saccharomyces cerevis'iae - genetics · exclusive groups based on their spectrum of suppression....

FRAMESHIFTS AND FRAMESHIFT SUPPRESSORS IN SACCHAROMYCES CEREVIS'IAE

MICHAEL R. CULBERTSON, LAWRENCE CHARNAS, M. TINA JOHNSON, AND GERALD R. FINK

Department of Genetics, Development and Physiology, Cornell University, Ithaca, New York 24853

Manuscript received March 11, 1977

ABSTRACT

Using ICR-170 as a mutagen, we have induced a set of mutations in yeast which exhibit behavior similar to that shown for bacterial frameshift mutations. Our genetic study shows that these mutations are polar; the polarity can be relieved by internal suppressors; they revert with acridine half-mustards and are not suppressed by known nonsense suppressorq. However, they are suppressed by other dominant external suppressors, which fall into two mutually exclusive groups. Five genetically distinct suppressors were obtained for one of these groups, using co-reversion of two frameshift markers. Three of these are lethal in combination with each other and show a reduction in the GLY3 tRNA peak on a Sepharose 4B column. A fourth suppressor shows an altered chromatographic profile for GLYI tRNA. We suggest that this group of suppressors represent mutations in the structural genes for the isoaccepting glycyl-tRNA's. Two other suppressors (one linked to the centromere of chromosome 111) were found to suppress a second group of frameshifts. Genetic and biochemical studies show that the nonMendelian factor [PSI+] increases the efficiency of some frameshift suppressors.

UTATIONS which add or delete bases from DNA have drastic consequences Mbecause they shift the frame of the genetic message, normally read in groups of three, producing a sequ.9nce of altered amino acids in the protein. Frameshift mutations were first described and analyzed in bacteriophage Tq (CRICK et al. 1961 1. Subsequent studies have focused on the unique features of frameshift mutations and the types of mutagens which induce them, in an attempt to determine the molecular events which give rise to mutations of this type.

Studies on bacteria have shown that a class of planar, heterocyclic compounds with a polyamine side-chain (called ICR compounds) cause frameshift mutations (AMES and WHITFIELD 1966). These studies have shown that most known base- substitution mutations fail to revert with ICR compounds, whereas both f l and -1 frameshift mutations can be reverted. The base changes induced in the DNA by these mutagens have been inferred from an analysis of the altered proteins produced by revertants of ICR-induced mutations. Studies on the hisD gene of S. typhimwium showed that ICR-191 frequently adds o r deletes a base from a monotonous run of G/C pairs (YOURNO and HEATH 1969; YOURNO 1971). For example, hisD3018, an ICR-induced mutation, resulted in the addition of a G/C

Genetics 86: 745-764 August, 1'377

746 M. R. CULBERTSON et al.

pair to a run of four GJC pairs. ICR-191 induces this mutation to revert by deletion of a G/C pair from a sequence of five G/C’s. ICR-191 causes both -1 and 4-1 frameshifts, whereas N-methyl-N’-nitro-N-nitrosoguanidine can delete but not add a G/C base pair.

A. detailed genetic analysis of hisD revertants showed that one class of ICR- induced frameshifts was suppressible by external suppressors (RIDDLE and ROTH 1970, 1972a, 1972b). This class can be divided into two groups of suppressible mutations, each group having its own suppressors. One type of suppressor suppresses the four base codon CCC. and the other GGG. Suppression of these +I insertions could result from a mutation in a tRNA which permits insertion of a n amino acid in response to a four base code word. Support for this hypothesis came from experiments showing that this class of suppressor mutations affects the chromatographic behavior of prolyl and glycyl-tRNA (RIDDLE and ROTH 1972a,b). Direct confirmation of the involvement of tRNA in frameshift suppression was obtained by showing that strains of Salmonella carrying the frameshift suppressor sufD produce a glycyl-tRNA with the nucleotide quadruplet CCCC at the anticodon position, instead of the triplet CCC found in wild type (RIDDLE and CARBON 1973). The addition of this extra base to the anticodon of the tRNA is presumed to permit recognition of the four-base code word GGG- and thereby correct the reading frame.

Although frameshifts have been found in eucaryotes, there is little information on the mutagens which induce this type of mutation. I t is known that frameshift mutations can be induced by UV in the CYCl gene of yeast (SHERMAN and STEWART 1973; STEWART and SHERMAN 1974; SHERMAN et al. 1974). There is little information on whether ICR compounds induce frameshift mutations. ICR- 170 has been shown to be a strong mutagen for Drosophila (CARLSON and OSTER 1962), Neurospora (MALLING 1967), and yeast (BRUSICK 1970). Many authors have speculated that this mutagen causes either frameshifts or nonsense mutations because of the noncomplementing nature of the mutants made with it.

In this report we show that a large proportion of the mutations induced by ICR-170 have properties similar to those described €or bacterial frameshift mutations; they are polar, revert strongly with acridine half-m-ustards, and weakly with diethyl sul€ate. and fail to be suppressed by UAA, UGA. and UAG suppressors. Moreover, 20 of the 21 ICR-induced mutants which revert with ICR-170 are suppressed by external suppressors. The suppressors fall into two mutually exclusive groups based on their spectrum of suppression. Both groups of suppressors are dominant and fail to suppress nonsense, missense, and deletion type mutations at his4 and other loci. Biochemical analysis shows that one group of suppressors has altered glycyl-tRNA. Our results suggest that in yeast ICR-170 induces both frameshift mutations and frameshih specific suppressors.

MATERIALS A N D METHODS

Strains: All mutations were induced in 01 S288C (referred to throughout as wild type). A list of ICR-I70 induced mutations at his4 is shown in Table 1. Diploids were constructed by selection using complementary auxotrophic markers or by direct micromanipulation of zygotes. Genetic procedures and media have been described previously (FINK 1970).

FRAMESHIFT MUTATIONS IN YEAST

TABLE 1

ICR-170 induced mutations of the his4 region

747

A- B- C-* B- C- C- Allele no. Group Allele no. Group Allele no. Group

12 I 7 I 700 I1 38 I1 204 I1 704 I1

504 I1 205 I 705 IV 505 I1 2% I 706 IV 506 I 208 I 707 I 507 I 209 I 708 I 5 09 I1 21 0 I1 710 IV 511 I1 21 1 I1 71 1 I1 512 I1 212 I1 712 I11 513 I 213 I 713 I11 514 I1 717 I 515 I1 1225 v 516 I1 517 I1 518 I 519 I1 520 I

* A-, B- and C- refer to the functions missing in the ICR-170 induced mutants. Groups were defined by reversion and suppression studies as shown in Table 3.

Mutagenesis: A number of mutagenic agents were used in these studies: ICR-170 (2-meth- oxy-6-chloro-9 [3- (ethyl-2-chloroethyl) amino propylamino] acridine.2.HCl), DES (diethyl sulfate), EMS (ethylmethane sulfate), NQO (nitroquinoline-N-oxide) . Forward mutations to auxotrophy were obtained by treatment of yeast strains with ICR-170 or EMS. The ICR-170 mutagenesis was performed according ta the method of BRUSICK (1970). After 12 h r starvation in 0.1 M phosphate buffer (pH 7), cells were exposed for 60 min to 0.025 mg/ml ICR-170 and plated on YEPD medium. Survival varied between 5-10%. EMS mutagenesis was performed as described by FINK (1970). Mutants were identified by replica-plating from YEPD to minimal medium.

Reversion tests with chemical mutagens were carried out by the petri plate test described by FINK and LOWENSTEIN (1967). Briefly, the mutagen was placed on a confluent lawn of an auxotrophic mutant growing on complete medium. A 5-20 pl drop of the mutagen to be tested was added to the surface of the plate. ICR-170 was used as a 0.5 mg/ml solution, DES and EMS were used without dilution, and NQO was used as a saturated solution. After 15 hr growth in the presence of the mutagen, revertants were visualized by replica plating the mutagen treated strains to minimal medium. Revertant colonies were visible after 5-6 days of incubation at 30". Muta- gens used in this way were ICR-170, DES, EMS, and NQO. ICR-I 70 was chosen for our studies because it gives more revertants in the petri plate test than does ICR-191.

Histidinol dehydrogenase assay: Nistidinol dehydrogenase was measured by the radioactive assay described by CIESLA et al. (1975).

Transfer RNA chromatography: Bulk transfer RNA was isolated from wild type and suppressor-bearing strains and deacylated according to the method of RIDDLE and ROTH (1972b). Aminoacylation was catalyzed by tRNA synthetase prepared from S288C according to the method of BOGUSLAWSKI et al. (1974). tRNA from mutant and wild-type strains were aminoacylated separately prior to column chromatography with 3H- and 14C-glycine (specific activities 42 Ci/mmol, 106 mCi/mmol) or 3H- and 14C-proline (specific activities 2.5Ci/mmol, 260 mCi/ mmol) (Schwartz/Mann), according to the method of RIDDLE and ROTH (1972b). Following


aminoacylation, each tRNA was purified from the reaction mixtures on DEAE-cellulose and then the 3H and 14C labeled tRNA's were mixed, precipitated twice with ethanol, and co-chromatographed on either a 2.0 cm x 40 cm Sepharose 4B (Pharmacia) column, as described by HOLMES et al. (1975) o r on a RPC5 column as described by PEARSON, WEISS and KELMERS (1971). Frac- tions were precipitated in 5% trichloracetic acid (w/v), collected on filters, and counted in POP-toluene (0.4% PPO, 0.04.1% POPOP w/v). The strains used for analysis of tRNA's are shown in Table 2.

RESULTS

The results are presented in three sections. The first is a genetic study of the ICR-170 induced mutants, the second reports the isolation and analysis of the frameshift suppressors, and the third the biochemical characterization of the suppressors.

Genetic study of ICR-170 induced his4 mutations

General properties: Several hundred mutants unable to grow on minima: medium after mutagenesis with ICR-170 were isolated, purified. and characterized. Thirty-nine were his4 mutants, and these strains were characterized iurther by complementation, recombination, suppression and reversion analysis. All of the 17 mutants in the his4A region are noncomplementing; all 10 in his4B show polarized complementation, complementing his4A mutants but not his4B or C mutants. Eleven of the twelve his4C mutants are noncomplementing within his4C. One his4C mutant (his4-710) exhibits intragenic complementation as do many other missense mutations in his4C. Only one of the 39 mutants is suppressed by SUPIV-0. None is suppressed by SUPIV-a. As an additional check fo r nonsense suppression his4-12, -38, -504, -506, -519, 204, -211, -712. and 713 were tested for co-reversion with known nonsense mutations (FINK and STYLE^ 1974). No evidence for co-reversion with nrgil-17, leu2-1, lysl-1 (UAA) ; met8-1. tyr7-1 (UAG) ; or Zeu2-2 (UGA) was found. Less extensive tests of the other 30 ICR-170-induced his4 mutations in combination with various UAA, UAG. and UGA alleles also failed to show co-reversion. We conclude that. with the exception of his4-1225, none of the ICR-170-induced his4 mutations is a nonsense mutation.

Recombination studies in which the ICR-170 induced mutations in hzs4A

TABLE 2

Strains used in isolation of transfer R N A

S288C LC2 GF5470-1C TJ12 TJ25 TJ5 TJ26 GF5 7 13-1 A

a oi his4-38 a his4-712 leu2-3 a his4-519 leu2-3 lysl-I a his4-519 leu2-3 lysl-I a his4-519 leu2-3 lys1-1 a his4-519 leu2-3 lys1-I a his4-713

S U f +

SUFI-I SUFZ-I SUF3-I SUF4-I SUF5-I SUF6-I SUF7-I

FRAMESHIFT MUTATIONS I N YEAST 749

23 I

FIGURE 1.-The map of ICR-I70 induced mutations of the his4 region of yeast. The position .of the mutations on the genetic map was determined by deletion mapping as described by FINK and STYLES (1974). The dark solid line represents the chromosome. The horizontal lines beneath the chromosome represent the extent of deletions. The numbers above the line designate the positions of the ICR-170 induced his4 mutations. The circled mutations are suppressed by SUFI, SUF3, SUF4, SUF5 and SUF6.

and B were crossed with h's4 deletions allowed the assignment of the mutations to positions within the his4 region (Figure 1 ) . Further characterization was obtained by intercrossing mutants whose sites of mutation lay within the same segment on the deletion map. This analysis showed that the sites of mutation are not distributed at random. Many mutations appear to have occurred at the same site. All eight mutations in segment XI11 failed to recombine with each other. Other sites within his4 also appeared to be especially susceptible to mutagenesis by ICR-170.

The ICR-170 induced mutants were characterized by their ability to revert with various mutagenic agents (see Table 3). All of the ICR-170 induced his4

TABLE 3

Reversion and suppression characteristics of the ICR-170 induced his4 mutants

DES' Number

ICR EMS Spontaneous mutants of

Suppression NQO

Group I - - 15 - + Group I1 + f + 18 SUFI, SUF3, SUF4, SUFS, SUF6 Group I11 + 5 + 2 SUFZ, SUF7 Group IV - + + 3 Group V + + + 1 UAA -

* DES = diethyl sulfate. NQO = 4-nitroquinoline-N-oxide. EMS = ethylmethane sulfonate.

750 M. R . CULBERTSON et al.

mutants reverted spontaneously at low frequency. Fifteen of the mutants (Group I) failed to revert at appreciable frequencies with UV light, ICR-170, DES, or 4-nitroquinoline N-oxide. Twenty of the mutants (Group I1 and 111) reverted at high frequencies with UV light, ICR-170, and 4-nitroquinoline N-oxide and at low frequency with DES or EMS (see Figure 2). Three of the mutants (Group IV) reverted with DES, EMS, 4-nitroquinoline N-oxide, and UV light but not ICR-170. One mutant (Group V) reverted with ICR-170, DES and EMS at high frequencies.

Internal suppressors of the ICR ifiduced mutants: Revertants of oneof the his4A polar mutants, his4-38, were analyzed extensively to determine the nature of the reversion event. Reversion analysis is often misleading because the spectrum of reversion events is limited by a selection which demands a functional protein. We have shown previously that a functional his4A region is not a prerequisite for relief of polarity into his4C since a strain carrying a deletion of all known sites in his4A and B grows well on histidinol (FINK and STYLES 1974). Therefore, in an attempt to obtain revertants free from the constraints of function, we selected for

FIGURE 2.-Co-reversion of a strain carrying two Group I1 mutations. Strain 5463-8B, a hid-519 Ieu2-3, was treated with ICR-170 or DES on a complete medium plate according to the procedure of FINK and LOWENSTEIN (1967). Each plate was then replica plated to minimal + leucine (top plates). After incubation for one week at 30°, these plates were replica plated to minimal medium (lower plates) and, after three days incubation at 30", the co- revertants (His+ Leu+) could be scored.

FRAMESHIFT MUTATIONS I N YEAST 75 1

the growth of any strain containing a change relieving polarity and allowing his4C (histidinol dehydrogenase) function.

Revertants of hid-38 capable of growth on minimal + histidinol were analyzed to determine the event which led to relief of polarity. Roughly half of the reevertants grew on minimal medium. Many of these were leaky or had a tem- perature-sensitive histidine requirement. The other half of the revertants failed to grow on minimal but had obtained the ability to grow on histidinol. One strain of this type, hid-14, was shown by ihese tests to contain a deletion which extends beyond the original hid-38 site (Figure 1 ) . The revertants capable of growth on histidinol were examined further by recombination tests with known deletions and point mutations at hisd. R.xombination analysis showed that these strains contained the original his4-38 and a new mutation in region I of the h’s4 map. This conclusion is based on the detailed analysis of two of the strains. his4-38R25 and hid-38R26, but the preliminary tests of many other revertants capable of growth on histidinol indicate that they behave similarly. Strains h i s 4 38R25 and h’s4-38R26 recombine with deleticn his4-34, but not deletion his4-30 or the original mutation hid-38. The simplest interpretation of these data is that his4-38R25 and hid-38R26 are double mutants containing a new mutation in region I as well as the original hid-38 mutation and that this new mutation suppresses the polarity of h ‘s4-38 permitting growth on tiistidinol. VVe attempted t o obtain support for this hypothesis by separating the components of the putative double mutant. hid-38R26 was crossed to wild type, and the resulting diploids were put through meiosis. The meiotic progeny were plated on histidine medium. Seven hundred and fifty haploid His- progeny resulting from the cross were tested for their ability to recombine with hid-38, and for their ability to grow on histidinol. No progeny were found which recombined with his4-38, but two were found which failed to grow on histidinol. These strains were indistinguish- able in their reversion, recombination, and suppression responses from the original his4-38 and we think they are likely to be his4-38 recombined out of the double mutant. So far we have been unable to separate the internal suppressor of his4-38 from the double mutant.

Properties of external frameshift suppressors Zsolation of the suppressors: Representatives from each of the groups in Table

3 were tested for their ability to revert to His+ by external suppression. Initially, mutants from each group were tested by picking the His+ revertants, purifying them. crossing them to wild type and examining the meiotic progeny by tetrad analysis. If the His- phenotype segregated His+ and His- spores 2:2, 3: 1, and 4: 0 in the tetrads, then the His- allele was designated tentatively as suppressible. There was no evidence for suppression of the 15 Group I mutants or the 3 Group IV mutants. The Group V mutant is a UAA mutation and gives rise to ochre suppressors in this reversion test. All Group ‘I1 mutants and both Group I11 mutants test-ed (hid-712, -713) reverted by extcrnal suppression. One of the UV induced suppressors of his4-38, designated SUFI-1, was found to sup- ress many ICR-170 induced mutations at his4 and other loci. This finding

TABLE

4

Cro

sses

to c

hara

cter

ize

co-r

euer

iant

s 8 a 4

Segr

egat

ion

c;

Cro

ss

his4

le

u2

Con

r I us

im

E;: ~

E

(1)

his4

-519

le

u2-3

SU

F(X

) x

His

4+

Leu

2f s

ui+

2

2,

3:1,

4:0

2:

2, 3

:1,

4:O

!a

12)

his4

-519

le

d-3

SU

FtX

) X

hi

s4-5

19

leu

23 s

uff

2:

2 2:

2 vl

-

~~

~ ~~

~~

__

1 su

ppre

ssio

n U

\

,

I,

(3)

his4

-519

le

u2-3

SU

F(X

) x

his4

-38

Le

d+

sui+

2:2

2:2,

3:1

, 4301

z su

ppre

ssio

n of

Gro

up I1

but

not

Gro

up I1

1 (4)

his4

-519

le

u2-3

SU

F(X

) x

his4

-712

le

u2-3

su

ff

2:2,

1:3

, 0:4

2:

2 (

m (5

) hi

s4-5

19

leu2

-3

SUF

(X)

X

his4

-519

le

u2-3

SU

FI-

1 2

2,

3:1,

4:0

2:

2, 3

:1,

4:O

’ di

ffer

ent f

rom

SUF

1-1

II

f?,

FRAMESHIFT MUTATIONS I N YEAST 75 3

prompted a more efficient and systematic analysis of the suppressors of ICR-170 induced mutations.

We adopted a convenient method to distinguish revertants which have the external suppressors from those which do not. Several Group I1 mutants were coupled with leu2-3, an ICR-170 induced, ICR-170 revertible mutation which is suppressed by SUFI-I . The reversion and suppression properties of leu2-3 suggested that it has the same codon change as the Group I1 his4 mutants. For this reason a suppressor of the his4 allele is likely to suppress leu2-3 leading to a His+ or Leu+ phenotype. Thus, the procedure for identifying suppressors aniong revertants of his4-519 leu2-3 double mutants was to revert to either His+ or Leu+ and then to check for co-revertants. Only those strains which had co- reverted to His+ Leu+ were analyzed further. His+ Leu+ revertants carrying the putative suppressors were characterized by a series of crosses shown in Table 4. From the results of these crosses it was possible to determine whether a strain which had reverted to His+ Leu+ contained an external suppressor and whether that suppressor was different from SUFI-I. In all cases tested the His+ Leu+ co- revertants arose by external suppression.

We next sought to answer the question: How many genetically distinct suppressors are there? The crosses used to answer this question are formally identical to cross 5 in Table 4 except that every suppressor was crossed by every other suppressor. The results of these crosses are shown in Table 5 and summarized in Table 6. These data show that five genetically distinct suppressors of Group I1 mutations were obtained by this procedure. All five suppressors are dominant. These suppressors are unlinked to his4, leu2 or mating type on chromosome 111.

As can be seen from Table 5 , tetrads from pairwise crosses of SUFl, SUF4,

TABLE 5

Linkage and lethal segregation of the suppressors

4 spore 3 spore

4+ 3+,1- 2+,2- 3+ 2+, 1- 2-, 1+

SUFI X SUF3 2 5 1 0 4 0 SUFI X SUF4' 1 0 (4 1 14 0 SUFI X SUFS 3 16 4 1 8 1 SUFI X SUF6* 2 0 0 1 18 0 SUF3 X SUF4 3 12 4 3 7 1 SUF3 X SUFS 4 12 2 0 5 1 SUF3 X SUF6 5 6 e 3 8 1 SUF4 X SUP5 2 3 1 0 1 1 SUF4 X SUF6' 2 0 0 1 8 0 SUFS X SUF6 7 17 6 3 8 0 SUFZ X SUF7 3 8 5 4 3 1

The crosses were formally all the same: his4 leu2 SUFX x his4 SUFY. The asci were tabulated according to the number of His+ spores in the ascus. Thus, a 44- ascus

had 4 his+ : 0 His-, a 3+, 1- had 3 His+ : 1 His-, etc. Since both parental strains were His+, the appearance of H i s spores signals recombination between the suppressors.

* These crosses gave many 4 spored asci in which only the 2 His- spores germinated.


TABLE 6

Properties of frameshift suppressors

No. independent Group Lethal Mutagen occurrences suppressed combinations

SUFI uv 2 I1 SUFI, SUF6 SUF3 ICR-170 2 I1 none SUFI spontaneous 1 I1 SUFI, SUF6 SUF5 ICR-170 3 I1 none SUF6 spontaneous 1 I1 SUFI, SUFI SUF2 ICR-I 70 13 I11 none SUF7 spontaneous 1 I11 none

and SUF6 show that the combinations SUFl-SUF4, SUF4SUF6, and SUFl- SUF6 are lethal. For example, in crosses of SUFl his4-519 x SUF4 his4-519 asci with four viable spores show 4 His+:O His-, a parental ditype segregation for the suppressors. This cross fails to produce asci with 4 viable spores that segregate 3 f : 1-. Although four spores are present, only 3 germinate, producing asci with 2 His+: 1 His- spores. We interpret the three spored asci as tetra- types in which the missing spore is SUFl SUF4 his4-519. Consistmt with this interpretation is the absence of asci with 2 f : 2- segregation for histidine. Such nonparental ditype asci would have two spores with the lethal genotype SUFl SUF4, and these would fail to germinate. We find many four spored asci in which only two His- spores germinate. This pattern of lethality is absent from crosses involving SUF3, SUF5, and wild type and is present in all intercrosses of SUFI, SUF4, and SUF6 regardless of the genetic background. Moreover, when SUFl is reverted to a nonsuppressing form, the lethality disappears from crosses with SUF4 and SUF6. These results lead us to believe that the lethality is a con- sequence of the combination of the two suppressor mutations in a haploid cell.

SUFl. SUF3, SUF4, SUF5. and SUF6 have the same spectrum, suppressing all Group I1 mutations but none of the mutations in Group I, 111, IV. or V. All 39 ICR-170-induced his4 mutations were checked by tetrad analysis for suppression by SUFI-1. his4-38, his4-519 and all ICR-170 induced his4C mutations were checked by tetrad analysis for suppression by SUF3, SUF4, SUF5, and SUF6. A rapid test for suppression of all his4 alleles mapping in his4A and his4B was devised. Strains of genotype hid-29 SUFX were constructed tor each of the suppressor strains and these strains were crossed by each his4 mutant to be tested. his4-29 is a deletion of his4A and his4R which is not suppressed by any of the frameshift suppressors. Since his4-29 fails to recombine with any known his4A and his4B sites, the appearance of His+ spores signals suppression in a cross of his4-29 SUFX by his4 suf +. Crosses of this type were placed on pre-sporulation medium, replica-plated to sporulation medium (on which diploids undergo meiosis) and then replica-plated to minimal medium. The appearance of large numbers of His+ spores from a cross of a his4 mutant by his4- 29 SUFX (and not from a control cross by his4-29 suf+) was taken as evidence for suppression. In fact, crosses by his4-29 suf+ never gave more than 1 or 2

FRAMESHIFT MUTATIONS IN YEAST 755

colonies per patch. The validity of the spot test was verified by performing tetrad analysis on selected crosses. These spot tests and the tetrad analyses show that all group I1 mutants (Figure 1 and Table 1) are suppressed by SUFI, 3 , 4 , 5 , and 6. We also tested 85 EMS induced his4 mutations (FINK and STYLES 1974). This group of EMS-induced his4 mutations consisted of missense mutations and all three types of nonsense mutations (UAA, UAG, and UGA) . None of the 85 was suppressed by any of the frameshift suppressors. SUFI, 3,4,5, and 6 were tested for their ability to suppress known UAG, UAA and UGA mutations at other loci. Among the alleles tested were: UAA - leu2-I, ZysI-I, arg4-17, ade2-I; UAG - met8-I, tyr7-I, trpl-I, and UGA - leu2-2. None was suppressed by any of the suppressors.

Efficiency of suppression and the influence of [PSI+]: The efficiency of SUP- pression by SUFI, SL'F4, and SUF6 is increased by the [PSI+] element. [PSI+] is a nonMendelian factor which increases the efficiency of UAA suppressors but not UAG suppressors (Cox 1965,1971; LIEBMAN, STEWART and SHERMAN 1971). All of our ICR-170 induced mutants are [PSI-], so that it was possible to con- struct two sets of strains which could be used tc test the effect of [PSI+] on frameshift suppression. One set of strains had each of the suppressors in combination with hid-38 and [PSI-] (e.g., hjs4-38 SUFI [PSI-] and the other set had each of the suppressors in combination with his4-38 and [PSI+] (e.g. . hid-38 SUFI [PSI+]). [PSI-] strains carrying his4-38 and any of the suppressors fail to grow on minimal medium at 37" even though they grow well at 30". We assume that the inability to grow is a reflection of lower efficiency of suppression at 37". Growth at 37" in the presence of [PS[+] was taken to mean increased efficiency of suppression. SUFI his4-38, SUF4 his4-38, and SUF6 hid-38 all grow on minimal medium at 37" in a [PSI+] background but not in [PSI-] background. Moreover, when his4-38 SUFI [PSI-] is crossed by hid-38 SUFI [PSI+], ability to grow on minimal medium at 37" segregates in a "Mendelian fashion (all tetrads were 4 growers: 0 nongrowers).

The effect of [PSI+] on the efficiency of suppression was shown by direct enzyme assay. The his4C gene product, histidinol dehydrogenase, was assayed in a radioactive assay (see METHODS). The results in Table 7 show that efficiency

TABLE 7

Eficiency of suppression in [PSI-] and [PSI+ J strains

Strain

S288C ICR38 5571-?A LC2 5571-6B 5474-1 1 A 5474-1 1C 5565-40

Histidinol dehydrogenase activity Genotype (cpm/OD cells/lO sl) Efficiency (%)

wild type [PSI- 1 3500 100 his4-38 sufI+ [PSI- ] 110 - his4-38 sufl + [PSI+] 95 - his4-38 SUFI-I [PSI- ] 21 7 3 his4-38 SUFI-I [PSI+] 980 25 his4-519 S u f i + [PSI- ] 98 - his4-519 SUFI-I [PSI- ] 450 10 his4-519 SUFI-I [PSI+] 1170 33


of suppression is between 3 and 10% for SUFl [PSI-]. In the presence of [PSI+ J suppression increases to 25% for his4-38 and to 33% for his4-519. Both his4-529 and his4-38 map in his4A so that the assay of his4C in his4-38 SUFI-I or his4- 519 SUFI-1 strains should be a measure of relief from polarity rather than restoration of function. Efficiency would be underestimated by measuring his4C if the amino acid inserted by the suppressor into his4A failed to function as well as that present in the wild-type protein. Frameshift suppressors which insert glycine (GGGG) for glycine (GGG) should not engender such problems.

Suppressors of Group I I I mutat’ons: SUF2 and SUF7 are dominant suppressors of Group I11 mutations (his4-712 and his4-713). Mutations in the SUF2 gene are frequent among ICR-170 induced revertants of his4-712. All 13 of the independently induced revertants of his4-712 mapped in SUF2. None of the alleles of SUP2 or SUF7 suppresses missense or nonseiise mutations of any other ICR-170 induced mutation. No influence of [PSI+] was found on either SUF2 or SUF7.

SUF2 maps on chromosome I11 approximately 0.5 centimorgans from the centromere (see Figure 3 ) . As can be seen in Table 8, cinly 5 meiotic tetrads in

-h is4

- l e u 2 c r c d c l O ‘SUF2

-pet 18 c ry I

/-mat I ‘tsml

-thr4 ‘tsm 5

‘SUP61 - r a d 18 -tup I ‘pea I

-MAL2 FIGURE 3.-A map of chromosome I11 of yeast. SUFZ is less than 1 map unit from the

centromere.

FRAMESHIFT MUTATIONS IN YEAST

TABLE 8

Linkage of SUF2 to markers on chromoso"e 111

75 7

Cross: 5659 P N T

Cross: 5634 P N T

Cross: 5514 P N T

cddO-SUF2 leu2SUF2 trpl-SUF2 metl4-SUFZ his44UF2 metl4-cdcl0 met14-leu2 metl4-trpl his4-leu2

149 0 3* 132 0 21

61 74 5 75 74 3*

103 0 2 90 0 18 105 0 11

113 0 72 77 75 0 68 66 18 72 74 6

85 0 21

Cross 5634: a his4-712 SUF2-I leu2-3 x (Y his4-712 cdc10 thr4 ade2 Cross 5514: a ade2-1 X a his4-712 leu2-3 SUF2-I Cross 5659: a his4-712 cddO SUFZ-I thr4 add-I X (Y his4-712 trpl leu2-3 met14

* These three tetrads were tetratype for the cdcl0-SUF2, met14SUF2, and trpl-SUFZ combinations but not for the metl4-trpl or metl4-cdcl0 combinations.

a total of 252 analyzed were recombinant for cdcIO and SUF2. In all five tetrads the recombinant spores had the orientation of markers consistent with the order his4-leu2-cdclO-SUF2. The markers included in diploid X5659 allowed US to determine on which side of the centromere SUF2 mapped. trpl and met14 are very closely linked to their centromeres on chromosomes 1V and XI, respectively. Tetrads resulting from a crossover between SUFB and its centromere should show a tetratype spore array for the combinations SUF2, trpl and SUF2, metI4, and a Dai-ental or nonparental ditype spore array for metl4, trpl . If a crossover occurs between SUFB and its centromere, then we can ask whether in that tetrad any of the markers on either arm of chromosome I11 have concomitantly recombined with the centromere. The diploid is heterozygous for leu2 and cdcI0 on the left arm of chromosome 111 and mating type and thr4 on the right arm. In all three tetrads which were recombinant for SUP2 and the centromere, mating type and thr4 had recombined with the centromere, but leu2 and cdcl0 had not. These results suggest that SUF2 is cm the same arm of chromosome I11 as thr4 and mating type, approximately 0.5 centimorgans from the centromere.

Frameshifts in the external suppressors: Several mutagens were examined for their ability to induce external suppressors. SUFI, 3 , 4 , 5 and 6 could be isolated among revertants induced with UV light and ICR-170 but not among those induced with DES, EMS, or nitrosoguanidine. The inability of DES to make suppressors of Group I1 mutants was examined in more detail in the co-reversion test (see previous section) using double mutant strains which have leu2-3 in combination with various his4 Group I1 mutations. We have shown that SUFI, 3 , 4 , 5 , and 6 suppress both the histidine and leucine requirement of these His- Leu- double mutants. Revertants of His- Leu- double mutants to His+ Leu+ are more likely to result from mutations in a suppressor gene than from two independent reversion events, one in the his4 gene and one in leu2. Thus, an


efficient method permitting the screening of mutagens for their ability to make Group I1 suppressor mutations is to test compounds for their ability to revert the double mutants to His+ Leu+. Strains containing leu2-3 in combination with his4-38, -519, -504, -211, -212, -700, -711 and -712 w a e tested for reversion in the presence of a variety of mutagenic agents. All double mutant strains except leu2-3 his4-712 could be reverted to His+ Leu+ by ICR-170 or UV light. Several of the co-revertants were picked and analyzed for suppressors by backcrosses. In all cases tested co-reversion was a result of a suppressor mutation which simul- taneously suppressed both the histidine and leucine requirements. SUFI. SUF3. SUF4. SUFS, and SUF6 were all represented in the co-reverants generated this way. As can be seen in Figure 2. DES fails to induce co-reversion of the His- Leu- strains. None of the nine strains tested could be reverted to His+ Leu+ with DES, regardless of whether the selection was made for His+, Leu+ or His+ Leu+. The inability of DES to co-revert Group I1 mutants is not related to functional aspects of his4 and leu2 in the presence of DES. Strains containing his4-644 leu2-1 (both UAA mutations) readily revert to His+ Leu+ with DES.

Biochemical consequences of frameshift suppression Frameshift suppression in yeast, as in bacteria, appears to result from alter-

ations in transfer RNA. All of our thinking has been predicated on the elegant bacterial work which showed that ICR-like compounds cause mutations in monotonous runs of G to make the four base codon GGGG (RIDDLE and ROTH 1970, 1972a, 197213) and that this type of frameshift is corrected by suppressor mutations which permit glycine tRNA to read the aberrant four base code word (RIDDLE and CARBON 1973). Do the yeast suppressors act via glycine tRNA? TO answer this question we performed a number of experiments in which the glycyl tRNA of suppressor strains was compared with the glycyl tRNA of wild type. In all of our experiments the chromatographic behavior of isoaccepting glycyl- tRNA’s from the suppressor strains and from wild type were compared by ana- lyzing their elution profiles on Sepharose3B. The tRNA froin the suppressor strains was aminoacylated with 3H-glycine and that from wild type with 14C- glycine. After aminoacylation, the two tRNA’s were mixed and co-chromatographed in a double label experiment (see METHODS). Three isoaccepting species, referred to by order of elution as GLYI, GLY2, and GLY3, were separated on Sepharose-4B. Similar pattems were obtained on RPC5 columns, although the order of elution was reversed. tRNA from all of our suppressor strains was compared with wild type by this double label experiment.

The three frameshift suppressors which are related by genetic criteria (SUFI , SUF4, and SUF6) are also related by their effect on the GLY3 tRNA (Figure 4A, 4D and 4F). Each of these suppressor mutations causes a pronounced reduction in the amount of GLY3 isoacceptor activity as compared with that pr.?sent in wild type (SUF1-52%, SUF4-49%. SUF6-39% ) . Several controls were run to rule out possible column artifacts. First, chromatography of wild type and mutant tRNA was performed with the labels reversed as compared with our standard experiment (in which the mutant tRNA was labeled with 3H-glycine).

I20

G

LY

2&

W

T(-

*-)

x S

UF

I-I

I.

E W

T (-

a-)

x

SU

FS

-I (

-0-1

60

L 2

0

W

rpP. .a W

T (

-a-)

x

SU

F4

-I (

-o-)

80

FR

AC

TIO

N

NU

MB

ER

-lo

ot

F

0

8

6o

4

%

Za

0

__

FI

GU

RE

4.

-Chr

omat

ogra

phy

of i

soac

cept

ing

glyc

yl-t

RN

A's

on S

epha

rose

4B

. Chr

omat

ogra

phy

was

per

form

ed a

s de

scri

bed

in M

AT

ER

IAL

S A

ND

ME

TH

OD

S. I

n ea

ch e

xper

imen

t ac

tiva

ting

enz

yme

from

wild

typ

e w

as u

sed

to a

min

oacy

late

tR

NA

fro

m w

ild t

ype

wit

h 14

C-g

lyci

ne an

d th

e tR

NA

fr

om s

uppr

esso

r st

rain

s w

ith

3H-g

lyci

ne. I

n (A

) th

e sa

lt gr

adie

nt a

s m

easu

red

by c

ondu

ctiv

ity a

nd t

he o

ptic

al d

ensi

ty p

rofi

le a

re b

oth

show

n (s

olid

lin

es).

The

sal

t gr

adie

nt a

nd t

he o

ptic

al d

ensi

ty p

rofi

les

wer

e th

e sa

me

for

each

col

umn.

Eac

h co

lum

n pr

ofile

sho

ws

wild

typ

e an

d (A

) SU

FI-

I,

(B)

SUF

I-I,

(C

) SU

F3-

I,

(D)

SUF

4-I,

(E

) SU

PS-

I, (

F) S

UF

6-I,

(G

) SU

F7-I

. Pa

nel

H s

how

s co

-chr

omat

ogra

phy

of w

ild t

ype

labe

led

wit

h 1%

-pro

line

and

SUF

I-I

labe

led

wit

h 3H

-pro

line.


When wild type tRNA was charged with 3H-glycine and the SUFl mutant tRNA with I4C-glycine, the GLY3 peak in the mutant was again reduced as compared with that of wild type. Co-chromatography of wild type tRNA labeled with 14C- glycine and 3H-glycine showed no differences in amount of I4C- or 3H-GLY3 tRNA. Thus the reduction in the GLY3 peak of SUFl is not dependent upon the radioisotope. Second, the fractions from an RPCS column containing the GLY3 tRNA from wild type and SUFI-1 were pooled and re-chromatographed on RPCS. A single GLY3 peak was obtained in which the degree of aminoacylation of SUFl (GLY3) tRNA was reduced by 49% as compared with wild type. tRNA prepared from a strain carrying another allele of SUFl (SUF1-2) also shows the characteristic reduction in GLY3 tRNA when chromatographed on an RPCS column together with wild type. These results support the conclusion that SUFl, SUF4 and SUF6 are all missing a portion of the acceptor activity of GLY3.

Strains carrying SUF5 are missing the normal GLYl species found in wild type (Figure 4E) and have instead a chromatographically different glycine tRNA. This new species is shifted on the Sepharose3B column in the direction of GLYB and is clearly separated from the wild type GLYl species by re-chromatography (Figure 5). Although GLYl tRNA is a minor species as determined by aminoacylation, the specific activity of the radioactive glycine used as a probe was sufficiently high to permit reliable determinations (the GLY 1 peak fraction contained 18,000 CPM above background for 3H and 2000 CPM for I4C with less than 1 % spillover of 3H into the I4C channel). Co-chromatography of wild type with SUFZ, wild type with SUF3, and wild type with SUF7 (Figure 4B, 4C, and 5G) failed to show any difference either in chromatographic behavior or in degree of aminoacylation of glycyl-tRNA.

We believe that the suppressor mutations SUFl, SUF4, SUF5, and SUF6 are directly responsible for the observed differences in glycine iso-accepting tRNA species. Other tRNAs fail to show alterations. We have examined proline (Figure 4H) and leucine (not shown) and these tRNAs appear unaltered in our suppressor-carrying strains. Two prominent peaks of prolyl-tRNA were resolved by chromatography on Sepharose-4B. The first peak is not symmetrical and probably contains several isoaccepting species of prolyl-tRNA.

- 0 S

FRACTION NUMBER

FIGURE 5.-Re-~hromatography of SUFS-I glycyl-tRNA. Fractions from the gradient shown in the insert in Figure 4E were pooled, dialyzed, precipitated with ethanol, and re-chromatographed on a Sepharose-4B column. The solid curve is A,,,.

FRAMESHIFT MUTATIONS IN YEAST 76 1

DISCUSSION

Evidence for frameshift mutations at his4: Several lines of evidence suggest that the ICR-170-induced, ICR-170 revertible mutations of Group I1 and I11 are frameshifts. These mutations have the following properties in common: (1) they are all polar; (2) the polarity can be abolished by internal suppressors; ( 3 ) they are not suppressed by nonsense suppressors nor do they co-revert with known UAG, UAA or UGA nonsense mutations; (4) they revert at high frequencies with ICR-170 and at low frequencies with EMS and DES; (5) they are all SUP- pressed by dominant external suppressors, some of which affect glycine tRNA; (6) ICR-170 makes both internal and external suppressors, hut DES and EMS make only internal suppressors. These observations taken together strongly suggest that the ICR-170 induced mutations of Group I1 are frameshifts and that their suppressors are frameshift suppressors.

Frameshifts in bacteria: The striking similarity between the properties of the Group I1 mutations and those of well characterized bacterial frameshift mutations adds an additional dimension to these studies. The picture that emerges from studies on bacterial frameshift mutations is that ICR compounds add and delete bases in runs of G/C pairs. The frameshifts which are produced by these insertions and deletions are polar and are reverted at high frequency by ICR compounds. 4-1 insertions are reverted at low frequency by alkylating agents but deletions of -1 are not. Since GG. is the code word for glycine and CC. is the code word for proline, ICR compounds affect sites coding for these amino acids in proteins (YOURNO and HEATH 1969; YOURNO 1971; and ROTH 1974). Bacterial suppressors which suppress these 4-1 insertions were shown by chromatography to fall into two classes: those which affect glycine tRNA and those which affect proline tRNA. Direct sequence analysis showed that glycine tRNA from a Salmonella strain containing the frameshift suppressor SUFD had the four-base sequence CCCC as the anticodon instead of the three-base sequence CCC found in wild type (RIDDLE and CARBON 1973).

Frameshift suppressors in yeast: These findings in bacteria lead us to expect that suppressors of our ICR-induced mutants might affect glycyl or prolyl-tRNA. In fact, four of the five suppressors of the G I O U ~ I1 mutants have alterations in the isoaccepting glycyl-tRNA’s. Three of the suppressors SUFZ, SUFB, and SUF6 affect GLY3. Each reduces the GLY3 isoacceptor activity by 40-50%, but no new species of glycyl-tRNA appears. Perhaps the suppressor tRNA’s are not aminoacylated in uitro under our conditions. Clearly. they must be aminoacylated in vivo to permit suppression.

There are many similcrities among SUFI, SUFI, and SUF6: ( 1 ) SUFI, SUFI. and SUF6 are all acted upon by [PSI+] ; (2) all three suppressor mutations affect the GLY3 peak; ( 3 ) any combination of two of these suppressors is lethal. These similarities could be explained in several ways. The GLY3 peak could contain the glycyl-tRNA‘s encoded by the three different structural genes sufI+, sufB+, and suf6+. These genes could represent identical DNA sequences or slight vari- ations in DNA sequence (e.g, sufI+, suf4+, and suf6+ could represent a threefold


redundant tRNA sequence or simply three different tRNA sequences which fail to be resolved in our chromatographic systems).

Suf5+ is likely to be the structural gene for the GLYl isoaccepting glycyl- tRNA. This conclusion is based on the alterations found in the SUFS strain. The GLYl peak of glycyl-tRNA is absent from SUF5 suggesting that there are no other genes contributing tRNA to this peak. In addition, the SUFS mutant has a new peak of glycine isoacceptor activity not present in wild type. This finding suggests that the SUFS mutation alters the sequence of the GLY 1 tRNA.

The induction of suppressors of Group I1 mutants by ICR-170 but not by DES or EMS is consistent with the model proposed for frameshift mutagenesis and suppression. Again, the parallels with the bacterial studies are striking. Analysis of Salmonella frameshifts have shown that both ICR compounds and alkylating agents can cause a -1 deletion; however, only ICR compounds can cause a $1 addition. Since frameshift suppressors arise by insertion of a base to make the four-base code word GGGG, DES and NG are unlikely to induce these suppressors at appreciable frequencies. In agreement with this view (see Figure 2), yeast frameshift suppressors arise at high frequencies with ICR-170 but not with DES, EMS or NG. These data together with our genetic and biochemical data suggest that the Group I1 mutations are frameshifts and that the suppressors act by inserting glycine for the code word GGG.

SUF2 and SL'F7 fail to suppress any of the sites suppressed by SUFI, 3 , 4, 5, or SUF6 and seem to comprise a distinct subset. These suppressors of Group 111 may be the class of proline suppressors found in Salmonella which read the four base code word CCCC. Unfortunately, we know less about these suppressors than the suppressors of Group 11. Since only two sites are suppressed by the Group I11 suppressors, the general characteristics of this group are as yet unclear.

[PSI+] enhances frameshift suppression for SUFI, SUF4, and SUF6. [PSI+] was already known to enhance suppression of UAA but not UAG. It has been suggested that [PSI+] might affect termination factors. This suggestion seems unlikely in view of our finding that [PSI+-] affects frameshift suppression. The affect of [PSI+] on frameshift suppressors as well as nonsense suppressors (both of which code for tRNAs) seem to localize the action of [PSI+] on some aspect of transfer RNA metabolism or interaction with the ribosome.

ICR-170 induced mutations: One must be cautious in using mutagen spe- cificity as the sole criterion for the identification of mutant types. Many ICR-170 induced mutants (Group I) could not be characterized by reversion and suppression tests. These mutants fail to revert at appreciable frequencies with most mutagens and could represent an array of complicated multiple base changes or frameshifts in sequences other than runs of G/C. More problematical is the fact that ICR-170 clearly makes nonsense (Group V ) and missense mutations (Group IV) at low frequencies. An additional complication is that in rare instances ICR-170 can revert a nonsense mutant. his4-1225 (UAA) is revertible by both ICR-170 and DES. It is possible that ICR-170 is acting on hid-I225 to eliminate the nonsense code word by deletion of an integral number of bases or at a tRNA gene where the anticodon can he changed by deletion of some contiguous bases to form a sequence capable of suppressing UAA.

FRAMESHIFT MUTATIONS I N YEAST 763

The spectrum of ICR-170 induced mutations in yeast is somewhat different from that of ICR-191 induced mutations in bacteria. S i x out of 48 ICR-191 induced mutations in the his operon of S. typhimurium are nonreverting, one being a deletion of the entire histidine operon. No completely stable class of mutations or extensive deletions were found in our sample of yeast mutants. In the Salmonella his operon, none of the mutants was revertible with EMS but not with ICR. In yeast, three mutants are of this phenotype and one of these com- plements like a missense mutation. It is possible that these differences result from structural differences between ICR-191 and ICR-170.

The existence of informational suppressors for frameshift mutations in yeast amplifies our ability to determine base changes by genetic means. If the yeast suppressors suppress 4-1 insertions as do the bacterial frameshift suppressors, then it will be possible to identify the insertions by a simple test of suppression.

This work was supported by Public Health Service Grant GM15408 to G. R. FINK. MICHAEL CULBERTSON is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by a grant from The Jane Coffin Childs Memorial Fund for Medical Research. The authors would like to thank JAMES HICKS. THOMAS JOHNSON, and ROSEMARY SCHIMIZZI for their help in preparation of the manuscript and CLAUDIA CUMMINS for the isolation of SUF7.

LITERATURE CITED

AMES, B. N. and H. J. WHITFIELD, JR., 1966

BOGUSLAWSKI, G., M. H. VODKIN, D. B. FINKELSTEIN and G. R. FINK, 1974

Frameshift mutagenesis in Salmonella. Cold Spring Harbor Symp. Quant. Bid. 31 : 221-225.

Histidyl tRNA's and histidyl tRNA synthetase in wild type and cytoplasmic petite mutants of Saccharomyces. Biochemistry 13 : 4659-4667.

The mutagenic activity of ICR-170 in Saccharomyces cereuisine. Muta- tion Res. 10: 11-19.

Comparative mutagenesis of the dumpy locus in D ~ O -

A sensitive radio-

BRUSICK, D. J., 1970

CARLSON, E. A. and I. I. OSTER, 1962 sophila melanogaster. Genetics 47: 561-576.

CIESLA, Z., F. SALVATORE, J. R. BROACH, S. ARTZ and B. N. AMES, 1975 chemical assay for histidinol dehydrogenase. Anal. Biochem. 63 : 44-55.

CRICK, F. H. C., L. BARNETT, S. BRENNER and R. J. WATTS-TOBIN, 1961 genetic code for proteins. Nature 192: 1227-1232.

Cox, B. S., 1965 PSI, a cytoplasmic suppressor of super-suppressors in yeast. Heredity 20: 505-521. --, 1971 A recessive lethal super-suppressor mutation in yeast and other PSI phenomena. Heredity 26: 211-232.

The biochemical genetics of yeast. pp. 59-78. In: Methods in Enzymology, Vol17A. Edited by H. TABOR and C. WHITE TABOR. Academic Press, Inc., New York.

A simplified method for testing mutagens in Sac- charomyces. J. Bacteriol. 100: 1126-1 127.

Gene conversion of deletions in the his4 region of yeast. Genetics 77: 231-244.

Separa- tion of transfer ribonucleic acid by Sepharose chromatography using reverse salt gradients. Proc. Natl. Acad. Sci. U.S. 72: 1068-1071.

General nature of the

FINK, G. R., 1970

FINK, G. R. and R. LOWENSTEIN, 1967

FINK, G. R. and C. STYLES, 1974

HOLMES, W. M., R. E. HURD, B. R. REID, D. A. RIMERMAN and G. W. HATFIELD, 1975

764

LIEBMAN, S. W., J. W. STEWART and F. SHERMAN, 1975 Serine substitutions caused by an ochre suppressor in yeast. J. Mol. Biol. 94: 595-610.

MALLING, H. W., 1967 The mutagenicity of the acridine mustard (ICR-170) and structurally related compounds in Neurospora. Mutation Res. 4: 265-274.

PEARSON, R. L., J. F. WEISS and A. D. KELMERS, 1971 Improved separation of transfer RNA’s on polychlorotrifluoroethylene-supported reversed-phase chromatography columns. Biochem. Biophys. Acta 228: 770-774.

RIDDLE, 5. and J. CARBON, 1973 A nucleotide addition to the anticodon of a glycine tRNA. Nature New Biol. 242 : 230-237.

RIDDLE, D. and J. ROTH, 1970 Suppressors of frameshift mutations in Salmonella typhimurium. J. Mol. Biol. 54: 131-144. -, 1972a Frameshift suppressors. 11. Genetic mapping and dominance studies. J. Mol. Biol. 66: 483-493. -, 1972b Frameshift suppressors. 111. Effects of suppressor mutations on tRNA. J. Mol. Biol. 66: 495-506.

M. R. CULBERTSON et al.

ROTH, J. R., 1974 SHERMAN, F. and J. W. STEWART, 1973

Frameshift mutations. Ann. Rev. Genet. 8: 319-346 Mutations at the end of the iso-1-cytochrome c gene of

yeast. pp. 56-86. In: Biochemistry of Gene Expression in Higher Organisms. Edited by J. K. POLLACK and J. W. LEE. Australian and New Zealand Book Co., Sidney.

Mutant of yeast defective in iso-1-cytochrome c. Genetics 77: 255-284.

Yeast frameshift mutations identified by sequence changes in iso-1-cytochrome c. pp. 102-127. In: Molecular and Environmental Aspects of Mutagenesis. Edited by L. PRAKASH, F. SHERMAN, C . W. LAWRENCE and H. W. TABER. Thomas, Springfield, Illinois.

YOURNO, J., 1971 Similarity of cross suppressible frameshifts in Salmonella typhimurium. J. Mol. Biol. 62 : 223-231.

YOURNO, J. and S. HEATH, 1969 Nature of the hisD3018 frameshift mutation in Salmonella

Corresponding editor: F. SHERMAN

SHERMAN, F., J. W. STEWART, M. JACKSON, R. GILMORE, J. PARKER, 1974

STEWART, J. W. and F. SHERMAN, 1974

typhimurium. J. Bacteriol. 100: 460-468.

saccharomyces cerevis'iae - genetics · exclusive groups based on their spectrum of suppression....

Documents