chromosome: Isolation of Xlac transducing bacteriophages," J. Bacteriol. 108, 5-9. 10. Gottesman, S. & Beckwith, J. (1969) "Directed transposition of the arabinose operon: A technique for the isolation of specialized transducing bacteriophages for any E. coli gene," J. Mol. Blol. 44, 117-127. 11. Hedges, R. W. & Jacob, A. E. (1974) "Transpositions of ampicillin resistance from RP4 to other replicons," Mol. Gen. Genet. 132, 31-40. 12. Heffron, F., Sublett, R., Hedges, R. W., Jacob, A. & Falkow, S. (1975) "Origin of the TEM j3-lactamase gene found in plasmids," J. Bacteriol. 122,250-256. 13. Berg, D. E., Davies, J., Allet, B. & Rochaix, J.-D. (1975) "Transposition of R factor genes to bacteriophage X," Proc. Natl. Acad. Sci. USA 72,3628-3632. 14. Howe, M. M. & Bade, E. G. (1975) "Molecular biology of bacteriophage Mu," Science 190, 624-632. 15. Kleckner, N., Chan, R., Tye, B.-K. & Botstein, D. (1975) "Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition," J. Mol. Biol. 97,561-575. 16. Hirsh, H.-J., Starlinger, P. & Brachet, P. (1972) "Two kinds of insertions in bacterial genes," Mol. Gen. Genet. 119, 191-206. 17. Sharp, P., Hsu, M.-T., Ohtsubo, E. & Davidson, N. (1972) "Electron microscope heteroduplex studies of sequence relations among plasmids of E. coli. I. Structure of F-prime factors," J. Mol. Biol. 71, 471-497. 18. Hu, S., Ohtsubo, E., Davidson, N. & Saedler, H. (1975) "Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: Identification and mapping of the insertion sequences IS1 and IS2 in F and R Plasmids," J. Bacteriol. 122, 764-775. 19. Ptashne, K. & Cohen, S. N. (1975) "Occurrence of insertion se-
Proc. Natl. Acad. Sci. USA 73 (1976)
4627
quence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications," J. Bacteriol. 122, 776-781. 20. Saedler, H., Reif, H. J., Hu, S. & Davidson, N. (1974) "IS2, a genetic element for turn-off and turn-on of gene activity in E. coli," Mol. Gen. Genet. 132,265-289. 21. Gilmore, R. A. (1967) "Super-suppressors in Saccharomyces cerevisiae," Genetics 56,641-658. 22. Hawthorne, D. C. & Mortimer, R. K. (1968) "Genetic mapping of nonsense suppressors in yeast," Genetics 60, 735-742. 23. Gilmore, R. A., Stewart, J. W. & Sherman, F. (1971) "Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast," J. Mol. Biol. 61, 157-173. 24. Liebman, S. W., Stewart, J. W. & Sherman, F. (1975) "Serine substitutions caused by an ochre suppressor in yeast," J. Mol. Biol. 94,595-610. 25. Perkins, D. D. (1949) "Biochemical mutants in the smut fungus Ustilago maydis," Genetics 34,607-626. 26. Mortimer, R. K. & Hawthorne, D. C. (1969) in The Yeasts, eds. Rose, A. H. & Harrison, J. S. (Academic Press, London), Vol. 1, pp. 409-410. 27. Sherman, F., Liebman, S. W., Stewart, J. W. & Jackson, M. (1973) "Tyrosine substitution resulting from suppression of amber mutants of iso-1-cytochrome c in yeast," J. Mol. Biol. 78, 157168. 28. Liebman, S. W., Sherman, F. & Stewart, J. W. (1976) "Isolation and characterization of amber suppressors in yeast," Genetics 82,251-272. 29. Brandriss, M. C., Soil, L. & Botstein, D. (1975) "Recessive lethal amber suppressors in yeast," Genetics 79, 551-560. 30. Brandriss, M. C., Stewart, J. W., Sherman, F. & Botstein, D. (1976) "Substitution of serine caused by a recessive lethal suppressor in yeast," J. Mol. Biol. 102, 467-476. 31. Mortimer, R. K. & Hawthorne, D. C. (1973) "Genetic mapping in Saccharomyces. IV. Mapping of temperature sensitive genes and use of disomic strains in localizing genes," Genetics 74, 33-54. 32. Gesteland, R. F., Wolfner, M., Grisafi, P., Fink, G., Botstein. D. & Roth, J. R. (1976) "Yeast suppressors of UAA and UAG nionsense codons work efficiently in vitro via tRNA," Cell 7, 381390. 33. Capecchi, M. R., Hughes, S. H. & Wahl, G. M. (1975) "Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro," Cell 6,269-277.
Genetic analysis of a transposable suppressor gene in Saccharomyces cerevisiae (gene mapping/nonsense suppression/transfer RNA)
HOWARD M. LATEN, JOHN GORMAN, FRANCES WEBB, AND ROBERT M. BOCK Department of Biochemistry and Laboratory of Molecular Biology, University of Wisconsin,
Madison, Wisc. 53706
Communicated by Oliver E. Nelson, Jr., October 8, 1976 ABSTRACT We have demonstrated in Saccharomyces cerevisiae the transposition of a gene coding for an efficient ochre (UAA) suppressor from a centromere-linked site on chromosome III to two new sites in the yeast genome. One site is on chromosome VI, very close to, if not allelic with, SUP)), one of eight genes coding for a tyrosine-inserting suppressor. The second site is on chromosome III, unlinked to the centromere and distal to the mating type locus. This site is very close to those mapped for the recessive lethal amber suppressors, SUP-RL1 and SUP61.
Gene transposition is a term that has been used to describe the phenomenon of gene movement as observed in the genomes of Zea mays (1-3), Drosophila melanogaster (4, 5), bacteria and their plasmids (6-12, 16-20), and temperate bacteriophages (13-16). For the purpose of this paper, we will define transposition as the movement of a gene from one locus on the yeast chromosomal complement to another, without regard to mechanism. Transposable elements in maize were first described by McClintock (1) and Brink and Nilan (2). These elements appear to modulate the expression of specific genetic information by repressing the action of adjacent genes (1-3). In D. melanogaster, spontaneous transposition of a controlling element from a location on the X chromosome to a new site on an autosome was reported by Green (4). The controlling element is integrated at the site of the white-crimson eye color gene, Wc (5), and when transposed, carries along a portion of the WC gene
(4).
In prokaryotic systems, the term transposition has been applied to both specific and nonspecific insertions of DNA from one genome into another or from one genetic locus into another. The transposition of the lac region of the Escherichia coli chromosome to other sites on the chromosome via an Flac episome intermediate has been reported by -several authors (6-9). Analogous transpositions involving the arabinose operon have also been reported (10). Insertion of DNA segments of R factors (antibiotic resistant plasmids) into other plasmids (11, 12) and into bacteriophage X (13) has also been demonstrated. Other insertions include those of the mutator phage, Mu, into the E. coli K12 chromosome (14), those of an altered phage P22 into the Salmonella typhimurium chromosome (15), and those of the insertion sequences (IS) into E. coli (16), plasmid (17-19), and bacteriophage X (16) genomes. Saedler et al. (20) have shown that, like the eukaryotic controlling elements, IS2 can control gene expression, specifically that of the galactose operon in E. coli. Our observations of gene transposition in Saccharomyces cerevisiae occurred during the characterization of several classes of yeast ochre suppressors isolated in our laboratory. This paper describes the genetic examination of one of the suppressor Abbreviations: SUP, suppressor; cM, centimorgan; PD, parental ditype; NPD, non-parental ditype; T, tetratype.
genes as it is transposed to new sites in the S. cerevisiae genome.
EXPERIMENTAL PROCEDURES Strains. GB700:2 1-16 was isolated in our laboratory by the selection for simultaneous reversion of the ochre (UAA)
mutations ade2-1, canl-100, his5-2, lysl-l, and trp5-48. The 2:2 segregation of this mutation at meiosis establishes it as a suppressor, designated SUPI16. SUPI 16 is linked to the mating type locus on chromosome III, and its frequency of second division segregation, 0.10, indicates it is proximal to the mating type locus, 5 centimorgans (cM) from the centromere. In addition to suppressing the above alleles, SUP 16 suppresses the ochre alleles arg4-17 and cycl -9. It has no effect on the amber
(UAG) alleles tyr7-1 and trpl-1. This pattern of suppression strongly suggests that SUP 16 may code for a class I, set I sup-
pressor. Class I, set I suppressors are a collection of efficient yeast ochre supersuppressors (21, 22) that insert tyrosine in response to the UAA codon (23). Eight such loci have been identified (21, 22) but none map on chromosome III. Another ochre suppressor, SUQ.5, a serine inserter, is an efficient suppressor only in the presence of the cytoplasmically inherited factor, *+ (24). Because all strains used in this study are '', it is unlikely that S UPI 16 is related to SUQ5. HL2038B was one of 20 independently derived strains selected after ethyl methanesulfonate treatment as a canavanine resistant revertant of strain GB700:2 1-16. GB700:2 1-16 is sensitive to the arginine analogue, canavanine, by virtue of the suppression of canl-100. The strains were shown to carry additional canavanine resistance mutations, presumably in the canl locus (Laten, unpublished results). The pattern of suppression of SUPI 16 in these strains was not altered. A complete list of strains used in this study is given in Table 1. Gene Designations. SUPI 16 refers to the suppressor gene in general, or as found in strain GB700:2 1-16. SUPr¶16 refers to the suppressor gene in all strains where transpositions of the gene are suspected or demonstrated. Genetic Methods. Strains were constructed by utilizing standard techniques of crossing, sporulation, and ascus dissection. Nutritional requirements were determined by growth on synthetic medium containing 0.67% Bacto-yeast nitrogen base (without amino acids), 2% glucose, 2% Bacto-agar, and appropriate supplements. Segregation of the suppressor was monitored by the suppression of two or more ochre nutritional markers. The ochre alleles used in this study include ade2-1, his5-2, lysl-l, trp5-48, and arg4-17. Mating type was determined by complementation of segregants with the multiplymarked aa strain, WY91, on unsupplemented medium. Gene Mapping. Distances between genes were calculated from the following equation (25): 100(6NPD + T) d 2(PD + NPD + T)
4623
4624
Genetics: Laten et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 1. Strain designations and genotypes
Strain no.
Genotype*
Source or parentage
GB700:2 I-16 GB701-14 HL2038B
a SUPI 16 (ade2-1 his5-2 lysl-l trp5-48 canl-100) leul-12 a ade2-1 his5-2 lysl-l trp5-48 canl-100 urab a SUP Jr6 (ade2-1 his5-2 lysl-l trp5-48) leul-12 canl-100-Rt
HL2038B-3D X2928-3D-1A X3424-6C X1049-9C JK373-21C JK421-9B GB502-4B
a
SUPtr16 (ade2-1 his5-2 lysl-l trp5-48) leul-12
Gorman and Bock Gorman and Bock See experimental procedures
HL2038B X GB701-14 Yeast Gen. Stock Ctr., Berkeley, Calif. Yeast Gen. Stock Ctr., Berkeley, Calif. Yeast Gen. Stock Ctr., Berkeley, Calif. Gorman
GB502-25B GB502-30A HL103-2B
adel gall leul his2 ura3 trpl metl4 ilv3 leu2 his6 ade2 arg4 lysl gal2 asp5 his2 trpl ura3 lys7 his8 ade2 arg8 trp5-48 ura3-0 ce trp5-48 a ura3-0 arg4-1 7 lysl-1 trpl-1 tyr7-1 as lysl-I arg4-17 a ade2-1 lysl-1 tyr7-1 a ura3-0 ade2-1 lysl-1 trpl-1 a (ade2-1 his5-2 lysl-l trp5-48)
HLZlA-11C
a
SUPit%6 (ade2-1
HLZlA-23C
a
SUPI 16 (ade2-1 arg4-1 7 lysl -1)
HL103-2B x GB502-11C
HLZ1C-7C HLZ1A1-2C HLZ1A3-1D HL301-2A HL302-9
a
SUPtr6 (ade2-1 trp5-48)
HL103-2B x JK421-9B
GB502-11C
a cs a a
Gorman Gorman and Bock Gorman and Bock Gorman and Bock Gorman and Bock HL2038B-3D X GB701-14
SUPi-6
lysl-l)
HL103-2B X GB502-11C
SUPit16 (lysl-l arg4-17) a SUPir16 (ade2-1 lysl-l) tyr7-1 a adel his2 ura3 trpl metl4 tyr7-1 arg4-1 7 lysl-1 a ade2 his6 leu2 ura3 ilv3 trp5-48 a his8 arg8 asp5 trpl ura3 ade2-1 lysl-l
HLZlA-23C x GB502-11C HLZlA-11C x GB502-25B X2928-3D-1A X GB502-4B X3424-6C X JK373-21C X1049-9C x GB502-30A
a
HL303-1B
Parentheses indicate suppressed alleles. t R denotes reversion of this allele.
*
where d is the distance in cM, and PD, NPD, and T are the number of parental ditype, nonparental ditype, and tetratype asci, respectively, for the marker pair.
RESULTS Several of the strains derived by ethyl methanesulfonate mutagenesis from GB700:2 I-16 were crossed with GB701-14. After sporulation of the diploids and dissection of the asci, the ascus types, PD, NPD, and T with respect to S UP, 16 and mating type were noted. Segregation of the suppressor gene was monitored by the suppression of the ochre alleles ade2-1, his5-2, lysl-l, and trp5-48. Although the strains carry the additional ochre
allele, canl-100, the presence or absence of this marker was not scored. Tetrads from 20 crosses were analyzed. In 14 of the crosses, SUP-16 was still centromere-linked, proximal to the mating type locus on chromosome III, as in GB700:2 I-16. However, tetrad analysis of the segregants from six of the crosses indicated that the SUP 1.6 gene was no longer linked to the mating type locus, a/la, as evidenced by a significant number of T and NPD asci. A list of the ascus types derived from one of the six crosses appears in Table 2A. Two genes are considered unlinked when the ratio of PD to NPD asci approaches 1:1. Perkins (25) has derived an equation that predicts the frequency of crossing-over between gene x and its centromere [X] when the frequency of crossing-over between gene y and its
Table 2. (A) Linkage relationships of SUPi% 6 from tetrad analyses of the segregants from the cross: HL2038B-3D x GB701-14, and (B) second division segregation frequency of SUPi-16
B*
Expected second
Calculated second
division division segregation segregation Frequency of frequency of frequency of tetratype asci known marker suuptr
A*
Number of asci PD
NPD
T
PD:NPD
(T)
(Y)
(X6
SUPIr16-a/a
15
10
14
1.5:1
0.36
0.38
0.00
SUPr,6-leult
16
18
5
0.9:1
0.12
0.02
0.10
Marker pair
(A) Segregant genotypes were determined (see Experimental Procedures), and the ascus types for the appropriate marker pairs noted. (B) The second division segregation frequencies for SUP I6 were calculated from the equation X = (T - Y)/[1 - (3/2) 1]. t The tight linkage of leu I to the centromere of chromosome VII, Y = 0.02, minimizes the absolute error of the calculated value of X.
*
Proc. Natl. Acad. Sci. USA 73 (1976)
Genetics: Laten et al.
4625
GB700:2 1-16
HL2038B X GB701-14 GB701 -14 X HL2038B-3D
HL103-2B X JK421-9B | X3424-6C X JK373-21C
GB502-1 1 C X H L1 03-2B
GB502-1 1C X HLZ1A-23C
HLZ1A-11C X GB502-25B HLZ1C-7C X HL302-9
X2928-3D-1A X GB502-4B
I
GB502-30A X X1049-9C
H L312
HL301-2A X HLZ1A 1-2C
HL303-1B X HLZ1A3-1D
I
HL31 1
HL313
FIG. 1. Lineage of HL311, HL312, and HL313. Solid lines connect segregants with matings from which they derive. Dotted line denotes a mutagenesis. The genotypes of all strains are listed in Table 1. centromere
[Y] and the frequency of tetratype asci [T]
are
known: X
=
(T
-
Y)/(1
-
3Y
[1]
The results of the application of this equation to the data in Table 2A are given in Table 2B. They indicate that the new SUPr_16 locus, although no longer on chromosome III, is still centromere-linked. To map the location of the transposed SUPs'16 gene in HL2038B, we constructed strains that carried several centromere-linked markers in addition to appropriate suppressible mutations. Strains carrying SUPI'16 along with ochre markers thereby ensuring that its segregation could be monitored by the suppression of two such alleles were also constructed. Fig. 1 outlines the lineage of the strains used to map the transposed suppressor gene. The genotypes and sources or parentage of all strains are given in Table 1. Three crosses involving 14 genetic loci, 12 of which were centromere-linked, were made. In cross HL313, SUP [16 showed no linkage to any of the markers in the cross: hWs8, asp5, trpl, ura3, arg8, or a/a (data not shown). In cross HL311 (Table 3), however, SUPr,31 shows linkage to the his2 locus on chromosome VI and is calculated to be 26 cM from this locus. An analysis of the asci in HL311 (data not shown) indicates that there were no crossovers between trpl and its centromere and metl4 and its centromere. This facilitates an ordering of these asci and reveals that the frequency of crossing-over between the suppressor gene and its centromere is 0.06. This is consistent with the findings presented in Table 2B, and confirms the transposition of SUP 116 to a site proximal to his2, 3 cM from the centromere. Although the mapping of the SUPI,31' locus in HL311 was straightforward, the data reveal one feature that is not consistent with results expected from a conventional genetic cross. This feature is the presence of two NPD asci with respect to the his2-SUP/!,361" marker pair, The frequency of T asci, from Table 3, roughly reflecting the frequency of single crossovers between the two genes, is 0.17. Because the formation of an NPD ascus requires a four-strand double crossover, and such crossovers constitute only 25% of the total number of double crossovers, the frequency of NPD asci in the absence of interference is expected to be [0.17]2[0.25] or 0.007 [only positive chiasma interference has been observed in yeast (26), and such interference would result in the decrease of this frequency; chromatid interference has been observed in yeast only rarely (26)]. From the binomial expression, the probability of two NPD asci
resulting from chromosomal recombination in this cross is 0.03. If one of the NPD asci were disregarded, the distance between his2 and SUP3' would shorten to 18 cM. One possible explanation for this finding is that one of these asci resulted from the transposition of SUP16 to a new site unlinked to hs2. To test this hypothesis, suppressor positive segregants from both NPD asci were crossed with appropriate nonsuppressor strains. The results demonstrated that in both cases SUPI-16 was still linked to his2 (data not shown). Finally, from the data from cross HL312 (Table 4), the 6 gene shows linkage to a/a. The map distance between the two genes is 37 cM. The data also indicate that SUP-2 is loosely linked to leu2, a centromere-linked gene on chromosome III separated from a/a by the centromere. If SUPI2 were proximal to a/ca it would exhibit much closer linkage (fewer NPD and T asci) to leu2. The suppressor gene is therefore distal to a/a and constitutes another SUPI16 site, the second on chromosome III. Table 3. Tetrad mapping of SUPfJ6 from the segregants of cross HL311
Linkage relationship or intergenic distance
Number of asci
Marker pair
PD
NPD
T
(cM)
ade1-SUP~r311
17
14
4
Unlinked
his2-SUPIJr3l1
27
2
6
ura3-SUPtr31l 1-16 trpl1-SUP~r3j metl 4-SUPtr3ll
14
12
9
Unlinked
17
16
2
Unlinked
18
15
2
9
6
20
11 6
6 7
17 22
Unlinked Unlinked Unlinked Unlinked
tyr7-SUPfjr311 a/10-SUPJr361 a/a-his2
26*
Segregant genotypes were determined (see Experimental Procedures), and the ascus types for the appropriate marker pairs noted. Map distances were calculated from the equation d = 100 (6NPD + T)/2(PD + NPD + 7). The a/a-his2 marker pair is a control that demonstrates non-linkage of a/a to his2. * This figure reduces to 18 cM with the statistical elimination of one NPD ascus (see Results).
4626
Genetics: Laten et al.
Proc. Natl. Acad. Sci. USA 73 (1976)
Table 4. Tetrad mapping of" I 6 from the segregants of cross HL312
Marker pair
PD
NPD
T
Linkage relationship or intergenic distance (cM)
his6-SUpIr312 I-16
13
12
11
Unlinked
leu2-SUPfr3j2
19
6
11
Linked
ura3-SUPtr3f2 ilv3-SUPtr3l2 'I"1-16 a/a-SUPtr312
12
15
9
Unlinked
8
15
13
Unlinked
19 15
2 0
15 17
Number of asci
a/ca-leu2*
37 27
See Table 3 for description of methods. The a/a-leu2 marker pair is a control that demonstrates the expected linkage of a/a to leu2. * Because the leu2 marker was suppressible, the genotypes of the segregants with respect to leu2 could not be determined in four out of the 36 asci. Two of these asci were either PD or T with respect to the a/a-leu2 marker pair and the other two were either T or NPD. Backcrosses necessary to determine the ascus types of these four asci were not made.
This finding, however, is not consistent with the data presented in Table 2B that demonstrate SUPr116 to be centromere-linked in HL2038B, nor is it consistent with the observation that SUPr16 is not linked to the mating type locus in any cross previous to HL312 (results not shown). The best explanation for these observations is that the SUP"2 site on chromosome III is the result of the transposition of the suppressor gene from chromosome VI (the site mapped in HL311) during the sporulation of the ZIC-7 ascus.
DISCUSSION The data presented demonstrate the transposition of the SUPI 16 gene from a site on chromosome III to a centromere-linked site on chromosome VI. In addition, the transposition of the SUP"I16 gene presumably from this new site to a site 37 cM distal to a/la on chromosome III is also demonstrated. We have no evidence to suggest that our data reflect the transposition of a controlling element analogous to those reported in maize (1-3), Drosophila (4), and E. coli (20); however, the nature of those factors responsible for the observed high frequency of transposition in both the presence and absence of ethyl methanesulfonate is not known. The alternative explanation that additional suppressor mutations have been generated is unlikely in view of the observation that the suppressor phenotype segregates as a single genetic marker in all crosses reported. That the observed transpositions are due to double mutations whereby one mutation results in the reversion of the suppressor SUPI 16 while the second mutation creates a new suppressor, SUPX, is also very unlikely. The method of screening for the canavanine-resistant revertants of GB700:2 I-16, one of those revertants being HL2038B, selected those strains which retained the function of an efficient ochre suppressor. The strains selected contained either the original suppressor, SUPI 16, or a new suppressor, SUPX,, but not both. The presence of two efficient ochre suppressors greatly retards growth in haploids (21). That the frequency of mutation under the conditions of this mutagenesis was not abnormally high is evident from the observation that,
among 97 canavanine-resistant revertants of GB700:2 1-16, none of the four point mutations, ade2-1, his5-2, lysi-1, and trp5-48 had reverted, there were no additional mutations (other than suppressible ochre mutations) in these loci, nor were there any additional nonsuppressible conditional lethal mutations in any of the several enzymes involved in the synthesis of adenine, histidine, lysine, tryptophan, or leucine (unpublished results). These observations together with independent observations of the stability of the SUPI 16 gene, make it clear that an unreasonably high frequency of mutation for both the reversion of SUPI 16 and the creation of SUPX would be required to generate the observed six out of 20 strains in which the suppressor is at a new locus. Thus, the preferred interpretation is that of gene transposition at a high frequency, reminiscent of the observations of high frequencies of transposition in maize. In this study, we have not attempted to answer the question of whether or not the observed transpositions are site specific. However, the site mapped for SUPir311 places it very close to the SUPlJ locus, one of eight genes that code for a tyrosineinserting suppressor in S. cerevlsiae (21-23, 27, 28). In addition, our finding of a second division segregation frequency of 0.06 for SUP ?.31 is identical to that reported for SUPI I by Hawthorne and Mortimer (22). Whether SUPII and SUP'r36 are at the same locus remains unclear. SUP-62 is on chromosome III distal to the mating type locus. A gene coding for the recessive lethal amber suppressor, SUP-RL1 (29), has been found to map between the thr4 and MAL2 loci, 30 cM distal to a/a (30). SUP61, also a recessive lethal amber suppressor, maps 39 cM distal to a/a (31), and may be identical to SUP-RL1. SUPuJ361 maps 37 cM distal to a/a (see Table 4) and could be at the same locus as either SUP-RL1 or SUP61. The contrasting characteristics of the mutations make it unlikely that S UPI 16 and SUP-RLI or SUP61 are lesions in identical genes. However, both these genes probably encode tRNAs and could exhibit a significant amount of sequence homology. Gesteland et al. (32) have demonstrated that two of the eight genes coding for the tyrosine-inserting suppressors SUP4 and SUP6, and the gene coding for SUP-RL1 code for tRNAs. In addition, Capecchi et al. (33) have shown that the tyrosine-inserting suppressor SUP7 and the serine inserter, SUQ5, also code for tRNAs. The sequence homologies that exist between tRNA species and the apparent redundancy of tRNATYr in S. cerevtsiae may be significant in the mechanism of the observed transpositions. This possibility is currently being
investigated. Note Added in Proof. By using the Poisson distribution, the expected frequency of NPD asci for the his2-SUPr3l marker pair is 0.005. The probability given as 0.03 in the text should be 0.01. This research was supported by National Science Foundation Grant GB-41275 and by National Institutes of Health Grant GM12395. 1. McClintock, B. (1951) "Chromosome organization and genic expression," Cold Spring Harbor Symp. Quant. Biol. 16, 1347. 2. Brink, R. A. & Nilan, R. (1952) "The relation between light variegated and medium variegated pericarp in maize," Genetics
37,519-544. 3. Fincham, J. R. S. & Sastry, G. R. K. (1974) "Controlling elements in maize," Annu. Rev. Genet. 8, 15-50. 4. Green, M. (1969) "Controlling element mediated transpositions
of the white gene in Drosophila melanogaster," Genetics 61, 429-441.
Genetics: Laten et al. 5. Green, M. (1969) "Mapping a Drosophila melanogaster 'controlling element' by interallelic crossing over," Genetics 61, 423-428. 6. Cuzin, F. & Jacob, F. (1964) "Deletions chromosomiques et int6grations d'un episome sexuel F-lac+ chez Escherichia coli K12," C.R. Hebd. Seances Acad. Sci. 258,1350-1352. 7. Beckwith, J. & Signer, E. (1966) "Transposition of the lac region of E. coli. I. Inversion of the lac operon and transduction of lac by ,80," J. Mol. Biol. 19, 254-265. 8. Beckwith, J., Signer, E. & Epstein, W. (1966) "Transposition of the lac region of E. coll," Cold Spring Harbor Symp. Quant. Biol. 31,393-401. 9. Ippen, K., Shapiro, J. & Beckwith, J. (1971) "Transposition of the lac region to the gal region of the Escherichia coli chromosome: Isolation of Xlac transducing bacteriophages," J. Bacteriol. 108, 5-9. 10. Gottesman, S. & Beckwith, J. (1969) "Directed transposition of the arabinose operon: A technique for the isolation of specialized transducing bacteriophages for any E. coli gene," J. Mol. Blol. 44, 117-127. 11. Hedges, R. W. & Jacob, A. E. (1974) "Transpositions of ampicillin resistance from RP4 to other replicons," Mol. Gen. Genet. 132, 31-40. 12. Heffron, F., Sublett, R., Hedges, R. W., Jacob, A. & Falkow, S. (1975) "Origin of the TEM j3-lactamase gene found in plasmids," J. Bacteriol. 122,250-256. 13. Berg, D. E., Davies, J., Allet, B. & Rochaix, J.-D. (1975) "Transposition of R factor genes to bacteriophage X," Proc. Natl. Acad. Sci. USA 72,3628-3632. 14. Howe, M. M. & Bade, E. G. (1975) "Molecular biology of bacteriophage Mu," Science 190, 624-632. 15. Kleckner, N., Chan, R., Tye, B.-K. & Botstein, D. (1975) "Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition," J. Mol. Biol. 97,561-575. 16. Hirsh, H.-J., Starlinger, P. & Brachet, P. (1972) "Two kinds of insertions in bacterial genes," Mol. Gen. Genet. 119, 191-206. 17. Sharp, P., Hsu, M.-T., Ohtsubo, E. & Davidson, N. (1972) "Electron microscope heteroduplex studies of sequence relations among plasmids of E. coli. I. Structure of F-prime factors," J. Mol. Biol. 71, 471-497. 18. Hu, S., Ohtsubo, E., Davidson, N. & Saedler, H. (1975) "Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: Identification and mapping of the insertion sequences IS1 and IS2 in F and R Plasmids," J. Bacteriol. 122, 764-775. 19. Ptashne, K. & Cohen, S. N. (1975) "Occurrence of insertion se-
Proc. Natl. Acad. Sci. USA 73 (1976)
4627
quence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications," J. Bacteriol. 122, 776-781. 20. Saedler, H., Reif, H. J., Hu, S. & Davidson, N. (1974) "IS2, a genetic element for turn-off and turn-on of gene activity in E. coli," Mol. Gen. Genet. 132,265-289. 21. Gilmore, R. A. (1967) "Super-suppressors in Saccharomyces cerevisiae," Genetics 56,641-658. 22. Hawthorne, D. C. & Mortimer, R. K. (1968) "Genetic mapping of nonsense suppressors in yeast," Genetics 60, 735-742. 23. Gilmore, R. A., Stewart, J. W. & Sherman, F. (1971) "Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast," J. Mol. Biol. 61, 157-173. 24. Liebman, S. W., Stewart, J. W. & Sherman, F. (1975) "Serine substitutions caused by an ochre suppressor in yeast," J. Mol. Biol. 94,595-610. 25. Perkins, D. D. (1949) "Biochemical mutants in the smut fungus Ustilago maydis," Genetics 34,607-626. 26. Mortimer, R. K. & Hawthorne, D. C. (1969) in The Yeasts, eds. Rose, A. H. & Harrison, J. S. (Academic Press, London), Vol. 1, pp. 409-410. 27. Sherman, F., Liebman, S. W., Stewart, J. W. & Jackson, M. (1973) "Tyrosine substitution resulting from suppression of amber mutants of iso-1-cytochrome c in yeast," J. Mol. Biol. 78, 157168. 28. Liebman, S. W., Sherman, F. & Stewart, J. W. (1976) "Isolation and characterization of amber suppressors in yeast," Genetics 82,251-272. 29. Brandriss, M. C., Soil, L. & Botstein, D. (1975) "Recessive lethal amber suppressors in yeast," Genetics 79, 551-560. 30. Brandriss, M. C., Stewart, J. W., Sherman, F. & Botstein, D. (1976) "Substitution of serine caused by a recessive lethal suppressor in yeast," J. Mol. Biol. 102, 467-476. 31. Mortimer, R. K. & Hawthorne, D. C. (1973) "Genetic mapping in Saccharomyces. IV. Mapping of temperature sensitive genes and use of disomic strains in localizing genes," Genetics 74, 33-54. 32. Gesteland, R. F., Wolfner, M., Grisafi, P., Fink, G., Botstein. D. & Roth, J. R. (1976) "Yeast suppressors of UAA and UAG nionsense codons work efficiently in vitro via tRNA," Cell 7, 381390. 33. Capecchi, M. R., Hughes, S. H. & Wahl, G. M. (1975) "Yeast super-suppressors are altered tRNAs capable of translating a nonsense codon in vitro," Cell 6,269-277.
