GENE DUPLICATION IN SACCHAROMYCES CEREVZSZAEl

P. E. HANSCHE AND V. BERES

Department of Genetics, University of California, Davis, California 95616

P. LANGE

Lehrstuhl fur Genetik, Institut fur Biologie, Universitat Tiibingen, 7400 Tiibingen, West Germany

Manuscript received July 12, 1977 Revised copy received December 5, 1977

ABSTRACT

Five independent duplications of the acid-phosphatase (aphtase) structural gene (acpl) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the dupli- cations arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acpl, and all known genes distal to acpl on the right arm of chromosome 11, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome IZ was found to be trans- mitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome I1 were found to be unstable, being lost a t a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found.

G E N E duplication has been postulated to be an absolute prerequisite to evolu- tion of the functional proteins that compose the metabolic machinery of living

organisms (e.g., HOROWITZ 1965; KOCH 1972; JENSEN 1976). Gene duplication is also presumed by some to have played a central role in evolution of the mech- anisms that regulate that machinery (BRITTEN and DAVIDSON 1971 ; DAVIDSON and BRITTEN 1973). In view of the central role postulated for gene duplication in evolution, surprisingly little experimental evidence bears directly on the mechanism (s) through which duplicate genes arise. Even less experimental evi- dence bears on the role of those mechanisms in the evolution process.

It is inviting to consider as pertinent evidence the recently published studies of the mechanism of gene duplication in prokaryotes. Those studies indicate that bacterial genes are duplicated by a process involving recombination (HILL and COMBRIATO 1973; RIGBY, BURLEIGH and HARTLEY 1974; ANDERSON, MILLER and ROTH 1976), and that spontaneous duplications in bacteria arise almost as frequently as spontaneous mutations (HILL and COMBRIATO 1973; JACKSON and

' This work was supported in part by National Science Foundation Grant No. BMS 75-10108.

Genetics 88: 673-687 April, 1978.

6 74 P. E. HANSCHE, V. BERES A N D P. LANGE

YANOFSKY 1973; ANDERSON, MILLER and ROTH 1976). At the same time, dupli- cated segments of the bacterial chromosome apparently persist in the bacterial genome only temporarily, being excised at a frequency of a few percent per chromosomal replication (JACKSON and YANOFSKY 1973; ANDERSON, MILLER and ROTH 1976). That fact, plus the paucity of repetitive DNA sequences in the bacterial chromosome, casts doubt on the evolutionary significance of the mech- anism that generates this type of gene duplication in bacteria.

The classical study bearing on the mechanism of gene duplication in eukaryotes concerns Bar eye in Drosophila (BRIDGES 1936). The mechanism gen- erating tandem duplications in that case has been demonstrated to be “unequal crossing over.” Unequal crossing over has been postulated also as the mechanism that generated the tandem duplicate ancestral genes that code for the (Y and 6 hemoglobin polypeptides of man (ZUCKERKANDL and PAULING 1965). The tan- dem, multiply duplicated genes of the human immunoglobin system are also presumed to have arisen via unequal crossing over (HOOD and TALMADGE 1970). The most conspicuous example of tandem duplications that presumably arise from unequal crossing over is the highly and moderately repetitive DNA that composes 20 to 50% of eukaryotic genomes. All the same, unequal crossing over cannot account for all of the duplicate genes presumed to have arisen in eukaryotic populations. Most documented duplication events appear to have re- sulted from gene transposition (translocations or inversions), followed by chro- mosomal or chromatid assortment. For example, GOPINATH and BURNHAM (1956) documented several gene duplications arising via that mechanism in experimental populations of corn. NEWMEYER and TAYLOR (1967), TURNER et al. (1969), PERKINS (1974) and PERKINS and BARRY (1977) have also docu- mented several duplications in Neurospora that were generated by gene trans- position. Similar cases have been documented in Aspergillus (KAFER 1975; PARAG and ROPER 1975). GOTTLIEB (1974, 1977) has postulated that this same mechanism has been responsible for the independently segregating, duplicate phosphoglucoisomerases in various species of Clarkia. He suggests that trans- position may be the primary mechanism through which structural genes are duplicated in some higher plant species.

The purpose of this study was to establish the mechanism(s) of gene dupli- cation in asexually reproducing populations of Saccharomyces cereuisiae.

MATERIALS A N D METHODS

FRANCIS and HANSCHE (1972) and HANSCHE (1975) described the general conditions of the selection experiments, and the methods for determining aphtase (acid phosphatase) activity, pH optimum, and K,. In the present experiments, the PO, source was 15 pg PGP (P-glycero- phosphate) per ml. The medium was buffered at pH 6.8. The specific reproductive rate of the chemostat population was about 0.05 h r l . In four experiments, chemostat populations were exposed to chronic UV light of sufficient intensity to induce 10% cell death per generation.

Detection of acid phosphatase by staining: strains grown on solid YEP were replica plated onto basal plates (Difco yeast nitrogen base without amino acids, with 0.1 g KNOJl substituted for KH,PO,, adjusted to pH 4 with HCI and 2% agar). Amino acids were added when required.

GENE DUPLICATION IN S. cerevisiae 6 75

After 3-2 days, the basal plates were overlaid with melted top agar (50") containing 0.84% agar adjusted to pH 4 with HC1 and a staining solution consisting d 0.6 mg of a-naphthyl- phosphate per ml and 1.4 mg of Fast Garnet per ml. The staining solution was added after the top agar cooled to 50". Strains with aphtase activity stained red within 10-30 min. Strains lack- ing aphtase activity remain white. Staining does not effect cell viability.

Nomenclature: In lieu of a published nomenclature for translocations or duplications in yeast, we follow the nomenclature suggested by PERKINS (personal communication) which is consistent with that used for translocations and duplications in Neurospora. Thus, we symbolize the karyotype of haploid strains carrying the translocated duplicate region of the right arm of chromosome I1 (that includes the acpl locus) reported by HANSCHE (1975) as Dp(IlR+U)I. The symbol ZIR+U indicates that a region of the right arm of chromosome I1 has been trans- located to an unknown chromosome, U . The 1 distinguishes this particular translocated dupli- cation of a portion of the right arm of chromosome ZZ from four others reported below. The symbolization of genotypes carrying such translocated, duplicate chromosome regions distin- guishes the chromosomal locations of alleles of duplicated genes. For example, (I1 acpl-1 I U ACPI-2) identifies the genotype of a Dp(IlR+U)l strain having the recessive allele, acpl-I, of the aphtase structural gene located on chromosome 11, and a dominant allele, ACPI-2, of the aphtase structural gene located in the duplicate region of the right arm of chromosome I1 that has been translocated to an unknown chromosome, U .

Sampling: Cells were collected from the chemostat at weekly intervals and grown on solid minimal medium with 15 mg pGP/ml for PO, source. The five largest cell colonies were isolated and assayed for aphtase activity. Isolates of the highest activity were characterized.

Principal strains: Ela, a acpl-1; Eh, a acpl-I; S288C, a gal2 mal me1 ACPl; 505, cy

ACPl-2; 5074 acpl-1 Dp(l lR+U)l; 509, a ACPI-2; 512, a acpl-1 DP(IIR+U)l, and 640, a/a abs/abs ACPl-2/ACPl-2. Except for 640, all strains have been described by HANSCHE (1975). Strains 505, 507, 509 and 512 were respectively discussed as spores 1, 2, 3 and 4. The symbol, abs, stands for a recessive gene that inhibits the abscission of daughter from parent cells, with clumps resulting (HANSCHE 1975). Strains with genetic markers: X334t-8C7 a leu2 his5 lys7 leu1 argl - + + + - -; C20-2C, a adel ural, lys2, t yr l , his7; C18-14C, a ade2 ural 1ys2

-E gal trp3 try1 his7; 900, a adel ade2 gall tsm134 lys2 tyrl his7 u r d ; 901, a adel ade2 ilsl gall lys2 tyrl his7 ural; XA99-13C, a lis4 cyh10 and XA99-13B7 (Y lts4 cyhl0. The first five strains with markers were obtained from Mortimer, University of California, Berkeley, California and the strains with the cyhlO marker from Manney (Kansas State University, Manhattan, Kansas) and Singh (University of Rochester, Rochester, New York). Strain KO-I4D, a phoC phoE, was obtained from Oshima, Osaka University, Japan.

RESULTS

The gene that codes for the structure of acid phosphatase maps into the right arm of chromosome I1

Our experimental system was designed to detect and select duplications of chromosomal regions that include the acid phosphatase (aphtase) structural gene, acpl (FRANCIS and HANSCHE 1972; HANSCHE 1975). Therefore, we mapped the locus of the aphtase gene to determine its proximity to chromosomal markers that might be useful in establishing the length of duplicated chromo- somal regions detected in our experiments. Strain El, of genotype acpl-l was used to map this gene locus. Strains of genotype acpl-l have negligible in uiuo aphtase activity detectable in our most sensitive assay (FRANCIS and HANSCHE 1972; HANSCHE 1975). The segregation of ascal spores obtained from crosses of strain El with various genetically marked disomic strains indicated that acpl-l

6 76 P. E. HANSCEIE, V. BERES AND P. LANGE

TABLE 1

Tetrad data indicating gene-to-gene linkages on the right arm of chromosome I1

Gene pair Calculated map

PD NPD T distance (cMs)

tsml34-acpl acpl-lys2 tsml34-lys2 acpl-tyrl lys2-tyrl

21 0 2 4.3 64 0 26 14.4 15 0 8 17.4 18 1 48 40.3 28 0 39 29.1

The gene order from tsm134 toward the right telomere is tsm134, acpl, lys2, t yr l , his7. * Distance was calculated by the method of PERKINS (1949).

was probably located on chromosome I I . The segregation of ascal spores obtained from further crosses of strain El with various strains carrying one o r more of the chromosome IZ markers i lsl , cyhl0, tsml34, Zys2, tyr l , glcl , and his7 place acpl on the right arm of chromosome ZZ about 14 cMs to the left of lys2 and about 4 cMs to the right of tsnl34 (Table 1 and Figure 1). Our estimates of map dis- tance between tsml34 and lys2 and between lys2 and tyrl are consistent with previously reported estimates of the location of these two genes (PLISCHKE et al. 1975). The segregation of ascal spores obtained from crosses of strain El with strain KO-14D (ToH-E et al. 1973) confirm that acpl-1 is allelic to the gene symbolized phoE by TOH-E et al. (1973) and symbolized pho5 by PLISCHKE et al. ( 1975). Following internationally accepted nomenclature of yeast genetics (PLISCHKE et al. 1975), we suggest the more specific notation, acpl, to differ- entiate this gene from those that may code for other acid phosphatases or that may code for alkaline phosphatases in this organism.

I I I I I I I I + + +

I 120 c M I I I

FIGURE 1 .-Recombinational map of chromosome 11, depicting the location of each of the genes used in this study and the duplicated region of the right arm of the chromosome, symbolized Dp(llR+U)I.

GENE DUPLICATION IN S. cereuisiae 677

Map of a translocated duplicate region of the right arm of chromosome 11, Dp(IIR+U) 1

HANSCHE (1975) reported the selection of a haploid strain of S. cereuisiae (strain 409) carrying an extra copy of the aphtase structural gene, acpl. The possibility that the independently segregating extra copy of the structural gene was a result of aneuploidy or polyploidy was excluded by three independent tests. This conclusion was based on deviations from expected meiotic and mitotic segregation ratios, the details of which are laid out in that report. However, it is relevant; to note here that all the data reported were consistent with the extra copy of the aphtase structural gene, acpl, of strain 409 being associated with a trans- located duplication of a region of the right arm of chromosome ZZ that includes the aphtase structural gene, the karyotype of strain 409 being Dp(ZZR-+U)l (MATERIALS AND METHODS). The experiments reported here were designed to establish the length of this translocated duplicate region of chromosome ZZ in strain 409.

To establish the length of the duplicate region of chromosome ZZ in Dp(ZZR-tU)l strains derived from strain 409, various strains with markers on chromosome ZZ, and elsewhere, were mated with strain 507 (MATERIALS AND

METHODS). Strain 507 carries the recessive mutant allele acpl-l on chromosome ZZ and the dominant mutant allele ACPI-2 on the translocated, duplicate region of chromosome ZI; its karyotype is Dp(ZZR-+U)l and its genotype is (ZZ acpl-2 I U ACPI-2). Zygotes from these test crosses were sporulated and ascal spores were scored for markers.

On the one hand, among the 47 asci scored for ade2 (chromosome X V ) , all segregated 2:2. Among the 20 asci scored for ural (chromosome XZ), all segre- gated 2:2. Among the 10 asci scored for mating type (chromosome ZZZ) all seg- regated 2:2. Further, asci scored for cyhl0, gall and tsml34, located proximal to acpl on the right arm of chromosome ZZ, all segregated 2:2 (Table 2). Each of the segregation ratios observed for these six markers is contrary to the segrega- tions expected (at least e/3 of the asci segregated 4: 0 and 3: 1) if the Dp(ZZR+U)I strains were polyploid (HANSCHE 1975). Each of the segregation ratios observed for the three markers proximal to acpl on the right arm of chromosome ZZ is also contrary to the expectation of at least e/3 of the asci segregating 4: 0 and 3: 1 if Dp(ZZR+U)l strains were disomic for chromosome ZZ (HANSCHE 1975). How- ever, all of these data are consistent with Dp(ZIR-+U)I strains being monoploid, as was their progenitor, S288C. The data provide evidence that Dp(ZZR-+U)Z strains carry only a single second, third, eleventh and fifteenth chromosome.

On the other hand, the zygotes from testcrosses with these same Dp(ZZR-+U)l strains segregate 4: 0,3: 1 and 2:2 at the acpl locus, 4.5 cM to the right of tsml34 on the right arm of chromosome ZZ, as did all other marker loci tested distal to acpl on the right arm of chromosome ZZ (Table 2) , i.e., lys2, tyr l and his7. These results are contrary to the expected segregation ratios (exclusively 2:2) were Dp(ZZR4U)I strains carrying only one copy of each of these genes residing on the right arm of chromosome ZZ distal to tsml34. These results (the three ascal

6 78 P. E. HANSCHE, V. BERES A N D P. LANGE

TABLE 2

Segregation of chromosome I1 markers in asci from crosses involving strains carrying a translocated duplicate region of the right arm of chromosome 11, symbolized Dp (IIR-tU) 1

Chromosome I1 genotypes of parental strains Normal X Dp(1lR-t U) i+

Segregation ratios ( + :-) * 4:O 3:l 2:2 1:3 0:4

Right-arm tests (starting at the centromere) cyhlO x CYHIO gall x GAL1 tsm134 x TSMl34 acpl-I x acpl-I ACPI-2 x acpl-I

t y r l x T Y R I his7 X HIS7

lysZ x LYSZ

Left-arm test ilsl x ILSI

7 1 1 19

27 27

11 30 7 13 60 22 13 26 8 14 30 4

1 1 5 4 1

* Recombination between genes on Dp(IIR+U)I and the right arm of chromosome I I would be expected to increase the frequency of 4:O asci at the expense of 2:2 asci for genes located on Dp(IIR-+U)I.

+Since the translocated duplicate region of chromosome I I arose in the haploid strain 525, Dp(IIR+U)I ( I I ACPI-21U ACPI-2), known to be wild type for the rest of the above markers, all genes on the duplicated region are expected to be wild type.

segregation types 4: 0, 3: 1 and 2:2 and the frequencies in which they arose) indicate that monoploid Dp(ZZR+ U ) l strains carry two independently segre- gating wild-type alleles of acpl, Zys2, tyrl and his7, only a single copy of which is normally located on the right arm of chromosome ZZ distal to tsml34.

Since the segregation ratios for each of the six other loci tested, ade2, ural, a, cyhl0, gall and tsml34, have ruled out Dp(ZZR-+U)l strains being polyploid, and since the segregation ratios of the chromosome ZZ markers to the left of acpl on the right arm of chromosome ZZ, cyhl0, gall and tsml34 and the segregation ratio of the iZsl marker on the left arm of chromosome ZZ (Table 2) rule out Dp(ZZR+ U ) l strains being disomic for chromosome ZZ, the simplest remaining explanation consistent with all of these results is that Dp(ZZR+U)l strains carry a translocated duplicate region of the right arm of chromosome ZZ. All of these results are consistent with those previously reported by HANSCHE (1975). In addition, they indicate that the left terminus of this duplicated region of chromo- some ZZ is no more than 4 cM to the left of acpl, between acpl and tsm134 (Table 2 and Figure 1 ) . They also indicate that the duplicated region probably extends to the telomere of the right arm of chromosome ZZ.

Dp(ZZR-+U)l strains of the genotype (ZZ acpl-l LYS2, T Y R I , HZS7IU ACPI-2 LYS2 T Y R l HZS7) were crossed with a normal strain of genotype acpl-l Zys2 tyrl his7, to establish the amount of recombination that takes place between alleles of genes on the translocated duplicate region of chromosome ZZ and those located on the right arm of normal second chromosomes. The segrega- tion ratios (Table 3 ) are consistent with the hypothesis that synapsis between

GENE DUPLICATION IN S. cerevisiae

TABLE 3

6 79

Estimate of the amount of recombination between genes on the right arm of chromosome I1 and genes on the translocated duplicated region of the right arm of chromosome I1

of Dp(IIR+U) 1 sfrains*

Recombinant chromatid detected

No. of spores Percent recombinants Total Recombinant Observed Expectedt

ACPI-2, lys2 223 4 1.79 0.6 lys2, TYRI 223 7 3.14 1.21 tyr l , HIS7 200 9 4.5 2.01 ACPI-2, lyS2, TYRI 223 3 1.34 0.175 ACPI-2, lys2, tyr l , HIS7 223 1 0.4.1. 0.29 L YS2, t yrl , HIS7 223 4 1.79 0.58

*The genotype of the cross used to make this estimate was acpl-I, lys2, tyrl, his7 x (I1 acpl-I, LYS2, TYRI, HIS71U ACPI-2, LYSZ, TYRI, HIS7).

The expected frequency of recombinants is equal to only 1/24th of the map distance between genes, since: (a) synapsis between the translocated region of chromosome I1 and the homologous region of a second chromosome is expected to occur only two-thirds of the time. (b) Given (a) only 1/2 of the second chromosomes carry recessive markers required for the detection of recombi- national events. (c) Given (b) single crossovers between the translocated region of Chromosome I1 and the marked second chromosome are detectable only when the recombined chromatids migrate to the same pole during anaphase I (half time). (d) Given (c) such crossovers are detected only when a recombinant and a nonrecombinant chromatid migrate to opposite poles a t anaphase I1 (half the time). (e) Given (d) half the recombinant chromatids are masked by nonrecombinant chromatids.

the right arm of the normal second chromosome homologues and the translocated duplicate region of the right arm of chromosome ZZ occurs randomly; i.e., two thirds of the time.

The fact that during meiosis the translocated duplicate region of the right arm of chromosome ZZ synapses randomly (apparently about two thirds of the time) with homologous regions of the right arm of normal second chromosomes makes it possible to test whether the duplicated region was translocated interstitially into the arm of an unknown chromosome or transpositioned to the terminus of an unknown chromosome. Were the translocation interstitial, one half the asci in which single crossovers arose between the right arm of chromosome ZZ and the translocated duplicate region of the right arm of chromosome ZZ should produce ascal spores that segregate 3: 1 (+;-) for spore viability. This is because a defi- ciency for the region of the unknown chromosome distal to the interstitial trans- location would arise in one spore whenever a recombinant and a nonrecombinant chromatid assorted together at anaphase ZZ. Thus, were the translocation inter- stitial, about 1/3 of all asci from such crosses should segregate 3: 1 for spore viabil- ity. Contrary to that expectation, viability of ascal spores with Dp(ZIR+U)I strains is normal (greater than 90%). This indicates the duplicated region of chromosome ZZ is not interstitially translocated, but is integrated into the term- inus of one of the arms of an unknown chromosome.

680 P. E. H A N S C H E , V. BERES A N D P. L A N G E

A second spontaneous duplication of the acpl gene Diploid strain 640 (MATERIALS AND METHODS) was used in a selection experi-

ment analogous to that in which Dp(ZZR-+U)l arose and replaced the original haploid strain of S. cerevisiae (HANSCHE 1975). By the five-hundredth genera- tion, the original strain had been replaced by a “mutant” with aphtase hyper- activity in vivo. The mutant isolate, 698, from that experiment was sporulated. Spore A1 was crossed with acpl tester strains to establish whether aphtase hyper- activity in uiuo segregated with the aphtase structural gene. Segregation of ascal spores from this cross suggested that spore A1 may carry an independently seg- regating duplicate of the aphtase structural gene.

Spore A1 was subs.equently crossed with strains carrying various markers. Zygotes were sporulated. On the one hand, all of the 12 asci assayed for ade2 (chromosome X V ) segregated 2:2. Of the five asci assayed for mating type (chromosome Z Z Z ) , all segregated 2:2. All 12 of the asci scored for cyhlO (just to the right of the centromere of chromosome Z Z ) segregated 2:2 (Table 4). All 40 asci scored for abs (unmapped) segregated 2: 2. All of these results are incon- sistent with strain 640 being polyploid. Further, the segregation of cyhlO is inconsistent with strain 640 being disomic for chromosome ZZ. However, these results are consistent with spore A1 being monoploid. On the other hand, the segregation of tsml34, distal to cyhlO on the right arm of chromosome ZZ, and the segregation of alleles of all loci tested to the right of tsm134 indicate that strain A1 carries two independently segregating copies of this region of the right arm of chromosome ZZ (Table 4). Apparently the left terminus of the duplicated region

TABLE 4

Segregation of chromosome I1 markers in asci from crosses of strains marked on chromosome I1 with strains (derived from 698) that carry two independen f l y segregating aphtase

structural genes, one on the translocation Dp (IIR+U) 5

Chromosome II genotypes of parental strains Normal X Dp(1IR-t U)5

Right-arm tests (starting at the centromere) cyhI0 X CYHIO tsmI34 x TSMI34 acpl-I X ACPI-2 ACPI x acpf-I acpl-I x acpl-I lys2 x LYSZ lys2 x lysZ tyrI X TYRI his x HIS7

Left-arm test ilsi x ILSI

Segregation ratios ( + :-) * 4:O 3:l 2:2 1:3 0:4

12 7 1 4

10 12 3 6 14 2

8 12 21 2

4 1 13 4 10

11 3

* Recombination between genes on Dp(IIR-+U)5 and the right arm of chromosome I I would be expected to increase the frequency of 4:O asci at the expense of 2:2 asci for genes located on Dp( I I R+ U)5.

GENE DUPLICATION IN S. cereuisiae 681

lies between cyhI0, located just to the right of the centromere, and tsm134, located about 60 CM to the right of the centromere (Table 4). The duplicated segment does not include the centromere, nor any known gene locus on the left arm of chromosome ZZ (Table 4). We have symbolized this translocated dupli- cate region of chromosome ZZ as Dp(ZZR+U)5.

The fact that more than 90% of the ascal spores from crosses of normal strains with Dp(ZZR+U)5 strains are viable indicates that this duplicated region of chromosome ZZ (like that of Dp(ZZR+U)I strains) is integrated into the terminus of one of the arms of the 17 chromosomes of this organism.

The segregation of ascal spores from crosses between Dp(ZZR+U)I strains of genotype (ZZ acpl-IIU ACPI-2) and Dp(ZZR+U)5 strains of genotype (ZZ acpl-IIU ACPI-2) (4:0, 3:l and 2:2) indicate that the duplicated region of chromosome ZZ in these two strains are not incorporated into the terminus of the same chromosomal arm.

Estimate of spontaneous rate of duplication of the aphtase structural gene In two experiments, populations of about IO9 cells were subject to selection for

aphtase hyperactivity in uiuo for an average of about 1,000 generations before the normal progenitors were replaced by spontaneously arising “mutant” strains with duplicate copies of the aphtase structural gene. A third experiment of the same magnitude had no detectable duplication of the right arm of chromosome ZZ. Those observations give us a current estimate of the spontaneous rate at which this gene is duplicated of between and duplications per mitosis.

UV induction of three additional duplications of the aphtase structural gene The mechanism through which regions of the right arm of chromosome ZZ were

duplicated in these two experiments seemed to involve transpositioning of a chromosome segment followed by chromatid assortment during mitosis. Since PERKINS (1974) has shown that W induces a high frequency of gene trans- positioning in Neurospora, we postulated that UV might induce gene duplication by the same mechanism in Saccharomyces.

In three independent experiments, normal haploid strains subjected to chronic UV (while being selected in a chemostat for aphtase hyperactivity) were replaced by mutant haploid strains A86, A151 and 802, each with aphtase hyper- activity in uiuo. Four asci from crosses with each of these three “mutant” strains were scored for mating type (chromosome ZZZ) . All segregated 2:2. N’ ineteen asci from a cross of an a b 2 strain with strain A86 were scored for ade2 (chromo- some XZ). Eighteen segregated 2:2. One segregated 3:l. Strains A151 and 802 were crossed with an abs strain (unmapped). Twenty asci from each cross were scored for abs, and all 20 segregated 2:2 in each case. These results are incon- sistent with strains A86, A151 or 802 being polyploid. Tables 5 , 6, and 7 present the results of crosses of these “mutant” haploid strains with various strains carrying chromosome ZZ markers.

The data in those tables indicate that in all three cases the genetic basis of aphtase hyperactivity resides in a transpositioned duplicate copy of the aphtase

682 P. E. H A N S C H E , V. BERES A N D P. L A N G E

TABLE 5

Segregation of chromosome I1 markers in asci from crosses of strains marked on chromosome I1 with strains (derived from strain A86) that carry the translocated duplicate region

of chromosome I1 designated Dp (IIR-U) 2

Chromosome I1 genotypes of parental strains Normal X Dp(llR+ U)2

Right-arm tests (starting at the centromere) cyhl0 x CYHIO tsml34 X TSMl34 acpl-I X acpl-1 acpl-I x ACPI-2 ACPI-2 X acpl-I

t yr l x TYRI his7 x HIS7

lys2 x LYS2

Segregation ratios ( + :-) 4:O 3:l 2:2 1:3 0:4

15 29 12 1

6 23 1 24 54 6 11 21 2 3

4 1

The translocated duplicate region of chromosome I 1 is expected to be a wild type. * Recombination between genes on Dp(llR+U)2 and the right arm of chromosome I 1 would

be expected to increase the frequency of 4:O asci a t the expense of 2:2 asci for genes located on Dp(IIR+U)2.

TABLE 6

Segregation of chromosome I1 markers in asci from crosses of strains marked on chromosome I1 with strains (derived from strain A15 l ) that carry the translocated duplicate region

of chromosome I1 designed Dp (IIR+U) 3

Chromosome I1 genotypes of parental strains Normal X Dp(1lR-t U)3

Segregation ratios ( + :-) * 4:O 3:l 2:2 1:3 0:4

Right-arm tests (starting from centromere) cyhl0 x CYHIO tsm134 X TSM134 TSM134 X tsm134 tsm134 X tsm134 acpl-l X acpl-1 acpl-I X ACPI-2 ACPI-2 x acpl-I lys2 x LYS2 LYS2 x lys2 tyr l x TYRI his7 x HIS7

Left-arm test i M x ILSI

41 5 5 1 2 6 1

5 20 39 15 3 9 1

21 27 8 3 3 3 5 7 1 6

1 2 1 7 1

19

The translocated duplicate region of chromosome I1 is expected to be a wild type. * Recombination between genes on Dp(IlR+U)3 and the right arm of chromosome 11 would

be expected to increase the frequency of 4:O asci at the expense of 2:2 asci.

GENE DUPLICATION IN S. cereuisiae 683

TABLE 7 Segregation of chromosome I1 markers in asci from crosses of strains marked on chrosmosome I1

with strains (derived from strain 802) that carry the translocated duplicate region of chromosome I1 designated Dp(IIR-+U)4

Chromosome I1 genotypes of parental strains Normal X Dp(llR+ U14

Segregation ratios ( + :-) * 4:O 3:l 2:2 i:3 0:4

Right-arm tests (starting at the centromere) cyhIO x CYHIO CYHIO x cyhIO ism134 X TSMI34 7 acpl-1 x acpl-I acpi-I x ACPI-2 32

lys2 x LYS2 17 ACPI-2 X acpl-I 28

lys2 x lys2 tyrl x TYRI 3 TYRI x tyrI 1 his7 x HIS7 8 HIS7 X his7

Left-arm test ilsl x ILSI

23

54 72 28

6 1 9 6

2

35 27 6

55 8 5 5

21 2

27

12

2

The translocated duplicate region of chromosome I I is expected to be a wild type. * Recombination between genes on Dp(IIR-+U)4 and the right arm of chromosome ZI would

be expected to increase the frequency of 4:O asci at the expense of 2:2 asci.

structural gene. All three cases involved duplication of the portion of the right arm of chromosome ZZ including and distal to the acpl gene. The 2:2 segregation of the centromere marker cyhlO and of the left arm marker ilsl in these strains indicates that the duplicated region does not include either the centromere or the left arm of chromosome ZZ; i.e., these strains carry only a single copy of the ilsl and cyhlO genes. The left terminus of the duplication symbolized Dp(ZZR-+U)2 (Table 5 ) like that of Dp(ZZR+U)l (Table 2) was shown to lie within 5 CM of the left end of the acpl gene, between acpl and tsml34. The left termini of the two other UV-induced duplications, symbolized Dp(ZZR-+ U)3 and Dp(ZZR+U)4, lie further to the left, between tsml34 and cyhlO (Tables 6 and 7). The fact that more than 90% of the ascal spores from crosses of normal strains with each of these three strains are viable indicates that these duplicated regions of chromosome ZZ (like those in Dp(ZIR-+U)I and Dp(ZZR+U)5 strains) are integrated into the termini of one or more of the arms of the 17 chromosomes of this organism.

From these three experiments in which UV was employed to induce gene duplication, we estimate that the UV treatment used induces about a 10-fold increase in the rate of duplication of the right arm of chromosome ZZ (to some- where between and duplications per mitosis).

684 P. E. HANSCHE, V. BERES A N D P. LANGE

DISCUSSION

Five independent duplications of the aphtase structural gene arose in these experimental populations of S. cerevisiae. The study further demonstrates a method for inducing and selecting duplicate copies of specific genes in S. cerevisiae.

In each of the five duplications detected, the event involved all known gene loci distal to the aphtase structural gene, acpl, on the right arm of chromosome ZZ. In each case the duplicated segment of the right arm of chromosome ZZ appar- ently has been transpositioned to the terminus of one of the arms of the other 16 chromosomes of the genome of this organism. This suggests that the duplication process involved, first, a break in the right arm of one of the chromatids of chro- mosome ZZ; then a transpositioning of that chromatid to the terminus of a chromatid of a nonhomologous chromosome; and, finally, the joint assortment, during anaphase of mitosis, of the normal chromatid of chromosome ZZ with the chromatid that carries the translocated segment of the right arm of chromosome ZZ. This process is modeled in Figure 2.

The five independent duplications of major portions of the right arm of chromo- some ZZ detected in our study vary in the fidelity with which they are transmitted

T R A N S LO C A T ION - D U P L I C A T I O N

N O R M A L MONOPLOID GENOME

A B C E F

A B C D E F Inrl

T E R M I N A L TRANSLOCATION O F REGION C - B C v E F C

&4=+ D E F A B .

U CH ROM AT1 D ASSORTMENT

r .

D E F ' C i -+J-J--+w-J+3 DUPLICATION O F REGION C

OR r - - 1

A B I I D E F ---J -0-

L O S S O F REGION C FIGURE 2.-This model depicts the postulated sequence of events involved in the transposi-

tioning and duplication of terminal segments of the right arm of chromosome ZZ.

GENE DUPLICATION IN S. cereuisiae 685

TABLE 8

Aphtase+, aphtase; and sectoring colonies from single-cell isolates from a chemostat inoculated with strain A145-D, of genotype acpl-1, Dp(IIR+U)2 for aphtase hyperactivity

Sample No. of No. of ahi te No. of Percent white Loss rate g- date sectors colonies colonies colonies percent/mitosis+

10 7/30, 0 3 58 4.9 21 1 25 6 509 33.4 2.M 10 8/25 1 0 70 1.4 21 1 1 685 0.3 0.00 10 9/7 1 0 94 1.05 21 0 0 541 0.00 0.00

The assay technique stains aphtase+ colonies red; aphtase- colonies are white. * No. of generations that single-cell isolate was grown before being plated. (Calculated from the

relation N e = N,e/lt) . Percent loss/mitosis was calculated from the relationship percent loss/mitosis = - (l/g)

ln(flo/fzl), where flo was the frequency of red colonies in generation 10 and f 2 1 was the fre- quency in generation 21).

from one generation to the next. For example, Dp(ZZR+U)I is transmitted mitotically with a very high fidelity; not a single loss being detected among the lo5 colonies screened (HANSCHE 1975). Dp(ZZR+U)S, however, is lost at a fre- quency of about (Table 8). The same phenomenon was reported by NEW- MEYER and TAYLOR (1967) and NEWMEYER and GALEAZZI (1977) in duplicated chromosomal segments of Neurospora, and by PARAG and ROPER (1975) in Aspergillus. The mechanism involved in the loss is understood no better than the mechanism through which these large chromosome segments are integrated into the termini of nonhomologous chromosomes. It is significant, however, that selec- tion for increased fidelity of transmission is effective. After about 100 generations of selection for aphtase hyperactivity in the chemostat, strain A145-D genotype (ZZ acpl-IIU ACPI-2) and karyotype Dp(ZZR+U)B was replaced by a “mu- tant” strain, A145-DS7 that transmits the duplicated region of the right arm of chromosome ZZ with at least 1,000 times the fidelity of strain A145-D (Table 8).

The genetic basis of the difference between these two Dp(ZZR+U)2 strains in stability of the translocated duplicate region of chromosome ZZ was explored. The 4:O segregation for aphtase activity of the ascal spores from the cross A145-D x A145-DS indicates that fidelity of transmission is achieved without further transpositioning of the duplicated region of the right arm of chromosome ZZ. The fact that ascal spores from the cross E l x A145-DS segregate exclusively 2:2 for aphtase activity, and that all aphtase plus ascal spores are stable, indi- cates that the genetic event that caused the stabilization of this transformed region of the right arm of chromosome ZZ occurred at or near the site of integra- tion. This suggests that the integration of these chromosomal segments into chro- mosome termini in fungi is not restricted to a single-step process.

No results from this study can yet be interpreted as evidence of tandem gene duplication via unequal crossing over between sister chromatids. Unequal cross-

686 P. E. H A N S C H E , V. BERES A N D P. LANGE

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