Current Genetics 3, 133-143 (1981)

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~

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© Springer-Verlag 1981

Putative Frameshift Suppressors in

Schizosaccharomycespombe

H. Hottinger* and U. Leupold Institute of General Microbiology, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland

Summary. Nine genetically distinct suppressors o f ICR170-induced ade6 and ade7 mutations have been identified in Schizosaccharomyces pomba The nine suppressors of ICR-170-induced and spontaneous origin have been assigned to the three chromosomes by haploidization and meiotic analysis. They do not suppress missense or nonsense mutations and are therefore likely to be frameshfft suppressors. Based on the spectrum of suppression, the nine suppressors fall into two mutually exclusive groups. Group I comprises the twodominant suppressors sufl and suf11. Group II consists of the seven dominant suppressors suf2 through suflS. The suppressors of both groups are inefficient and all lead to a marked reduction of growth rate. Within suppressor groups, combinations of suppressors lead to drastic reductions of growth rates and to an increased efficiency of suppression. Freely segregating modifiers of suppression increasing and decreasing the efficiency of supression have been found for all the suppressors. The two omnipotent suppresso~ sup1 and sup2 increase the efficiency of suppression of some frameshift suppressors. The suf5 locus is unstable and reverts at very high frequency both meiotically and mitotically. Key words: Frameshift suppression -Schizosaccharomyces pombe

Introduction External frameshift suppressors have been described in bacteria (Yourno et al. 1969; Riddle and Roth 1970,

* Presentadress: Department of Molecular Biophysics and Biochemistry, Yale University, P.O.: Box 6666, New Haven, Connecticut 06511, USA Offprint requests to: U. Leupold

1972a, b) and in the yeast Saccharomyces cerevisiae (Culbertson et al. 1977, 1980; Cummins et al. 1980). These suppressors all seem to act at runs of repeated C or G residues (Yourno and Kohno 1972; Riddle and Roth 1970, 1972 a, b; Culbertson et al 1977). Kohno and Roth (1978) have reported a new type of frameshift suppressor which acts at runs of A residues in messenger RNA. Three groups of frameshift mutants seem to be specifically suppressed by frameshift suppressors. One group contains the four base codon CCC., another group the codon GGG- and the third group the codons AAA(A/G). Suppressors of the first two types of codon have been shown to affect the chromatographic behavior ofprolyland glycyl-tRNAs in Salmonella typhimurium (Riddle and Roth 1972a, b). In yeast one group pf suppressors has altered elution profiles of the glycyl-tRNAs (Culbertson et al. 1977, 1980). Direct evidence for the involvement of tRNA in frameshift suppression comes from a sequence analysis of an altered glycyl-tRNA from the suppressor carrying strain sufD of Salmonella. This strain produces a glycyl-tRNA which has the nucleotide quadroplet CCCC at the anticodon position, instead of the triplet CCC found in the wild-type strain (Riddle and Carbon 1973). The codons AAA(A/G) seem to be suppressed by a frameshift suppressor which affects a lysine tRNA. This suppressor sufG of Salmonella appears to be alMic to a nonsense suppressor (UA(A/G)) and maps very close to a position known to code for a lysine tRNA (Kohno and Roth 1978). All these frameshift suppressors seem to restore the proper reading frame of +1 insertions by inserting the correct amino acid in response to a four base code word. A mutation of the +1 addition type in the anticodon of the corresponding tRNA will permit this reading process to occur. 0172-8083/81/0003/0133/$02.20

134 In this study we report the genetical characterization o f suppressors in the fission yeast Schizosaccharomyces pombe with properties similar to those found for bacterial and yeast frameshift suppressors. ICR-170-induced ade6 and ade7 mutants o f S. pombe were reverted b o t h spontaneously and b y ICR-170 induction and were screened for the presence o f external suppressors. The ICR-170induced ade6 and ade7 mutants used in these experiments have been previously described (Munz and Leupold 1970) and many o f t h e m seem to be o f the +1 frameshift type. Based on the spectrum o f suppression, we have identified two different groups o f suppressors. All o f the suppressors are dominant and suppress neither nonsense nor missense mutations at ade6 and ade7.

Materials a n d Methods a] Strains. The ICR-170-induced ade6 and ade7 mutations used in these experiments have been described in a previous publication (Munz and Leupold 1970). The suppressible ade6 and ade7 frameshfft mutations identified in the present study are listed in Table 1. Nonsense and missense mutations and their suppressors as well as the strains used in mapping studies and haploidization experiments have been described (Hawthorne and Leupold 1974; Thuriaux et al. 1975; Kohli et al. 1977).

b) Genetical Methods. The genetical methods and media have been described by Gutz et al. (1974) and Kohli et al. (1977). The media used included YEL, YEA and MMA (yeast extract medium in liquid form or solidified with agar and minimal medium agar) with and without supplements for growth, MEA (malt extract agar) for sporulation and SPA (synthetic sporulation agar) for the synthesis of diploid~ For the synthesis of diploids, Magdala red (MR) was added to YEA and MMA to allow detection of diploid colonies, and m-fluorophenylalanine was added to MMA to induce haploidization of diploids. Spore suspensions were prepared by treating cross material with snail enzyme (Industrie Biologique Fran~aise) in water (final dilution 1 : 250) and incubating the suspension overnight at 30°C.

cJ Mutagenesi~ Reversion tests with ICR-170 were carried out according to the plate test described by Fink and Lowenstein

(1967). Some minor adaptions were necessary for satisfactory results with Schizosaccharomyces pombe. A. 0.1 ml sample of a late exponential culture, frown in YEL supplemented with 75 mg/1 adenine, was spread on a YEA+adenine plate and incubated at 30°C for 24 h. The 24 h culture was replica-plated to a fresh YEA+adenine plate and the cells were allowed to grow for 8 to 9 h at 30°C after which the mutagen was added to the plate. ICR-170 was spotted onto the 8-9 h old replica-plates as a 5-20 #1 drop of a 1 ~g/~zlaqueous solution. The treated plates were incubated for 24 h in the presence of the mutagen at 30°C. Replicas of the treated plates were made on MMA. After 8-10 days incubation the revertant colonies could be isolated. All the experiments with ICR-170 were carried out in the dark or in dim red light. ICR-170 (2-methoxy-6-chloro9 [3-(ethyl-2-chloroethyl)aminol acridine. 2 • HC1) was kindly provided through the courtesy of the Chemotherapy Laboratory of the Institute for Cancer Research in Philadelphia.

tL Hottinger and U. Leupold: Frameshift Suppressors in S. pombe

Results

Revertants of the ICR-170-Induced Mutants The ade6 and ade7 mutants o f S. pombe form a red pigment if grown on media with limiting amounts o f adenine (Gutz et al. 1974). This feature was used in the analysis o f frameshift suppression. Revertants o f ade7 with less than wild-type activity will still show the accumulation of the red pigment and will grow more slowly on minimal medium. Frameshift suppressor mutations can be expected to be inefficient suppressors. These suppressors will lead to a red to pink colony colour and reduced growth rate in the absence or limitation o f adenine in the growth medium. Analysis o f only vigorously growing revertants would severely limit the spectrum o f expected reversion events. We therefore analyzed prototrophic revertants o f widely varying plate age, ranging from vigorously growing 4 day old revertants to extremely poorly growing 21 day old revertants. At least five independent spontaneous reversion experiments with a total o f approximately 6 x 109 cells were carried out per strain. Prototrophic colonies growing on MMA were picked and reisolated. To determine the nature o f the reversion event t h e y were crossed to wild t y p e and the meiotic progeny examined in a random-spore analysis. In the case o f an event at the ade7 locus, the progeny are all expected to grow on MMA. In the event of extragenic suppression ade7 auxotrophs will segregate out; the number ranging from a few (linkage) to 25% (absence o f linkage). We arbitrarily set the resolution o f our analysis at about 1 map unit, i.e. extragenic suppressors linked more closely to ade7 would not have been resolved. The spores were plated on YEA, incubated at 30°C and then replica-plated to MMA. After further incubation the plates were scored. Vigorously growing revertant colonies appearing after 4 to 6 days incubation were invariably white prototrophic locus revertants. They occurred with varying frequency in all strains tested. Incubation during 6 and more days led to the isolation o f locus revertants with a pink to red colony colour. Pale pink colonies grew vigorously on MMA, more strongly pigmented colonies required more time to grow. Such pigmented locus revertants could not be isolated from all o f the strains tested, but with the exception o f ade7-C49, all o f the strains for which external suppressors have been found also reverted in this manner. A third t y p e o f revertant requiring at least 10 days incubation proved to be o f the external suppressor t y p e . These colonies were red to pink and o f a small to very small size. The white, vigorous locus revertants can be explained by assuming correction at the site o f the original lesion or in the immediate vicinity restoring complete wild-

H. H0ttinger and U. Leupold: Frameshift Suppressors in S. pombe type activity. The pigmented locus revertants probably have a second frameshift mutation located proximal or distal to the first one and suppressing the effect of the primary lesion. Correction of the reading frame, however, has led to only partial restoration of activity in these revertants.

External Suppression of the Frameshift Mutants The first spontaneous external suppressors were isolated from the two heteroallelic strains ade7-C3 and ade7-C8 after a period of 14 days incubation. The two suppressors were crossed to a set of UV induced ade7 mutants of the nonsense (UGA, UAA) and missense type, to a set of ICR-170 induced ade6 mutants, to each other and finally to the two mutants carrying the heteroalleles adeT-C3 and ade7-C8. This first screening of different mutants helped ascertain the novel nature of the suppressors. The suppressor ofade7-C8, designated sufl, did not suppress any of the other mutants tested, whereas the suppressor of adeT-C3, designated suf2, was found also to suppress the ade6-C67 allele, Furthermore, the two suppressors were not allelic. To answer the question of the number of genetically distinct suppressors the three strains adeT-CS, ade7-C3 and ade6-C67 were reverted spontaneously and with ICR-170. 43 independent ICR-170 induced external suppressors of ade6-C67 (14) and ade7-C3 (29) were examined. The ade7-C8 allele failed to revert significantly with ICR-170 in three experiments, but a later experiment with an allele ade7-C50 suppressed by sufl led to the isolation of induced suppressors of this group. A total of 7 suppressors induced in ade7-C50 and of 44 suppressors isolated spontaneously from ade7-C8 were examined. The spontaneous and induced suppressors obtained from ade7-C8 and ade7-CSO (group I) were systematically crossed to each other, as were those obtained from ade6-C67 and ade7-C3 (group II). Random spores from these crosses were spread on YEA and the resulting colonies were replica-plated to MMA. The plates were examined after a further period of incubation of 4 days. The presence of auxotrophs indicated that distinct nonallelic suppressors had been crossed. The resolution of the random-spore analysis was set to enable a separation of suppressors about 0.5 map units apart. The examination was greatly facilitated by the fact that all the frameshift suppressors are inefficient in suppressing pigment formation and that even on enriched medium, almost all of the suppressor strains grow significantly more slowly than strains carrying the suppressor wild-type alleles. Colony colour and colony size allowed an unambiguous classification on YEA. Replica-plating to MMA

135 helped to substantiate the classification and allowed scoring of the auxotrophs. Suppressors induced in ade-C67 fell into 7 different genes. Those induced in ade7-C3 fell into 6 different genes. Representatives of the genes obtained in an ade6C67 background were crossed to representatives of the genes found in the ade7-C3 background. The two sets coincided and resulted in 7 distinct suppressor genes: suf2, suf3, suf4, suf5, suf6, suf7 and suf8 (of which suf8 was found only in the ade6-C67 background). Suppressors induced in adeT-CSO and spontaneously isolated from ade7-C8 were located in 2 genes; sufl and suf11. Crosses of group I suppressors (sufl, suf11) with the group II suppressors (suf2 - sufS) confirmed the existence of 9 distinct new suppressor genes.

Spectrum of Suppression o f Group I and Group [I Suppressors Suppression of Frameshift Mutants. Sufl and suf2 do not suppress ade7-413, a nonsense allele of the UAA type. The suppressors of group I and group II were crossed to ade7-413 and tetrads were isolated. The presumptive double mutants sufx ade7-413 were backcrossed to their suppressible alleles to test for the presence of the suppressor. Strains of the genotype sufx ade7-413 fail to grow on MMA and in a cross sufx ade7413 x + ade7 the appearance o f pink sufx ade7 spores signals suppression of the ade7 allele by sufx. Strains combining the suppressors with the insensitive ade7413 allele were crossed to 40 ICR-170 induced ade7 mutants and to UV-induced missense and nonsense mutants. Included in these tests were 4 UV-induced ade7 alleles suppressed by extragenic suppressors which were isolated and classified by Barben (1966) and which were tentatively proposed to be of the missense type by Hawthorne and Leupold (1974). Spores were plated directly on YEA and on MMA. After incubation for 5 days the YEA plates were in addition replica-plated to fresh MMA plates and incubated for 7 days. The two parallel tests for prototrophic recombinant growth on MMA were carried out to ensure that weakly suppressed Strains could unambiguously be detected as being suppressed. Suppressor carrying strains form small colonies on YEA that can be distinguished from colonies not carrying the suppressor solely on the basis of reduced colony size. In case there are signs of growth on MMA plates directly spread with spores this can be checked on the MMA replicas. On these replicas only distinctly smaller colonies should show growth. None of the four UV-induced missense mutants tested could be suppressed by the group I and group II suppressors. Of the 40 ICR-170 induced ade7 mutants the group I suppressors suppressed 4 alleles and the

I-L Hottinger and U. Leupold: Frameshift Suppressors in & pombe

136 *

o*o*

**

•

*o*o•

•

e~

o,ro,¢

v0o

o

momoo

~)

',;',',

I~

",

I,°I 300

i 750

II

Ill

250 500

Fig. 1. Map of the ade7 gene using ICR-170-induced and UVinduced mutants~ The map is roughly drawn to scale. • Group I suppressible alleles; o group II suppressible alleles; * UV-induced alleles serving as reference markers in mapping the suppressible ICR-170-induced mutant~ The thick line represents the ade7 gene. The thin lines beneath the gene gives the distances between sites or clusters of sites~ The numbers above the thin lines are prototrophie spores per 106 spores obtained from 2-point crosses. The values given here are representative means rounded to the nearest 50 between: (1) 2 sites; (2) a site and a cluster; (3) 2 cluster~ The arrangement of the sites corresponds to the most probable sequence deduced from the 2-point crosser For a more detailed map see Hottinger (1980). The high recombination frequencies given by C8 in crosses with all other mutant sites tested may well be due to a specific marker effect characterizing this site. The true map location of C8 may therefore lie much nearer or even within the fine-structure map defined by a total of 33 sites of UV-induced mutations and ending with the mutant sites 275 to the left and 50 to the right (Leupold 1961)

group II suppressors 8 alleles. The alleles suppressed b y the two groups o f suppressors do not overlap. Figure 1 shows a map o f the ade7 gene including the two groups o f ICR-induced mutants along with a few references sites from a map previously published b y Leupold (1961). Sufl and suf2 were further introduced into an insensitive ade6 background to allow a rapid screening o f ade6 mutants for suppressibility. The two strains o f the genotype sufl acle6-712 and suf2 ade6-712, where ade6-712 is a nonsense mutant o f the UGA type, were crossed to 56 ICR-170-induced ade6 mutants. Sufl did not suppress any o f these mutants; suf2 suppressed 12 o f the 56 ade6 mutants tested. The other group II suppressors were tested for their ability to suppress the 12 suf2 sensitive ade6 alleles. Suf3, suf4, suf5 and suf6 in an ade6-712 background were crossed to the 12 mutants and showed suppression. A parallel test crossing sufx ade6-C67 with + ade6 and scoring the progeny for growth on MMA also showed that all o f the group II suppressors suppressed the 12 ade6 mutants. In case o f suppression 50% o f the progeny clones are expected to grow on MMA, i n the absence o f suppression 25% are expected to grow. Table 1 gives a complete list o f the suppressible ICRinduced ade6 and ade7 mutants along with some o f the suppressible UV-induced missense mutants used in these experiments. This table also shows that none o f the 4 group I and 20 group II suppressible ade6 and ade7 mutants tested b y Munz and Leupold (1970) is suppressed b y the nonsense suppressors sup1 (an omni-

potent suppressor which codes for a ribosomal protein) and sup3-e (a UGA suppressor which codes for a serine tRNA; Kohli et al. 1979, 1980). Nor is any o f the three ade7 mutants tested during the present work, twosensitive to group I and one sensitive to group II suppressors, suppressible b y the presumptive missense suppressors sup4, sup5, sup6 and sup 7 (Barben 1966) (data n o t given).

Suppression o f Nonsense Mutant~ Group I and group II suppressors were tested for their ability to suppress nonsense mutants. Strains carrying sufl through suf8 on the background of ade7-C8 or adeT-C3 were crossed with a strain carrying the UAA allele ade7-413 and tetrads were isolated. In addition strains carrying sufl and suf2 alone were crossed with strains carrying the UGA allele ade6-712 (both suppressors) or the U A A allele ade7-413 (only suf2). The tetrad data o f these crosses are given in Table 2. In further experiments other nonsense mutations (ade6-704, adeT~4, arg1-230, glu1-57 and leu3-155) were tested for sensitivity to group I and group II suppressors either b y random-spore analysis or tetrad analysis. None o f the UAA mutations argl-230, ade7-413 and the UGA mutations ade6-704, ade6-712, ade7-84, glu1-57 and leu3-155 were suppressed b y the group I and group II suppressors tested. The allele adeT-572, which is suppressed b y the omnipotent nonsense suppressors sup1 and sup2 b u t not b y the UGA- and UAAspecific nonsense suppressors, was also found to be insensitive to group I and group II suppressors.

Efficiency o f Suppression Efficiency of Suppressors and Effects Upon Growth. The colony size and colour o f suppressor carrying strains growing on solid media were examined. Ade6 and ade7 mutants of S. pombe accumulate a red pigment on growth medium with a limited supply o f adenine (Gutz et al. 1974). Partial restoration o f the activity o f the ade6 or ade7 gene product will lead to a reduced accumulation o f the red pigment. The degree o f suppression can thus indirectly be judged from the reduction o f the red colour o f colonies o f suppressor carrying strains growing on adenine limited media. Comparing suppressors in different suppressible ade7 backgrounds was not possible, because o f the colour variations causedby different ade7 alleles. The relative efficiency o f suppression was determined b y comparing the effect o f the suppressors on a mutually suppressible allele. A relative measure o f the growth rate o f suppressor strains was obtained b y comparing the colony size o f different strains on YEA. F o r example, in cross o f sufx ade7 x + + the four progeny genotypes can be compared directly for relative colony size o n YEA. Microscopic examina-

137

IL H 0 t t i n g e r a n d U. L e u p o l d : F r a m e s h i f t S u p p r e s s o r s i n S. pombe T a b l e 1. S u p p r e s s i o n ofade6 a n d acle7 m u t a n t s

Gene/allele

Strain

Suppressors

Origin

g r o u p II

group I

ade7-

C8 C19 C46 C50 C3 C12 C20 C35 C38 C43 C46 C49 465

ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR UV UV UV UV UV

+ + + + -

+ + + + + + + +

-

-

541 680

49C4-3 49C-5 59C4-6 61C7-1 49C2-2 49C4-9 49C6-7 5 9C1-4 59C3-19 59C4-3 59C4-18 59C4-29 465 486 519 541 680

-

-

C5 C8 C10 C13 C15 C16 C19 C27 C43 C54 C63 C67

4 9 C 2-6 49C3-5 49C3-7 49C3-10 49C4-7 49C4-12 49C5-3 49C7-4 59C1-6 59C3-24 61C7-2 61C8-7

ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR ICR

-

+ + + + + + + + + + + +

486 519

ade6-

sup3

supl

(UGA)

(UGA, UAA)

sup4, sup5

sup6, sup7

missense

missense

--

+

m

m

m

m

m

m

m

m

-

+ + + +

m

m

S u p p r e s s o r s o f g r o u p I: sufl, sufll; s u p p r e s s o r s o f g r o u p II: suf2, suf3, suf4, suf5, suf6, s u p a n d aufS. N o n s e n s e s u p p r e s s i o n o f t h e I C R - 1 7 0 - i n d u c e d m u t a n t s w a s t e s t e d b y M u n z a n d L e u p o l d ( 1 9 7 0 ) . S u p p r e s s o r s sup4, sup5, sup6 a n d sup7 as well as t h e ade7 alleles: 465, 486, 519, 541 and 680 have been described previously by Barben (1966)

T a b l e 2. T e t r a d a n a l y s i s o f n o n s e n s e s u p p r e s s i o n b y g r o u p I a n d g r o u p II s u p p r e s s o r s Tetrads with 3 viable spores

Tetrads with 4 viable spores

Cross 4-

sufl ade7-C8 suf2 ade7-C3 suf3 ade7-C3 suf4 ade7-C3 suf5 ade7-C3 suf6 ade7-C3 suf7 ade7-C3 suf8 ade7-C3 sufl ÷ suf2 + suf2 4-

x 4- ade7-413 x ÷ ade7-413 x ÷ ade7-413 x ÷ ade 7-413 x 4- ade7-413 x + ade7-413 x ÷ acle7-413 x ÷ ade7-413 "~ x ÷ ade6-712 x÷ade6-712 x÷ade7-413

3-:1+

2-:2+

1-:3+

~4+

3-

2-:1+

1-:2+

3+

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

3 0 1 0 0 0 0 0

4 3 1 1 1 0 0 1

0 0 0 0 1 0 1 1

0 0 0 0 0 0 0 0

4 1 1 3 2 0 2 0

6 8 4 10 6 7 6 4

2 4 1 1 5 6 3 2

0

0

10

0

0

0

1

1

0

0

0

9

0

0

0

4

0

0

0

0

10

0

0

0

1

0

0

T h e t e t r a d s a r e t a b u l a t e d a c c o r d i n g t o t h e n u m b e r oracle- a n d ade+ s p o r e s in t h e t e t r a d . * T h i s c r o s s g a v e m a n y t e t r a d s in w h i c h o n l y 2 o r 3 s p o r e s g e r m i n a t e d

138

H. Hottinger and IT. Leupold: Frameshift Suppressors in S. pombe

tion of plates spread with spores helped determine whether reduction of col0ny size was due to retarded germination of spores or to a real reduction of the growth rate.

colonies on YEA, required long periods of incubation and had plating efficiencies on MMA of only 10-15% of wild type. Strains carrying suppressor suf7 had a much higher growth rate which was comparable to that of strains containing the inefficient nonsense suppressor sufl. The order of the relative efficiency of pigment suppression was found to be positively correlated with the order based on the reduction of colony size: (1) suf2; (2) suf4; (3) suf3; (4) suf6, suf8; (5) suf5, suf7. Every suppressor was crossed with every other suppressor and the triple mutants sufx sufy ade7-C3 were isolated. The sufx sufy ade7-C3 double suppressor strains were tested for their genotype by crossing the presumed triple mutant with a strain carrying the suppressible ade7 allele alone and isolating the four resulting progeny genotypes. The combinations suf2suf3, suf2-s'uf4 and suf3-suf4 grew extremely poorly. The plating efficiencies were greatly reduced, e.g. suf2-.~uf4 had a plating efficiency of less than 5% on MMA. These double suppressor strains efficiently suppressed the red colony colour. Combinations suf5-suf6, suf5-suf7 and suf6-suf7 formed colonies distinctly smaller and much lighter in colour than the parental strains, but they were easily isolated and crossed with other strains in clear contrast to combinations such as

Group I Suppressors. The strains sufl ade7-C8, sufll ade7-C8, sufl+ and sufl l+ grew distinctly more slowly than the 4- ade7-C8 auxotrophs on supplemented MMA or on YEA. The colonies of the strains of constitution sufl ade7-C8 and sufll ade7-C8 were pink on MMA and YEA and could not be distinguished from each other on the basis of colony colour or size. The triple mutant sufl sufll ade7-C8 gave very small colonies on YEA that were about 50% of the diameter of the double mutants sufl ade7-C8 and sufll ade7-C8. These triple mutants were more efficient in suppressing the red pigment, the colony colour being a very pale pink. The plating efficiency of the double-suppressor spores was reduced on YEA and strongly reduced on MMA ( 5 0 60% of single-suppressor spores). Diploids of the general type sufx ade7-C8 + + glul hI * * ura5 leu2 + rneil were constructed. * * designates combinations of different ade7 alleles, suppressor sensitive (ade7-C8) or insensitive (ade7-C3), with sufx (sufl, sufl l) or suf ~. The homozygous diploids sufx ade7-CS/ sufx ade7-C8 grew much more slowly on YEA and MMA than the diploids sufx ade7-C8/+ ade7-C8 heterozygous for the suppressor. The diploids heteroallelic for ade7 and heterozygous for the suppressor, sufx ade7-C8/ + ade7-C3, grew well on YEA but extremely weakly on MMA. These results indicate that the two suppressors are dominant in suppressing the adenine requirement of the sensitive ade7 auxotrophs and that the complete substitution of the wild-type sufl or sufll gene product by the corresponding mutant product impairs normal cell growth.

Group H Suppressors. The group II suppressors were tested for relative efficiency of pigment suppression and growth rate in the suppressible background ade7-C3. The presence of the active suppressor reduced the growth rate significantly on YEA. The strains suf2 ade7-C3, suf4 ade7-C3, suf5 ade7-C3, suf6 ade7-C3 and suf7 ade7-C3 had the same growth rate on YEA and YEA supplemented with excess adenine as the strains suf2 +, suf4 +, suf5 +, suf6 + and suf7 ÷, respectively. The suppressors were assigned ranks according to their relative colony size and colour. Rank (1) was assigned to the suppressors forming the smallest colonies and the colonies with the least colour. Rank (5) was assigned to the suppressors yielding the largest colonies and the most pigmented colonies. The colony size order on YEA and YEA with excess adenine supply was: (1) suf2; (2) suf4; (3) suf3, sudS; (4) suf5, suf6; (5) suf7. Strains carrying suf2 and suf4 formed very small

suf2-suf4. Diploids of the same type as described for group I suppressors above were constructed for all group II suppressors. For these suppressors ade7-C3 was the sensitive allele used and ade7-C8 the insensitive allele. The doubly homozygous diploids sufx adeT-C3 / sufx adeT-C3 were found to be comparable to the haploids sufx adeT-C3 in growth behaviour and suppression. The diploids sufx adeT-C3 / + adeT-C3 heterozygous for the suppressor grew very well on all solid media, much better than the homozygous diploids. The effect was very pronounced for all suppressors and the diploids heterozygous for the suppressor grew as well as the control diploid + adeT-C3 / 4- + on complete medium. The doubly heterozygous diploids sufx ade7-C3 / 4- ade7-C8 grew vigorously on YEA, but the colonies were red rather than pink and on MMA growth was very weak. Replica-plating such diploid colonies from YEA to MMA revealed distinct, albeit weak growth. A single diploid of the type suf3 suf7 ade7-C3 + 4- h- / + + ade7-C3 ura5 leu3 rneil was isolated and grew vigorously on YEA and MMA. The haploid parental strain suf3 suf7 ade7-C3 grew on MMA, but extremely slowly and with very low plating efficiency. We concluded from these results that the group II suppressors are dominant and inefficient in suppressing the adenine requirement of the sensitive ade7 auxotrophs. Furthermore, the substitution of the wild-type sufx gene product by the corresponding mutant product negatively affects normal cell growth. This effect incre-

FL Hottinger and U. Leupold: Frameshift Suppressors in S. pombe Table 3. Modifiers of frameshift suppresion Strong Reducreduction tion or elimination

Increase supl

STAll

+

+

+

$Uf2

+

+

+

ggf~ gUf4

+ +

+ +

+ +

suf5 suf6

+ +

+ +

+ +

s'uf7

+

+

+

suf8

+

+

-

sufll

+

+

+

sup2

+ --

+

+ + +

+

The first three columns list the suppressors for which freely segregating modifiers of the type indicated were found (+) or not found (-). The effect of the modifiers on the efficiency of suppression was classified as: (1) strongly reducing or eliminating suppression; (2) reducing suppression; (3) increasing suppression of pigment formation. In the columns sup 1 and sup2 the + implies enhancement of suppression observed; - no detectable difference observed ases with the efficiency o f suppression of pigment formation and in suppressor combinations. It is completely eliminated in the diploids heterozygous for the active suppressors.

Group I and Group II Suppressor Combinations. Combinations of group I with group II suppressors were isolated and no combination was found to be lethal. The colonies formed on YEA were always very small, but the colony colours corresponded to the colour of one of the parental types. This seems consistent with the fact that in any such triple mutant only one of the two active suppressors can suppress the ade7 allele present. On the other hand their deleterious effects upon growth appear to be additive.

Modifiers o f Group I and Group H Suppressors Plates with colonies o f suppressor carrying strains growing on YEA frequently contained individual colonies which were less efficient in suppressing the red pigment accumulation and which grew more vigorously than the sufx ade7 colonies. Some o f these colonies appeared to be ade7 auxotrophs. The same strains, when grown on MMA, tended to include a few more efficiently suppressed colonies o f a lighter colour. These colonies as a role grew with the same rate as the sufx ade7 colonies or at a somewhat reduced growth rate. Colonies showing such altered phenotypes were examined to see whether such strains harboured modifiers of suppression. If a modifier is present in a suppressor carrying strains, a cross with the suppressible ade7 allele will reveal this.

139 For example, the cross sufx mody ade7 x + + ade7 would generate 4 genotypes which then could be identified by back-crossing to + + ade7 and to sufx + ade7. Potential linkage of the modifier and the suppressor requires adequate sampling and the special case o f a modifying mutation within or in the immediate vicinity o f the suppressor gene is indicated by the absence o f sufx + ade7 progeny. As indicated in Table 3 freely segregating modifiers enhancing or decreasing the efficiency o f pigment suppression were found for all the suppressors of group I and group II. In these experiments only the segregation of modifiers was demonstrated. We did not investigate the nature o f any of the modifiers o f suppression found. The omnipotent nonsense suppressors supI and sup2 which suppress both UAA and UGA nonsense mutants, but no group I or group II suppressible frameshift mutants, are believed to code for ribosomal proteins (Thuriaux et al. 1975). We investigated whether the presence o f the presumed ribosomal suppressors could positively or negatively influence suppression o f group I and group II suppressors. A temperature sensitive allele sup2-413 (absence o f growth at 35 ° C) was crossed with sufl ade7-CS, suf2 ade7-C3, suf7 ade7-C3 and sufll ade7-C8 enabling easy identification o f the triple mutants sup2 sufx ade7. The two group I and two group II suppressors tested, sufl, suf11 and suf2, suf7 all showed an increase in the suppression of pigment formation when combined with sup2. The close linkage of sup1 with the temperature sensitive mutant gene tpsl4 (allele tpsl 4-5 = tsl-1-5, Kohli et al. 1977) was use d to advantage in isolating sup1 sufx ade7 strains. The strains supl ++ ade7-413(ade7-413 is a UAA nonsense allele insensitive to sufx) and + su.fx tps14 adeT-C3 (ade7-C3 is a group II suppressible allele insensitive to sup1) were crossed and the strain sup1 sufx + ade7-C3 isolated. The two parental strains fbrm pink colonies on YEA and MMA that are easily distinguished on the basis o f colour. The desired triple mutants yield very small colonies capable of growth at 35 ° C. Clones o f this type were isolated and crossed to sufx ade7-413 to check for the presence o f supl and sufx. The group II suppressors suf2, suf5 and suf6 were tested: suf5 and suf6 showed enhanced suppression o f pigment formation in combination with sup1, whereas suf2 did not show a significant decrease of pigmentation. We conclude from the results o f these experiments (see Table 3) that the two recessive, omnipotent nonsense suppressors sup1 and sup2 have a weak enhancing effect on the efficiency of suppression o f the group I and group II suppressors. A very special situation arose in the case o f the suf5 locus. Strains carrying this suppressor on the background o f a suppressible ade7 mutation were found to be unstable and to revert to auxotrophy at high frequency both meiotically and mitotically. The meiotic reversion

140

H. H0ttinger and U. Leupold: Frameshift Suppressors in S. pombe

Table 4. Allocation of sufgenes to linkage groups

Chromosome I

sufl suf2 suf3 su:4 sufS suf6 m.¢7 suf8 sufll

Mapping technique II

III

+ (+)

+ + +

+ + + +

T Ha H H Tb H H RS, T RS

Abbreviations: Tetrad analysis, T; random spore analysis, RS; haploidization (induced), H. a The haploidization of suf2 resulted in a ×2 of 7.76 for linkage group II which is significant at the 0.1 level- The x2's for the other two linkage groups were: X2 = 3.12 (group I) and ×2 = 5.21 (group III); these values permit only a tentative allocation ofsuf2 to linkage group II b C. Gysler, personal communication

frequency in selfings was between 10 . 3 and 10 - 4 spores and the mitotic frequency between 10 - s and 10 . 6 cells. This exceeded by far the meiotic frequency o f the other group II suppressors o f about 10 - s to 10 - 6 spores and the even smaller reversion frequencies o f the group I suppressors of 10 - 6 spores. The mitotic frequencies o f all the other suf genes were o f the order 10 . 6 to 10 . 7 cells and were primarily due to occurrence o f antisuppressor mutations. 30 independent mitotic and 15 independent meiotic auxotrophic revertants of suf5 were isolated and tested for the site o f the reversion event. Backcrossing o f the isolates with a strain carrying the suppressible ade7 allele should reveal the presence o f unlinked or loosely linked antisuppressors. Crossing with an ade6 mutant reveals the presence of an additional lesion in the ade6 gene also leading to red auxotrophic colonies. All the revertants proved to be true ade7 auxotrophs. Five meiotic and five mitotic auxotrophic revertants were crossed to a strain carrying the suppressor suf2 and all o f the revertants were suppressed. The site o f the reversion must therefore lie at or near the suppressor locus. Two suf5 ade7 clones were repeatedly crossed to wild type and the suf5 ade7 double mutant reisolated in an attempt to eliminate potential mutators, but the strains remained unstable and reverted at the same high frequency.

described by Kohli et al. (1977). The diploids used for the haploidization were homozygous for a suppressor sensitive allele and heterozygous for the suppressors. The fact that the suf genes strongly reduce growth rates selected against suppressor carrying haploids on the haploidization medium. This limitation was partly overcome by screening large samples and reducing the number o f cells spread per plate after haploidization. In spite o f these precautions the haploidization of suf strains proved difficult and in the case o f suf2 the allocation to chromosome II must be regarded with some reservation. Suf3 is a member of linkage group I whilst genes suf4, suf6 and surf were assigned to linkage group II (see Table 4). Random spore data from crosses involving sufl and the chromosome III genes ade6 and argl indicated that sufl is linked to both ade6 and argl. The strain sufl + tps14 ade7-C8 was constructed and crossed with + argl + ade7-C8 (tps14-5 = tsl-5, Kohli et al. 1977). The tetrad types obtained for the three different marker pairs were: argl x s~fl, 19 PD : 2 NPD : 36 T; argl x tps14, 29 PD : 1 NPD : 25 T; sufl x t p s 1 4 , 2 7 P D : 5 NPD : 42 T. Linkage was only loose and the most probable arrangement of the markers was determined as tps14 - argl - sufl with the map distances 28 and 44 map units, respectively. The map distance tps14 - sufl o f 49 map units is a minimal estimate as evidenced by the relatively large number o f NPD's. The mating-type locus mat on chromosome II showed linkage with suf8: isolation of 20 tetrads from a cross suf8 ade6-C67 h - x + ade6-C67 h + resulted in 16 PD, 0 NPD and 4 T tetrads, giving a map distance o f 10 map units. A random spore analysis of a cross between a suppressor insensitive his7 mutant and suf8, suf8 + ade6-C6 7 h - x + his7 ade6-C67 h +, revealed the close linkage (1.5 map units) o f suf8 with his7. The arrangement o f suf8 and his7 relative to the mat locus could not be determined in this cross. The suf8 locus is proximal to mat and closely linked to his7. The suf5 locus also maps on chromosome II, in the immediate neighbourhood (0.6 cM) o f the centromere marker tps13 (tps13-24 = tsl-24, Kohli et al. 1977) as shown by a tetrad analysis o f the cross suf5 + + ade7-C3 h - x + tps13 his3 adeT-C3 h ÷ which yielded 77 PD, 0 NPD and 1 T tetrads for the marker pair suf5 x tps13 (C. Gysler, personal communication).

Discussion

Frameshift Mutations Genetic Mapping o f the S u f Genes Several suf genes were allocated to their respective chromosomes by employing the haploidization method

Munz and Leupold (1970) have discussed the ICR-170induced ade6 and ade7 mutations in S. pombe and concluded that many o f these mutants had the proper-

IL Hottinger and U. Leupold: Frameshfft Suppressors in S. pombe

141

ties characteristic of frameshift mutations such as: (1) absence of suppression by nonsense suppressors; (2) induction of reversion by ICR-170 but not by NG; (3) only polarized complementation or complete absence of complementation; (4) lack of temperature sensitivity and osmotic remediability. The results of the present experiments leading to the isolation of the two mutually exclusive groups of extragenic suppressors from several of these mutants support this conclusion. The two groups of suppressible ICR-170 induced auxotrophic mutations reverted to give three different types of prototrophs: (1) locus revertants, corrected at the site of the primary lesion or in the immediate vicinity, with completely restored wild-type growth on minimal medium; (2) locus revertants with only partially restored prototrophy probably due to a compensating frameshift mutation proximal or distal to the primary lesion; (3) extragenically suppressed mutants with only partially restored prototrophy. The dominant extragenic suppressors of ICR170-induced auxotrophs were themselves strongly induced by the frameshift mutagen ICR-170. Furthermore the two groups of suppressors did not suppress nonsense or missense mutations. Finally four suppressors of S. pombe tentatively described as missense suppressors by Hawthorne and Leupold (1974) could not suppress the presumptive framsehift mutations of group I and group II. These different fines of evidence taken together strongly imply that the ICR-170-induced ade6 and ade7 mutations of group I and group II are frameshifts and that their suppressors are frameshift suppressors.

delete bases in runs of G.C pairs (Yourno and Heath 1969; Yourno 1971 ; Riddle and Roth 1972b; Culbertson et al.1977). Yourno and Heath (1969) examined histidinol dehydrogenase from prototrophic double-frameshift revertants of an ICR-191-induced mutant of Salmonella and concluded from the amino-acid replacements observed that this mutant had resulted from the addition of an extra G.C pair in the DNA sequence coding for the enzyme, leading to an additional C residue in the mRNA. Later it was shown that mutants carrying this type of +1 insertion could be suppressed by suppressors which led to altered elution profiles of proline tRNAs. A second group of suppressors acting on a different set of +1 frameshift mutations was found to code for chromatographically altered glycine tRNAs (Riddle and Roth 1970, 1972a, b). In yeast Culbertson et al. (1977, 1980) and Cummins et al. (1980) found two mutually exclusive groups of suppressors, one o f which led to alterations in iso-accepting glycyl-tRNAs. These observations led us to expect that the two groups of frameshift suppressors in S. pombe reported in this study might also affect glycyl- or prolyl-tRNA.

Frameshift Suppressors The nine dominant suppressors of ICR-170-induced mutants investigated in these experiments fall into two mutually exclusive categories. The group I suppressors sufl and sufll suppress 4 ade7 mutations mapping at 3 distinct sites in the ade7 gene but do not suppress any of the 56 ade6 mutations tested. Group II suppressors (suf2 through sufS) suppress a set of 8 ade7 mutations mapping at 4 different sites in the ade7 gene and 12 ade6 mutations. Very striking similarities obtain when the frameshift mutations and frameshift suppressors first described in a bacterium, Salmonella typhimurium, and later in the budding yeast Saccharomyces cervesiae are compared with the mutations and suppressors reported here for the fission yeast S. pombe. In all three instances frameshift mutations leading to the isolation of the suppressors were induced with ICR mutagens, either ICR-170 or ICR-191. In each of the three organisms the suppressors were also strongly inducible with the same ICRmutagens. These ICR compounds seem to preferentially add or

Group I Suppressorz An interesting feature o f this group is the small number of suppressible alleles in a sample of 56 independent ICR-170-induced ade6 mutations. This could reflect a paucity of regions of the ade6 gene relatively rich in G/C pairs containing the codons recognized by the group I suppressors, or simply a small number of sites representing the codons recognized by group I suppressors. In S. cerevisiae the group III suppressors, tentatively assumed to be a class of proline suppressors, were found to act on only 2 alleles out of 39 his4 mutants tested (Culbertson et al. 1977; Cummins et al. 1980). Considering the nature of the two amino acids implied, glycine and proline, one would be inclined to expect a less frequent occurrence of proline in a normal polypeptide and thus suggest that the group I suppressors of S. pombe might affect proline tRNAs. The large number of revertants analyzed in this group and the small number of suppressors found makes it unlikely to expect that more than one or two additional suppressors will be found. The fact that sufl-suf11 double suppressors are not lethal clearly indicates the existence of at least one further gene. Group II Suppressors. Like the group I suppressors, the group II suppressors have adverse effects upon growth. The seven suppressors of this group affect the growth rate on yeast extract medium to varying degrees. Serious impairment of normal growth is observed in the presence of suf2 and suf4; more moderate effects are observed with suf6 and suf7. Suf3, suf5 and suf8 range between these two extremes. This indicates that the wild-type suppressor genes are of varying importance for the proper

142 maintenance of cell growth and function. Double-suppressor combinations are not lethal, but the effect on growth rate is drastic for all combinations, particularly for sul'2suf3, suf2-suf4 and suf3-suf4. This strong reduction of the growth rate of haploid suppressor strains and doublesuppressor combinations can be completely eliminated in diploids heterozygous for the suppressor and its wildtype allele. An interesting point is the positive correlation of the reduction of the growth rates and the efficiency of suppression, measured as the degree of suppression of pigment formation, which characterizes different suppressors. The greater efficiency exhibited by the more poorly growing suppressor strains is most easily explained by assuming that either more copies of these suppressor tRNAs are made or that the tRNAs from these genes are used more frequently because of a bias in codon usage. Both of these possibilities would lead to an impairment of normal growth when the suppressor gene is mutated to the suppressor active from. The observed increased efficiency of suppression of double-suppressor combinations probably simply reflects an increased gene dose in these strains. The second explanation appears to be less likely since two different observations suggest that group II suppressors affect the same isoacceptor (or at most two isoacceptors since the possible codon-anticodon interactions allowed by wobble restrict the number of anticodons that can read a given codon to maximally two). First the 20 group II suppressible mutants are suppressed by all the suppressors of this group. There are no mutants suppressed by just one or a few of the suppressors as in Salmonella (Riddle and Roth 1970). Second the group II suppressors revert at a high frequency to the suppressor inactive state in meiosis (approx. 10 - s spores). A possible explanation for this instability observed in meiosis is that recombination between the suppressor tRNA gene and redundant wild-type tRNA genes lead to a frequent loss of suppressor activity (Hofer et al. 1969, Munz and Leupold 1980). The frequency of reversion would depend on the number of identical or similar tRNA genes available for this type of heterologous recombination.

Modifiers of Suppression A characteristic feature o f tRNAs is the large number of genes required to ensure their proper functioning. Mutations in many different unlinked genes would be expected to affect the efficiency o f t R N A suppressors: (1) mutations in tRNA processing enzymes; (2) mutations in tRNA modifying enzymes; (3) mutations affecting the charging reaction; (4) mutations affecting the translation apparatus; (5) mutations in tRNA regulator genes. Freely segregating modifiers of suppression were observed for both groups of suppressors. Closely linked

H. H0ttinger and U. Leupold: Frameshift Suppressors in S. pombe modifiers or additional mutations in the suppressor gene leading to increased efficiency of suppression were not found. The loss of suppressor activity was either due to unlinked or loosely linked modifiers or to reversion events occurring at or near the suppressor locus itself. An interesting observation was the weak enhancement of suppression observed in some combinations of group I and group II suppressors with the recessive omnipotent nonsense suppressors supl and sup2 which are believed to code for ribosomal proteins.

The suf5 Gene Locus The extremely high rate of reversion of the suf5 locus from the suppressor active to the suppressor inactive state is something of an enigma. The group II suppressors all have a very high frequency of reversion in meiotic selfings, but the suf5 locus has a 100-fold greater rate of reversion than the other genes o f this group. The spontaneous mitotic frequency of reversion of suf5 is o f the order 10- 5 to 10-6 cells and the meiotic frequency 10. 3 t o 10 - 4 spores. In preliminary experiments we found that the majority of the suppressor inactive revertants were not due to unlinked antisuppressors and mutators, but appeared to be due to events occurring at the suppressor locus. At the sup4 of S. cerevisiae, spontaneous mitotic deletions occur at a high frequency as demonstrated by Rothstein (1979). A very high rate of spontaneous deletions at the suf5 locus may also account for the extreme mitotic and meiotic instability of this locus. The deletion hypothesis could be tested by a search for novel frameshift suppressors in revertants that have lost sufS. Complete absence of suf5 suppressors in such strains would support the deletion hypothesis. An alternative explanation could be that the suf5 locus is a tandem duplication containing two copies of the same tRNA gene sequence. The close proximity of two identical copies of the same tRNA gene would allow frequent heterologous recombination leading to suppressor inactive revertants. This kind of mechanism would account for both the mitotic and meiotic instabilities at the suf5 locus. In E. coli the occurrence of duplicate tRNA genes mapping at the same locus seems to be common (Russel et al. 1970; Inokuchi et al. 1979). Feldman (1976) has proposed tandem duplications for the arrangement of tRNA genes also in yeast. Recently dimeric precursors of tRNA molecules have been found in the yeast S. eerevisiae (Schmidt et al. 1980) and in S. pombe (Mao et al. 1980). A tandem repeat of the gene would also explain the preponderance of suf5 alleles among frameshift suppressors obtained by forward mutation. The mutations of 18 independently isolated, spontaneous and ICR-170-indu-

H. Hottinger andU. Leupold: Frameshfft Suppressors in S. pombe ced prototrophs derived from suppressible ade6 and ade7 mutants mapped in suf5, more than twice the number o f the next most frequently mutated locus suf2. This could be due to an increased target size in the case o f the suf5 locus. A high mitotic instability has also been observed in three out o f five group II frameshift suppressors o f S. cerevisiae (Culbertson et al. 1980). 50 suppressor inactive revertants derived from one o f the three suppressors, sufl, were analyzed in more detail and all were found to carry a local alteration at a second site at or near the suppressor gene which could be separated from the site o f the original mutation by recombination. The authors favour the simplest interpretation that reversion is due to mutations affecting the synthesis or function of the suppressing t R N A rather than to deletions o f tandemly duplicated DNA.

Acknowledgement~ We are indebted to Dr. Richard M. Peck of the Chemotherapy Laboratory of the Institute for Cancer Research in Philadelphia for the generous gift of ICR-170. We wish to thank Drs. Peter Munz, Ernst Schweingruber, Gabriel V6geli and JiJrg Kohli for their stimulating discussions The research reported in this paper is part of a dissertation submitted in partial fulfillment o f the requirements for the Ph.D. degree at the University of Bern, Switzerland. This work was supported by the Swiss National Science Foundation.

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Communicated by B.S. Cox Received February 6, 1981