tems. HAWTHORNE

GENE CONVERSION OF DELETIONS IN THE HIS4 REGION OF YEAST'

G. R. FINK AND c. A. STYLES

Genetics, Development and Physiology, Cornell University, Ithaca, New York 14850

Manuscript received January 11, 1974

ABSTRACT

A selection procedure has been devised which allows the recovery of deletions of the his4 region of Saccharomyces cerevisiae. Many deletions were obtained by this procedure and were characterized genetically and biochem- ically. These deletions permit the construction of a linear map of the his4A and his4B region. A deletion which was large on this genetic map reduced the size of the his4 gene product. Tetrad analyses revealed that deletions can affect the conversion frequency of other alleles at his4. Analysis of reciprocal recombination in conversion tetrads showed that the order of genes on chromosome I11 is: centromere-EeuZ-his4A-his4C. This means that in his4 transcription and translation proceed away from the centromere.

IABLE haploid strains carrying deletions are rare in yeast. Among 550 mutations induced at the hid locus by EMS, ICR-170, ultraviolet light (u.v.) ,

nitrous acid, and N-methyl-N'-nitro-N-nitrosoguanidine, none was a deletion (FINK, unpublished). Even in systems where large numbers of spontaneous mutations have been selected (ROMAN 1956; JONES 1972) deletions are rare. There have been several reports of deletion mutants obtained by special selection systems. HAWTHORNE (1963) reported a deletion of the mating type locus which was lethal in the haploid. Various selective procedures have produced viable deletions of the cycl gene ( SHERMAN and STEWART 1973).

In this paper we report a positive selection procedure for viable haploid strains carrying deletions in the his4 region in yeast. Many of the deletions appear to involve the loss of several hundred nucleotides. Analysis of tetrads from crosses in which strains carrying these deletions were mated to wild type shows that even large deletions undergo gene conversion. The deletion can be converted to wild type and the wild type can be converted to the deletion. In this respect deletions appear to be no different from single nucleotide changes. A deletion wholly within the his4 region does not affect the conversion frequency of a distal site in the region, whereas a deletion through the proximal end of his4 ( SHAFFER, EDEL- STEIN and FINK 1972) lowers the conversion frequency of the distal site. The use of deletions has enormous advantages for fine-structure studies because deletion mapping is more rapid and more accurate than the other generally available methods. In addition deletions should provide an invaluable tool in yeast for the elucidation of regulatory elements adjacent to structural genes.

Supported by NIH grant, No. GM 15.108-07.

Genebcs i'i: 231-244. June, 1974.

232 G . R. F I N K A N D C. A. STYLES

MATERIALS A N D METHODS

Yeast strains: The strains used in this study are derived from (1: S288C, which was obtained from h. R. K. MORTIMER. As described in a previous publication (FINK 1966), histidine- requiring mutants of yeast are unable to grow on histidinol in place of histidine. Mutations per- mitting growth on histidinol have been obtained in a gene called HOLI. Double mutants hisHOLI can grow on histidinol if they have a functional his4C region. All eight HOLI mutations in our collection are dominant in diploids, segregate 2:2 in crosses by wild type, and are unlinked to any of the known histidine genes.

Genetic analysis: All media, procedures for sporulation, tetrad dissection, and scoring of genetic markers have been described by HAWTHORNE and MORTIMER (1960).

Hislidinol dehydrogenase assay: A sensitive assay for histidinol dehydrogenase has been developed with the use of 3H-histidinol. Radioactive histidinol was converted to histidine by histidinol dehydrogenase, the two imidazoles were separated, and the amount of radioactive histidine was used as the measure of enzyme activity. 2.5 nanomoles of 3H-histidinol (75 mCi/ mmole), 3 pmoles of Tris-HC1 pH 8.9 and 70 pg of protein from a crude yeast homogenate were placed in a total volume of 50 pl. The reaction was allowed to proceed at 30" for 30 mintes and was terminated by addition of 4 81 of chromatography solvent. 20 pl of the reaction mixture was spcltted on Whatman 1 paper. The paper sheet was then placed in a solvent system containing isopropanol: ammonia:H,O (70:2O:IO, vo1:vol:vol). Histidine and histidinol were located using non-radioactive standards as described earlier (FINK 1965). The histidine spots were eluted and the eluate was counted in a Nuclear Chicago scintillation counter. The amount of histidine produced by the reaction is proportional to time and enzyme concentration.

Nonsense mutations of his4: Nonsense mutations were identified by analysis of suppression patterns. Two procedures were used to determine whether a strain carried a nonsense mutation in the his4 region. Both tests showed that only polar his4A and B mutations are nonsense. In the first test, strains carrying the his4 allele in question were crossed to strains carrying known nonsense suppressors. The diploids were isolated, sporulated, and the segregation of the suppressor and the his4 allele determined by tetrad analysis. The appearance of 3: 1 or 4: 0 segregation (h i s+:h i s ) was taken as preliminary evidence for suppression. The putative double mutant, SUP his4, was backcrossed to wild type and shown to segregate both the suppressor and the his4 allele. In these studies SUP3, SUP4-I, SUP5 and SUPII , known ochre suppressors, and SUP4-3 and SUP7-2, known amber suppressors, were used (SHERMAN et al. 1973; MESSENGUY and FINK 1973). In the second test, multiply-marked strains were constructed containing the histidine mutation in question together with known ochre (Zeu2-I. arg4-17, l y s l - I ) and amber (metd-I, trpl-I , tyr7-I) mutations. These strains were reverted for one of the requirements and then tested for the presence of the others. Simultaneous reversion of the histidine allele together with the previously identified nonsense alleles was taken as evidence that the his4 allele was a nonsense mutation of the same type as the co-reverting allele (MORTIMER and GILMORE 1968).

For many his4 mutations the assignment to either the amber or ochre class could be made unambiguously. Some his4 mutations gave less clear-cut results. When strains carrying metd-I, leu2-I, and either his4-619 or his4-I20 were reverted for their histidine requirement, a few of the revertants grew extremely slowly without leucine and without methionine. We have been unable to confirm the existence of a suppressor in these strains because of spore inviability.

Leu2-I seems to be suppressed by a greater variety of nonsense suppressors than other ochre alleles. Strains which are leu2-I his4-I7 arg4-17 lysl-I or leu2-I his4-I66 arg4-17 lys l - I , when reverted for leucine, co-revert at high frequency for histidine or for arginine and lysine. HOW- ever, simultaneous reversion of leucine, histidine, and lysine or leucine, histidine, and arginine, or leucine. histidine, arginine and lysine are extremely rare.

Selection of deletions: The strategy employed in the selection of deletions depended upon several unique featuies of the his4 region. The A, B, and C segments control three different enzyme activities (Figure 1). These segments have some degree of functional autonomy since missense mutations in one of the segments (in A, for example) do not effect the enzymes spe- cified by the other two. The his4 region is transcribed and translated from A to C (Figure 1).

DELETION MUTANTS IN YEAST 233

A I B I C

PRAMP - BBMIl PRATP PRAMP HlSTlDlNOL - HISTIDINE

3 2 IO

FIGURE 1.-The reactions controlled by the his4 region. The A,B,C designations refer to segments of his4 responsible for catalyzing the 3rd, 2nd and loth steps (respectively) in the path- way of histidine biosynthesis. The basis for the selection (see text) is that polar his4A mutations prevent growth on histidinol. PRATP = phosphoribosyl-ATP; PRAMP = Phosphoribosyl- AMP; BBMII = N- (5’-phospho-D-ribulosylformimino) -5-amino-1- (5” phosphoribosyl) -4-imidi- zolecarboxamide.

By virtue of the polarity of translation and transcription from 5‘ + 3’, a nonsense mutation in his4A has no his4B or his4C activity. Whereas mutants carrying a missense mutation in his4A or his4B can grow on histidinol, a strain carrying a polar his4A o r his4B mutation will not grow at all on histidinol. Deletions were obtained by selecting revertants of polar his4A mutants on histidinol. In theory, an in-frame deletion of the polar mutation could abolish polarity. Such deletions should allow growth on the histidinol plates so long as they do not extend into his4C. The efficiency of the selection system is low and presumptive deletions must be tested further for ability to recombine and revert. Growth on histidinol is often the result of nonsense suppression of the polar mutation or reversion of the polar to wild type or to a his4A missense mutation. In a typical experiment approximately 107 cells of a strain containing a polar his4 mutation were spread on the surface of a minimal f histidinol plate. The colonies were irradiated with U.V. light to give a survival of 20% and stored in the dark at 30” until colonies appeared. The plates were replica-plated to a minimal plate to identify revertants which still required histidine. Colonies which grew on minimal + histidinol but not minimal were picked, purified by streaking, and retested by replica plating.

Recombination testing: In deletion mapping and allele identification in the gene conversion study, we have relied on mitotic reversion to prototrophy as a measure of the ability of two alleles to recombine. To increase the frequency of reversion, a histidine-requiring diploid was exposed to a dose of U.V. which gave 90% survival. Two sites were considered separable when the heteroallelic combination gave a higher frequency of prototrophs than either homoallelic combination. We have examined a few of the prototrophs produced by this method from a heteroallelic diploid and they all have arisen by gene conversion.

Testing of presumptive deletions: We have used two criteria to characterize the histidine- requiring strains which grew on histidinol-reversion and recombination. A strain which fails to revert and fails to recombine with two or more mutants which recombine with each other is called a deletion. There are, of course, other aberrations which could satisfy these criteria.

The initial screen for deletions was a test of reversion ability. Approximately 2 x I O T cells from a culture of the presumptive deletion were spread on the surface of a minimal petri plate and irradiated with U.V. light at a dose which gave 20% survival. If a mutant failed to revert in this test it was retested for reversion by plating and irradiating 5 x 107 cells on each of 10 plates. If no revertants appeared, then the strain was tested for its ability to recombine with various point mutations within the his4 region. To test recombination, the deletion strain was crossed by various point mutations. The diploids were selected by micromanipulation or (where the parents carried complementary auxotrophic markers) by selection on the appropriate medium. For each cross several independent diploid clones were tested for their ability to give rise to prototrophs on minimal medium after U.V. irradiation. The “spot test” procedure described by FINK (1966; see Figure 5) was used. After three days’ incubation a t 30” the plates could be scored for the purpose of prototrophic colonies. In this test we scored for the ability of point mutations to give prototrophic mitotic recombinants with the deletion. The homozygous parental strains, tested identically, provided important controls for reversion. For most mutant combina-

234 G . R. FINK A N D C . A. STYLES

DIPLOIDS USED IN CROSSES

A B C 290 0

39 ., -

290 0 Y

15 - 290 0 - ”

29

FIGURE %-Diploids used in gene conversion studies. The heterozygotes shown here contain two mutations in the his4 region. In addition each diploid was heterozygous for Zeu2-I and ade2-I.

tions this “spot test” gave an unambiguous answer: there was either recombination or no recombination. When a site is very close to the end point of the deletion or when a site has a high frequency of back mutation, the spot test is inconclusive. In such cases the heterozygote in question and the parental homozygote controls were plated on the surface of several minimal medium petri plates. If the ratio of colonies:

irradiated heterozygote-unirradiated heterozygote

irradiated homozygote -unirradiated homozygote 2x

was considerably greater than 1, then the point mutation was considered to be outside of the deleted region.

Gene conversion studies: Tetrads were dissected and the resulting spore clones analyzed for their auxotrophic requirements. The crosses which were analyzed are shown in Figure 2. Each diploid was heterozygous for two his4 mutations as well as ade2-1, leu2-2, and mating type. Leu2-I is approximately 15 map units from his4 and five map units from the centromere. Asci which showed aberrant segregation both for his4 and for one of the outside markers (< 0.2% of all tetrads tested) were considered false tetrads and were not included in the tabulations. The specific his4 allele present in each spore from every tetrad was identified by analysis of that spore’s ability to recombine with each of the parental strains. Double mutant strains which failed to recombine with either parental strain were shown to be able to recombine with other strains carrying different his4 alleles. Prototrophic convertants were tested for possible aneuploidy (1) by streaking out the strain and looking for vegetative segregation and (2) by crossing the strain to wild type and examining meiotic progeny of this cross for the appearance of his- segre- gants. All prototrophic convertants tested in these ways proved to be haploid and not aneuploid.

Molecular weight studies: Extracts were prepared by breaking cells in a Braun Homogenizer and centrifuging the resulting homogenate a t 30000 x g in a Sorval RCPB centrifuge. The extracts were then clarified by centrifugation at 100,000 x g in a Spinco Ultracentrifuge. 100 a1 of the supernatant solution was layered cn a 55201% sucrose gradient, centrifuged in a Spinco L265B for 16 hours a t 46000 rpm and analyzed according to MARTIN and AMES (1961). Fractions were collected and histidinol dehydrogenase was assayed as described earlier ( SHAFFER, RYTKA and FINK 1969).

DELETION M U T A N T S IN YEAST 235

RESULTS

Characterization of the deletions: The selection for revertants of polar his4 mutants able to grow on histidinol produced 22 strains which could be classified as deletions (Table 1). Of these, 18 were crossed by each other and by all known sites in his4 and his4B. The diploids resulting from these crosses were examined for their ability to produce mitotic recombinants. The recombination responses are consistent with the representation of each of the deletions as the loss of a single continuous stretch of the his4 region (Figure 3 ) . His4A and B are sub- divided into 13 segments based on these crosses. A mutant can be assigned unambiguously to one of these segments by “spot testing” as described in MATERIALS

AND METHODS. The region deleted always includes the original polar mutation and never includes his4C sites. In fact, as would be expected from their ability to grow on histidinol, all deletions complement his412 mutants. The complementation responses of deletions with individual his4A and his4B mutants are complex and do not form any obvious pattern. One noteworthy feature of these inter- actions, shown in Figure 4, is the complementation of several of the deletions with nonsense mutations in h i d A (e.g., hid-26 with -39, -644, -86).

In the spot test, most diploid combinations of deletion x point mutation gave many colonies where there was recombination and only the low background reversion of the point mutation where there was none. Point mutations very close to the end point of a deletion presented some difficulties. In these cases the spot test lacked the resolution to distinguish between a low frequency of recombination and reversion. In these instances strains were tested for recombination by the whole plate test described in MATERIALS AND METHODS. A diploid homozygous for the point mutation and one heterozygous for the point mutation and a deletion known to cover that point mutation served as controls. The diploid his4-38/ hid-15, for example, did not give a clear positive response in the spot test. As can be seen in Table 2, the whole plate test clearly indicates recombination between his#-38 and his4-15, but not between his438 and his#-26. The data shown in Table 2 point out that a site a short distance from the end point of hid-15 gives several orders of magnitude more recombinants than does hid-38.

A number of strains which grew on histidinol were not deletions. His4-39 yielded several strains which appear to be his4A missense mutations. One of these, his4-39R61, appears to map at or near the same site as hid-39. Two others,

TABLE 1

Origin of deletion strains

Polar mutation Deletion derivatives

his4-39 his4-15, -24, -27 his4-260 his4-580 his4-2, -8, -13

his4-26, -28, -29, -30, -32

his4-619 hi~4-3, -9, -31, -33, -34, -41, -42, -44, -49, -56, -60

The deletions on the right were derived from the polar histidine mutations 011 the left by mutagenesis with U.V.

236 G . R. FINK A N D C. A. STYLES

c 0 a .- 2

m

IC-

@-?- _ - -

(+ fR-

1 0 m

8

DELETION MUTANTS IN YEAST 23 7

A I B C I @$ e, @, m, 120, 6 260, 38. 166. I2

. . . . . . . . . . . . . . . . . . . . . . . .: ... l...., i ... ::.: .....................

470. @, @, @ @ ...............

eso .. ...... s,:;,:::. I17

~...........t.~ ........ '....3 IO6

207

IS I FIGURE 4.--Complementation responses of his4 mutants with three of the his4 deletions. The

mutants which fail to complement are represented as overlapping solid lines. All circled numbers represent nonsense mutations. Those with stripes are amber and those without are ochre. The heavy solid lines are the complementation responses of deletions. The complementation responses of the other deletions are too complex to be presented in a linear representation.

his4-39R19 and his4-39R25, peculiarly do not seem to map at the hid-39 site. These discrepancies are under investigation. In addition, several revertants of his4-39 had a normal his4 region but had new mutations in other histidine genes. For example, his4-39R19 had a his5 mutation. We suspect these strains originate by two mutational events; the first is a reversion of his4-39 + HIS4 and the second is from HIS5 -+ his5.

The effects of deletions of the his4 region on the physical properties of the his4 gene product were examined by enzyme assays and centrifugation studies. The his4C activity (histidinol dehydrogenase) was assayed in a number of the deletion strains. As can be seen in Table 3, the activity of this enzyme in many of the deletion strains was reduced as compared to the activity in wild type. His4-l5 has a nearly normal level of the his4C activity, but this activity is considerably less stable than that of wild type. The reduced activity of histidinol dehydro-

FIGURE 3.-Deletion map of the his4 region. The heavy solid line represents the yeast chromosome in the his4 region. Each number on that line stands for an independently isolated mutation. The letters designate the different functional segments within h i d . Sets of mutations that fail to recombine with one another are grouped vertically. All circled numbers represent nonsense mutations. Those with stripes are amber (UAG) and those without are ochre (UAA) . Mutations below the heavy horizontal line are deletions. The position of the mutational sites above the line were determined by deletion mapping. Distances between the sites are arbitrary.

238 G . R. F I N K A N D C. A. STYLES

TABLE 2

Recombination frequencies* in t h whole plate test ( x 10-8)

Diploid Control Irradiated Diploid Control Irradiated

26/26 15/15 29/29 26/15 26/29 15/29 38/38 38/15 38/26

< i o - 2

< 1 w < 10-2 < 10-2 < 10-2 < 10-2 < I

1 <I

< 10-2 < 10-2 < 10-2 < 10-2 < IG-2 < 10-2

4.4 70.0 3.5

38/29 29/260 15/260

260/260 26/260 26/12 15/12

260/12 29/12

3.7 4.0

1780.0 6.4 4.0

1 5714

1

1 424.08

* All numbers in the table represent the average number of colonies, per 108 diploid cells plated on minimal medium. A culture of the diploid to be tested was grown on complete medium, washed twice, plated and

irradiated as described in MATERIALS AND METHODS. Revertants have never been observed in diploids homozygous for a deletion o r in diploids heterozygous for overlapping deletions.

genase in the deletion strains is probably a reflection of the association of the gene products of the his4 region in a multi-enzyme complex (SHAFFER, RYTKA and FINK 1969). Mutations which delete DNA from his4A and his4B are likely to affect the activity of his4C by disrupting the normal association of the his4 complex. Evidence for the physical deletion of material from his4 comes from an analysis of the sedimentation behavior of the histidinol dehydrogenase activity in the large deletion his4-29. As can be seen in Figure 5, the his4C activity of the deletion strain migrates more slowly when subjected to ultracentrifugation in a sucrose gradient than does the normal complex, a behavior suggesting a reduced molecular weight for the mutant enzyme.

TABLE 3

Enzyme levels in deletions

Strain

h i s 6 8 13 15 2.4 26 27 29 30 31 32

S288C

h+4c specific activity X 109

2.1 7.0

46.0 0.7 2.4

17.9 7.0 3.9 8.5 2.6

55.0

-

Histidind dehydrogenase in wild-type and mutant strains was determined by the assay described in MATERIALS AND METHODS. Specific activity is expressed as p moles of histidine/mg proteinfir. The strains were grown in minimal medium with histidinol.

DELETION MUTANTS IN YEAST 239

>- .5

2 a w *4

a

+ 0

m z W

g .3 a n

n

z Q 0.10

e

> I W

J 0

l-

I

0.05

40 30 20 IO FRACTION

FIGURE 5 .-Sedimentation properties of the his42 activity (histidinol dehydrogenase) in a 5-20% sucrose gradient. The solid line represents the activity measurement for his42 from each fraction from the gradient. E. coli alkaline phosphatase was used as a standard. The arrow points to the fraction of maximum phosphatase activity.

Gene conversion: All 23 deletion strains were crossed to wild type and shown to segregate 2 deletion spores : 2 wild-type spores in the majority of tetrads. Al- though the number of tetrads was small (8-10) in the case of any one deletion, the results indicate that his4 deletions, like his4 point mutations, are linked to leu2 on chromosome 111. Meiotic segregants of mating type opposite to that of the original isolate were mapped by the spot test procedure. In no case was there a difference in the map position or complementation response between the original isolate and the ascospore derivative of opposite mating type. The analysis of large numbers of meiotic progeny (> 4000 his- spores) resulting from crosses of deletion strains by wild type showed that only the original deletion could be recovered. When the double mutants his4-588 his#-594 or his#-580 his#-260 were crossed by wild type, meiotic segregants containing a single mutation (i.e., his4 588) could easily be recovered. These results are not consistent with the hypothesis that the deletions represent multiple point mutations.

In order to examine the gene conversation of deletions, several diploids (Figure 2 ) were sporulated and the resulting tetrads analyzed for the segregation of the

240 G. R. FINK A N D C . A. STYLES

TABLE 4

Meiotic events at the his4 region

Co-conversion Conversions his-leu

No. of - ++c c*+ - Diploid tetrads ++C C + + ++A A++ A++ +-+A R P N T

his4-39/290 44M 11 3 10 16 2 5 2 242 4 97 15/290 401 8 7 2 1 1 3 4 0 244 3 91 29/290 433 4 2 5 3 0 1 1 296 3 108

The frequency of gene conversion for the diploids shown in Figure 2. R stands for the number of asci showing reciprocal recombination.

genetic markers. The results are shown in Tables 4 and 5. The deletions tested (hid-15, -29) can be converted to wild type and the wild-type strand can be converted to the deletion.

Crosses were designed to provide information on the influence of deletions on the conversion frequency of another his4 allele. The conversion of his4-290, the most distal his4C site, was examined in crosses with a number of his4 deletions and a his4 point mutation. The results indicate that hid-29 diminishes the frequency o€ gene conversion of his4-290, whereas his425 does not. Although the proximal end point of his4-29 has not been defined, it does not affect the recombination of his4 with leu2.

Even though most of the events within his4 are gene conversions, the data permit an ordering of the his4 region with respect to leu2 and the centromere. As shown in Figure 6, a cross of leu2 A + X + + C can be considered a three-point cross. Since one-half of all conversion events are associated with reciprocal recombination of outside markers (HURST, FOGEL and MORTIMER 1972), the distribution of recombinants among the different convertant classes indicates whether the order is leu2 his4A his@ or leu2 his4C his4A. The data were analyzed as shown in Figure 6. The results in Table 6 clearly indicate that the order is

TABLE 5

Conversion frequencies for his4 mutations

Percent conversions Diploid A C

29/290 2.0 (a 1.6 ($) The his4A alleles are h.is4-39, his4-15 and his4-29, respectively; the his4C allele is his4-290 in

all three mosses. The conversion of his4-290 in cross 3 is significantly lower than in crosses 1 and 2. Using the method of BRANDT and SNFDECOR (SNEDECOR 1940) the probability (P) is less than .01, indicating significant heterogeneity.

DELETION M U T A N T S IN YEAST

CONVERSION WITH ASS 0 C I AT E D R EC I PRO CA L R EGO M 8 I NATION

24 1

leu 2 + A

leu2 + leu2 +

U+ c +

leu2 A + n

+ + A c + t C

FIGURE 6.-Tetrad types resulting from conversion with associated reciprocal recombination. On the left, the order: centromere-Zeu2-his4C-his4A is shown and on the right the order: centromere-Zeu2-his4A-his4C. If the order on the left were correct, then conversion of C but not A would be associated with reciprocal recombination between leu2 and his4A. If the order on the right were correct then conversion of A but not C would be associated with reciprocal recombination between leu2 and his4C. The data shown in Table 5 support the order: centromere-leu2- hislA-his4C.

cerztromere-Zeu2-his4A-his4C. The distribution of markers in the three reciprocal recombinants from these crosses and in ten others from several other crosses not shown here indicate the identical order.

DISCUSSION

The genetic map of the his4 region obtained by deletion mapping agrees in its overall features with the map obtained by the X-ray mapping procedure (FINK

TABLE 6

Recombination associated with gene conversion

Type of conversion A-* + + - , A c-, + + - + c

Diploid Fraction showing reciprocal recombination ..

his4-39/290 13/16 4/10 0/3 1/11 15/290 6/11 w2 1 /6 1 /8 29/290 1 /4 3/4 w2 1/4

TOTAL 20/3 1 7/16 1/11 3/23

Orientation of the his4 region with respect to the centromere. The conversion tetrads described in Table 3 were analyzed to determine the proportion of those tetrads in which the conversion chromatids also show reciprocal recombination between a his4 site and Zeu2.

242 G. R. F I N K A N D C. A. STYLES

1966). The order of regions within his4 by both procedures is his4A-B-C. More- over, both mapping techniques indicate the order 260 (644, 39, 4, 588), 806. Where the sites are far apart, the X-ray map seems to correspond to the deletion map. For sites which are close together the deletion map is assumed to reflect the correct order. Based on this assumption mutants less than 1.5 map units apart ( 1 map unit = 1 prototroph/106 survivors/200r) on our X-ray map can not be reliably ordered. Thus, the order 260-4-644-39 and 594-71 6-806 determined by X-ray mapping are superseded by the orders shown in Figure 3.

Many features of the conversions of deletions are similar to those described for point mutations ( FOGEL and MORTIMER 1969). Conversions of the type “deletion + +” occur as frequently as the type “+ + deletion”. The conversion of deletion his4-29 to wild type provides direct evidence that gene conversion can involve the replacement of a segment several hundred nucleotides long. Gene conversions represent a large proportion of the meiotic events within his4. Only 3 of the 1238 asci examined were reciprocally recombined for the input his4 alleles, whereas 97 involved conversion of either or both alleles (Table 3). Con- versions of deletions, like conversions of point mutations, lead to reciprocal recombination for the flanking markers approximately 50% of the time. As can be seen in Table 5, 27/47 asci which had a conversion of A were reciprocally recombined for his4C and leu2. In addition the fidelity of gene conversion is main- tained-deletions are faithfully converted and new mutations are not generated by the process.

The existence of deletions of his4A and B which retain his4C function rein- forces the conclusions from earlier studies that these sub-regions have functional autonomy. Analysis of strains carrying nonsense mutations in his4C showed that they contained his4A and his4B activities with a reduced molecular weight (SHAFFER, RYTKA and FINK 1969). In other words, the product (s) of the A and B regions were functional in the absence of the product of the his4C region. Many of the deletions have removed most of his4A and two, his4-24 and his4-29, in addition, have removed part or all of his4B. Despite the fact that the his4C activity is altered in these deletions strains, all deletion strains have measurable his4C function. The lowering of activity by deletions is most likely a result of the association of the three his4 gene products in an enzyme complex (SHAFFER, EDELSTEIN and FINK 1972). In view of this association, it is surprising that the residual portions of the complex remain active in nonsense and deletion mutants.

The frequency of conversion of a missense allele within his4 can be lowered by the presence of a deletion in the his4 region of the homologous chromosome. There are several possible interpretations of the depression of conversion by deletions. One hypothesis is that near the origin of transcription are specific recognition sites for the initiation of conversion. These sites can be envisioned as struc- tures in the DNA resulting from intrastrand pairing of complementary DNA sequences or from palindromic sequences (that is, from tandem or from reverse repeats of complementary sequences). The actual structure recognized by the recombination enzymes might be a complex formed by the juxtaposition of such recognition sites from each homolog. A deletion which altered the recognition

DELETION MUTANTS I N YEAST 243

site on one homolog would alter the complex, lowering the affinity of this DNA for the recombination enzymes, and consequently would be expected to lower the conversion frequency of sites on both strands. In our study, deletion his4-15, which lies wholly within the his4 region, did not significantly affect the conversion of the his412 missense allele hid-290, whereas deletion hid-29 which ex- tends into an unmapped region in or adjacent to his4A did. The conversion frequency of deletion hid-29 is low compared to that of deletion his4-15 and that of point mutation his4-39. These results could be explained if the recognition sites for initiation of conversion are located at the his4A end of the his4 region, where transcription and translation begin (SHAFFER, EDELSTEIN and FINK 1972). The deletion of recognition sites would lower the conversion frequency of the deletion strand as well as the his412 missense strand. Another explanation of our results is that exact pairing between homologs, a prerequisite for normal recombination and gene conversion, is not possible in deletion heterozygotes. De- letions would lead to altered pairing and would reduce recombination. The larger the deletion the greater the reduction in pairing and conversion.

Unfortunately, information is insufficient to decide whether large deletions depress conversion because of imperfect pairing per se or because of the absence of recognition sites for recombination enzymes. Deletions at both ends of his$, some entering through A and some through C, could provide a direct test of these models. If specific recognition sites do exist and are involved in the depression of conversion by deletions, then only deletions through A end of his4 would reduce gene conversion. Our selective procedure does not permit the isolation of deletions of the C region. If aberrant pairing is responsible for the depression of conversion in strains heterozygous for a large deletion, then strains homozygous for the deletion might show normal or even elevated conversion frequencies. Pre- liminary studies on a strain homozygous for deletion his429 and heterozygous for his4-290 (29/29-290) indicate that the conversion frequency of the distal allele, hid-290, returns to normal, a result consistent with the hypothesis that aberrant pairing results in altered conversion frequencies. Further work on new deletions and the genetic structure of the ends of the his4 region will be necessary to define the factors controlling gene conversion.

LITERATURE CITED

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FOGEL, S. and R. K. MORTIMER, 1969 Informational transfer in meiotic gene conversion. Proc. Natl. Acad. Sci. U.S. 69: 96-103.

HAWTHORNE, D. C., 1963 A deletion in yeast and its bearing on the structure of the mating type locus. Genetics 48: 1727-1729.

HAWTHORNE, D. C. and R. K. MORTIMER, 1960 Chromosome mapping in Saccharomyces: centromere-linked genes. Genetics 45 : 1085-11 I O .

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JONES, E. W., 1972 Fine structure analysis of the ade3 locus in Saccharomyces cereuisiae. Genetics 70: 233-250.

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MESSENGW, F. R. and G. R. FINK, 1973 Temperature-sensitive nonsense suppressors in yeast. Genetics 75 : 459464.

MORTIMER, R. K. and R. A. GILMORE, 1968 Suppressors and suppressible mutations in yeast. Adv. Biol. Med. Physics 12: 319-331.

ROMAN, H., 1956 A system selective for mutations affecting the synthesis of adenine in yeast. C. R. Lab Carlsberg, Ser. Physiol. 26: 299-314.

SHAFFER, B., J. RYTKA and G. R. FINK, 1969 Nonsense mutations affecting the his4 complex of yeast. Proc. Natl. Acad. Sci. U.S. 63: 1198-1205.

SHAFFER, B., S. ELIELSTEIN and G. R. FINK, 1972 His4 a gene complex of Saccharomyces cereuisiae. Brookhaven Symp. Biol. 23 : 250-270.

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p. 207. In: Statistical Methods. Iowa State College Press, Ames.

Corresponding editor: D. R. STADLW

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