Yeast amber suppressors corresponding to tRNA3Leu genes

.J. Mol.

Biol.

(1984)

178, 209-226

Yeast Amber Suppressors Corresponding tRNAr Genes SUSAN JOHN

W. LIEBMAN~, W. STEWART~,

ZBIGNIEW FRED

SRODULSKI~, SHERMAN’

AND

R. REEDY

CAROL GINA

to

BRENNAN’

1 Department of Biological Sciences University of Illinois at Chicago, Box 4348 Chicago, Ill. 60680, U.S.A. and ’ Department of Radiation Biology and Biophysics University of Rochester School of Medicine Rochester, N. Y. 14642, U.S.A. (Received

II

January

1.984, and in revised form

10 May

1984)

Amber suppressors previously isolated from the yeast Saccharomyces cerevisiae and belonging to the same phenotypic class (Liebman et al., 1976) were assigned to nine different linkage groups named SUP52 through SUPGO. One of these suppressors, SUP52, had been shown to cause the insertion of leucine and had been genetically mapped (Liebman et al., 1977). The following additional amber suppressors were mapped: SUP53 maps near the centromere of chromosome III closely linked to Zeu2; SUP54 maps on chromosome VII, 6 CM distal to trp5; SL’P56 maps on chromosome I, 5.4 CM distal to adel; SUP57 maps on chromosome VI, closely linked t,o metlO; and SUP58 maps on the left arm of chromosome XI, loosely linked to metl4. We show by protein analysis that) like SUP52, the suppressors SUP53 through SUP56 are leucine-inserters. Furthermore, by hybridization with a cloned tRaAp probe we demonstrate that at least SlJP53, SUP54, SUP55 and SlJP56 contain mutations in redundant tRNAj;‘” genes because they each generate a new XbaI site in a DNA fragment encompassing a tRNAp gene. These new XbaI sites are predicted by the known sequences of tRNA4’” genes if the CAA anticodon mutates to the amber suppressing anticodon CTA. It is likely that each of the nine suppressors in this phenotypic class contain similar mutations in different tRNAt;“” genes since we find that there are approximately nine unlinked redundant copies of tRNA4’” genes in haploid strains.

1. Introduction Numerous nonsense suppressors in the yeast Saccharomyces cerevisiae have been isolated and genetically mapped (for reviews, see Hawthorne & Leupold, 1974; Sherman et al., 1979; Sherman, 1982). These suppressors were initially classified according to their ability to suppress particular alleles conferring auxotrophy. 209 0052~~838/84/260209-18

$03.00/O

(Q 1984 Academic

Press Inc. (London)

Ltd

210

s.

15,.

LIEBMAS

E7’

AL.

Subsequently the action of many of these suppressors on bonafide UA4A and CAC: cycl mutant,s permitted a determination of the suppressible codons as well as the amino acids inserted by the suppressors. Such studies of VAG suppressors revealed that only eight loci (S’(7P2-SCj’PN and A’L~Pll) yield tyrosine-inserters (Lirbman et al.. 1976), that one unique locus (NUP61, alternatively called 81,:1’121,l) yields a recessive-lethal serine-inserter (Krandriss et al.. 1975>1976; Olson rt al.. 1981: lCtcheverr,v rt al.. 1982), and that at, least one locus (RI’P52) yields a lrucine-insert,er (Liebman et al., 1977). Strains containing SGPG-a. Si:P7-a. or Si’PGl-a were shown t’o contain transfer RNA capable of translating I’AG codons in an in vitro protein synthesizing system (Gesteland ef al.. 1976: Capecchi et al.. 1975). Nucleotide sequencing of suppressor tRh’As from SI!P:j-a (Piper rt al.. 1976) and Sf:P61-a (Piper, 1978; Etchevrrry et al., 11979) strains revealed anticodon base-pair changes in tyrosine and serine tRSA. respectively. Tn addition. the eight t’.vrosine-insert,ing suppressor genes have each been correlated to one of the eight EcoRI restriction fragments that hybridize to tyrosine tRSA 1)~ making use of restriction site polymorphisms genetically linked to t>he suppressors (Olson et aZ., 1979). In another molecular study. three serine-inserting l.TAA suppressors have been shown t)o correspond to cloned t)RSA& genes (Broach rt al.. 1981: Olson et al., 1981; M’aldrorr rt al., 1981). In an earlier study, Liebman et al. (1976) described the isolation of dominant amber suppressors and their classificat,ion into phenotypic classes accaording to their ability to suppress amber mutations. The most efficient suppressors were tyrosine-inserters. The suppressors described in t)he present st’udy are distinguished from the tyrosine-inserting suppressors by their reduced action on the amber allele cycl-179. One of t’hese suppressors, SCP52, had been shown to cause the insertion of leucine at, the site of amber codons and had been mapped to the left arm of chromosome X closely linked to the centromere (Liebman et al.. 1977). Here we show that’ the suppressors in this class fall into nine independent groups that are unlinked to each other or to any of the previously studied tyrosine-inserting amber suppressors. We determine the chromosomal loc*ations of five of these new suppressor loci and show that, like SI:P52, the suppressors ,!!I:PX?. SliP51z ACP55 and Sl’1’5Fi are leucine-inserters. The simplest explanation of t,he leucine-insertions is that) these suppressors code for redundant copies of tRNAke” genes. By considering the four published sequences of yeast tR,NAk’” genes (Kang et al.. 1980: Standring et al., 1981: Venegas et al.. 1979: Andreadis et al.. 1983). it can he seen that a new XbaI site in these tR?iAkeU genes would be created when the CAB ant*icodon is mutated to the amber suppressing ant,icodon CTA (see Fig. 1). In t,his paper. we demonstrate the creation of these new XbaT sites in several of our suppressor strains. In addition. assuming the ot’hel phenotypically similar suppressors (AI’P57 t)hrough 8CPGO) were mutations in other tRNA’;‘” genes; one would predicat at least nine copies of tRNA;“” genes in haploid strains. In agreement with t)his. we now show by hybridization of restriction fragments that t’here are approximately nine redundant tR?u’A$“” genes in haploid strains. Preliminary accounts of some of these results have been reported (Reed, 1979; Reed & Liebman, 1979: Srodulski & Liebman. 1983: Srodulski, 1982).

tRNAjLe"

gene

**~~TGATTCA.~GAAA~~O~ $

l ***TGATT&TAGAAA*m*

Suppressor

FIG. 1. Mutation of the anticodon of the coding strand of a tRNaq’” gene to a UAG supprewr gene sequence shown was found in all 4 tRNAp rrsultjing in an Xbal restriction site. The tRNAf;‘” genes that have hren sequenced (Kang et al., 1980; Standring et ul., 1981; Venegas et al., 19W: .indrradis of al., 19X3). The antieodon is in it,alics and a portion of the intervening sequenw is ~~nderlined. The Shni sequence that would br created by mutating the C”A.4 anticodon to the ITA4(: wppw-f’ssing atlt,i~~odon (‘UA is marked.

2. Materials (a)

Yeast genetic

and Methods

naa~rkers

and

nomenclature

The suppressors

analyzed in bhis study were previously isolated as revertants from the strain SL210-3A with the following genotype: XA4Ta ~~~1-179 m&-l aro7-l trpl-l ad&H t&l-f le&l his&2 l&-f (Liebman et al., 1976.1977). These revertant strains have been given “L” numbers, e.g. L-126, L-127. etc. The cycl-179. met&l, ~07-1, trpl-1, ade3-26 and ilvl-1 alleles are amber (VA(;) mutations, while the 1~~2-1, his5-2 and lysl-1 alleles are ochre (IJAA) mutations. The 886 dominant amber suppressors obtained were classified into 34 phenotypic classes according to their ability to suppress the amber mutations. The 43 most efficient, suppressors comprising classes I. 2 and 3 turned out to be tyrosine-inserters. The dist,inct~ion between the suppressors in classes 1, 2 and 3 was based on their different actions on the amber marker. &l-l. The classifications based solely on suppression of &:2-l turned out to be invalid because these differences were not inherited by meiotic progeny, In the present sludy, we are concerned with the 80 suppressors in classes 4 and ;T whicah suppress the amber allele ~~~1-179 less efficiently than do the t,yrosine-inserters. Like the t’yrosine-inserters, the class 4 and 5 suppressors act on t)he presumed amber nutritional markers m&-f, am?-1, trpl-1 and ade3-26. but not, on the ochre markers hidd?, lysl-1. !@I or l&?-I. The distirlctioll betSween t,he class 4 and 5 suppressors is probably spurious since it is based on suppression of iht-1. as were the false distinctions between classes 1. 2 and 3. (h)

Yeast gen,etic

‘methods

Conventional procedures of crossing, sporulation and dissection were employed for meiotic analysis (see Sherman & Lawrence, 1974). The suppressors were scored by suppression of homozygous suppressible markers. generally trpl-1 or ~07-1. The suppressors were initially assigned to their respective loci by genetic analysis with random spores. Haploids bearing the suppressors to be checked for allelism were crossed. These diploids were homozygous for the amber-suppressible markers trpl-1 and/or aro7-7 and heterozggous for the non-amber-suppressible markers Eys2-1 and 2~~1-2. Following sporulation the asci were digested with glusulase and sonicated to disperse the spores. Thr mixture of spores and unsporulated diploids wa,s t,hen diluted and plated onto complete medium. Finally, t,he plates were replica,-plated onto synthetic complete medium, medium lacking lysine (Lys-), and medium larking t,ryptophan (Trp-) or tyrosine (Tyr-) to SCOW for suppression of trpl-1 or aro7-1, respectively. The 2 heterozygous unlinked lysine markers were used to determine the approximat,e number of haploid rolonies present. becauseI iii”‘,. of the spores are expected to require lvsine for growt,h while the diploid cells 110 not require lysine. Therefore, t,he number of colonies t,hat did not, grow on Lys- medium js Tr,‘!,0 of the total number of colonies derived from meiotic spores. The presence of suppressors was d&ected on Trp- or Tyr- media. If the 2 suppressors are tightly linked. then all of the spore clones will carry a suppressor, the amber markers trpl-1 and aro’i-I will always be suppressed. and all of t,he haploids will grow on the Trp- or Tyr- media.

212

S. W.

LIEBMAN

ET

BL.

However, if the 2 suppressors are not linked. then one would expect 250/6 of all spores not t,o carry a suppressor and thus not, grow on ‘I’rpF or Tyr- media. Standard media (Sherman & Lawrence. 1974) were used for scoring various markers. The ,~~~9-~ gene, which prevent+z the maintenance or replication of t,he killer plasmid. was kin13l-v provided by Dr Reed Wickner (Eat. Inst. Health) and was scored by the zone of inhibition of sensitive cells on a buffered nutrient medium containing methylene blue (Somers & Bevan, 1969). Strains bearing the ~1~7-1 mutation (obtained from the Yeast Genetics Stock Center, Berkeley, Calif.) were propagated at 23°C and the ~1~7-1 mutation was scored by its lethal effect at 30°C. (v) l~~t~~rn~~~~~~rl c$ amino acid replacements on supprexszd iso- I-cytohwrw

CA

Strains containing the suppressed cycl-76 mutant gene were grown and the iso-lcytochromes c were prepared by the procedures described by Sherman el al. (1968). Methods for cyanogen bromide cleavage, the fractionation of cyanogen bromide digests on Sephadex QFiO (Stewart & Sherman, 1973), sequential Edman degradation and ~hromatogra~hie identification of the pheny~thi~~hydantoin and dansyl derivatives by thinlayer chromatography have been described (Stewart et OZ., 1971). (d) Hybridization Isolation of DNA from yeast, (Cryer et al., 1975), restrict,ion digestion of DNA, separation of digested DKA on agarose gels, and transfer of DNA to nit,rocellulose filters (Southern, 1975) were performed as described previously (Liebman et GE., 1981). Most blots were probed with Escherichia co& plasmid pJD137 (kindly supplied by Dr J. D. Johnson), ehich contains a veast tRxAieU gene on a 2.5 kbt EcoRI fragment of yeast DNA inserted into the EcoRT iite in pBR322 (Johnson et al., 1980). The yeast fragment contains a single Xbal site and the tRNAp gene is located on a 0.6 kb &oRI-XbaT piece with the 3’ end of the t,R?u’A very near the XbaI site. The 2.5 kb EcoRT fragment was originally isolated from a 6.3 kb HindI segment in which a 2.1 kb HindIIT-XbaI fragment contains the tRh’Ap gene (Beckman et al., 1977; Carrara et al., 1981). Some blots were probed with YEplY or gene linked to the LECB gene (Broach et ul.. 1979; YIp3.1; which contain a tRNAy Botstein et RI., 1979; Andreadis et al.. 1983). i4;‘.coli plasmid DNA was prepared by the procedure of Elwell et al. (1975). Nick translation was according to the Kick Translation Reagent Kit, purchased from Bethesda Research Laboratories. Blots were hybridized for 24 h at 65°C in 5 mM-Ka,EDTA. 40 m&I-NaOlI, 50 m&I-NaH,Po, H,O. 0.9 ,n-h’aci and 0.3% SDS with 100 pg denatured, sonicated calf thymus DNA/ml. Filters were washed 4 times at room ~mperature in lOni~-~a~,~I~*.~PO~~ 0.29b SDS. and 1 ~M-~a~EDTA. Plus intensifying Autoradiography was carried out at - 70°C with DuPont Lightning screens using Kodak XR-5 film.

3. Results

Of the original 80 suppressors belonging to phenotypic classes 4 and 5. 15 were inviable or would not sporulate in crosses; therefore, only the remaining 65 strains were investigated. A random spore test, described in detail in Materials and Methods, was used to determine which of these suppressors were allelic or closely linked. The test consists of crossing pair-wise combinations of the suppressors and (~et,erminin~ the fraction of random spore segregant~s t$hat do not, contain either t Abbreviations used: kb, lo3 base-pairs; SDS, centiMorgan = 15:, crossing over between 2 genes).

sodium

dodeeyl

sulfate;

c&f.

centiMorgan

(1

YEAST

tRXAt;‘U

AMBER

2 13

SPPPRESBORS

suppressor. If the suppressors are very closely linked, all the segregants should carry a suppressor; if they are not linked, approximately one-quarter of the segregants should not carry any suppressor. By crossing ail of the suppressors to a specific tester suppressor, analyzing the crosses, and continuing this process with a second suppressor not linked to the first one, the suppressors have been placed into nine linkage groups, XUP52 through SUPGO. This analysis involved 283 crosses and the data from 18 of these crosses showing suppressors that are the same or diRerent from each of the nine linkage groups are presented in Tabie 1. The major problem encountered in this test was t,hat some of the diploids did not. sporulate. When this occurred, suppressor segregants from various pedigrees were mated to a variety of tester strains in an effort to improve sporulation. The number of suppressors assigned to each linkage group by the random spore analysis is listed in Table 2. Occasionally. very low frequencies of suppressorless colonies were found in the random spore analyses of two suppressors. In these cases, the suppressors were presumed to belong to the same linkage group if t,he frequencies corresponded to the frequencies of suppressor-less colonies arising from homozygous crosses. For example, when spores from a cross between two independent SUP53 suppressors were examined, one out of 979 segregants appeared without, a suppressor. Similarly. when spores from a cross between an identical SCP53 suppressor allele TABLE

1

Random spore data demonstrating linkage assignments Test,er strain St,rairl

no.

(‘Kg-312 (‘RX-31% SL%!W31) SL298-31) (‘W-101> (‘RJ-lOIf (‘RlO-if% (!ftlo-l6(’ (‘Kll-tx‘ (“Rll-6(’ SL509- 1213. SId09-1213 SL*515-101) ~sLsl5-lon SL516-71) SL516-71) SL534- I I) SIAW I I)

Locus

~.

s UP52 s UP52 s VP ,5<‘i SliP53 s c P54 RUP54 9UP.55 SI!P55 &‘I!/‘56 8UPt56 SliP57 N~P,V S CPSR SliP58 si:Ps? SI’P59 j < SUP60 SUP60

Unknown SUpplWS”r strain ..L-119 L-116 L-l 16 L-322 L-303 L-301 L-280 L-303 Id-322 L-184 L-187 T‘-166 L-209 L-212 L-212 L-184 I,- 184 L-212

No. of Trp+ and Trp- spores 72 161 112 $7 133 116 65 84 152 104 33 249 231 251 153 115 304 331

--..

So. of Trpspores 0 39 0 23 0 26 0 26 0 50 0 53 0 52 0 26 0 53

Tester strains with the genotype Jf.4Ttc SlrPX trpl-I lys&-1 were crossed to “L” strains bearing unknown suppressors with the genotype MATa SCPY trpl-1 tysl-1. All of the suppressors suppress the amber Irpl-I allele. A mixture of spores itnd unsporula~ diploids were plated on complet,e medium and replica-plated to media lacking either lysine or tryptophan. The total number of Trp+ and Trp- spores on the complet,e plates were estimated as 133% of the Lys- colonies. The absence of ‘I’qC spews indicates that AIJF’X and SUPY APP identical or closely linked.

214

S. R’.

LIESMAN TART,E

Number

of amber

were examined, colonies frequency of three in 885. frequency even in crosses isolated suppressors that random spore analysis are (b) Mapping

suppressors

ET

AL.

2

assigned

to each

locus

lacking suppressors were found at an equally high Apparently, suppressor-less segregants arise at a high between identical suppressors. Thus, the independently occasionally give rise to suppressorless colonies in the probably identical.

of the SUP53.

SUP54,

SUP56.

SUP57

and SUP58

loci

Heterozygous crosses gave rise to clear 2 : 2 segregations of the five suppressors list’ed above. The suppressors were scored by their action on the homozygous amber markers trpl-7 or aro7-1. The five suppressors were assigned to their respective chromosomes by close linkage to t’he markers listed in Table 3. In addit,ion, each suppressor was mapped relative to its centromere by determining first and second division segregation frequencies using three or more of the centromerr markers adel, ura3, met74, leu2 and Zeul (Table 3). The close linkage of Xl/P53 and leu2 was based on 94 tetrads that included one non-parental dit’ype, three tetratypes and 90 parental ditypes, corresponding to a distance of 4.8 CM. However, t’he low frequency of tetratypes suggests that the single non-parental dit’ype tetrad found among the 94 examined is probably a rare event and a more reasonable estimate of the distance between SCP53 and leu.2 is probably less than 4.8 CM. The lru2 locus has been mapped (see Mortimer & Schild, 1980) and was located on the left arm of chromosome III about 4.9 CM from the centromere. Our data, however. place leu2 a little further from the centromere. at 11 CM, since led segregat’ed at second division in 21 out of 94 tetrads. In each of the 19 tetrads where SUP53 segregated at second division, led? also segregated at second division. suggesting that t’he two markers are on the same chromosome arm. There were two tetrads in which Zeu.2 segregat,ed at second division, but SI,TP53 segregated at) first division. These data indicate t,hat SUP53 maps between t’he centromere and led. The close linkage of SCTP54 and trp5 was based on 50 tetrads that included six tetratypes and 44 parental ditypes, corresponding to a distance of 6 CM. The six tetratype tetrads were also tetratype for the leul-SUP54 gene pair. but were parental ditype with respect to leul and trp5. Furthermore, 14 of the 15 tetrads that were tetratype for the leul and trp5 gene pair were also tetratype with respect to leul and SlTP54. The leul and trp5 markers have been mapped to the left arm of chromosome VII and are 3.1 and 18.5 CM, respectively, from the centromere (Mortimer & Schild, 1980). These data map the suppressor 6 CM distal to trp5 on chromosome VII.

YEAST

AMBER

tRSA\‘”

TAHLE Lin,kap Tetrad

(‘R-7 CR-7 CR-33 CR-33 (‘R-33 CR-33

SUPPRESSORS

215

3 data frequencies

Segregat,ion

PI)

NPD

T

90

1

3

44 29

0 0

6 22

33 58

0 0

I r, 7

Fl)

75

44

SL-510: SL-51 SL-510: SL-511 SL-510; SL-511 SIAm SL-532 SL-532 SI,-.iZP SL-533 SIG36 SL539 SL-535 SL535 SL-55X CR-34 (‘R-34 CR-34 SL-5’361 I SIA37 SL-536 SIA3i SL-536 SL-537

I

51 5x 31 4ti 31

0 0 0

15 0 15

7 10 10

0 3 7

2 30 32

17

19 57 3

2

10

2 3 7 10 5 9

3 2 8 6 12 5

8 8 3% 25 30 “8

9

17 14

FI) (first division segregation) and SD (second division 01’ the marker segregation relative to known centromere-linked frrqrwncy of PI) (parental ditype), NPD (non-parental I’rrkin’q (IS-L!)) equations:

segregation) were determined by comparison markers. CM were determined from the ditgpe) and T (tetratype) and the following

CM = .iO(T + BSPD)/( PD + SPD or CM = 50(SD)/(FD+SD). t A mow awuratr distance less than 4.8 c&I could $ I)ata from Mortimer & Schild (1980).

be calculated

+ 7’)

by neglecting

the

single

SI’D

tetrad.

The close linkage of SC:P56 and adel was based on 65 tetrads that included seven tetratypes and 58 parental ditypes, corresponding to a distance of 54 CM. In 61 of t,he 62 t’etrads examined, both the suppressor and adel segregated at the first division. In four other tetrads. adel and SCTP56 both segregated at t’he second division, suggesting t,hat they are on the same chromosome arm. In the remaining seven tetrads, adel segregated at first division with the suppressor segregating at second division. implying that adel is closer to the centromere than SII’P56. The frequencies of second division segregation, 1 7.7yj0 and 6.4%: indicate that SI’P5fi and adel map 8.8 CM and 3.2 CM. respectively, from the centromere.

216

S. W. LIEBMAX

ET

AL.

The adel locus has been mapped {Mortimer & Schild, 1980) and was located on the right arm of chromosome I, 4 cM from the centromere. These data map SUP56 to chromosome I, 5.4 CM distal to adel. The close linkage of SUP57 and met10 was based on 46 tetrads that were all parental dit,ype. Since the previously mapped tyrosine-inserting suppressor SUP6 also is tightly linked to metl0, we examined the possibility that SUP57 and SUB? were allelic by crossing the UAA suppressor SlJPG-o to the amber suppressor SUP57. Clearly, SUP57 represents a different locus from SCTPG? since two of nine tetrads examined were t)etratype for the two suppressors, i.e. one segregant suppressed both oehre and amber markers and another segregant did not suppress eit,her one. The data in Table 3 show t,hat~ StiP58 is approxima,tely 45 CZ from met14. In a separate cross, no linkage was det,ect.ed between SUP58 and met1 (Table 3). Since met1 is about 50 cM from the centromere on chromosome XI (Mortimer & Schild, 1980), it appears that, SIJP58 is on the opposite side (left arm) of chromosome XI. Also on the left arm of chromosome XT is a 100 cM segment containing several markers whose linkage has been established by aneuploid analysis (Mortimer & Schild, 1980). The orientation of this 100 cM fragment has not been established since none of its markers has been shown to be linked to any of the markers known to be linked to the centromere. According t,o the mapping data, SIJP58 resides somewhere in the unknown region between the centromere and the 100 cM fragment (see Fig. 2). Crosses were examined to orient this fragment by looking for linkage with one of its outside markers a.nd SCP5ti. No linkage was detected between SUP58 and cEy7 (Table 3); and attempts t,o detect linkage between SCP58 and ma,k9-1 gave ambiguous results, showing linkage in some crosses but, no linkage in other crosses. Analyses of crosses that were heterozygous for SUP58 and either heterozygous or homozygous for mak9-1 indicate that, mak9-I was an amber allele and was suppressed by SUP58. Although all the SUPS8 segregants were phenotypically Mak+, 20 other crosses showed that suppression of mak9-1 by other amber suppressorsis dependent on background genes. SUP52, SUP57 and SUP59 never suppressedm,akQ-1while SUP53, SUP54, Sc!P55, SCP56 and SIJP60 suppressed mak9-1 in some crossesbut not in others. (c) ~e~,et~ca.~al~~e~of the SCP55, SUP59 a& SUP60 Eoci We undertook a genetic analysis of diploids that were heterozygous for the centromere markers leul, leu2, adel, met14 and urad; homozygous for the amber marker trpl-1; and heterozygous for one or another of the suppressors SUP55, SUP59 and SUP60 (Table 3). A clear 2 : 2 segregation was observed for each suppressor, which was scored by the suppression of trpl-1. No linkage was detected between any of these suppressors and any of t,he auxotrophic markers, nor was centromere linkage detected for any of the suppressors. (d) Ver$‘icatinn of linkage

awigsments

The mapping data described in the previous sections partially verify the independence of each of the nine linkage groups uncovered by the random spore

YEAST

t,RNAr

AMBER

SUPPRESSORS

“17 XI I

i

SUP54

f-

mok9

trp5 leul

f-

I +-

c/y7

SUP58

X t SfJP52 K

met3

mefl

FIG. 1. A partial genetic map of 5’. cerevisiae showing the location of the mapped suppressors and markers used in this study. Centromeres are represented as closed circles and the left arm of each chromosome has arbitrarily been drawn above the centromere. Linkage distances established by tetrad analysis are drawn to scale and are represented by continuous lines. The dotted line represents linkage ~stabiish~d by aneuploid analysis and is not drawn to scale. Chromosome segments omitted from bhe diagram are indicated by the broken lines. When the order of 2 or more genes has not been determined, they are enclosed within parentheses. The map position of the SUP53 gene indicated in the Figure differs slightly from the position predicted from genetic analyses, and was established by physical analyses described in the text.

analysis. We have succeeded in mapping SUP52 (Liebman et al., 1977), 8UP53, XUP54, SlJPS6 and SUP58 and they are indeed each found at’ a unique position, unlinked to each other or to any of the eight tyrosine-inserting suppressors. Sl!P.57 was found to be linked to, but distinct> from the tyrosine-inserting suppressor 8UP6. Full verification of the independence of the nine linkage groups required further analyses of the unmapped suppressors SUP55, ~~~~~ and SUP60. The unmapped suppressor SUP55 segregated inde~ndently from SUP53, SUP54, SUP%, XUP57, SUP59 and SUP60 (Tabie 4). We showed that SUP55 is different from SUP58 and SUP52 because SfJP55 is not linked to met14 OF a centromere. while SUP58 shows linkage to metl4, and SUP52 is tightly centromere-linked (Liebman et al., 1977; Table 3). The unmapped suppressors SUP59 and RlJP60 were shown to be different from each other since they segregated independently in a single cross (Table 4). SUP59 and SUP60 were distinguished from SUP52, SUP53, SUP54 and SUP56 by their second division segregation frequencies and they were distinguished from SUPS7 and SUP58 on the basis of linkage distances to his2 and met14 (Table 3). Tn addition, we have shown that SUP55, SUP59 and SUP60 are distinct from all the t~yrosine-inserting suppressors. Random spore analysis showed that REIPB. 9

218

S.

\V.

ET

LIEUMAN

TAHLF:

Veri$cation

qf linkage

group

1

assignments

l,-1”Xx(‘K10-l6(’

(w-lor) (‘ltlo-ltx’ I,-270 x (‘RIO-16C L-288 x 1,-1x-” x 1,.212 x

AL.

x 1,-17x x (‘Rll-61% SL509-1IU x I,-%87 SL.ilA-7D SL;ili-7.4 SL531-11)

by tetrad analysis

1’1)

‘I

SI’I)

2 I .,

1 8 7 3 2 7 .4 3

2 3 I 0 2 I I 2

0 0 3 3 I

SITP4, ASITI’.5, SI?P7 and SlrPX were each unlinked to SC~P55. SC:P59 and A’(:/‘60 (Table 5) whereas the map positions of the three other amber tyrosineinserting suppressors. SLTl’3, SLTPll and S~!PA showed that they were distinct’ from SC!P&5, Sl!P.59 and ~S’L~l’60 (Table 3: Mm-timer & rSchild, 1980). TABLE

Random

Tyrosine-inserting suppressor strain

5

spore data showing no linkage between new suppressors tyrosine-inserting suppre.ssors No.

of AN+ and ilrospores

No.

and

of Are-

spores

8

41

61 90 111 139 12.5 5.5 197 I49 115 129 Tvrosine-inserting suppressor strains with genotype MATa SliPX ~07-1 lys-I strains bearing unknown suppressors with the genotype MATa SCPY aro7-1 supprwsors suppress the amber ~07-1 allele. A mixture of spores and unsporulated plated on complete medium and replicated to media lacking lysine or tyrosine. The on the complete plates were estimated as 133010 of the Lyscolonies. The presence indicates that SIIPX and SUPY are unlinked (see the text). t VAA alleles of the tyrosine-inserting suppressors SUP4 and St,‘P7 were used to to S(‘f’55 due to lack of sporulation in diploids containing the amber alleles. Using method described previously (Liebman rt al.. 1976), no linkage was found.

were 1~~2-1.

crossed to All of the diploids were number of spores of Arespores

determine the random

linkage spore

YEAST

tRNA4’” (e)

AMBER

SUPPRESSORS

Lack of interaction

219

effects

Earlier work has shown that combinations of two UAAor two UAG-suppressors of the eight tyrosine-inserting suppressors in a haploid strain result in severely retarded growth or inviability (Gilmore, 1967; Liebman & Sherman, 1976). This “interaction effect” presumably results from oversuppression due to the combined action of two suppressors. On the contrary. pair-wise combinations of less efficient UAA suppressors do not affect growth rate. probably because even the combined efficiency of two of these suppressors is low (Ono rt al., 1979n,b). Likewise, in t,he current study we find that combinations of two of the nine suppressors do not noticeably affect growth rat’e (Table 6).

(f’) Leucine

substitution

caused by SCP53,

SUP54,

SUP55

and SUP56

The amber mutant ~~~1-76, which contains a UAG codon corresponding to amino acid position 71 in iso-1-cytochrome c (Stewart & Sherman, 1973). was coupled with each of the suppressors SUP53, SUP54, SUP55 and SCP56. Tso- 1 -cytochromes c were prepared from these strains and the amino acid inserted hy each of the suppressors was determined by sequencing the amino-terminal region of the smallest cyanogen bromide-cleaved peptide encompassing position 71. As shown in Table 7, each of the suppressors caused the insertion of a leucine residue at the site corresponding to the UAG mutation. (a) Correlation

of suppressor

genes with tRNAf”

genes

Total yeast DNA from the nine different isogenic suppressor strains and from their suppressorless parental strain was digested with restriction enzymes that do not cut within the sequenced tRNAi’” genes. The DNA was then fractionated on agarose gels, transferred to nitrocellulose and hybridized with plasmid pJD137 (described in Materials and Methods) that contains a yeast tRNAf;‘” gene along with 2.5 kb of neighboring yeast DNA. Blots of parental DNA cut with EcoRI contained one major band at the expected size of 2.5 kb and eight minor bands; HindIII-cut DNA contained the expect’ed major band at 6.5 kb and six to eight minor bands, some of which overlapped the major band. In XbaI-cut, XbaI-HindIII-cut and XbaI-EcoRI-cut D?;A. two major and eight minor bands were detected (Fig. 3, Table 8). Two

TABLE

Lack

of interaction

of the

two

6 SUP57-and

suppressors

SUP58 concn

Genotype SI’PSX SI’P.57 9 CP.5 7 s 111’.5X

Doubling

time 2.2 2.5 2.5

(h)

of cells at

stationary phase 3.47 3.25 3.27

x lo8 ml x 108 ml

x 108 ml

220

S. W:. LIEUMAN

ET

TABIX

7

sequencesof cy~oyen

NH,-tern&& Strain

no.

AL.

bromide-cleaves

peptides

Sequence 71 S~r-(~Iu-‘llyr-Ileu-‘~hr-Asn-Pro-

SL291-2.4 SL296-61) f:II14-:31) (‘R18-x (‘R20-3(’

Lines above and below thr r&dues respectiveiy, were identified, Altered detected at various positions including detection from sample to sample and in detected at positions other than 72 was

Srl~-La?~-‘l’yr-I~eu-l’tlrf ----%! -Lrzc-Tvr-Leu-Thr-Asn-f’rct--I----G-&?-Tyr-Leu-Thr-A--.?-..----q-r ) t I - 1ieu -‘l’vr-l,eu-Thr-Asn-Pro---iST-IX-QFTzi;;;;---

indicate that dansyi derivatives and ~henylthiohydan~ins, residues at-~ indicated in italica. In addition, tyrosine was residue 3 in most but not all samples; the variability in repeated analysis of the same sequence indicated that tyrosine a contaminant.

major bands were expect,ed when the Dh’iz was cut with XbaT although only one of them should be homologous t,o tRKAyU since the 2.5 kb yeast fragment on pJ1)137 contains an Xbal site. These results suggest that there are nine tRNAy genes in our haploid yeast strain, a finding that is in agreement with t’he nine suppressor loci uncovered in our genetic studies. An examinat,ion of t,he tRh’Ap” homologous fragments on the Xbal, XbaIHindIII and Xb~~-EcoRI blots reveals several differences between the 8UP53, ST.‘:P54, SliPriS and SUP5fi suppressor strains and their isogenic parent (Fig. 3. Table 8). Tn contrast, the suppressors and their isogenic parent gave identical results when their DNA was digested solely with UindTII or EcoRI. Apparently, as predicted above (Fig. I), the SCP53, SVP54, SCP65 and SUP56 mutations each gene&e a new XbnT site in distinct tRNAt;‘” homologous DT\;A fragments that is not found in the suppressorless isogenie parent,. These new suppressor XbaI sites should cause a parental fragment to be replaced by two smaller fragments whose sizes add up to the size of the missing fragment. Indeed, this occurred several t,imes. However, in other cases one or both of the smaller fragments were probably not detected because they were too faint or overlapped with other bands (see Table 8). The restriction site alterations in the suppressor strains allow us to correlate specific DNA fragments with specific suppressor foci. In our strains, the wild-type gene sups+ is -on a IO.2 kb XbnT fragment, a 8.6 kb XbaI-EiindIII fragment and a 4.8 kb XbaI-EcoR’I fragment; the wild-t,ype gene sup54+ is on a 4.2 kb XbaT fragment; the wild-type gene szcp~r-f is on an 11.2 kb XbaI fragment. on a 8.4 kb XbaI-HindTII fragment and on a 3.3 kb EcoRI-XbaT fragment; and sq5fif is on a 23 kb XbaI fragment and a 14 kb Xb~I-~in~dII1 fragment. The five &her suppressors, I’it’l’58, SCPST. SirP:S. SfiP59 and SUP60 could not be correlated with specific t,RNAy” fragments, because they did not show any detectable restriction site alterations on our blots.

YEAST

tRXA4’”

AMBER

1‘AHLE

Summary

of fragments

221

SUPPRESSORS

8

hybridizing Sizes bands

to tR,VAi””

genes

(kb) of the missing (- ) and extra (+ ) in the suppressor strains relative to the isogenir parent SL210-39

Fragment sizes (kh) of SL210-3A 23, 15. 11-2, 102, 10.2. 4.8,

4.2, 4.0, 3.0, 1 .o 14.0.

9.8, 8.6, 8.4. 7.0, 4.9, 3.0, 2.1. 1.7.

I.0 i.3. 3.3, 1.8. 0.6

4.8, 4.3. 2.6, 2.1, 1.0, 0.65

~ IO.2 +6.8 +3.4

-4.2 + I.1

-8.6 +6.8

4.8

-8.4 + 7.8

-4.3 + 1.1

~ 14.0 + IO.2

-3.3 + 04.5

The major bands intensely hybridizing to pJD137 are indicated in boldface. Overlapping fragments at IO.2 kb on the XhaI blot and at 8.6 kh and 8.4 kb on the XhaI-Hind111 blot could he distinguished from each other because they were altered separately in suppressor mutants.

(h) Correlation

of a cloned tRSAi””

gene with, SUP53

Our genetic mapping (see above) has shown that Sc’Y53 is tightly linked to the LEUZ gene near the centromere of chromosome III. Indeed, plasmid YEp13 containing the LEUZ gene (Broach et al., 1979) was also found to contain a t,RNAi’” gene (Dobson et aZ.! 1981: Andreadis et al., 1983). In order to determine if this tRNA\‘” corresponds to SlTP53 we used YEp13 to probe our blots. As expected, the 10.2 kb Xbal, 8.6 kb XbaT-Hind111 and 4.8 kb XbaI-EcoRI fragments assigned above to the wild-type sup53’ gene did hybridize strongly t’o the YEplY probe. Furthermore, in the presence of the SUP53 mutation, these fragments were replaced by smaller pieces. clearly establishing that YEpl3 is homologous to the fragments containing NC:P53. Tn addition to these fragments, a strongly hybridizing 3.4 kb fragment was also present in all DNA cut with XbaI-EcoRI. Assuming that SFP.53 is the tRSA\“” gene that is found near LEU2, the fragment sizes we obtain are consistent with the positions of the EcoRI and HindIII sites previously reported (Dobson et al.. 1981) in the LEU2 region of S288C type yeast strains (see Fig. 4). The positions of XbaI sites in this region were not mapped. Our data predict that XbaI sites should occur as shown in Figure 4. In particular, the left XbaI site is predicted to occur about 1.7 kb to the left of the Hind111 site within the transposable element Tyl-17 which is distal to tRNAi’“. Indeed, element Tyl-917, which is similar to Tyl-17, is known to contain an XbaI site in just this position (P. Farabaugh, personal communication). The only discrepancy in these data is that our genetic mapping implies that SUP53 is proximal to LEC2, while the physical location of t’he tRKAi”” gene is

222

S. R. I

2

LIEBMAN

3

4

5

ET 6

.4L. 7

8

9

IO

1.7

I.0

1-

Fro. 3. Autoradiographs of digested yeast DSA probed with tRNAS;“” genes. Blots were probed with E. coli plasmid pJD137 (Johnson rt ~1.. 1980). Lanes I and 6 contain D8A from SL210-3A (YATa cycl-179 mrtX-1 rwo7-1 trpl-l u&3-26 ilal-l leu%-l h%.+2 lysl-1). DNA samples from the following suppressor strains, directly isolated from SL210-3A (Liebman rt al.. 1976,1977), were run in the following lanes: SI:P53, lanes 2 and 7: S’CP.5~. lanes 3 and 8: SUP.5.5. lanes 4 and 9: and SUP56. lanes 5 and 10. DNA was digested with XhaI and Hind111 (lanes 1 to 5) or with XbaI (lanes 6 to 10). Lanes 1 to CJand 6 to 10 are from separate gels. The sizes of the restriction fragments are designated in kb. The differences in intensity of the various bands reflect the degree of homology with the probe, efficiency of transfer and overlapping bands. The intense IO.2 kb band in lane 6 contains overlapping fragments, one of which is missing in lane 7. Restriction patterns of ACP52 and SUP57 to SUP60 DNA are not shown since they could not be distinguished from the pattern of normal DNA (lanes 1 and 6).

distal to LEU2. One possible explanation might be that the LEU2 and SUP53 genes are inverted in our strains relative to the orientation found in strain S288C. The proximity of a Tyl element to these genes makes this possibility more plausible. If Tyl-17 bounded one end of the inversion, then the size of the 7.1 kb EcoRI fragment hybridizing to a LEG2 probe should be altered (see Fig. 4). However: when EcoRI-cut DNA from strains S288C, L-127 (SUP53), and SL210-3A (the normal parent of the suppressor strains) were probed with YIp33, which contains the XhoI-Sal1 LE:C2 fragment, identical bands appeared in each of the strains at the expected sizes of 7.1 kb and 3.4 kb. While we cannot rule out the possibility that Tyl-I 7 and the distal EcoRT site are included in the inversion, it appears more likely to us that the genetic map does not give an accurate picture of the physical locations. The evidence suggests that SUP53 is the tRNA’;‘” gene that is located slightly distal to LEU2 and which is found on YEp13.

P%3

EX

( XbaI)

H

SX 4.8

4

E

3.4

n

P

E

7.1

M

E

)

IO.2

4

(~‘6~1)

*

8.6

4

4

S

w 3.4

w

FIG;. 4. Kwtriction map of the ~St~P.53 region. Data above rrsl~wtivelv. from Uobson et cd. (1981) and this investigation. I’ragments in kb. The open box is Tyl-17. thr adjacent filled box /,EC’%. The restriction sites are indicated as follows: E, EcoHI; Shol. The p&ions of the X&I sites shown in parentheses were thr wstriction map and discussed in the text. (‘ut DNA was kwltaining. rwpwtiwly. the XhoI-Pstl and Xl,trl-SW1 fra~men~,s

and below the restriction map are. The numbers indicate the sizes of tht is sup5.3’ and the cross-hatched box is H. liindII1; P, PstT; S, AMI: and X. predicted from the data shown below prottrd with &her YEp13 or YIp33 shown at t,he bottom of the Figure.

4. Discussion Tn this investigat,ion, previously isolated (Liebman rt aE., 1976j amber suppressors have been placed into nine linkage groups. Using t,hr iso- 1-(~yto{!hrorn~~ c assay, several of these su~pr~?ssors, SfiTP52 through SVF.%. have been identified as leucine-insert,rrs. We genetically mapped SCP53. SCPB4. A’I’P+56, SCP57 and SUPS8 (see Fig. 2) and found that none of them was linked to t,hr previously mapped leucine-inserting UAA suppressors (One et al.! 19796). This is in contrast to the earlier observation that SI.:P52 is tightly linked to the leut:ine-insert,ing C‘AA suppressor Sl,:PB (Liebman et al., 1977; One et nl., 19796). ,Since the other le~lcine-inserting amber s~lppressors are not linked to leucineinserting I:AA suppressors, the possibility that a single gene can be converted b> single base-pair c*hanges from the wild-t,ype allele t’o both a UAC: (SI’P5%) and a ITA,\ (SITP89) lcucine-inserting suppressor is unlikely. More likely, in this single (base two adjacent genes encode leucyl-tRT\;A, one of which can be converted to an amber and the ot.her to a UAA suppressor. Thr suppressors within a given linkage group may each be at the same tRYAt;“” gene. or they may represent closely linked. perhaps even tandemly repeated loci. In ari at tempt to distinguish between these possibilit’ies, we looked for rtJc>ombination bcatween linked supprcssom. Although crosses between suppressors c~lassified as linked gave about, two per 1000 segregant,s lacking a suppressor. (arosses bet,ween ident,ical suppressor alleles also gave suppressorless segregants at approximately this same frequency, suggesting that alleles assigned t.o one linkage group art’ within one gene. There arc’ several possible explanations for the high I)ac~kground of suppressor loss. Micient suppressors are unstable because the>

224

S. W. LIEBMAN

ET

AL

grow at reduced rates, mutate to suppressors of lower efficiency (Liebman & Sherman, 1976), and adversely affect sporulation (Rothstein et al., 1977). Thus, growth and sporulation may select for either the loss of the suppressor or a reduction in its efficiency; this would account for the high background of suppressorless segregants. Another possible explanation for the high frequency of suppressor loss is heterologous recombination between the non-homologous redundant suppressor genes. Such heterologous recombination between redundant suppressors at non-homologous positions of the genome has been reported in Schizosaccharomyces pombe (Munz et al., 19X2). Recently J. C. Crowley, H. Y. Steensma and D. B. Kaback (New Jersey Medical School, Newark, NJ: unpublished results) cloned an 8 kb XhoI fragment (pLF41) that hybridized yeast tRNA and that is located 5 kb centromere distal from the ADEI gene. A genomic Southern blot (Brennan, G. and Liebman, S. W., unpublished results) showed pLF41 predominantly hybridized to a 23 kb XbaI fragment from wild-type cells that was replaced by 18 kb and 5 kb fragments in SCP56 strains, presumably due to an XbaI site created by the SUP56 mutation. These results establish that pLF41 contains the wild-type sup56 gene and the restriction map of the region (Crowley, J. C., Steensma, H. Y. and Kaback, D. B., unpublished results) locates this gene between 8 kb and 13 kb from A DEl. on the distal side of centromere T. This position is consistent with the genetic map reported here (Fig. 2). Finally, our results clearly show that at least SUP53. SUY54, SUP55 and SfTP5fi are mutations in tRNA\“” genes because they each generate a new XbaI site in distinct tRNAj;‘” homologous DNA fragments. The other phenotypically similar suppressors, SIJP52, SUP57, SUP58, SUP59 and SI/P60, which did not generate detectable XbaI sites, are also probably tRNAi’” mutations because one of these suppressors, SUP52, is a known leucine-inserter (Liebman et al., 1977) and because we find nine bands homologous to the tRNAj;“” probe on Southern blots of total yeast DNA cut with a variety of restriction enzymes. However, since the probe used contains more than just the tRNA\“” gene, it is possible that some of the hybridization bands could be due to another redundant element on the probe. If the SUP52, SUP57, SUP58, SUP59 and SUP60 suppressors were generated by a new XbaI site at the tRNAk’” anticodon, the new site might not’ detectably change the fragment size if the tRNAke” gene were located at the extreme end of the fragment as it is in the pJD137 clone. Alternatively, if the intervening sequences of the tRNAi”” genes differed from those already sequenced, the CAA to CTA anticodon change might not create an XbaI site, since the hypothetical XbaI site contains one base-pair from the intervening sequence (see Fig. 1). Finally, we cannot exclude the unlikely possibility that these suppressors arise from tRNA\“” mutations outside of the anticodon or that they correspond to mutations in other tRNA species altogether. We greatly appreciate the technical assistance provided by S. Consaul and K. Brockman (llniversity of Rochester), We thank M. Cavenagh. A. Hopper, J. D. ,Johnson, P. ,Johnson, B. Nichols, S. Picologlou. E. Jones and K. Downs for helpful advice and suggestions and P. Farabaugh, J. C. Crowley, H. Y. Steensma and D. B. Kaback for sharing unpublished results.

YEAST This GM24189 Career D.H.H.S., at the by the

tRNA4’”

AMBER

SUPPRESSORS

22:i

investigation was supported in part by Public Health Service research grants and GM12702 from the National Institute of Health, in part by the Research Development Award CA00780 (to S.W.L.) from the National Cancer Institute. in part by the U.S. Department of Energy contract no. DE-AC02-76EV03490 University of Rochester, where it is designated report no. UR-3490-2370, and in part Cniversity of Illinois at Chicago Research Board.

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