Topical Reversion at the HIS1 Locus of Saccharomyces cerevisiae A Tale of Three Mutants

Corresponding author: R. C. von Borstel, Department of Biological Sciences, CW405 Biological Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9, Canada, rc.von-borstel{at}ualberta.ca (E-mail).

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS CONCLUSIONS LITERATURE CITED

Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself. Thirteen mutations of his1-1 now have been identified with respect to base sequence. Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, née his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, née his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, née his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion. Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's. Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype. Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis. Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.

IN the yeast Saccharomyces cerevisiae it is often difficult to study topical reversions, that is, mutations that arise in the same genetic locus as the mutant. The difficulty arises because the topical frameshifts, nonsense, and missense mutations often are swamped out by a plethora of extragenic suppressors, so that an analysis of each revertant becomes an overwhelming exercise. With one interesting exception,¹ his1 revertants have been reported as arising only at the locus itself (KORCH and SNOW 1973 ). No his1 mutants were found that responded to chain termination suppressors. This puzzling finding was investigated exhaustively with the genetic techniques available in 1973. Our interest in topical reversion led us to study spontaneous reversions of his1 mutations in the KORCH and SNOW collection.

We have analyzed, in depth, reversions of a nonsense, a missense, and a frameshift mutation at HIS1. We have confirmed the observation of KORCH and SNOW 1973 that these reversions of his1 mutants are indeed topical. The revertants are detected among cells grown in glucose in multiple cultures, where revertants appear from the background only when the limiting metabolite (in these cases, histidine) is exhausted. We have used the P₀ of the Poisson distribution for calculation of the spontaneous mutation rate (cf. VON BORSTEL 1978 ; VON BORSTEL et al. 1971 ), which avoids most types of selection because mutational events rather than mutant frequencies are measured.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS CONCLUSIONS LITERATURE CITED

Strains and mutants:
According to the customary nomenclature for mutations of S. cerevisiae, the first mutant allele of HIS1 discovered was called his1-1, and as mutants were discovered, the enumeration grew until KORCH and SNOW 1973 mapped his1-315. Unfortunately, two of the more used numbers, namely, his1-1S and his1-7S, were duplicated for the original mutant alleles that KORCH and SNOW 1973 designated his1-1F for the his1-1 isolated by C. RAUT (unpublished results) and his1-7F for the his1-7 of R. K. MORTIMER (unpublished results) used by FOGEL and HURST 1967 . SEE KORCH and SNOW 1973 for a full historical account of the naming of the mutant alleles of HIS1. Here we mention the "F" (for FOGEL) in Table 1, and it is removed from this designation henceforth. The names his1-1S and his1-7S are retained for the two mutants found by KORCH and SNOW 1973 .

In Table 1 we rename the mutations in a locus to take into account their position in the base sequence as well as the type of mutation that took place. With slight departures, such as keeping the three-letter name for the phenotype itself, this is, in accordance with rules being established for the human genome (GLICKMAN 1997 ). The human genome nomenclature is being changed now to take in at a glance the most information possible about each mutant base pair. We have altered the rule by enclosing the nature of the mutation in parentheses to permit it to be a removable segment, whereas the proposal for the human genome is to affix the change into the name itself.

For the reversion experiments, the mutations were in different genetic backgrounds (Table 2). The strains containing his1-1 and his1-19 originally came from the collection made by KORCH and SNOW 1973 and were constructed into genetic backgrounds with other mutant loci from our own collection by SAVAGE 1979 (see also MANIVASAKIM 1993).

Media:
The complex medium of general use is YEPD (1% yeast extract, 2% Difco peptone, and 2% dextrose). The components of synthetic complete media (cf. VON BORSTEL et al. 1971 ) are filter-sterilized, except for the glucose and agar, which are autoclaved separately. The components of media are mixed together while the solutions are still hot. We have found that autoclaving the Yeast Nitrogen Base and the amino acids and bases together alters mutation rates.

Measurement of spontaneous mutation rates:
The growth-limiting concentration of histidine is 0.2 mg/L for his1-798 and his1-208 and 0.4 mg/L for his1-434. An average of 2500 cells is inoculated into each compartment of multiple-well assays, and cells go through at least eight or nine doublings in the medium before growth ceases. All experiments are carried out at 26°, and the routine spontaneous mutation rate is determined at 14 days. The incubation temperature must be monitored carefully, because there is a doubling in the spontaneous reversion rate, for most strains, with each 5° increase in temperature from 10 to 30° (R. C. VON BORSTEL and C. M. STEINBERG, unpublished data).

The P₀ component of the Poisson distribution is used to calculate the spontaneous mutation rate. Thereby, the spontaneous reversion rate (M) is

VON BORSTEL et al. 1971

VON BORSTEL 1978

If a large number of compartments is used, experiments are highly repeatable, as shown in Table 3 for reversion of the missense mutant, his1-798.

The his1-798 reversion data are the result of seven replicate tests carried out over an 8-month period. The mean of the seven tests is 6.3443 x 10^-8 reversions/cell/generation, the standard deviation is 0.2968, and the coefficient of variation is 0.0468. The preferred measurement of error is the coefficient of variation, which gives an accurate statement of the precision of the P₀ method. Calculation of the standard error or standard deviation requires the multiplication of the variability of the numerator and the denominator, and this leads to a perceived exaggeration of the error limits.

For very low reversion rates, as observed with his1-434, we grow the mutants to ~1–5 x 10⁷ cells/ml in YEPD medium and then place 1 ml each on 100–160 individual plates of histidine-dropout medium. After cessation of growth, the histidine-independent revertants arise from the background. Because there is still some histidine present in the inoculum that is plated, complete depletion of histidine (and thus cessation of growth) occurs ~6–8 cell generations later (ca. 1–2 x 10⁹ cells). Thereby, fewer than 2% of the mutants could have arisen in the original culture medium.

For examination of late-appearing mutants, particularly those arising after nearly 30 days, the mutants were examined for growth rate by growing them in a histidine-dropout medium, using synthetic complete medium for the control. The cells were counted with a hemocytometer every 2 hr for at least three doublings of the control in synthetic complete medium.

Extraction of genomic DNA from yeast for PCR:
A single yeast colony was transferred into 100 ml of fresh lysing solution (1 M Sorbitol, 20 mM EDTA, 10 µl/ml ß-mercaptoethanol, 2 mg/ml Zymolyase 20T) and incubated for 5 min at 37°. One hundred microliters of PCI (70% phenol, 29% chloroform, 1% isoamylalcohol) was added and incubated at 60° with vigorous shaking for 3 min. The aqueous phase was transferred to a fresh 1.5 ml microfuge tube for a standard powdered glass DNA extraction. The final DNA recovery from the powdered glass was in 20 µl of TE (10 mM Tris-HCl, 1 mM ETDA, pH 8.0).

Preparation of sequencing template by PCR and purification on agarose gels:
In earlier studies (LEE et al. 1988 , LEE et al. 1992 ), shuttle vectors containing the mutant HIS1 coding sequences were recovered from S. cerevisiae, amplified in Escherichia coli, and subsequently sequenced using the dideoxy chain-termination method developed by SANGER et al. 1977 (³⁵S-ATP). Direct sequencing of yeast colony PCR products for the HIS1 locus was first done by RITZEL et al. 1989 . Amplication of sequencing template by PCR was done according to the following protocol: the outside primers H1O (5'-GCTGCCAAGTGAGTCACCTCTACC-3') and H1X (5'-GCATGAAGACGGTAGTAAAGC-3') were used to amplify the his1 allele template for sequencing. The PCR reaction mixture included 3 µl of 10x PCR buffer (500 mM KCl, 100 mM Tris-HCl, 50 mM MgCl₂), 2 µl of the DNA extract, 1 ng of each primer and 2 µl of Taq polymerase solution, made up to 30 µl with sterile Milli-Q water. Twenty microliters of paraffin oil was added to each sample prior to PCR to reduce evaporation. The primers were made by the Microbiology Services Unit in the Department of Biological Sciences, University of Alberta (Edmonton, Canada).

The above reaction was cycled at 1 cycle (95° for 5 min), 30 cycles (95° for 30 s, 60° for 30 s, 73° for 90 s), 1 cycle (73° for 5 min) followed by a hold at 6°. The PCR was done in a Stratagene Robocycler 96 with thin-wall 200-µl tubes (Rose Scientific, Edmonton, Canada). Twenty microliters of 30% glycerol:0.25% bromophenol blue loading dye was added to each sample. From each sample, 110 µl was loaded on a 1% agarose:0.5x TBE (45 mM Tris-borate, 1 mM EDTA) minigel:0.5 µg/ml of ethidium bromide in a minigel box (Tyler Research Instruments, Edmonton, Canada) and electrophoresed at 60 V. The DNA bands were visualized on a UV transilluminator and cut out of the gel with minimal UV exposure. The DNA was isolated from the gel slices following the GeneClean II protocol with TBE modifier. DNA was resuspended in 20–50 µl of water depending on the size of the band. Confirmation of successful DNA extraction was made by electrophoresing a sample of the purified DNA on a 1% agarose:0.5x TBE gel.

Sequencing of the his1 mutants using ThermoSequenase with Redivue ³³P terminators:
The primers H1A (5'-ATGGATTTGGTGAACCATCTAACC-3'), H1B (5'-GTCGACGTAGACTTAGCAATCG-3'), H1C (5'-GTTAGTTCCATGATTGAGAG-3'), and H1E₂ (5'-GCTCTGGGAATTGGTGATGC-3') were used for sequencing the coding strand. The primers H1X (5'-GCATGAAGACGGTAGTAAAGC-3') and H1R (5'-TCTGTTCTATCTTATACACGACAA-3') were used for sequencing the noncoding strand. Dideoxy sequencing (SANGER et al. 1977 ) was done following the ThermoSequenase protocol from Amersham Life Science (Oakville, CA), with annealing temperatures 5° below the T_m for each primer and 50 cycles. Sequencing products were separated by denaturing polyacrylamide gel electrophoresis on 6% 57 acrylamide:3 bis-acrylamide with 8 M urea and 0.5x TBE. Urea was removed from the gels by soaking in 20% ethanol for 5 min followed by vacuum drying at 80° for 1 hr. Fuji RX autoradiography film (Fisher Scientific, Toronto, Canada) was exposed to the dried gel for 12–36 hr.

Sequence analyses were done manually by comparison with the wild-type HIS1 sequence. Mutations were confirmed by analysis of the second DNA strand.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS CONCLUSIONS LITERATURE CITED

Revertants of his1-208 (AT) (née his1-1):
The mutant his1-208 is caused by a mutation that creates an ochre chain termination codon. The reversion rate for this allele is 1.7 x 10^-9 reversions/cell/generation. The sequence changes in the revertants of his1-208 provide clear evidence that this ochre chain termination codon UAA does not revert by mutations of the anticodon of the tRNA (Table 4). Each histidine-independent revertant of his1-208 was a single base missense or back mutation within the three-base coding region. All seven possible single base mutations that replace the chain-termination codon with an amino acid codon will support growth. Any mutation to the amber UAG or opal UGA codons would not be expected to grow. None of the revertants were leaky; that is, late-arising revertants have not been observed among spontaneous mutants of his1-208. All base substitutions for the UAA codon function as well as any other.

View this table: In this window In a new window   Table 4. Revertants of the his-208 (A T) (his1-1 ) termination codon 5'-TAA-3'

There are a number of ways of explaining the lack of chain-termination suppression in HIS1, the most obvious of them being that tRNA suppression of chain termination is usually an inefficient process, and perhaps the codon has to be translated efficiently in order for the enzyme to be active. The efficiency has to be extremely low, because late-arising revertants of his1-798 can be as slow as 10% of the growth rate of the normal cells, and perhaps less. It would be interesting to test other opal, amber, as well as other ochre mutations at different locations within the locus. However, none was available from the KORCH and SNOW 1973 collection.

Revertants of his1-798 (GA) (née his1-7):
This mutation is a transversion lying near the 3'-end of the locus. It reverts most frequently by intragenic missense suppression. All reversions occurred on the upstream side of the primary mutation, excepting back mutation at the mutant base. The revertants are classified into three groups: nonfeeders, feeders, and undefined (untested) (Table 5). Two revertants in Table 5 are of particular interest because they are rare: HIS1-798 ( 798), which is a back mutation (a restoration to the wild-type genotype), and HIS1-798 ( 199-201), a deletion of an entire codon.

View this table: In this window In a new window   Table 5. Revertants of his1-798 (G A) (his1-7)

For mutagen testing assays, his1-798 could be useful because it would permit measurement of mutagen-induced transition or transversion types at numerous places within the locus. As would be expected when missense suppression makes many mutation sites available throughout the gene, the spontaneous reversion rate is high, being in the range of 10^-7 mutations/cell/generation.

Feeders and nonfeeders are about equal in frequency. The feeders are an interesting example of excretion of a metabolite enabling nearby cells to grow into colonies. The metabolite that is excreted was found to be histidine, as determined by analysis of the supernatant of liquid cultures. HIS1 encodes the first enzyme of the histidine pathway, phosphoribosyl adenosine triphosphate:pyrophosphate phosphoribosyltransferase (E.C.2.4.2.c) (FINK 1964 ). Feeding of nearby cells suggests that an overproduction of histidine is controlled by the first step of the pathway. This indicates that the capability for feeding is because of a loss of feedback inhibition by histidine. The feeders are located in three main groups in the locus. It is interesting that one base change can elicit overexpression, whereas the same base mutated in a different way, or a reversion in the same codon, may result in a nonfeeder. These are at amino acids 69, 123, 156, and 266, corresponding, respectively, to base pairs at 205 and 206, 367, 472 and 473, and 796 and 798. There is no unique contiguous part of the gene that is mutated in the cross-feeding of revertants of his1-798 that might encode a feedback inhibition site. This differs from the suggestion put forth by KORCH and SNOW 1973 that the alleles permitting excretion of histidine were located in a particular portion of the locus. Later work (LAX and FOGEL 1978 ; LAX et al. 1978 ; FOGEL et al. 1978 ) showed that there was more than one location, but still these authors advanced the suggestion that regulation of excretion was under the tight control of structural elements within the gene. Enlightenment may be provided by a comparison of the genetic data with a structure derived by X-ray crystallography.

The spontaneous reversion rate for allele his1-798, which is discussed as a model system for spontaneous mutation rate measurement (see MATERIALS AND METHODS), is 6.3 x 10^-8 reversions/cell/generation. Revertants that are feeders could confound mutation rate measurements based on revertant counts, but this problem is avoided when mutational events are counted using the P₀ fraction of the Poisson distribution. Late-arising revertants of his1-798 are shown in Table 6.

Instead of reaching a plateau by the 14th day at 26°, a few spontaneous revertants continued to appear until this experiment was terminated at 30 days. Most of the revertants tested that arose after the 14th day grew more slowly than those which arose previously. A few mutants, namely, revertants 3, 13, and 15, grew at the same rate in the presence or absence of histidine. Revertants 3 and 13 are petite mutants and grew slowly with respect to the controls. The only one of these three revertants that was not a petite mutant was revertant 15. Late-arising mutants that grow rapidly have been observed with reversions of the trp5-48 mutant (data not shown). These revertant colonies are invariably slow-growing mutants, where secondary mutations make the cells grow rapidly. When patches of slow-growing cells are streaked on the plate, papillations appear, constituting rapidly growing cells. The colony from revertant 15, shown in Table 6, was not isolated early enough to obtain a subset of slow-growing revertants. On the other hand, revertant 15, appearing on day 30, may be the first case of adaptive mutagenesis that we have observed. When the DNA sequence is obtained, this revertant will be examined for two or more alterations within the base sequence of HIS1.

The mutants described by Cairns and his colleagues (CAIRNS et al. 1988 ; CAIRNS and FOSTER 1991 ; FOSTER and CAIRNS 1992 ) as being caused by "directed mutagenesis" arose late and were ascribed to mutations taking place in stationary phase in E. coli. RYAN 1955 noted that DNA synthesis was taking place during "stationary phase," and this could account for some of the mutations, although he too believed that reversions were arising during stationary phase (RYAN 1959 ). This Ryan-Cairns deviation from the expectation that mutations take place during genomic DNA replication in E. coli has been observed in S. cerevisiae as well (VON BORSTEL 1978 ; HALL 1992 ; STEELE and JINKS-ROBERTSON 1992 ). The current most popular explanation for the Ryan-Cairns deviation in E. coli is that recombinational events are taking place during stationary phase, which, for the most part, lead to frameshift mutations [ HARRIS et al. 1994 , FOSTER and TRIMARCHI 1995 ; but see FOSTER 1998 ; ROSENBERG et al. 1998 ].

All late-arising spontaneous reversions we have studied can be explained as slowly growing mutants due to leakiness of revertants or as fast-growing mutants that are due to a secondary spontaneous revertant arising within a pool of slowly growing cells (cf. HALL 1990 ). And the last colonies to arise comprise cells that grow more slowly than all previously arising colonies.

Revertants of his1-434 (AAG) (née his1-19):
This mutation is a deletion of a base along with a transition at the 5' end of the run of adenines where the deletion occurred. The mutation has an ochre chain termination codon as the next codon in the sequence. The maximum allowable distance over which a reversion can take place is the addition of one base within six bases upstream. Upstream from that position any +1 addition or -2 deletion turns the codon prior to these six bases into an opal chain termination codon. The revertants that have been found to date are shown in Table 7. The two slowly growing mutants arose by day 5, so no late-arising mutants accrued in this experiment. Ten of the revertants were additions of one base pair, and two were deletions of two base pairs. The remaining seven also had base substitutions; five of these were at one or both ends of the run of adenines, and two were outside of the run of adenines.

View this table: In this window In a new window   Table 7. Frameshift revertants of the frameshift mutant his1-434(AA G) (his1-19)

The reversion rate for his1-434 is very low (9.4 x 10^-12 reversions/cell/generation). To maximize the opportunity for observing late-arising mutants that grow rapidly, a reversion experiment was carried out and observed for 30 days at 26°. The growth rates of each revertant were measured and compared in synthetic medium with and without histidine (Table 7). It is important to note that two revertants shown in Table 6 grew more slowly in medium without histidine, but like the other revertants, they arose within the time frame we call early (within 5 days at 26°), and thus must have occurred during log phase growth on the plate.

In a run of identical bases, the dynamics that produce a -1 reversion also can produce a +1 mutation (STREISINGER et al. 1966 ; FOWLER et al. 1974 ; STREISINGER and OWEN 1985 ; BEBENEK et al. 1992 ). The high frequency of base substitution-associated frameshifts seen here was not observed in either of two comprehensive analyses of hypermutability in homonucleotide runs in yeast (TRAN et al. 1996 , TRAN et al. 1997 ). Therefore, it is somewhat astonishing to observe that, in our observations, about 40% of all revertants also had base substitutions, most of which were at the end of the run of identical bases (Table 7). Something like this had been observed by BEBENEK et al. 1992 in vitro when they provided imbalanced frequencies of different deoxyribonucleosides in the substrate for replication of a segment of DNA containing a +1 mutation.

The reasons for the frameshift along with a base change could relate to mismatch repair. For example, we speculate that a misincorporated base leaving the replication fork may bind to a homolog of the E. coli MutS protein. This binding may then hold the primer on the template in a slipped position, thus allowing a base to be added or deleted with a higher probability than would have occurred without the misincorporation. Another possibility is that there is a steric hindrance to mismatch repair in some cases of very close mismatches, as suggested by MANIVASAKAM et al. 1996 . Yet another possibility (S. ROSENBERG, personal communication) is that a depletion of mismatch repair components may occur locally, allowing multiple mismatches to persist.

	CONCLUSIONS

TOP ABSTRACT MATERIALS AND METHODS RESULTS CONCLUSIONS LITERATURE CITED

1. The data presented here are consistent with the notion put forward by KORCH and SNOW 1973 that HIS1 mutations are reverted within the locus itself. Nevertheless, we have not excluded all possible trans-acting suppressors.

2. An ochre nonsense codon that was not suppressible by any external suppressor was reverted. All possible single base revertants encoding amino acids within the codon were identified.

3. Cells containing reversions of his1-798 that encoded different amino acids for the same codon could exhibit different feedback inhibition phenotypes.

4. The -1 frameshift mutation in a run of A's reverted by the conventional purine addition or double base subtraction most of the time, but a surprising number of the reversions were associated with base substitutions at one or both ends of the run of A's.

5. Some of the reversions are late-arising mutations. It is not a certainty that these late-arising reversions occurred during stationary phase because most of them were slow-growing revertants.

	FOOTNOTES

¹ SNOW 1980 reported one type of extragenic suppressor of his1 mutants that depends on interaction of two nonhomologous proteins. This finding presaged the useful two-hybrid system of FIELDS and SONG 1989 ; SONG et al. 1991 for demonstrating protein interactions between nonhomologous proteins. Now that the Fields-Song two-hybrid method is available, the protein interaction found by SNOW 1980 should be investigated further.
This paper is dedicated to Jan Drake on the Occasion of his1st-65th Birthday. It is also a reminder for him to bite the bullet gracefully.

	ACKNOWLEDGMENTS

This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and by a contract from Pro-Neuron, Inc.

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