REVERSION AT THE HIS1 LOCUS OF YEAST' SEYMOUR FOGEL, CAROL LAX2

AND

DONALD D. HURST3

Department of Genetics, University of California, Berkeley, California 94720 Manuscript received July 21, 1977 Revised copy received April 17, 1978 ABSTRACT

The his1 gene (chromosome V ) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions.-The spontaneous reversion rates of seven hisl mutations were studied. The reversion rates of the alleles a t the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles.-Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven hisl mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven hisl mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.

HE critical detailed analysis of reversions at selected loci can provide useful Tin formation concerning the organization and function of the eucaryotic genome. I n particular, special analytical opportunities to examine the organization of regulatory regions within genes are provided by loci coding for bifunctional enzymes that catalyze the first step in biosynthetic pathways. The his1 gene of Saccharomyces cereuisiae manifests these properties. It specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis (FINK 1964, 1965), an aggregate probably composed of six identical subunits (KORCH 1970, 1973; KORCHand SNOW1973). The enzyme is bifunctional; it catalyzes the condensation of ATP and phosphoribosylpyrophosphate, and also exerts a regulatory function in histidine synthesis. RASSE-MESSENGUY and FINK(1974) isolated regulatory mutants at a site in o r near the hisl locus. Although these triazolalanine-resistant mutants (TRAZ)

' This work supported by grants RG1-GM17317

and 5701-GM003F7 from the Publx Health Service These data are included m a thesis submitted in partial fulfillment of the requirements for the P h D degree at the University of California, Berkeley, California 94720 Permanent address Biology Department, Brooklyn College, Brooklyn, New York 11210 Genetics 90: 489-500 Noiemher, 1978

490

s. FOGEL, c. LAX and

D. D. HURST

are effectively unaltered in the catalytic properties of their phosphoribosyl transferase; nonetheless, they code for an enzyme that exhibits feedback resistance. Consequently, the internal histidine pools of strains carrying such analogueresistant mutants are increased three- to fifteen-fold over wild type, and several excrete histidine into the surrounding medium, However, the detailed molecular basis f o r the regulatory control exerted by the hisl enzyme in yeast is currently unknown. In contrast, the Salmonella hisG gene, corresponding to hisl of yeast, codes for a bifunctional enzyme whose regulatory activities are more fully characterized. Convincing evidence exists that the enzymatic protein in conjunction with histidyl tRNA binds to the histidine operon at the operator region. The bound complex exerts negative control over the transcription rate of the other histidine biosynthetic genes and thus ultimately determines the cellular levels of all coordinated gene products (KOVACH et al. 1969; KOVACHet al. 1970; ROTHMANDENESand MARTIN1971; VOGELet al. 1972; BLASIet al. 1971; GOLDBERGER 1974). GOLDBERGER (1974) designated such self-regulating systems as “autogenous.” The control mechanism of hisG is not applicable to his1 or to several other autogenously regulated, bifunctional, first-enzyme systems in yeast ( BOLLON and MAGEE1971; MEURIS 1974). Generally, the biosynthetic pathway genes in yeast are distributed throughout the genome. They are not organized into transcriptionally controlled clusters as in procaryotes (FINK1964, 1965; MORTIMER and HAWTHORNE 1973). A description of the hisl enzyme’s regulatory role in yeast will, in part, depend on a fuller understanding of the genetic organization of the hisl locus. An extensive genetic analysis of hisl mutants (KORCH1970; KORCH and SNOW 1972) uncovered several distinctive locus properties. Among the 65 mutants included in this fine-structure analysis. nonsense mutants were conspicuously absent. In addition, alleles mapping within the proximal one-sixth of the gene exhibited spontaneous and induced mitotic reversion rates to prototrophy one to two orders of magnitude higher than alleles located elsewhere within the locus (FOGEL and HURST 1967; KORCH1970; KORCHand SNOW1973). Revertibility differences among various hisl alleles are not easily reconciled on either the basis of their mutagenic origin (since most mutants were UV or EMS induced) or the supposition that they represent different alterations in various regions of the gene. Recombination and interallelic complementation studies suggest that most alleles are base-pair substitutions (KORCHand SNOW1973). However. the possibility that some mutants could represent triplet additions or deletions or frameshift mutations cannot be excluded. This study critically re-examines the spontaneous reversion rate of seven hisl mutant alleles. We focus on the following problems: (1) Do various hisl alleles exhibit qualitatively distinguishable reversion spectra related to their fine-structure map positions? (2) Is reversion from auxotrophy to prototrophy, o r restoration of apparent wild-type catalytic function separable from maintenance of normal regulatory function?

REVERSION OF

his2

49 1

I N YEAST

MATERIALS A N D M E T H O D S

Strains: The haploids and hybrids used in this study were synthesized from strains in the Berkeley stock collection. These include his1 alleles I * , 6 , 7 , 16, 30, 204, 270, 315 and the double mutant 270,315. Alleles hid-6, 16 and 30 were obtained from C. KORCH.The various mutants 30 was originate as follows: alleles 6,7 and 2004 were UV-induced mutants (R. K . MORTIMER); recovered by the nystatin counter-selection method after EMS treatment (R. SNOW1966); 270 and 315 were EMS induced (B. Z. DORFMAN); and 1 was obtained by C. RAUTand sent to US by D. HAWTHORNE. The standard wild-type strain X2180, the parent of hisl-7t provided the standard wild-type HIS1 allele. hid-290 was contributed by G. FINK.The various diploid strains and their genotypes are listed below: GENOTYPE

HYBRID

21793

a ura3 a +

LA8

a um3 " a +

23604 LA2

a

" ~

a

Q

LA76

a ura3 a +

a

+

+ hom3

~

LA75

23599

~

homd

+

hom3

+

a

22524

a ura3 a +

22546

a ura3 a +

~

v

~

v

~

v

~

arg6

his?-7

arg6

hisl-7 hisl-315 hisl-315

hisl-6 hisl-6

r,

ura3

a

hisl-7 hisl-7

homd

+

hom3

+ +

hom3

+

trp2

+

+

+

trp2

+

trp2

arg6

+

arg6

trp2

+

+

+

arg6

hisl-1 hisl-1

+

+

arg6

t T 2

hisl-204 + hisl-204 a r g 6

trp2

arg6

trp2

+

adel

adel ade 1 ade 1

+ leul

+

hisl-30 hisl-30

hisl-270 hisl-270

leul

ade I adel ade 1

adel leul

+

+ +

& + ade I ade 1

+ leul

adel adel

ade6

+

Media: Complex complete media (GNA) : 1% Difco yeast extract, 2% Difco bactopeptone, 2% glucose, 0.67% Difco beef extract, 2% agar, supplemented with twice the quantities of nutrilites listed for SC media. Synthetic complete media (SC): 0.67% Difco yeast nitrogen base without amino acids, 2% glucose, 2% agar, supplemented with 0.02% adenine-SO,, 0.05% L-arginine-HCl, 0.02% L-histidine-HC1, 0.05% L-leucine, 0.02% L-methionine, 0.3% L-threonine, 0.05 % L-tryptophan, 0.02% uracil. Omission media: SC media lacking a single nutrilite. In some instances medium lacking two nutrilites was used, e.g., in random spore analysis. Sporulation media (KAc) : 1% potassium acetate, 0.05% glucose, 0.5% Difco yeast extract, 2% agar, supplemented with twice the amount of nutrilites listed for SC. * Allele hisl-l corresponds to the mutant cited by FOCELand HURST(1967) and MACNI(19F3). KORCHand SNOW (1973) refer to this specific mutant as h i d - I F . It should not be confused with allele hisl-IS, which they derived from R. K. MORTIMER'S strain JB 13. JB 151, the original hid-7 isolate, is designated hisl-3 by KORCHand SNOW(1973).

+

492

s. FOGEL, c . LAX and D. D. HURST

Zygote isolation and tetrad analysis: Recovery of zygotes and tetrad micro-manipulation followed conventional procedures (MORTIMER and HAWTHORNE 1969) with slight modifications. When parental haploid growth requirements were complementary, diploids were selected from mass matings by their ability to grow on selective media. Tetrads were dissected by micromanipulation on the surface of GNA agar plates and these plates were employed directly as masters in replica-plating to appropriate omission media. This procedure eliminates unnecessary CAMPBELL and LUSNAX, transfers and allows for detection of post meiotic segregants (FOGEL, in preparation). Random spore analysis: About 2 x 106 cells were incubated four h r in 0.5 ml Glusulase (Endo Laboratories, Garden City, New York) to digest ascal walls. The Glusulase-treated SUSpension was diluted in 10 ml distilled water. The digested sporulated cultures were sonicated (Bronwell Biosnik IV, Bronwell Scientific Inc.) in an ice bath at 100 watts for four minutes. The suspension was diluted and plated on GNA o r omission media to yield 50-100 clones per plate. After 72-96 h r incubation, the random spore clones were replica-plated to relevant omission media. X-irradiation procedure: Mitotic gene conversion was induced by X raying plates seeded with appropriate heteroallelic diploid cells. The beryllium-window X-ray source (Machlet OEG of the Donner Laboratories, Berkeley. The 60) was generously provided by R. K. MORTIMER dose at the surface of the plate was 250 rads per second at 50 KV and 25 ma (unfiltered). Prototrophs appeared on plates after four to five days. Ultrauiolet irradiation procedure: Heteroallelic drains were grown on GNA agar plates (33 clones/plate), replica-plated to the appropriate omission medium and irradiated with a Hanovia Letharay germicidal lamp #683A-7. The dose rate at the wave length corresponding to the principal mercury line was 40 ergs/mmz/second. Prototrophic revertants were screened after four to five days. Homoallelic hisl spontaneous reversion rate determination: The reversion rate of various hisl alleles was determined using the following modification of RYAN’S(1963) procedure. One thousand or more independent clones of each strain were grown overnight on GNA agar plates (33 clones/plate) . These were transferred by replica-plating to histidine omission medium. The revertant papillae were counted after seveii days. The distribution of revertants is described by the Poisson equation:

where the proportion of clones containing X events is given as P, or the probability of observing X revertants per clone; m is the average number of revertants per clone; e is the base of the natural logarithm and X ! is X factorial. The proportion of clones without papillae (Po)was ascertained. From the zero term of the above equation

the average number of revertants per clone, m, was estimated. The reversion rate, p, the number of reversions/cell/division is defined as p=-

m d

where d is the number of divisions. The cell number per clone, N , was determined from hemocytometer counts of cells washed from the surface 01replica-platings. N is equal to the number of cell divisions, minus one. Since N is large and therefore approximately equal to d, the homoallelic hisl retersion rates for diploid cells were calculated from: p=-

m N

REVERSION

OF

his1

IN YEAST

493

Since the strains were diploid, halving the recorded values would convert them to a per locus basis. The P ( o l method of determining the mutation rate as used here might be expected to underestimate the true rate, because only about 10% of the cells in a clone are transferred by replica plating. Conceivably, there are many replica prints without papillae that were included i n the P ( o ) category that would have displayed at least one papillae had the entire colony been transferred. Relative mutation rates should not be affected by these considerations. Homoallelic reuertant recouery procedures: Diploid clones, homoallelic for various hisl alleles, were grown overnight on GNA plates, replica-plated to histidine omission media, and then incubated for three to seven days. Prototrophic revertant papillae appeared on a background of nongrowing cells. A single random papilla per clone was taken and restreaked to histidineless media. Selection for fast- o r slow-growing colonies was avoided. The revertants were allowed to grow for 24-48 hr and then replica-plated to sporulation media. Sporulation was typically abundant, i.e., 50% o r greater after three to six days. Four-spored asci were also numerous. Four o r more tetrads were dissected from each revertant. Ascosporal survival was excellent (>90%). Tetrads yielding four surviving ascosporal clones were analyzed, and the genotype of each clone was ascertained by replica-plating to all relevant omission media. Characterization oj hisl revertants: The tetrads described above were screened routinely for their histidine nutritional requirement. The identification of the hisl allele was determined as follows: (1) Histidine auxotrophs: All ascosporal clones exhibiting histidine auxotrophy were crossed to various hisl tester strains, including the parental hisl allele from which each revertant was derived. Each diploid was then tested for intragenic complementation and UV or X-rayinduced mitotic conversion frequencies. Complementation was defined as the ability of the diploid to grow confluently after 24-72 h r in the absence of exogenous histidine. (2) Histidine prototrophs: Random prototrophic ascosporal haploids were backcrossed to the parental hisl allele, and the resultant diploids were subjected to tetrad analysis. In all instances a 2 f : h segregation pattern fcr a histidine requirement characteristic of heterozygosity a t a single genetic locus was observed. Bioassay for histidine excretion: Strains t o be checked for histidine excretion were grown overnight on GNA agar plates, replica-plated to histidine omission media and sprayed (FOGEL, unpublished) with a suspension of the nonrevertible, histidine-requiring yeast strain, his4-290. Alternatively, histidine omission agar plates were first sprayed with the receptor strain and then replica-plated. In either instance, growth of the receptor auxotroph strain around the prototropic clone was used as a measure of the histidine released. RESULTS

Recovery and genetic analysis of hisl homoalldic prototrophic revertants Spontaneous hisl homoallelic reversion rates: Spontaneous mitotic homoallelic reversion rates were determined for seven alleles that span 80% of the his1 gene map. The alleles, 30, 315, 6, and 7, in that order, map within the extreme proximal region, while three alleles, 204, 207, and 1, are localized and ordered within the distal region of the fine-structure map (KORCHand SNOW1973). The homoallelic reversion rates expressed as prototrophs/cell/division and the fine-structure map position of the various alleles are summarized in Table 1. The proximal alleles exhibit spontaneous reversion rates about 50-fold higher than those of distal alleles. The apparent polarity in spontaneous reversion rates for proximal and distal alleles is consistent with the induced rates reported f o r the same mutants by KORCH(1970). I n a comparison of homoallelic reversion rates, where map position was determined by x-ray- or MMS-induced mitotic gene conversion, he found that six of eight his1 alleles positioned in the proximal

s. FOGEL, c. LAX and

494

D. D. HURST

TABLE 1 Relations of homoallelic hisl sponianeous reuersion rates io position on fine structure map

hisl-x

spontaneous reversion rate

allele

(prototrophs/cell/division)

30 31 5 6

0.68 x 1 0 : ;

0.17 x 10 x 10;; x 10

2.0 2.5

7

204 270

2.3 2.3 7.1

i

x 101; x 10x 10

* Fine structure map from KORCH(1970).

tRelative position of hisl-270

from present study.

one-sixth of the gene reverted to prototrophy about ten times as often as did 29 other allelic mutants distributed elsewhere. Hence, the capacity to regain prototrophic function by a given hisl allele either spontaneously. or by mutagenic induction, appears related to its map position. To determine if other effects are restricted to alleles as a function of map position, various homoallelic revertants were examined in detail. Genetic analysis of hisl homoallelic prototrophic reuertants: To avoid selection against possible revertant types, prototrophic papillae were selected from diploid rather than from haploid strains. The rationale for this approach was based on FOGEL and HURST’S earlier (unpublished) finding that some homoallelic diploids yield a unique class of apparent revertants. These arise as a consequence of a second-site mutation where the double mutant displays allelic complementation with the parental mutant. Independent vegetative prototrophic clones were recovered from the seven homoallelic combinations given in Table 1 and each revertant was subjected to tetrad analysis according to standard procedures. In characterizing individual revertants, consideration was given to: the segregation of the histidine requirement among the four ascosporal clones from each tetrad; the phenotype of each spore after hybridization to the input parental allele; and the histidine excretion capacity of the respective haploid segregant clones and their corresponding hybrids. The generalized segregation patterns outlined in Table 2 served as the diagnostic basis for classifying the various revertant types. Designated as HZSl-x-w in Table 2 are revertants that display a regular 2+:2m Mendelian segregation pattern. I n each ascus, two spores carry a prototrophic

REVERSION

OF

his2

495

I N YEAST

TABLE 2 Meiotic analysis of histidine independent clones from homoallelic hisl-x/hisl-x diploids Revertants without HIS segregants

Revertants with HIS segregants

2+:2-*

____ 2 + :2-t

2+:2-

2+ :2-

_ _ _ _ . _ _ _ _ _ ~ _ _ .

(a) tetrad segregation

Of:+$

(his+ :his-) (b) diploid phenotype of each spore class when backcrossed to hisl-x ( c ) genotypes of (b) (d) genotypes of homoallelic revertant

2

HISI-x-e hisf-x HISI-x-w hisl-x :2 2 ---- : 2 hisi-x hid-x hisl-x hisi-x HISI-x-w --__ hisi-x

HIS1 -x-e hisl-x

2+ :2-

2

hisl-x-c h i d - x : 2 -hid-x hisl-x hisf -x-c hid-x

-___

* Revertant segregating an allele phenotypically equivalent to wild type, HISI-x-w, and derived from hisl-x. t Revertant segregating a prototrophic excreter allele, HISI-x-e, derived from hisi-x. $ Revertant segregating an auxotrophic allele, hisl-x-c, complementary to and derived from hisl-x.

allele phenotypically indistinguishable from the standard wild type, while the two other spores contain a mutant identical to the input parental allele, hid-X. Other prototrophic revertants also segregate 2+:2m, but they harbor a histidineindependent allele characterized by histidine excretion. By analogy to the Salmonella system (SHEPPARD 1964), these might be feedback resistant or regulatory deficient. Prototrophic excreters derived from his2 -x are symbolized HIS1 x-e. The third mitotic revertant category yields only histidine-requiring ascosporal clones (0$ :4m). Here, the newly derived allele is intragenically complementary to, though derived from, h i d - x . Accordingly, such alleles are designated hisl-x-c. I n this allelic notation system, HIS and his signify prototrophy and auxotrophy, while the suffixes, w , e, and c, respectively indicate phenotypic equivalence to wild type, histidine excretion, and interallelic complementation with the parental allele. The original allele from which the particular revertant originates is also indicated. The generalized term for a parental allele is given as x. The tetrad segregation patterns for the various categories of homoallelic revertants are displayed in row a of Table 2. Row b indicates the histidine requirement of the four diploids obtained in each instance by crossing each spore with h i d - x . The genotype of each diploid is shown in row c, and the genotype deduced for each original revertant is given in row d. Table 3 summarizes the total number of individual revertants analyzed for each homoallelic combination and the proportion of w ,e and c types recovered for the various his2 alleles. Three alleles yielded only HZSI-x-w revertants: hisl-6, located in the proximal, highly revertible segment of the locus, and hisl-270, located in the distal, less revertible segment. Two other alleles, hisl315, located in the extreme proximal region, and hid-2 in the extreme distal

s. FOGEL, c. LAX and

496

D. D. HURST

TABLE 3

Distribulion of homoallelic hisl-x/hisl-x revertants Revertant type:

HISI-x-w Allela

Homoallelic hybrid

Revertants analyzed

hid-30 hisl -31 5 hisl -31 5 hisl-6 hisl-7 his1-7 hisl-204 hisl-270 his11

LA 76 z 3604 LA 2 LA 75 LA 8 Z 1793 Z 2524 Z 2526 3599

40 49

hid-x

34 (75%) 39 (80%)

40 40

I40 246

5 5 44

z

HISI-X-E hisl-x

hisl-x-c

--

hid-x

4 (10%) 10 (20%)

{MI* 40 (100%) 90 (64%)

33 {216}* 5 (100%) 5 (100%) 9 (21%) 35

0 (23%) 0 0

(79%)

~~

* All revertants yield histidine independent segregants, excretion property not ascertained.

region, yield similar HISl-x-w revertants. as well as the class characterized by histidine excretion, HISl-x-e. Without exception, when HISl-x-e alleles are backcrossed to the parental allele, the resultant diploids are histidine-independent, though they do not excrete sufficient histidine to yield a positive bioassay. Twenty percent of hid-315 revertants were histidine excreters, while the corresponding value of hisl-1 was 80%. Since revertants of these types are freely generated from parental alleles mapping at opposite ends of the his1 gene, no correlation between map position and reversion spectra was apparent. Unexpectedly, some prototrophic revertants from hid-7 and hid-30 failed to produce wild-type segregants upon meiotic analysis. For example, among 386 independent homoallelic revertants from hisl-7, 47 (or 12%) were in this category. No prototrophic spore clones were recovered after re-examining about 20 tetrads per revertant. When the four histidine-requiring spore clones from any ascus of this unique class were backcrossed to the parental allele, two resultant diploids exhibited homoallelism f o r hid-x, and two diploids grew at a rate approximately comparable to wild type or histidineless medium. Designated hisl -x-c, these interallelic complementers define a novel and previously undescribed revertant category. Recovery of hisl -x-calleles depends on selecting prototrophic papillae from homoallelic diploids rather than from haploid strains. Homoallelic hisl-7 and hisl-30 yielded similar mutational patterns, with revertants in all three categories. hisl-7 and hisl-30 map close together in the proximal region. Since the HISl-x-e o r hisl-x-c prototrophic revertants cannot be due to backmutation to the wild-type HIS1 base sequence, it was important to determine whether the prototrophic phenotypes reflect mutational events at the hisl locus or mutational events elsewhere in the genome that modify or suppress the original histidine requirement. In either instance, the expected segregation patterns are: 2 HISl-x-w: 2 hisl-x; 2 HISl-x-e: 2 hid-x or 2 hid-x-c: 2 hisl-x. However, since the markers hom3 and/or argb, which closely flank h i d , were heterozygous

REVERSION

OF

his1

IN YEAST

49 7

and segregating in all homoallelic diploids and their corresponding revertants, unlinked modifiers or suppressors could be unequivocally excluded. About 2500 tetrads representing 709 revertants were analyzed. Without exception h o n 3 and arg6 exhibited linkage to the altered histidine phenotype at values comparable to the published map distances for the hom3-hisl-arg6 intervals (MORTIMER and HAWTHORNE 1973; FOGEL and HURST 1967). This consequence is predicted only if the reversional event occurred at or very near the his1 locus. DISCUSSION

Aimed at developing a better understanding of the structural and functional aspect of genetic organization at the his1 locus in yeast, the present reversion analysis centers on two problems. First, it examines the conspicuous polarity between proximally and distally distributed alleles with respect to spontaneous reversion rates in mitotic cells. Then it correlates a n allele’s position on the genetic fine-structure map with the relative frequencies and varieties of observed spontaneous reversions. These reversions, uniformly attributable to genetic changes within or near the locus, display altered catalytic and/or regulatory functions. The previously reported polarity of spontaneous and induced reversion rates among various his1 alleles (KORCH1970; KORCHand SNOW1973) is verified by the present study. Moreover, the polarity appears somewhat exaggerated when the spontaneous rates involving the same alleles are compared. The 100-fold disparity in reversion rates for different regions of the locus appears unrelated to ( 1 ) the mutagenic agent that induced the parental allele, (2) mutational events elsewhere in the genome. (3) recombinational events in the vicinity of the his1 locus, and (4)the classes of revertants recovered. Of the seven alleles studied, mutants 30,270 and 315 derived from EMS treatment, while mutants 1, 6, 7 and 204 arose after UV mutagenesis. All are probably missense mutants with the possible exception of 1, which has been consid(1966). For all revertants ered a frameshift mutant by MAGNIand PUGLISI analyzed, the events leading to the prototrophic phenotype occur at or adjacent to the his1 locus. No instances of prototrophy associated with mitotic recombination for markers flanking his1 were found. We partitioned revertant alleles into three phenoltypic categories, “wild type”, histidine excreters and auxotrophs complementary to the input parental allele. If reversional polarity depends on the number of alternative ways that proximal alleles can be restored to prototrophic function compared to the limited potentialities available to distal alleles, then the distribution of revertant types might reflect a n allele’s map positioln. However, it is clear that HZSl-x-w and HZSI-x-e revertants are not differentially diagnostic for proximal and distal gene segments. Three of the folur highly revertible alleles ( 7 , 30 and 315) yielded prototrophic revertants that were phenotypically distinguishable from wild type upon tetrad analysis. These revertants exhibit interallelic complementation responses with the parental alleles, or they result in histidine excretion. But the combined frac-

498

s. FOGEL, c. LAX and

D. D. HURST

tion of total events, maximally 25%, fails to account for the 100-fold difference in reversion rates observed between the proximal and distal gene segments. Also, the proximally situated, highly revertible hisl-6 allele yielded only HISZ-x-w revertants, though it maps close to 7,30 and 315, while hid-1 yielded the largest fraction of histidine-excreting prototrophs, 80%. hisl-1 displays minimal revertibility (7.1 x and is the most distal site examined. It differs from alleles 6, 7, 30 and 315 since it fails to exhibit interallelic complementation with any known allele (KORCHand SNOW1973; FOGEL, unpublished). The reversion data concerning the other two complementing distal alleles (204 and 270) is very limited. Taken collectively, the data indicate that the frequency distribution of revertants classified as HISl-x-w and HISl-x-e is not related to map position in any simple manner. An apparent correlation exists between map position and the occurrence of hid-x-c revertants. Only proximal alleles yielded revertants of this type. These novel complementing alleles are previously undescribed and were recovered only by selecting prototrophic revertants from diploid rather than from haploid strains. However, the recovery frequency of hisl-x-c alleles, maximally 15%, is also insufficient to account for the observed reversional polarity. It may be emphasized that among 129 revertants involving alleles 6 and 315, no complementers were recovered. If we assume that complementers arise from proximal alleles with a frequency of about 15%, some 19-20 complementers should have been detected. Similarly, no complementing alleles were found among 54 revertants stemming from distal alleles, 204,270 and 2, though eight to nine were expected. I n both instances, the absence of hisl-x-c alleles is highly significant. Thus, reversion to the hid-x-c type may be allele, as well as region, specific. Our inability to correlate the recovery frequencies and/or distribution of hisl-x-c, HISl-x-e and HISZ-x-w alleles with the observed spontaneous reversional pollarity is presumptive evidence against the notion that the high revertibility of proximal alleles reflects the number of different second-site alterations capable of restoring prototrophic function. However, the screening procedure employed to partition different revertant types was doubtless insufficiently sensitive to distinguish among the different molecular alterations possible. For example, revertants classified as HISI-x-w may represent second-site alterations within the locus. Such mutants may well specify only slightly altered enzymes that do not affect growth rates o r lead to histidine excretion. Some triazolalanineresistant mutants (TRAY) mapping at his2 display such features. Notably, some exhibit seemingly normal catalytic properties, yet possess elevated internal histidine pools indicative of abnormal regulatory function, though they fail to excrete histidine at levels detectable by the bioassay screening procedure (RASSEMESSENGUY and FINK1974). In the present study such revertants would be spuriously classified as HISl-x-w, or wild type revertants. Thus, it seems plausible to assume that a continuous spectrum of effects exists between H I S l - x - w and HIS1 -x-erevertants.

REVERSION

OF

his1

IN YEAST

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The genetic separability of the catalytic and regulatory properties of the h i d specified gene product comprises anather major consideration of the present work. Prototrophic histidine excreters, HZSZ -x-e, specify catalytically sufficient enzymes that are nontheless regulatory deficient. We emphasize that the prototrophic excreters recovered here are dominant in catalytic function when hybridized to the input parents1 allele. Yet, the regulatory deficiency is recessive. This follow; since HZSI-x-e/hisl -x and HZSZ-x-e/+ do not excrete histidine. The relationship between the excreter alleles analyzed here and the TRAY regulatory mutants that map at his1 remains unresolved. All triazolalanine resistant mutations (TRAZ) are dominant in hybrids carrying a standard wildtype allele, may lead to enhanced levels of expression for the other enzymes in the pathway and may cause excretion of histidine (RASSE-MESSENGUY and FINK 1974). Whether any or all HZSI-x-e revertants are triazolalanine resistant is currently unknown. HZSZ-x-e alleles were recovered from some but not all homoallelic mutants. Hence these mutants are probably implicated in the regulatory function of phospharibosyl transferase. However, for other mutants, such as hisl-6, the absence of excreter alleles among 40 homoallelic revertants is viewed as highly significant. The recovery of his1 -x-c revertants from only proximal alleles yields additional information. If the complementational response is a coinsequence of second-site alterations within the his2 gene, then a novel type of intracistronic complementation has been uncovered. I n this instance, the gene product from a mutant allele ( h i d - x ) is complemented by the product of a particular kind of second allele ( h i d - x - c ) that harbors an identical parental site along with an additional mutant site. This interpretation suggests a protein-protein interaction at the heteropolymer level wherein coimplementatim may stem from altered aggregation of the six phosphoribosyl transferase subunits (KORCH 1970) or from altered enzyme stability or activity. The excellent technical assistance of K. LUSNAKis gratefully acknowledged. We also thank D. MALONEY and D. CAMPBELL for their helpful suggestions and R. K. MORTIMER for critically reading the manuscript. LITERATURE CITED

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Reversion at the HiS1 locus of yeast.

REVERSION AT THE HIS1 LOCUS OF YEAST' SEYMOUR FOGEL, CAROL LAX2 AND DONALD D. HURST3 Department of Genetics, University of California, Berkeley, Ca...
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