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OF Zys2 MUTATIONS in the YEAST SACCHAROMYCES CEREVZSZAE

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OF Zys2 MUTATIONS in the YEAST SACCHAROMYCES CEREVZSZAE PATTERNS OF GENETIC AND PHENOTYPIC SUPPRESSION OF Zys2 MUTATIONS IN THE YEAST SACCHAROMYCES CEREVZSZAE BHARAT B. CHATTOO', EDWARD PALMER, BUN-ICHIRO ON02 and FRED SHERMAN Department of Radiation Biology and Biophysics University of Rochester School of Medicine and Dentistry Rochester, New York 14642 Manuscript received April 19, 1979 ABSTRACT A total of 358 lys2 mutants of Saccharomyces cerevisiae have been charac- terized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppres- sor; an omnipotent suppressor SUP46; and a frameshift suppressor SUFI-1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to re- quire certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require sup- pressors that were expressed at high levels. Many of the mutants suppressi- ble by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phe- notypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phe- notypic misreading. STUDIES with the iso-I -cytochrome c system of Saccharomyces cerevisiae have revealed nonsense suppressors that insert tyrosine, serine or leucine resi- dues at the site of either UAA or UAG mutations (GILMORE,STEWART and SHERMAN1971; LIEBMAN,STEWART and SHERMAN1975; 1976; BRANDRISSet al. 1976; LIEBMANet al. 1977; ONO, STEWARTand SHERMAN1979a,b; SHERMAN, ONO and STEWART1979). These suppressors have been instrumental in identi- 1 Present address: Sarabhai Research Centre, Post Box 162, Baroda 390 007, India. 2 Present address: Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, 700 Japan. Genetics 93: 67-79 September, 1979. 68 B. B. CHATTOO, et d. fying nucleotide sequence at the mutant sites of numerous suppressible markers and aiding in the identification of mutationally altered tRNAs with suppressor activity. Suppressible markers characterized with the defined suppressors have proved to be invaluable in a wide range of investigations. In particular, certain of the nutritional markers responding to suppressors that insert only certain amino acids have aided in the isolation of specific classes of suppressors (ONO, STEWARTand SHERMAN1979b). In this paper we show that suppressible mark- ers also can be useful for revealing relationships between genetic suppression due to altered tRNAs, genetic suppression presumably due to altered ribosomes and phenotypic suppression due to misreading. In order to extend the range of available suppressible markers, we have char- acterized a large number of Zys2 mutants. Recently Zys2 mutants have become easily obtainable by a selection procedure based on the observation that, unlike normal strains, Zys2 strains can utilize a-aminoadipate as a principal nitrogen source (CHATTOOet al. 1979). The following properties of 358 Zys2 mutations are reported in this study: suppressibility by UAA and UAG suppressors insert- ing tyrosine, serine or leucine; suppressibility by a putative UGA suppressor; suppressibility by an omnipotent suppressor probably having abnormal ribo- somes; suppressibiilty by a frameshift suppressor; phenotypic suppressibility by the aminoglycoside antibiotic paromomycin; osmotic remediability ; and temper- ature sensitivity. While most of the suppressible Zys2 mutants uncovered in this study were similar to the suppressible mutants commonly observed, some excep- tional Zys2 mutants exhibited restricted ra,nges of suppressibility and others ex- hibited extended ranges not previously reported. The patterns of suppressibility of the Zys2 mutants suggested a similarity in the action of omnipotent suppres- sion and phenotypic suppression. MATERIALS AND METHODS Strains: All of the lys2 mutants examined in this investigation were of independent origin and all of them arose spontaneously from the strain AS2-2A (a trpl-1). These Zys2 mutants were selected on AA medium as previously described by CHATTOOet al. (1979). The supres- sor tester strains, listed in Table 1, were obtained from other studies (GILMORE,STEWART and SHERMAN1971; LIEBMAN,STEWART and SHERMAN1975; 1976; LIEBMANet al. 1977; ONO, STEWARTand SHERMAN1979a, b) or were specially constructed for this study. Nonsuppressible Zys2 markers were sometimes introduced into suppressor-bearing strains by simply plating the strain on AA medium and testing the resultant colonies (CHATTOOet al. 1979). A putative UGA suppressor was isolated by the co-reversion of the leu2-2 and his4-166 markers that were kindly provided by G. FINK (Cornell University). This suppressor does not act on standard UAA and UAG markers, but it does act on the Zed-2, his4166 and lysl-101 markers, which are also suppressed by omnipotent suppressors. Evidence suggesting that this SUP-UGA suppressor acts on UGA mutations will be presented elsewhere. Media: The standard complex and synthetic media used in this study have been described by SHERMANand LAWRENCE(1974). The synthetic minimal medium contained 0.67% Bacto-yeast nitrogen base without amino acids, 2% dextrose, 2% Bacto-agar when needed for solidification, 30 mg per 1 lysine, 20 mg per 1 tryptophan, and other supplements (SHERMAN and LAWRENCE1974) when required. The main AA medium used in this study contained 0.167% Bacto-yeast nitrogen base without amino acids and without ammonium sulfate, 2% dextrose, 0.2% D,L-a-aminoadipic acid, 20 mg per 1 L-lysine, 20 mg per 1 L-tryptophan and GENETIC AND PHENOTYPIC SUPPRESSION IN YEAST 69 TABLE 1 List of tester strains Strain number Pertinent genotype SL334-6C LY SUPS-a lys2-1 leu2-1 his5-2 aro7-1 cycl-179 AA-1 a SUPl6-a lys2 lysl-l leu2-1 aro7-l trpl-1 metd-l canl-100 cycl-179 BClO5-1 B Q! SUP52-a lys2-1 metd-l ilvl-I aro7-I AA-2 $- 01 SUP44 lys2 leul-12 ilv3 his5-2 trp5-48 cycl-13 AA-3 P+ (Y SUP16-o lys2 leu2-1 his5-2 ade2-1 canl-100 ilvl-2 cycl-72 AA4 $+ (Y SUP27-0 lys2 leu2-1 his5-2 ade2-1 canl-I00 ilvl-2 cycl-72 B0506-7D $+ (Y SUP-UGA lys2-1 leu2-2 his4-166 aro7-1 ura4-l AA-5 $+ a SUP46 lys2 leu2-2 his4-I66 metb-1 AA-6 9- 01 SUFI-I lys2 his4-519 The lys2 markers were introduced in the AA strains by selection on AA medium. 2% Bacto-agar (CHATOOet al. 1979). Other AA media contained the above ingredients, plus other standard supplements that were required by various auxotrophis strains. Genetic suppression: The suppressibility of the lys2 mutants was determined by crossing each mutant to the a tester strains listed in Table 1. These tester strains contain one or another of the suppressors, a nonsuppressible lys2 marker, and usually one or more auxotrophic markers complementary to the marker in the parent strain AS2-2A (a trpl-I). (Some of the UAG suppressor strains also contain the UAG mutation trpl-I, which does not pre- vent prototrophic selections of the diploid strains.) Diploid strains were isolated from the mating mixtures on a synthetic medium supplemented with lysine. The suppression of the lys2 mutations was determined by examining the level of growth of these diploid strains after two to three days incubation on a synthetic medium lacking lysine. Equivalent results were obtained by spotting the mating mixtures instead of the diploid strains on lysineless medium. In addition to examining the suppressibility in strains heterozygous for the suppressors and heteroallelic for the lys2 markers, the diploid strains were sporulated and the sporulated cul- tures were spotted on a lysineless medium supplemented with the requirements of the other markers. This latter test is equivalent to testing haploid strains having the suppressors coupled with the lys2 markers. Phenotypic suppression: Identification of the lys2 mutants phenotypically suppressed by paromomycin was carried out by the procedure described by PALMER,WILMHELM and SHER- MAN (1979a). Approximately 107 cells of each strain were spread on separate plates of a synthetic medium lacking lysine and containing tryptophan. A paper disc saturated with approximately 7 pmoles of paromomycin was placed in the center of each and the plates were incubated at 30" for seven days. In some instances the degree of phenotypic suppression was estimated more quantitively by the level of growth in liquid synthetic media lacking lysine and containing various amounts of paromomycin (PALMER,WILHEZM and SHERMAN1979b). Ten ml of synthetic media, in 125 ml side-arm flasks, was inoculated to an initial titer of 106 cells per ml, and the growth of the cultures vigorously shaken at 30" was determined after 36 hr by measuring the turbidity with a Klett-Summerson colorimeter containing a No. 60 (560-650 nm) light filter. Osmotic remediability and temperature sensitivity: Cell suspensions of the lys2 mutants were spotted on synthetic media supplemented with tryptophan and containing either 1.5 M KC1, 1.0 M KC1, 0.5 M KC1 or no additions of KC1. The plates were incubated at a",30" and 37" for five days to identify mutants that exhibited temperature-sensitive growth and osmotic remedial growth, as well as those that exhibited growth that was dependent on the interac- tion of these two factors.
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