allelic complementation in the first gene for histidine ... - Europe PMC

ALLELIC COMPLEMENTATION IN THE FIRST GENE FOR HISTIDINE BIOSYNTHESIS IN SACCHAROMYCES CEREVZSZAE. I. CHARACTERISTICS OF MUTANTS AND GENETIC MAPPING OF ALLELES1p2 CHRISTOPHER T. KORCH3

AND

RICHARD SNOW4

Department of Genetics, University of California, Dauis, CA 95616 Manuscript received July 16, 1971 Revised copy received February 2, 1973 Transmitted by G. R. FINK ABSTRACT

A number of hisl mutants were tested for suppressibility, for reversion by EMS, ICR-170, and nitrous acid, for their allelic complementation response, and for their temperature sensitivity and osmotic remediability. None of 52 mutants tested was suppressible by a known ochre suppressor. This is a very surprising result compared with other studies of suppressibility in yeast and suggests that another function essential to the cell is associated with the hisl gene product, the polarity effect of a nonsense mutation destroying the activity of the hisl enzyme and this second function. Sixty-four hisl alleles were ordered by allelic mapping methods utilizing gamma rays, X-rays, and MMS. The three maps agree well in placement of alleles and have been oriented on chromosome V of yeast with respect to the centromere. The 18 noncomplementing alleles are localized in the distal half of the gene, whereas the complementing alleles are distributed more or less evenly. Mutations which revert to feedback resistance map in the proximal end. Also a t this end are mutations having a very high X-ray or MMS induced homoallelic reversion rate. This suggests that a number of missense mutations can occur in this region which result in innocuous amino acid substitutions in the enzyme. One X-ray map unit is estimated to correspond to about 131 base pairs or 43 amino acids, in agreement with results for the cytoand SHERMAN (1969). chrome-c protein obtained by PARKER

INTRAGENIC complementation, which involves subunit interactions in multimeric proteins, offers the possibility of genetically studying such interactions and, thereby, protein structure and function. At present there is no well-defined system established in which genetic studies can be combined with detailed biochemical studies on protein structure and function, which could lead to better understanding of intragenic complementation at the molecular level. This and Part of a thesis submitted by C. T. K. to the Graduate Division of the University of California (Davis) in partial fulfillment of the requirements for the Ph.D. degree. * Supported by grants from the National Institutes of Health (GM 13716), the National Science Foundation (GB87W), and the Atomic Energy Commission (Contract AT (04-3)-34, P.A. 151). C. T. IC. held a traineeship on a Genetics Training Grant frcm the National Institutes of Health (GM 701). * Present address: Laboratoire de Gn6tique Physiologique, Institut de Botanique, Universit6 Louis Pasteur, 8 rue Goethe, 67--Strasbourg, France. 4 Author bo whom reprint requests should be sent. Genetics 74: 287-305 June, 1973.

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C. T. KORCH A N D R. SNOW

the associated reports (KORCH1970, 1973) describe work with such a genetic and biochemical system which should permit an evaluation of current hypotheses concerning intragenic complementation. The first gene of the histidine biosynthetic pathway in Saccharomyces ceret:isiae was chosen because oi the abundance of mutations we have at this locus, and the regulatory nature of the enzyme encoded by the gene (FINK1965). In yeast, complemetation tests and allelic mapping can easily be performed. The gene histidine-i ( h i d ) ,is located on the long arm of the fifth chromosome, approximately 43 CMfrom the centromere. It is flanked proximally by the gene hom3 (formerly known as thr3) and distally by the gene argb (HAWTHORNE and MORTIMER 1968). FINK(1964,1965) established that his1 codes for the first enzyme in the histidine biosynthetic pathway, N-l- (5’-phosphoribosyl)-adenosine triphosphate: pyrophosphate phosphoribosyltransferase (E.C. 2.4.2.c). This enzyme catalyzes the formation of PR-ATP from ATP and PRPP with the release of inorganic pyrophosphate. The product, PR-ATP, is subsequently converted by nine sequential steps into L-histidine. Phosphoribosyltransferase is subject to feedback inhibition and repression by L-histidine (FINK1965). Allelic mapping in yeast can be carried out by three methods: meiotic recombination, spontaneous mitotic reversion, and induced mitotic reversion. The latter method was used in this study since it is the most efficient and accurate. The other two methods are associated with technical difficulties and yield results which are complicated by several factors (see MANNEY 1964a, b, for discussion). MANNEY and MORTIMER (1964) devised a genetic mapping method utilizing X-rays to induce mitotic reversion. (Gamma rays can be used similarly, with certain precautions discussed later.) This method is based on the observation that sublethal doses of X-rays induce prototrophic reversions in a heteroallelic diploid strain, and that the number of revertants induced, measured as the frequency of revertants/108 viable cells/rad, is directly proportional to the base pair separation of the alleles present in the diploid. The order of alleles obtained is the same as obtained with meiotic mapping (MANNEY and MORTIMER 1964; ESPOSITO 1967, 1968). The order can be deduced by mapping triads of two-factor crosses of the type m, x m2.m2 x m3, and ml x m3.The basic assumption is that X-rays cause lesions randomly in the DNA, and that a lesion occurring anywhere between the sites of the two alleles allows for a recombination event to follow. Hence, the greater the distance between the two sites, the more likely it is that a lesion will occur between them, and therefore the higher will be the induced reversion rate. We have reported a method of allelic mapping in yeast which utilizes methyl methanesulfonate (MMS) instead of X-rays to induce mitotic reversion (SNOW and KORCH1970). The principle of this mapping method is the same as for X-ray mapping, although MMS and X-rays cause reversion in different ways. We found that the number of MMS-induced revertants is not directly proportional to the dose (in minutes of exposure) but rather to the square of the dose. The MMS allelic map does not show as good additivity as the X-ray map, possibly because of the rather high specificity of the MMS for guanine nucleotides in DNA (BROOKESand LAWLEY 1964).

HISTIDINE MUTANTS O F YEAST

289

In this part of the study of intragenic complementation,the his2 mutants used were characterized for suppressibility by a nonsense suppressor (SUP1 1), temperature sensitivity, osmotic remediability, ability to complement interallelically, and their ability to be reverted to prototrophy by several mutagens. Sixty-four his2 alleles were also mapped by the induced mitotic mapping procedures discussed above using x-rays, gamma rays, and MMS. This allowed not only the ordering of the alleles but also observations on the distribution of the various classes of mutations within the gene. The complementation studies are discussed in detail elsewhere (KORCH,1970,1973). MATERIALS A N D METHODS

Media: “Complete” medium (YEPD): 1% Difco yeast extract, 2% Difco peptone, 2% dextrose. 2X YEPD: twice the concentrations of the YEPD constituents. Minimal medium (M): Difco Yeast Nitrogen Base with 2% dextrose but without amino acids. Supplemented minimal medium: minimal medium supplemented with the following (mg/l): adenine sulfate, 20; uracil, 10; leucine, 30; lysine, 20; histidine, 20; threonine, 80; tyrosine, 20. For diagnostic replica plates, one of the preceding supplements was omitted. Presporulation medium: 0.8% Difco nutrient broth, 0.25% Difco yeast extract, 5% dextrose. Sporulation medium: 0.9% potassium acetate, 0.25% Difco yeast extract, 0.1% dextrose. Petite medium: 3% glycerol, 0.025% dextrose, 1% Difco yeast extract, 2% Difco peptone. When necessary, the above media were solidified by adding 2% agar. Source and selection of mutants: The mutagenic origin of the his1 mutants used in this study is as follows: alleles IF, is,2 to 6, 204, and 315 were obtained following treatment with UV light; alleles 7S, 8 to 10, 12, 14 to 48 following treatment with EMS; allele 49 arose spontaneously; and alleles 50 to 56, 59, 62, 63, 65 to 69 were obtained following treatment with nitrous generously gave us the his1 alleles I S , 2 to 6 acid of the wild type S288C. DR. R. K. MORTIMER and 170 histidine auxotrophs obtained by total isolation after EMS mutagenesis; of the latter 13 were his1 mutants. W e also received from him strains X1687-12B (a trp5-48, arg4-17, hk5-2, ade2-1, ade?), S395D (a hisl-IF, k u l , trp2) and various strains containing the nonsense suppressor SUPII. His1 alleles 7 s through 69 were originally isolated by the nystatin counter selection method (SNOW1966). The alleles hisl-1 and his13 of FOGEL and HURST (1967), designated here as his2-1F and hisf-3 are different from alleles hid-IS and hid-7s. Hisl-204 and hisl-315 were kindly given to us by DRS.S. FOGEL and D. HURST. To allow for prototrophic selection of diploids for the complementation tests, adenine, leucine, or tyrosine markers were crossed into the hid strains. Characterization of mutants: Nonsense suppressibility of all but 11 his1 alleles (12, 16, 18, 29, 34, 38,39,42,42,48, 66) was tested i n two ways. I n the first, diploids of the genotype SUP11 ade2-1 HIS1 pet1/ ade2-1 hid-x PET1 were sporulated and random spore clones isolated. SUP11 (also known as S, Sd, and S I I ) is a class I1 ochre suppressor (HAWTHORNE and MORTIMER 1968) which suppresses the ade2-1 mutation recessively, thus making it possible to select random spores bearing the suppressor by picking, from a sporulated culture plated on minimal supplemented with histidine, those clones no longer requiring adenine for growth. If the his1 allele in the diploid is suppressible, all the selected clones should be histidine-independent; if not, there should be approximately equal numbers of histidine-dependent and-independent clones. The segregation of the nonsuppressible nuclear petite mutation, petl, was used as a control for the randomness of spore clone selection. Alternatively, nonsense suppression was checked by ascus dissection of diploids of the above genotype, followed by diagnostic replica plating. Alleles I F , 204, and 315 were tested by S. FOGEL and D. HURST(personal communication) and were found not to be suppressible by a nonsense suppressor. Temperature sensitivity and osmotic remediability were tested by replica-plating from YEPD master plates to plates of minimal with p H unadjusted (about pH 4.5), minimal a t p H 8.0, and

+

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C . T. KORCH A N D R. SNOW

minimal at p H 4.5 containing 1 M KCI. For each master, sets of three such plates were incubated at 150,30", and 36°C for several days to a week. Alleles were tested for revertibiilty using a modification of the method developed by FINK and LOWENSTEIN (1969). Cells were grown to about IO*/ml i n liquid YEPD, then 0.1 m l of this culture was spread onto a YEPD plate which was incubated for 24 hours a t 30"C, then replicated to minimal. The agar of this minimal plate was sliced in half; to one half was added 0.1 ml of ICR-170 (4 x IC-* gm/ml) and to the other half 5 p l of EMS applied onto small millipore filters. These plates were the controls without mutagen pretreatment. The cells on the second YEPD plate were allowed to grow for 6-8 hours, at which time the mutagen was added as above l 24 hours. Then the plates were replicated to minimal and the plates reincubated for a t ~ t a of medium. These were the experimental plates with mutagen pretreatment. The p H of the YEPD and minimal plates used with sodium nitrite was adjusted to 4.5 to stabilize the sodium nitrite during the critical 24-hour period of treatment. Genetic mapping procedures: For gamma and X-ray treatment, cultures of heteroallelic or homoallelic diploids were grown in 4 ml of YEPD ( 2 YEPD ~ for gamma ray treatment) in 16 X 125 mm test tubes, from inocula of a few hundred cells. The small inocula were used to ensure a minimum of revertants at the start of growth. Cultures were incubated in a reciprocal shaker a t 30°C for 3 days, which was sufficient time for the cells to reach stationary phase. Cells were then washed twice in O.24M potassium phosphate buffer (pH 6.3), counted, and diluted to about 3 x lO7/ml in the buffer. For gamma ray treatment, I ml suspensions of cells in 13 x 100 mm test tubes were arranged in a random pattern in a wire rack and given 1, 2, 3, o r 4 KR in a cobalt-60 source. No attempt was made to ensure that the cells were oxygenated during exposure. After exposure, the samples, diluted appropriately, were added to 3 m l of warm 0.7% top agar, and the contents poured onto plates of minimal medium. An untreated sample for each cross was also plated. Viability of the untreated and 4 KR samples was measured by diluting and plating on minimal medium supplemented with histidine. Plates were incubated for 4 days a t 30°C before counting. For X-ray treatment, samples in buffer were spread on minimal plates which were then irradiated with 1, 2, 3, or 4 KR of X-rays. Appropriate dilutions and platings were made to assess viability before and after treatment. Plates were again incubated for 4 days at 30°C before counting. Treatment with MMS was carried out as described previously (SNOWand KORCH1970). All platings were done in duplicate. Viability was not significantly affected after any of these treatments. Plate counts of prototrophic colonies of treated and untreated samples were used to determine the linear regression of induced prototrophs/l08 viable cells on dose (rads for gamma rays or x-rays, or minutes squared for MMS treatment). The least squares regression coefficient was used as the measure of map distance. A map unit is defined as 1.0 induced prototroph/l08 viable cells/unit dose. Only regression coefficients with Student's t-values greater than 3.18 (the 95% confidence limit) were used i n mapping the his1 alleles by all three methods. For the x-ray and MMS maps, data were obtained on homoallelic reversion rates to permit many of the heteroallelic reversion rates (regression coefficients) to be corrected before the maps were constructed. It was found empirically best to correct the heteroallelic rates by subtracting one-fourth of the homoallelic rates of the two alleles in the cross. Subtracting one-fourth did not result in negative map distances, as sometimes occurred when one-half was subtracted. With the regression coefficient for each cross as a measure of the genetic distance between alleles involved, the alleles were positioned in the gene by the results of two-factor crosses. This procedure is based on the observations that the sum of two distances in a triad adds up approximately to the third. The genetic maps were constructed in the following systematic manner. First, for each of the triads so ordered, a n additivity coefficient was calculated. This coefficient is defined as the sum of the two smaller distances divided by the largest. Second, the triads were categorized into groups with additivity coefficients deviating from unity by 5% or less, 5-10%, 10-20%, 20-30%, or greater than 30%. Third, map building began with those triads having an additivity coefficient between 0.95 and 1.05 (i.e., i: 5% from unity), and later was expanded to include those triads with greater and greater deviations from unity.

29 1


To resolve ambiguities of allele order the following rules were used. First, when the two smaller map distances of a triad were about the same magnitude (differing by less than a factor of IO), the position of a n allele relative t o alleles already mapped was the average distance from those alleles. Only triads which agreed in order were used, even if they were in different additivity coefficient categories. Of all the possible triads the difference in magnitude of the two smaller distances was less than IO-fold in 91.5%, 90.3%, and 83.8% of the cases for the x-ray, gamma ray, and MMS data, respectively. Those triads with one map distance that was negligible were not included in this accounting. Second, in the case where the two smaller map distances of a triad differed in magnitude by a factor greater than IO, only the smallest of the three map distances of the triad was used as the map distance of the new allele from the nearby, already positioned allele. However, the order indicated by the triad was used. Third, in the instance of conflicts i n the positioning of a n allele, the order used was that indicated by the majority of the triads involving that allele. This was done even if the average of the additivity coefficients for the minority of triads was closer to unity. Fourth, the majority rule was also used to resolve ambiguities of order in the case where two map distances of a triad were very large, and the third distance was very small. Radiation dosimetry: For gamma rays, sixteen 1.0 ml samples of Fricke solution (WEISS, ALLENand SCHWARZ 1956) were arranged in a convenient manner and irradiated for an appropriate period and the average dose determined. The exposures were corrected for decay of the cobalt-60 source. X-rays were emitted by a Machlett OEG-60 tube run at 50KV, 25ma. Samples were 15.6 cm from the window of the tube. The tube was calibrated with Fricke solution against which in turn had been calibrated with an ion the X-ray machine of DR. R. K. MORTIMER, chamber specifically designed to measure the low-energy X-rays emitted by this tube. The tube was also calibrated with a Victoreen dosimeter with a thimble designed to measure low-energy X-rays (Victoreen model No. 652). The average value of these two calibrations was 164 rad/ second. Source of chemicals: The nitrogen mustard ICR-170 (~-methoxy-6-chloro-9-[3(ethyl-2chloro-ethyl) amino-propylamino] -acridine2HCl was generously supplied by DR.H. J. CREECH, Institute for Cancer Research, Philadelphia, Pennsylvania. EMS (ethylmethanesulfonate) and MMS (methylmethanesulfonate) were obtained from the Eastman Kodak Organic Laboratories, Rochester, New York. RESULTS

Mutant characteristics: Three independent tests for suppressibility of 51 his1 alleles by random spore analysis and a fourth by ascus dissection showed that none were suppressible by the ochre suppressor, S U P l l . Allele 49 was considered not to be suppressible, on the basis of evaluation of incomplete asci. Table 1 summarizes the information gained from tests of temperature sensiTABLE 1 Temperature sensitivity an$ osmotic remediability of his1 mutants Allele

1s 28 30 32 42 68

Minimal, pH 4.5 i50c 3a0c 3vc

+++ +++ + +++ ++ +++ -

-

The levels of growth responses are: weak but definite growth; -, no growth.

Minimal, pH 4.5, 1 M KC1 15°C 30°C 36'C

+ -++++++ + +++ ++ - -

Minimal, pH 8.0 15°C 30°C 36°C

+++ +++ + +++ - ++ +++ -

-

+++, strong growth; ++, intermediate growth; +,

292


tivity of the his2 alleles. Alleles I S , 28,30, 42, and 68 are temperature-sensitive at pH 4.5 and they are affected by changes in the osmotic conditions of the medium. Allele 68 will grow only at 15°C in the presence of 1M KC1. Allele 30 was unable to grow in the presence of 1M KCl even at 15°C. The optimum growth temperature of alleles I S and 42 was raised by 1M KC1. The temperature range of growth for allele 28 was extended by 1M KCI. Allele 32 is classified as cold-sensitive and osmotic-sensitive,i.e., it grew only at 30°C in the absence of 1M KC1. The wild-type strain (S288C) grew under all conditions. The results of revertibility tests with EMS, ICR-170, and nitrous acid are presented in Table 2. EMS reverted 13 of the 40 mutants induced by it and 6 of the 15 nitrous acid mutants. ICR-170 may revert alleles 18,55, and 66, but the results were dubious. Among the 5 temperature-sensitive alleles tested, only alleles 30 and 42 were reverted (by EMS). Nitrous acid reverted the EMS-induced mutant /”S.Two of these revertants were histidine excreters, as indicated by the halo of many very small colonies surrounding the large revertant colonies, the number and size of which decreased with distance from the revertant clone. By analogy with SHEPPARD’S work ( 1964) with thiazolealanine-resistant mutants of Salmonella, this implies that hisl-7S is able to revert to feedback resistance and thus excrete histidine into the medium. The alleles were also tested for complementation. No complementation was found for alleles 2,4,15,17,19,26,31,35,39,41,45,47,49,52,53,54, 68, and I F . Of 15 noncomplementing alleles tested for mutagenic reversion, only allele 39 was revertible (by EMS). Allele 68 is a temperature-sensitive mutant under osmotically remedial conditions (1M KCl) . Genetic mapping results: In Figure 1 examples are presented of the regressions of induced prototrophs per IO6 viable cells versus dose (kr) and dose squared (minutes2) for the same triad of crosses done by the X-ray and the EMS mapping methods. The two methods lead to the same relative order and spacing of the alleles. Table 3 summarizes some general characteristics of the mapping data f o r the his1 alleles which is presented in detail by KORCH( 1970). The detailed data have for those wishing to see them. The been submitted to the editors of GENETICS mean Student’s t-value of the regression coefficients for the heteroallelic crosses is very high and significant in all cases at the 99.9% confidence level, implying excellent agreement with the hypothesis of a linear relationship between the induced heteroallelic reversion rate to prototrophy and dose (dose squared in the case of the MMS data). The Student’st-values were only less than the 95 % confidence level value in some cases where the regression coefficients were very small. Among those crosses tested more than once by both the MMS and the X-ray method, the mean “coefficient of reproducibility” (see Table 3) of the regression coefficients was significantly smaller for the X-ray method. The mean additivity coefficients for the triads of crosses was approximately unity for the X-ray and gamma ray data (0.998 and 0.955, respectively), but not for the MMS data (0.891). However, in all cases the values did not differ significantly from unity. As discussed earlier (SNOWand KORCH1970), the MMS data are more linear


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400

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FIGURE1.-Representative triads of regressions of induced prototrophs on dose. (a) X-ray triad. Regression coefficient (R) i s in prototrophs/l08 viable cells plated/rad. (b) MMS triad. R is in prototrophs/lOS viable cells plated/minut&.

when the regressions of prototrophs are plotted against dose squared rather than against dose. But in the case of the MMS data the mean y-intercept of the regression of prototrophs on dose squared was larger than for the other two methods. This suggests that the true power to which the dose should be raised is slightly less than 2, since this average intercept value indicates attempts to fit straight lines to curves with decreasing slopes. By the gamma ray method, some crosses showed a leveling off of the regression curve at the 3 and 4 kr doses. This is likely TABLE 3 Summary of genetic mapping data Number of crosses Heteroallelic

Mapping

method

(--)

Homoallelic

X-ray 185/42 Gamma ray 154/3 MMS 101/9

Student's Mean y-axis t-value intercept of regression prototrophs Mean coefficients coefficients of Mean 10 cells reproducibility

(?)

22.4** ' -0.60 +l.W 13.8** 15.1** f4.28

0.30 0.64

Number of triads used

163

213 68

Triad additivity coefficients

___

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0.998 t. 0.280 0.955 t 0.315 0.891 C 0.312

-

Student's t-value

3.56" 3.03' 2.83'

* Significant at 99% level.

* * Significant at 99.9% level.

The coefficient of reproducibility is defined as R,, - R,,,/Rmean, where R,, was the largest the sum of all regressions regression coefficient observed for a cross, R,,, the smallest, and R,,, observed for the cross. The coefficients of additivity are the sum of the two smallest regressions divided by the largest.


295

due to the loss of radiation sensitivity caused by the anoxic conditions that probably developed during irradiation. This has been noted in the literature before (JAMES and WERNER 1965; MORTIMER, BRUSTAD and CORMACK 1965). The mitotic genetic maps of the his1 alleles constructed by the three methods are shown standardized to the same length in Figure 2. Correction of the heteroallelic reversion rates for the homoallelic reversion rates was done where data was available. This simplified the ordering of the alleles. This correction was not applied to the gamma ray data because only three homoallelic crosses were tested. The number of alleles mapped by the three methods were 44, 61, and 41 by the gamma ray, X-ray, and MMS methods, respectively. The lengths of the three maps in appropriate map units are 3.3 (gamma ray), 9.0 (X-ray), and 117 (MMS). The three maps were oriented on chromosome V with respect to the flanking genes hom3 and arg6 by determining the order of alleles IF, 3,204, and 315. These alleles were ordered meiotically in the his1 gene with respect to these and HURST (1967). The mitotic and meiotic map order flanking genes by FOGEL of these alleles is the same. Comparison of the maps constructed by the three methods shows that the order of alleles is very similar. The orientation of alleles with respect to the flanking markers is the same in all three, the left side (nearest hom3) containing alleles 3, 6, 7 , 9, 27, 40,48, 315. Alleles is,I F , 2,4, 17,19,35,39,61, and 49 are closer to arg6. Starting from allele 40 at the hom3 end, the order for the following 23 alleles is the same in all three maps: 40,315,6,27,9,3,7,48, 51, 56,59,67,19, 2, IS, 29, (iP,4 1 ) , 38,17, 61, 35, and 39. Alleles 1F and 41 are very close and their order has not been definitely determined. In the center of the maps, between alleles 48 and 19, the order of alleles 10,15,18,20,33,36,37,46,50,55, and 66 is difficult to determine because they were positioned with respect to alleles I S , 2 and 19 and not with respect to an allele in the center of the gene and to the left of 19. The distribution of alleles with high homoallelic X-ray reversion rates is not random. Figure 3a shows that except f o r 18 all these alleles lie at the left end of


MAP P O S l T l O N

FIGURE 3.-Plots MMS data.

of homoallelic reversion rates against map position. (a) X-ray data. (b)

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the gene. Three peaks are marked by alleles 6,7S, and 30. The homoallelic reversion rate for the MMS data shows the same polarity (Figure 3b). In both cases the terminal mutation at the hom3 end is allele 40 which has a reversion rate less than 3,6,7S, 9 and 27, but somewhat greater than IS,17 and 39. In all three maps, the 18 noncomplementing alleles occur in the arg6 half of the gene. The alleles which can revert to feedback resistance (3,7S, and 315) are in the hom3 end. The possible significance of these unusual distributions of mutants is discussed later. DISCUSSION

Genetic mapping: The X-ray map shown in Figure 4 represents the most likely order for the 64 alleles mapped at the his1 locus. Alleles 28, 32, and 42 were placed on this map using their X-ray map distances but using the gamma ray map order, because of the difficulty of placing these temperature-sensitive alleles by X-ray mapping. The X-ray map was used as the standard allele order of the gene because it was constructed from the most extensive set of data, is the most accurate based on the t-values of the regression coefficients, has an average additivity coefficient closest to unity, and because X-rays (like gamma rays) are thought to act randomly on the DNA of the cells, in contrast to MMS (BROOKS and LAWLEY 1964). 0.0

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298


I n the MMS map the relatively barren central region and the distances to allele 40 from alleles 315, &,27,9,3, and 7s are proportionately greater than in the other two maps. This might be due to a difference in the way recombination is stimulated by MMS or by ionizing radiations. SNOWand KORCH(1970) discussed a model to explain this difference. The hypothesis is that an X-ray can cause two single-strand breaks, one in each of two paired double-stranded DNA molecules. A break would presumably be a prelude to a recombination event, and thus the regression of prototrophs on dose should be a linear function of the dose. MMS, on the other hand, is thought to cause only one single-strand break for each molecule of this monofunctional alkylating agent which attacks the DNA. It is known to alkylate primarily the N-7 position of guanine, but it also alkylates the N-3 position of adenine at a much lower frequency (BROOKS and LAWLEY 1964). Hence, two alkylating events are postulated to be necessary for MMS-induced recombination, accounting for the fact that the regression of prototroph frequency on dose rises approximately as the square of the dose. A corollary of the later hypothesis is that the MhIS map distances could be distorted because of the guanine concentration in different regions of the gene, unlike the X-ray map distances which should be independent of base sequence. The great expansion between allele 40 and its neighbors as well as in the central region of the MMS map (relative to the X-ray and gamma ray maps) may be due to a relatively higher guanine content than average. The major problem encountered with the gamma ray mapping method was a noticeable leveling off of heteroallelic reversion rates at the higher doses. Conditions of anoxia probably developed in the buffer suspensions of yeast before and during irradiation and thereby reduced the sensitivity of the cells to gamma rays. The crosses for the X-ray map were done under conditions which did not allow anoxia to develop. Support of this explanation is provided by the fact that the Xray map is longer than the gamma ray map by a factor of 2.7, the same as the factor 2.77 by which the sensitivity of a yeast culture to such ionizing radiations as x-rays and gamma rays is reduced when the culture is deprived of oxygen (MORTIMER, BRUSTAD and CORMACK 1965). PARKER and SHERMAN(1969) established that 1 X-ray map unit (1 induced prototroph/lOs viable cells/r) corresponds to 129 base pairs or 43 amino acids. To arrive at this value they used two cytochrome-c mutations which are known by peptide analysis to be separated by a specific number of amino acids. We have attempted a similar estimate for the his1 gene. The most accurate estimate of the size of his1 from the X-ray data is 7.6 map units, from allele 40 on the left side to allele 39 on the right. (Allele 49 appears to be further to the right, but we have been unable to map it consistently, and triads involving it have always had low additivity coefficients. Gamma and X-ray mapping places it at the extreme right end, but MMS mapping puts it to the left of allele 17.) Phosphoribosyltransferase, coded for by hid, has a probable molecular weight of about 2.2 . lo5 daltons, and is probably composed of six subunits (VOLL,APPELLA and MARTIN1967; KORCH1970,1973; WHITFIELD 1971; R. M. BELL,personal communication, 1970). Thus the molecular weight of a subunit


299

is estimated as 3.6 . lo4 daltons. The average molecular weight of an amino’acid residue for the Salmonella phosphoribosyltransferase is 109 daltons (VOLL, 1967). This gives 332 as the estimated number of amino APPELLA and MARTIN acids in the polypeptide encoded by hisl, and hence 43.7 amino acids or 131 nucleotide base pairs per X-ray unit, the same value obtained by PARKER and SHERMAN (1969). If allele 49 is considered to mark the extreme right end of the gene, the estimate is 37 amino acids or 111 base pairs per map unit. Significance of the distribution of the diferent mutant classes: The distributions of the different types of his1 alleles within the gene can be summarized as follows. (1) There are about 50% more alleles in the hom3 half of the gene than in the arg6 half (37 us. 26); (2) the majority of temperature-sensitive alleles (4 us. 2) and complementing alleles (37 us. 9) are similarly located in the hom3 half; (3) the proportion of alleles revertible by EMS and nitrous acid as well as those revertible in homoallelic diploids by MMS and X-rays is distinctly higher in the hom3 half than in the arg6 half of the gene (16 us. 1) j (4) all the 17 noncomplementing alleles map in the arg6 half of the gene, the opposite of revertibility and general distribution of the hisl alleles. Also, of the 15 noncomplementing alleles tested for mutagenic revertibility only allele 39 responded (to EMS). DONALD HURST (personal communication, 1970) found that alleles 3 and 315 could revert to feedback resistance, as shown by their becoming histidine excreters. These three alleles all map in a small region of the hom3 half of the gene, the same region which shows very high homoallelic reversion by MMS and X-rays. The noticeable polarity of homoallelic and mutagen-induced revertibility within his1 suggests that in the hom3 end of the gene, revertants can be produced either by restoration of the original wild-type base sequence or by mutation to an acceptable missense sequence (with an essentially wild phenotype) more frequently than mutations in other regions of the gene. This would be in agreement with the finding that three alleles within this “hotsp~t’~ of revertibility can revert and simultaneously give rise to feedback resistance, which implies that a base change has occurred that results in some activity of the gene product but alters its normal properties. Also this distribution suggests that the hom3 end of the gene is the location of the feedback site of the enzyme. Interestingly, in the hisG gene of Salmonella typhimurium, which also codes for phosphoribosyltransferase, almost all of the feedback-resistant mutants map in a small region of the gene which is in about the same relative position on the hisG genetic map as are the three alleles 3,7S, and 315 on the his1 map (ST. PIERRE 1968). The localization of the 18 noncomplementing hisl alleles to the arg6 half of the gene implies a uniqueness of this region. It might be expected that at least some noncomplementingalleles would be frameshift mutations which completely destroyed the activity of the protein. The reports that nitrous acid causes frameshift mutations are important here (WHITFIELD, MARTINand AMES 1966; SCHWARTZ and BECKWITH1969), since 4 of the 18 noncomplementing mutants were induced with it. This might explin the noncomplementation property of

300


these four, but it seems unlikely that the distribution of noncomplementingalleles can be explained by half the gene's being susceptible to frameshift mutations while the other half is not. Also, none of the noncomplementing mutants was 1967; MAGNI reverted by ICR-170, a possible frameshift mutagen (MALLING and PUGLISI 1966) nor by nitrous acid. Hence it is likely that the uneven distribution of noncomplementing alleles is related to the distribution of the functional sites in the protein. On the genetic map there is a large cluster of noncomplementingmutants containing 10 of the 18 alleles, lying between 5.6 and 6.2 X-ray map units from the hom3 end of the gene. Between 4.5 and 5.2 map units there is a group of four more of these alleles and between 7.2 map units and the arg6 end of the gene another group of four more. These three groups may mark regions critical for the activity of the enzyme. The temperature-sensitive mutations ZS, 28,32,42, and 68, map in the central region of the gene (3.3 to 5.1 X-ray map units from the hom3 end). These alleles, probably missense mutations, may be in a region of the gene important for the structural conformation of the functional site(s) but which does not have a specific function in catalysis or feedback inhibition. Such a suggestion has been made by LANGRIDGE (1968a, b, c) from his work with temperature sensitivity of suppressed nonsense mutants of P-galactosidase. Absence of nonsense mutations: The lack of nonsense suppressible (ochre) alleles at the his1 locus is very unexpected considering the number of such alleles (1967, 1968) and found in other genes of S. cerevisiae (Table 4). ESPOSITO MORTIMER and GILMORE (1968) found that approximately 30% of auxotrophic mutations suppressible by suppressor SUP 12, used in this study. In Table 5 , TABLE 4 Comparison of nonsense suppressibility in his1 and other genes of S. cerevisiae. Except for ade3 and ade6, the suppressor involved was SUP 1 1 . Number of suppressible alleles

Total number of alleles

trp5

21

51

41

uv

his4ABC de3 ude6

13 14 9 10 8 5 14 94 0

39 33 38 43 20 27 50 301

33 42

EMS, NG

Gene

arg4

leu1 ade7 Others* Total his1

-

52

Percent suppressible

24 23

40 18 28 31.2 a

Mutagenic origin of alleles

Spontaneous Spontaneous

uv uv uv uv

JONES1964 E~POSITO 1967 MORTIMER and GILMORE 1968 MORTIMER and GILMORE 1968 MORTIMER and GILMORE 1968 MORTIMER and GILMORE 1968

W, EMS,

"his report

mo,, Spontaneous ~~

~

Reference

MANNEY1964a MORTIMER and GILMORE 1968 FINK1965

~

* Summary of several genes, excluding those listed above.

301


TABLE 5 Comparison of suppressibility by SUP 11 among noncomplementing alleles of hisl and other genes in S. cerevisiae

Gene

trp5 his4ABC his4BC his4C leu1 arg4 Total hisl

Number of nonTotal complementing number of suppressible noncomplementing alleles alleles

Percent suppressible

18

26

69

8 4 1 5

16 5 8 12 14 81 15

50 80 13 42 50 53 0

7 43 0

Reference

MANNEY 1964a MORTIMER and GILMORE 1968 FINK1965 FINK1965 FINK1965 MORTIMER and GILMORE 1968 MORTIMER and GILMORE 1968

This report

the proportion of suppressible alleles among noncomplementing alleles is shown for several loci including hisl. Except for his1 the proportion is much higher than among a random sample of mutants. DRS.G. FINK,D. HURST, and S. FOGEL have also never found nonsense suppressible alleles in their studies involving the his1 gene (personal communications, 1969). Several possibilities could explain this absence of nonsense mutations at hisl. Four possible explanations that involve technical difficulties are (1) the nystatin selection technique, which involves testing on minimal and minimal plus histidine media, might discriminate aganst suppressible mutants; (2) an ochre suppressor might have been present in the strain (S288C) in which the his1 alleles were isolated; (3) an ochre suppressor might have been present in the hisl tester strain (S395D) against which the his1 mutants were tested for allelism; (4)the his1 gene product from a suppressed nonsense mutation might be temperaturesensitive at 30°C, the growth temperature used in the tests. The first possibility is unlikely because among the histidine mutants induced by EMS and selected by nystatin, 9.8% were his1 mutants, while among the 170 mutants obtained by total isolation by DR.MORTIMER, 7.6% were his1 mutants. and GILMORE(1968), approximately one-third of a According to MORTIMER random collection of mutants should be nonsense mutants. If there were a bias against his1 nonsense alleles due to nystatin selection, one would expect the proportion of hisl mutants among all histidine mutants to be lower among those selected with nystatin than among those selected after total isolation. This was not found. The second explanation was eliminated because the allele ade2-1, a n ochre allele, was crossed into all but two of the his1 strains as one of the markers to select diploids for complementation tests and genetic mapping, and its segregation in ascus dissections was always 2+:2-. The third possibility was checked by crossing strain S395D to a strain carrying several suppressible ochre mutations (X1687-12G) and observing the segregation

302

C. T. KORCH AND R. S N O W

of the various markers in asci. The segregations were normal. Therefore, an ochre suppressor was not present in S395D. The fourth explanation was tested by picking 13 hisl strains carrying the suppressor SUP11 from the ascus dissections mentioned before, growing them up on YEPD at 30°C, then replicating them onto minimal and minimal plus histidine. These plates were incubated at 3°C intervals from 12°C to 39°C. The plates were checked after one, two, four, and seven days. None of the strains showed any growth on the medium lacking histidine at any of the temperatures used. Since 11 of the 13 strains used carried noncomplementing hisl alleles, and since this class of alleles is most likely to include suppressible alleles, we discarded the idea of a suppressed gene product which was temperature-sensitive. In yeast, the majority of nonsense mutations are ochre mutations (HAWTHORNE and MORTIMER 1968; MANNEY 1968). A fifth possibility is that perhaps at the hisl locus ochre mutations do not occur, but amber mutations do. This possibility is not rigorously excluded, but in view of the data presented in Tables 4 and 5, and that of HAWTHORNE and MORTIMER (1968) and MORTIMER and (1968), it seems most unlikely. GILMORE The most likely explanation for the absence of nonsense mutations is that a second vital function is lost simultaneously with the hisl function when a nonsense allele is induced, and hence the recovery of such mutant strains is precluded. This might occur, for instance, if the polarity effects of a nonsense mutation in his1 interfered with the translation of an adjacent gene product of a polycistronic messenger molecule. Other possibilities which might be imagined are that phosphoribosyltransferase could be part of an enzyme complex having functions addition to the catalysis of the first step in the histidine biosynthesis, a nonsense mutation making the whole complex nonfunctional. Alternatively, the polypeptide coded by the hisl gene might be used as a subunit in two different proteins, one being phosphoribosyltransferase, the other having an essential but unknown function. The possibility that some second, supplementable function unrelated to histidine biosynthesis is lost when a nonsense mutation occurs in the his1 gene was tested indirectly by considering the coincidence of double mutations among 170 histidine auxotrophs isolated by DR.R. K. MORTIMER. This group was selected on the basis of growth on complex medium (YEPD) and lack of growth on a supplemented minimal medium lacking histidine. Of the 13 his1 mutations in this group, none had an additional requirement. These hisl mutants, selected by total isolation, occurred in the same frequency as hisl mutants selected with nystatin, and indicate that there was not a systematic bias against pleiotropic mutants in the selection regimen used to acquire the his1 mutants we used. We prefer the explanation that the postulated second function is not supplementable, but related to histidine biosynthesis, for two reasons. First, from previous examples in other organisms, genes translated together as an operon and therefore subject to the polarity effects of nonsense mutations are related, as for example the genes of the histidine operon in Salmonella (AMESand HARTMAN 1963) and the genes for the second, third, and last step of histidine biosynthesis


303

in S. cereuisiae (FINK1965). Second, the functions associated with enzyme complexes which are sensitive to nonsense mutations in one of the genes coding for a function of the complex are also related, as for example the enzymes of histidine biosynthesis just mentioned (SHAFFER, RYTKAand FINK1969). There are eight known histidine loci in S. cerevisiae, his4 having three functions, thus accounting for the ten functions in the pathway. Since his1 is not linked to any other known histidine gene, we propose that the hypothetical additional function associated with his1 involves either a histidine permease or histidyl-tRNA synthetase, or perhaps both. The loss of either of these functions would be a lethal event for a haploid cell if only one copy of each gene exists. The loss of the permesse would not permit histidine auxotrophs to grow, except perhaps in the presence of extremely high concentrations of histidine. The loss of the histidyl-tRNA synthetase would also be lethal because histidine could not be added to growing protein chains. It is interesting to compare the genes which code for phosphoribosyltransferase in S. cereuisiae (hisl) and in Salmonella typhimurium (hisG) because the following differences are evident. First, in Salmonella all the histidine biosynthetic genes are linked as an operon (AMESand HARTMAN 1963) unlike yeast where almost all of the genes are unlinked (FINK1964, 1965). Second, there is no interallelic complementation among mutants of hisG (LOPER et al. 1965) ; whereas, it occurs quite frequently among mutants of h i d . Third, among his1 mutants no nonsense mutations have been found; and yet, in the hisG gene such mutations are very prevalent and, in fact, most of the mutations in this gene are of the frameshift, nonsense, and deletion types (LOPERet al. 1964). Most of the his1 mutations are of the missense type, as shown by their ability to complement interallelically (KORCH 1970, 1973). Until recently only a few missense mutations had been identified among the hisG mutations (B. AMES,personal communication, 1970). If complementation tests were to be performed now among these latter mutants, complementation might be observed. The his1 gene or gene product has been postulated above to be associated with a second function vital to yeast. The phosphoribosyltransferase from Salmonella and from yeast are similar as far as their K, for ATP, their molecular weight, their pH optimum, their partial purification by a heat step, their stabilization by L-histidine (the feedback inhibitor), their loss of sensitivity to L-histidine with aging, and their inhibition by L-histidine and its methyl ester. However, the yeast enzyme is not inhibited by thiazolealanine as is the Salmonella enzyme and the K, of the yeast enzyme for PRPP is an order of magnitude larger than that of the salmonella enzyme (KQRCH1970,1973). Thus it seems likely that Salmonella typhimurium and Saccharomyces cerevisiae differ in the regulation of the activity of both the phosphoribosyltransferase and the genes which encode this enzyme. We are very grateful to DOREENE MCCOMBS, MARJORIE INGRAHAM, CATHRYN SPROTT, and KELLYHOWARTH for their competent technical assistance, The valuable criticisms received from ROBERTK. MORTIMER, DAVIDHOAR,ROBERT GRANT,and BARBARA STEPHENSON were greatly appreciated. We thank SEYMOUR FOGEL, DONALD HURST,GERALD FINK,and ROBERT K. MORTIMER for yeast strains and for unpublished results for certain experiments.

304

C. T. KORCH AND R. SNOW LITERATURE CITED

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LOPER, J. C., M. GRABNER, R. STAHL,Z. HARTMAN and P. E. HARTMAN, 191% Genes and proteins involved in histidine biosynthesis in Salmonella. Brookhaven Symposium 17: 15-52. 1966 Mutagenesis of super-suppressors in yeast. Cold Spring MAGNI,G. E. and P. P. PUGLISI, Harbor Symp. Quant. Biol. 31 : 699-705. MALLING,H. V., 1967 The mutagenicity of the acridine mustard (ICR-170) and the structurally related compounds i n Neurospora. Mutat. Res. 4 : 265-274. MANNEY,T. R., 1964a Tryptophan synthetase mutants of yeast: Action of a super-suppressor i n relation to allelic mapping and complementation. Ph.D. thesis, University of California, Berkeley, California. -, 1964b Action of a super-suppressor i n yeast i n relation to allelic mapping and complementation. Genetics 50: 109-121. -, 1968 Evidence for chain termination by super-suppressible mutants in yeast. Genetics 60: 719-733. 19M Allelic mapping in yeast by X-ray induced mitotic MANNEY,T. R. and R. K. MORTIMER, reversion. Science 143: 581-582. MORTIMER, R. K. and R. A. GILMORE,1968 Suppressors and suppressible mutations in yeast. Adv. in Biol. and Med. Phys. 12: 319-331.


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MORTIMER, R. K., T. BRUSTAD and D. V. CORMACK, 1965 Influence of linear energy transfer and oxygen tension on the effectiveness of ionizing radiations for induction of mutations and lethality in Saccharomyces cereuisiae. Radiation Res. 26 : 465-482. PARKER, J. H. and F. SHERMAN,1969 Fine-structure mapping and mutational studies of gene controlling yeast cytochrome c. Genetics 62: 9-22. D. 0. and J. R. BECKWITH,1969 Mutagens which cause deletions in Escherichia coli, SCHWARTZ, Genetics 61: 371-376. SHAFFER,B., J. RYTKAand G. R. FINK,1969 Nonsense mutations affecting the his-4 enzyme complex of yeast. Proc. Natl. Acad. Sci. (Wash.) 63: 1198-1205. SHEPPARD,D. E., 1964 Mutants of SalmonelLa typhimurium resistant to feedback inhibition by L-histidine. Genetics 50: 611-623. SNOW,R., 1966 An enrichment method for auxotrophic yeast mutants using the antibiotic “Nystatin.” Nature 211: 206-207. SNOW,R. and C. T. KORCH,1970 Alkylation induced gene conversion in yeast: Use in f i e structure mapping. Molec. Gen. Genetics 107: 201-208. ST. PIERRE,M. L., 1968 Mutations creating a new initiation point for expression of the histidine operon in Salmonella typhimurium. J. Mol. Biol. 35: 71-82. VOLL,M. J., E. APPELLAand R. G. MARTIN,1967 Purification and composition studies of phosphoribosyl-adenosine triphosphate: pyrophosphate phosphoribosyltransferase, the first enzyme of histidine biosynthesis. J. Biol. Chem. 242 : 1760-1 767. 1956 Use of the Fricke ferrous sulfate dosimeter WEISS,J., A. 0. ALLENand H. A. SCHWARZ, for gamma ray doses in the range 4 to 40 KR. Proc. Inter. Cong. Peaceful Uses of Atomic Energy 14: 179-181. WHITEFIELD, H. J., JR., 1971 Purification and properties of the wild type and a feedbackresistant phosphoribosyladenosine triphosphate: pyrophosphate phosphoribosyltransferase, the first enzyme of histidine biosynthesis in Salmonella typhimurium. J. Biol. Chem. 246: 899908. WHITEFIELD,H. J., R. G. MARTINand B. N. AMES, 1966 Classification of aminotransferase (C gene) mutants in the histidine operon. J. Mol. Biol. 21 :335-355.