coincident gene conversion events in yeast that involve a - genetics
TRANSCRIPT
Copyright 0 1986 by the Genetics Society of America
COINCIDENT GENE CONVERSION EVENTS IN YEAST THAT INVOLVE A LARGE INSERTION
JOHN E. GOLIN,*,' S. CARL FALCO* AND JEANNE P. MARGOLSKEEt**
"Central Research and Development, E. I . d u Pont de Nemours and Company, Wilmington, Delaware 19898, and +Department of Biochemistry and Biophysics, University of Calijornia,
San Francisco, Calijornia 9 4 1 4 3
Manuscript received December 13, 1985 Revised copy accepted August 16, 1986
ABSTRACT
In yeast, spontaneous gene conversion events involving sites that are far apart (16 cM) occur 1000 times more frequently in mitotic cells than is expected for two independent acts of recombination. It has been proposed that a major por- tion of these could be due to a long, continuous heteroduplex intermediate. We have examined this possibility in further detail by introducing, via transforma- tion, a large plasmid insertion between the LEU1 and TRPS loci and studying its behavior among coincident convertants involving the flanking sites. Among such convertants, there is frequent loss of the plasmid when it is present in hemizygous or homozygous configuration. Our results could support the long heteroduplex model for coincident recombination events, but only if novel as- sumptions regarding the formation and fate of mismatched DNA are made. Therefore, an alternative model that proposes multiple, concerted recombination events is discussed.
N yeast, mitotic recombination appears to differ from its meiotic counterpart I in several ways including the timing within the cell cycle (FABRE 1978; ESPOSITO 1978; ROMAN 1980; GOLIN and ESPOSITO 1981) and the structure of the intermediates (see ESPOSITO and WAGSTAFF 1981 for review). In addi- tion, it has recently been demonstrated that, although spontaneous mitotic recombination events are very infrequent, they can encompass a large region of a chromosome (ESPOSITO 1978; GOLIN and ESPOSITO 1981, 1984; MONTE- LONE, PRAKASH and PRAKASH 1981). Thus, in yeast, mitotic gene conversion events involving sites 16 cM apart occur 1000 times more frequently than is expected for two independent events. Such enhancement is not found among meiotic or X-ray-induced recombinants (ESPOSITO and WAGSTAFF 198 1 ; Ro- MAN and FABRE 1983; ROMAN 1984). Furthermore, the effect shows distance dependence (GOLIN and ESPOSITO 1984). Sites farther apart show less enhance- ment. It has been proposed that a major portion of the coincident events could be due to a long, continuous heteroduplex (ESPOSITO 1978; GOLIN and ESPOS- ITO 1984). The data, however, do not rule out alternative possibilities. For
' Present address: Department of Biology, Catholiq University, Washington, D.C. 20064. * Present address: Department of Genetics, Hebrew University, Jerusalem, Israel.
Genetics 114: 1081-1094 December, 1986
1082 J. E. COLIN, S . C. FALCO AND J. P. MARGOLSKEE
TABLE 1
JG 400
IG 302
HO a leul-c trp5-c cyh2 metl3c ade5 ade2 lys2-2 tyrl-2 his7-2 ura3-1
HO a leul-12 trp5-d CYH2 metl3d ADE5 ade2 lys2-I tyrl-1 hisl ura3- 313
JG33-18B X JG 34-38A JG34-38A with pJM53 (URA3) inte-
grated by transformation between leul-12 and trp5-d
JG 231 X JG 33-18B
HO a leul-c pJM53 (URA?) trp5-C CYH2 metl3d ADE5 ade2 LYS2 tyrl-1 hisl ura3-313 his7
JG 300RS 8-16 X JG 34-38A
IC 231 X IC 300-RS-8-16
COLIN and ESPOSITO (1 977)
COLIN and ESPOSITO (1 977)
GOLIN and ESPOSITO (1977)
Random Lys’ spore from JG300
pJM53 inserted in the leul-c, trp5- c homologue “
” ” ” Homozygous for pJM53
example, pairing of chromosomes in mitosis could be rate-limiting for the initiation of recombination. Once such rare pairing occurs, multiple recombi- nation events might take place in a distance-dependent fashion.
In this report, we describe experiments in which coincident gene conversion was analyzed at loci on either side of a large (1 1.6 kilobase pair (kbp)] insertion. The data presented in this paper could accommodate the long heteroduplex model, although a series of observations requires novel assumptions regarding the formation and fate or recombination intermediates. For this reason, an alternative to the long heteroduplex model is presented. This model proposes that coincident conversion is due to concerted, multiple recombination events.
MATERIALS AND METHODS
Strains: The pertinent strain genotypes used in this study are found in Table 1, and the linkage relationships of the markers used in this study are illustrated in Figure 1. The detailed genotype and geneology of JG44 are found elsewhere (GOLIN and ESPOS- ITO 1977, 1984).
Plasmid Plasmid pJM53 is a YIp5 derivative (BOTSTEIN et al. 1979) that has the yeast URA3 gene inserted into the AvaI site of pBR322 and a 6.1-kbp fragment of yeast DNA inserted into the Hind111 site. The yeast segment was obtained using a cloned LEUl gene to probe a bacteriophage lambda library 0. P. MARCOLSKEE and I. HERSKOWJTZ, unpublished results). The plasmid can integrate by homologous recom- bination into a site 8.8 kbp distal to LEUl (see Table 1).
Media: Media used in this study are identical to those reported earlier (GOLIN and ESPOSITO 1977), except that cycloheximide was used at a concentration of 1 mg/liter. E. coli harboring pJM53 were cultured in Luria broth plus 50 pg/ml of Ampicillin.
Growth and plating of vegetative cells: The diploids used in recombination exper- iments were cultured as described previously (GOLIN and ESPOSITO 1981), except that 12-ml cultures were employed.
Calculation of recombination rates: Recombination rates were calculated using the
COINCIDENT GENE CONVERSION IN YEAST 1083
pJM53 (UffA3I
LEU1 1 TRP5 MET13 + 12 d + CYHZ + d ADE5
3.04 7 10 0.3 79 15 86 0 ; ; I I I 1 I I I I I I
e : : I I I
1 1 I I I I
FIGURE 1.-The arrangement of genetic markers. The diploids used in this study carry a series of markers on chromosome Vll that are used to monitor recombination. The site of pJM53 integration is indicated by an arrow. In addition to the pJM53 insertion, there are three pairs of noncomplementing alleles at LEUl , TRl5 and MET13. The ADE5 and CYHZ loci are heterozygous for recessive mutations. Numerals indicate distance in centimorgans.
method of LEA and COULSON (1948). A description of this method's salient features is found elsewhere (COLIN and ESPOSITO 1984).
Dissection of asci: Ascal dissection was performed by conventional means. Sporulated diploids were treated with 0.2 ml of 1:50 dilution of glusulase (DuPont) for 5 min to dissolve the ascal walls. Following this, tetrads were spread on a YPD plate. The plate was placed on a microscope fitted with a micromanipulator, and ascospores were teased apart with a dissection needle. The ascosporal colonies were allowed to germinate and grow for 2-3 days at 30" before genotypic analysis.
Selection of independent recombinants: To select clonally unrelated Leu+ Trp' prototrophs, cultures of the diploids were plated on synthetic complete medium. Single colonies were transferred to 2-ml YPD liquid cultures which were incubated overnight and then plated on leucine, tryptophan omission medium. Since diploids are heteroal- lelic at the loci in question, prototrophs resulting from mitotic gene conversion are obtained. Only one prototroph was picked per original colony to ensure independent origin.
Genetic analysis of prototrophs: Because of its diploid nature, a prototroph resulting from conversion between heteroalleles contains a chromosome with a genotype (at that locus) that remains undetermined. To ascertain the full genotype at the convertant locus, random ascospores were collected and analyzed as follows. The Leu+ Trp+ pro- totrophs remain heteroallelic at the unlinked LYSZ locus. During sporulation, Lys+ colonies arise as a result of gene conversion events (which occur at rates ca. 200 times the mitotic levels). Random Lys' ascosporal colonies were recovered when the sporu- lated master plate was replicated to lysineless medium. On the average, half of these were Trp+, whereas the other half harbored the masked chromosome. For example, when a diploid was TRP5ltrp5-c, half of the Lys+ colonies were TRP5 and the other half were trp5-c. The complete genotype of each recombinant was determined by anal- ysis of 18 Lys+ meiotic segregants. These segregants were backcrossed to tester strains for the leul, trp5 and met13 heteroallelic pairs to establish their genotypes at these loci. The Lys+ segregants were also used to determine the genotypes of the colonies at loci distal to TRP5 (CYHZ, MET13 and ADE5).
DNA preparation: Plasmid DNA was prepared from bacterial strains as described by DAVIS, BOTSTEIN and ROTH (1980) and was purified by cesium chloride centrifugation as described by MANIATIS, FRITSCH and SAMBROOK (1982). Yeast chromosomal DNA was isolated using the method described by FALCO et al. (1982).
Yeast integrative transformation: The transformation of strain JG34-38A was car- ried out as described by HINNEN, HICKS and FINK (1978).
Nick translation of probes: Nick translation of pJM53 DNA was carried out using a nick-translation reagent kit (BRL, Bethesda). Labeled DNA was separated from un-
1084 J. E. GOLIN, S. C. FALCO AND J. P. MARGOLSKEE
incorporated nucleotides on a 0.9 X 15-cm column of Sephadex G-50 (Pharmacia) equilibrated with 10 mM Tris-HCI, pH 8.0, 50 mM NaCl and 0.1 M EDTA.
Hybridization analysis: DNA hybridization was carried out by the method of SOUTH- ERN ( 1 975). Total yeast DNA was digested with restriction enzymes (BRL Laboratories) of choice and separated on 0.8% agarose gels by electrophoresis (40 mA, 16 hr). DNA was transferred to nitrocellulose filters (SOUTHERN 1975); following this, the filters were treated for 30 min at 42" in a solution of 50% formamide, 5 X SSPE, and 1 % Sarkosyl before hybridization. The DNA was then hybridized to heat denatured, labeled probe pJM53 in the above solution for 15 hr before washing four times (30 min, 50") in 0.1 X SSPE, 0.1% SDS. The washed filter was dried and exposed to Kodak X-0-Mat R film with DuPont Cronex Hi-Plus intensifying screens at -70" to reveal hybridized fragments.
RESULTS
Coincident mitotic gene conversion in diploids hemizygous for a plasmid insertion: The purpose of this study was to determine whether the high fre- quency of coincident mitotic conversion events is the result of a continuous heteroduplex structure. Our experimental approach was based on observations from a number of organisms, including yeast, which demonstrated that the presence of nonhomologous DNA can sometimes reduce recombination in neighboring regions (LICHTEN and FOX 1983; HAMZA et al. 1981; SMOLIK- UTLAUT and PETES 1983). Thus, if coincident gene conversion events at LEUl and TRP5 were due to a continuous structure, placing a large insertion or deletion between the loci might reduce the observed frequency of double recombinants. To accomplish this end, we placed the plasmid pJM53 via in- tegrative transformation between the two loci to create a large insertion flanked by a 6.1-kb direct repeat. The yeast strains JG44 and JG300 are isogenic except for the hemizygous plasmid insertion present in the latter (Table 1). The JG302 and JG400 strains are closely related, although not isogenic to the others. The diploids contain heteroallelic, noncomplementing mutations at a number of loci including TRPS and LEUl on chromosome VIZ. Recombination is assayed by plating out cells on the appropriate omission media and counting the colonies which arise. The colonies are not due to back mutation at either of the sites (GOLIN and ESPOSITO 1977).
The linkage relationships of the markers in question were verified by analysis of more than 50 tetrads (data not shown). The diploids were tested for the stability of the plasmid insertion. Approximately 500 colonies were picked from nonselective media and tested; all were Ura+. To determine whether the plas- mid was present in single or multiple copy number, Southern hybridization analysis (SOUTHERN 1975) was performed. These results, shown and explained in Figure 2, indicate that a single plasmid copy is present in each strain. To determine whether the presence of the insertion had any effect on the rate of formation of Leu+ Trp+ recombinants, ten cultures of the JG300 diploid and the isogenic but non-plasmid-bearing strain JG44 were grown and plated on leucine, tryptophan and tryptophan-leucine omission media as described in MATERIALS AND METHODS. The rate of gene conversion at each locus (LEU1 and TRPS) and the rate of coincident conversion were determined. In addition, Leu+ Trpf colonies were picked and analyzed for their genotypes at the URA3
COINCIDENT GENE CONVERSION IN YEAST 1085
FIGURE 2.-Characterization o f strains by hybridization analysis. To determine whether pJM53 was present in single or multiple copy, yeast DNA was prepared and digested overnight with PvulI, a restriction enzyme with one site in pJM53 outside the 6.1-kb pair yeast fragment. The DNA was then run on a gel, blotted and probed using the procedures described in MATERIALS AND METHODS. The presence of pJM53 in more than one copy per chromosome would have been indicated by a fragment of plasmid unit length. Lane 1 , PuulI cut pJM53; lane 2, JG34-38A; lane 3, JG231; lane 4, JG300-RS-8-16.
insertion, the TRP5 locus and the LEUl locus. Since the Leu+ Trp+ recombi- nants remain diploid, these prototrophs contain chromosomes with complete genotypes that remain undetermined. To ascertain the full genotype, random spore analysis was used (see MATERIALS AND METHODS).
The results, which are found in Tables 2 and 3, can be summarized as follows: First, the rate at which Leu+ Trp+ events arise in JG300 was only slightly less than the rate of comparable events in JG44. Therefore, the pres- ence of a plasmid insertion has little, or no, effect on the rate of coincident gene conversion at these loci. Second, the genotypes of the coincident recom- binants recovered in JG300 were similar to JG44. For example, Leu+ Trp+ events are three to five times more likely to be associated with homozygosity of markers distal to TRP5 than are Trp+ events which did not involve LEUl. Of the 53 Leu+ Trp+ events analyzed, 17 (30%) were homozygous for the CYH2, MET13 and ADE5 loci, in contrast to only 6% of the total Trp+ popu- lation. There is also a marked polarity for recovery of the leul-c allele among coincident convertants that is not found among single locus (Leu+) recombi- nants (see PLOTKIN 1978; COLIN and ESPOSITO 1981). This phenomenon was also found previously for the non-plasmid control (COLIN and ESPOSITO 1984; J. COLIN, unpublished results). Similar results were obtained in the analysis of many independent Leu+ Trp+ events described later. Third, most of the Leu+ Trp+ recombinants (68%) were Ura- and must therefore have lost the URA3 gene present in the plasmic insertion. The rest were primarily URA3lura3. URA31URA3 recombinants were rarely (1 in 53) recovered.
To verify the results obtained from this analysis, 172 independent Leu+ Trp+ recombinants were collected. One hundred twenty-five (73%) were Ura-. Ninety of the 172 were tested for their genotype at the CYH2 and ADE5 loci. A total of 23 (26%) exhibited homozygosity for these markers. Therefore, the
1086 J. E. COLIN, S. C. FALCO AND J. P. MARGOLSKEE
TABLE 2
Total Trp' Diploid Culture no. x lo5
JG300
10
10
3.5 2.7 1.7 2.2 3.5 1.4 1.3 2.2 1.7 1.5
1.7 0.2
7.1 5.1 3.6 1.9 3.1 3.2 2.9 3.0 4.2 3.7
3.2 0.5
rota1 Leu+ Total Leu+ x I O 5 Trp+ X 10'
3.3 0.1 20 0.3
3.8 0.2 2.8 0.2 3.4 0.2 2.3 0.4 2.5 0.1 3.4 0.5 2.4 0.2 2.4 0.2
2.8 0.2 0.4 0.1
20 1.4 14 0.6 5.3 0.6 2.2 0.3 3.5 0.3 3.9 0.5 3.8 0.2 3.4 0.6
10 0.2 5.2 0.1
3.9 0.3 0.5 0.2
4.1 6.8 9.0
11 4.5
behavior of the independent recombinants and those derived from culture are very similar.
Coincident gene conversion was also examined in JG400. This strain is hem- izygous with respect to pJM53, but the insertion is in the other homologue (Eeul-c, pJM53, trp5-c). As was the case with JG300, the presence of pJM53 had little, if any, effect on the rate of coincident Leu+ TrpC recombinants (data not shown). The distribution of URA3 genotypes is, nevertheless, mark- edly different. Of 60 independent Leu+ Trp+ recombinants analyzed, only ten (-16%) were Ura-. Of the remainder, 38 (75%) were URA3/ura3, while 12 (25%) were URA3/URAJ. The basis for the difference between JG300 and JG400 remains unclear at the present time.
Analysis of recombination in strains homozygous for an insertion: The pJM53 insertion is lost during coincident gene conversion in homozygous (JG302) diploids. Coincident Leu+ Trp+ recombinants arise in JG302 at about the same frequency that they do in the hemizygous strains (data not shown). Tetrad analysis of 57 independently derived coincident convertants revealed that 35 (-61 %) were either LJRA3/ura3 or ura3/ura3 (Table 4) and had there- fore lost one or both copies of the plasmid.
COINCIDENT GENE CONVERSION IN YEAST
TABLE 3
1087
Genotype at pJM53
- ura3 URA3 uRA3 Total Allele recovery - - ura3 ura3 um3 observed (no.) Genotype at L e d , TRPS
1. LEUl leul-c
1 leu-I2 (5)
TABLE 4
URA3 genotypes of Leu' Trp+ events collected from a pJM53 homozygote
ura3 an a 1 y z e d URA3 ura3 ura3
- - - No. of events LIRA3 URA3
57 22 28 7
Analysis of putative Ura+ revertants: A striking feature of Leu+ Ura- re- combinants is the appearance of nearly confluent growth on uracil omission media after 48-72 hr of incubation. When such material was streaked out on YPD (nutrient) media and the subclones tested for their phenotype, all were Leu+ Ura+ Trp+. When the original Leu+ Trp+ Ura- colony was streaked and tested, however, less than 0.1% of the subclones were Ura+ (see rate deter- mination data described below). Southern hybridization analysis of DNA pre- pared from single colony subclones of the initial Ura- recombinant and sub- sequent revertants demonstrated that pJM53 was absent in the former, but
1088 J. E. COLIN, S. C. FALCO AND J. P. MARCOLSKEE
1 2 3 4 5 6
FIGURE 3.-Hybridization analysis of Ura+ revertants. To determine whether a Ura+ revertant is due to the presence of integrated pJM53, Southern blot hybridization was performed on Leu+ Trp+ recombinants. The initial Ura- Leu+ Trp+ colony and subsequent Ura+ revertants were streaked on YPD media to obtain single colony isolates. These were used as a source of DNA. The DNA was cut with Xhol. an enzyme making a unique cut within the 6.1-kbp yeast fragment. The presence of an intact plasmid in the original JG300 strain (lane 1) and two revertants (lanes 3 and 5 ) is shown. The initial Ura- Leu+ Trp+ recombinants (lanes 4 and 6) have a hybridization pattern identical to the isogenic, non-plasmid-bearing control strain JG44 (lane 2).
TABLE 5
Revertant PD NPD TT
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10
14 12 15 21 20
5 18 17 10 29
0 0 0 0 0 8 0 0 0 0
0 1 1 0 2 5 2 1 0 4
a PD = parental ditype, NPD = nonparental ditype, TT = tetratype. For any cross, an excess of PDs us. TTs and NPDs indicates linkage.
present in the latter (Figure 3). The hybridization patterns of the revertants were identical to those obtained from the initial JG300 (pJM53 hemizygote) diploid. The map position of the putative Ura+ revertants derived from ten independently isolated Leu+ Trp+ recombinants was determined. To do this, single colonies isolated from revertant patches were sporulated, and the re-
COINCIDENT GENE CONVERSION IN YEAST
TABLE 6
1089
Total UraC Cfu/culture Culture x 1 0 5 x 10-7
1 1 . 1 2.3 x 107 2 20 1 . 1 x 107 3 1.3 0.9 x io7 4 0.5 7.4 x 107 5 15 5.4 x 107 6 22 1.4 x 107 7 9.3 3.9 x 107 8 3.3 4.7 x 107 9 110 2.3 x io7
Median frequency 3.3 Median rate 0.5
sulting tetrads were dissected. The results, shown in Table 5, demonstrate tight linkage of URA3 to LEU1 in nine of ten cases analyzed.
The rate of reversion from Ura- to Ura+ was also determined. T o do this, Leu+ Trp' Ura- colonies were suspended in 1 ml of YPD media and were allowed to grow overnight before harvesting and plating. The results, presents in Table 6, indicate a rate of about one Ura+ revertant/105 cell-forming units. This suggests that reinsertion takes place after an initial lag of over ten gen- erations.
DISCUSSION
The data presented in this paper can be summarized as follows. First, the presence of a large region of nonhomology (the plasmid insertion pJM53) has little or no effect on the frequency of coincident gene conversion events in- volving loci flanking both sides of the insertion. Second, in hemizygous strains, the plasmid insertion is lost (resulting in a Ura- phenotype) at high frequency (70%) from Leuf Trp+ recombinants when it is between the leul-12 and trp5- d alleles, but at a fourfold lower frequency when it is inserted between leul-c and trp5-c. Third, the plasmid is also lost at high frequency from diploids which bear it in homozygous configuration. Finally, reversion from a Ura- to a Ura+ phenotype frequently occurs. Genetic mapping and DNA hybridization experiments indicate that the Ura+ revertants result from restoration of pJM53 at its original location.
Two models have been put forth to account for the high frequency, distance- dependent coincident mitotic gene conversion of widely separated genes in yeast (GOLIN and ESPOSITO 1984). The first proposes that co-conversion is due to a single long heteroduplex recombination intermediate, whereas the second proposes that it is due to multiple concerted recombination events following some rate-limiting step, such as pairing of homologous chromosomes. These models can now be considered in light of the observations presented above.
LICHTEN and Fox (1983) found that regions of nonhomology bracketing a
1090 J. E. GOLIN, S. C. FALCO AND J. P. MARGOLSKEE
0
1
'T-- 2
I - > FIGURE 4.-A model to explain the behavior of pJM53 in coincident recombination events.
The figure shows the first of two mechanisms proposed to account for the behavior of pJM53 in coincident recombination events involving the LEU1 and TRP5 loci. The figure illustrates plasmid loss by excision of a single-stranded loop from a symmetric heteroduplex at the two-strand stage of mitosis. In this diagram, the plasmid is drawn with the duplicated yeast chromosomal region indicated as - and the YIp5 sequences as - - -. Each line represents a single polynucleotide chain. The "+" indicates the URA3 gene present in YIp5. If the loop is excised with great preference, the -/- class could be found in excess, whereas the +/+ class would be comparatively rare.
genetic interval on bacteriophage X reduce recombination in that interval, particularly in replication-restricted crosses. This observation is consistent with the idea that the nonhomology prevents branch migration of a Holliday-type heteroduplex recombination intermediate into the interval. However, in the same study, evidence from multifactor crosses carried out under replication- permitted conditions suggested that nonhomologies could be included in re- gions of heteroduplex DNA. In yeast, most mitotic gene conversion occurs before DNA replication (ESPOSITO 1978; FABRE 1978; COLIN and ESPOSITO 1981; ROMAN 1980; ROMAN and FABRE 1983). Thus, it was of interest to investigate the effect of a large nonhomologous insertion on coincident gene conversion of flanking markers. The observation that the nonhomology had little or no effect on the frequency or character of coincident conversion events indicates either that a nonhomology can be included in a region of heterodu- plex DNA in yeast or that long heteroduplexes are not the source of the coincident conversions.
The frequent loss of the plasmid insertions from hemizygous strains can be accommodated by either the long heteroduplex model or multiple concerted recombination events model. The long heteroduplex model could account for the Ura- events because the creation of extensive heteroduplex DNA spanning the LEUl-TRP5 region would result in a single-stranded loop that might be preferentially excised from the JG300 diploids (Figure 4). BENZ and BURGER (1973) proposed such a mechanism to account for selective loss of the wild- type allele from crosses between wild-type and rZZ-deletion mutants of bacte-
COINCIDENT GENE CONVERSION IN YEAST
1
1091
1
tion by intrachromosomal recombination. figure illustrates loss of the plasmid inser-
riophage T4. No such process has been observed previously in yeast, however. The multiple events model could explain loss of the plasmid insertion by a separate, but associated, intrachromosomal event: homologous recombination between the direct repeats (Figure 5) . There is ample precedent for the latter mechanism in yeast (SCHERER and DAVIS 1979; ROEDER and FINK 1980), al- though association of intrachromosomal and interchromosomal events has not been reported.
The frequent loss of the plasmid insertion among Leu' Trp+ recombinants from an insertion homozygote is more difficult to explain within the context of the long heteroduplex model. Figure 6 illustrates how misalignment of the insertion could lead to a series of single-stranded loops. One or both of these could be cleaved to give recombinants. Misalignment of the insert on both chromosomes is required to produce a ura3/ura3 recombinant. The fact that the plasmid is lost from the homozygous diploid as frequently as from the hemizygous diploid requires that the misaligned structure drawn in Figure 6 occurs more frequently than the properly aligned structure. This seems un-
1092 J. E. COLIN, S. C. FALCO AND J. P. MARGOLSKEE
1 -
FIGURE 6.-Loss of plasmid in a homozygote by misalignment. The figure illustrates how pJM53 could be lost from a homozygote strain by a mechanism analogous to that proposed in A. Misa- lignment of direct repeats in a heteroduplex could create two single-stranded loops. Preferential excision would lead to cells which are either Ura-/Ura- or Ura+/Ura-.
likely. The multiple events model can explain loss of the plasmid from the homozygous diploid by the same intrachromosomal recombination mechanism as proposed for the hemizygous strain.
Reversion from Ura- to Ura+ by restoration of pJM53 to its original chro- mosomal location is also more difficult to explain by the long heteroduplex model. It must be proposed that a precisely excised single-stranded DNA re- gion either persists and reintegrates by single-strand assimilation (RADDING 1982) or replicates and reintegrates later through a double-stranded interme- diate. The multiple events model proposes that the excised double-stranded circle persists and is later reintegrated by a reversal of the excision reaction.
Thus far, we have assumed that most mitotic gene conversion occurs before DNA replication. Genetic analysis clearly indicates that this is the case (ESPOS- ITO 1978; FABRE 1978; GOLIN and ESPOSITO 1981; ROMAN 1980; ROMAN and FABRE 1983). This interpretation can be extended to coincident convertants with two important reservations. First, genotypic analysis of coincident recom- binants to date cannot rule out a GP contribution. Second, recent evidence suggests that although X-ray-induced gene conversion occurs at the two-strand stage, associated intergenic exchange may be completed following DNA repli- cation (ROMAN and FABRE 1983; ROMAN 1984). This could provide an alter- native explanation for the high frequency loss of the plasmid insertion from a long heteroduplex recombination intermediate. A major difference between the two- and four-strand stages is the presence of sister chromatids in the latter. If coincident conversion in long heteroduplexes was associated with high frequencies of unequal crossing over between the direct repeats of sister strands, Ura- recombinants could be produced. Although sister chromatid
COINCIDENT GENE CONVERSION IN YEAST 1093
exchanges of this type have been reported in yeast, they appear to be relatively rare (JACKSON and FINK 1981). In addition, such a mechanism for generating Ura- recombinants does not account for the frequent reversion to Ura+ ob- served.
In summary, the data presented in this paper do not eliminate the long heteroduplex model as an explanation for coincident mitotic gene conversion events. However, a model proposing multiple associated recombinant event appears to better fit the data. The analysis of recombination in strains bearing insertions which do not have direct repeats is a critical test of the models. The long heteroduplex model predicts that these will also be lost with high fre- quency during recombination events involving LEU1 and TRP5 when the in- sertion is present in hemizygous configuration. In contrast, the multiple events model predicts increased stability because intrachromosomal recombination be- tween direct repeats cannot occur.
We thank DEBBIE CHALEFF, IRA HERSKOWITZ, BOB LAROSSA, HERSCHEL ROMAN and especially FRANK STAHL for their incisive comments on several drafts of this manuscript. Work at the University of California, San Francisco, on the construction of pJM53 was supported by National Institutes of Health research grants to IRA HERSKOWITZ.
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Coincident gene conversion during mitosis in Saccharomyces.
Hybrid DNA formation
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JACKSON, J. and G. R. FINK, 1981 Gene conversion between duplicated genetic elements in yeast.
LEA, D. E. and C. A. COULSON, 1948 The distribution of numbers of mutants in bacterial populations. J. Genet. 49: 264-284.
LICHTEN, M. and M. S. Fox, 1983 Effects of nonhomology on bacteriophage lambda recombi- nation. Genetics 103: 5-22.
Molecular Cloning: A Laboratory Manual.
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MANIATIS, T. , E. F. FRITSCH and J. SAMBROOK, 1982 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
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MONTELONE, B. A., S. PRAKASH and L. PRAKASH, 1981 Spontaneous mitotic recombination in
PLOTKIN, D. J., 1978
RADDING, C. M., 1982
DNA rearrangements associated with a transposable element
Recombination in diploid vegetative cells of Saccharomyces cerevisiae. Carls-
A comparison of induced and spontaneous gene conversion in mitotic and
ROMAN, H. L. and F. FABRE, 1983 Gene conversion events and associated reciprocal recombi- nation are separable events in vegetative cultures of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA S O 6912-6916.
SCHERER, S. and R. W. DAVIS, 1979 Replacement of chromosomal segments with altered DNA sequences constructed in vitro. Proc. Natl. Acad. Sci. USA 76: 4951-4955.
SMOLIK-UTLAUT, S. and T. D. PETES, 1983 Recombination of plasmids into the Saccharomyces cerevisiae chromosome is reduced by small amounts of sequence heterogeneity. Mol. Cell. Biol.
Detection of specific sequences among DNA fragments separated by gel
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COINCIDENT GENE CONVERSION EVENTS IN YEAST THAT INVOLVE A LARGE INSERTION
JOHN E. GOLIN,*,' S. CARL FALCO* AND JEANNE P. MARGOLSKEEt**
"Central Research and Development, E. I . d u Pont de Nemours and Company, Wilmington, Delaware 19898, and +Department of Biochemistry and Biophysics, University of Calijornia,
San Francisco, Calijornia 9 4 1 4 3
Manuscript received December 13, 1985 Revised copy accepted August 16, 1986
ABSTRACT
In yeast, spontaneous gene conversion events involving sites that are far apart (16 cM) occur 1000 times more frequently in mitotic cells than is expected for two independent acts of recombination. It has been proposed that a major por- tion of these could be due to a long, continuous heteroduplex intermediate. We have examined this possibility in further detail by introducing, via transforma- tion, a large plasmid insertion between the LEU1 and TRPS loci and studying its behavior among coincident convertants involving the flanking sites. Among such convertants, there is frequent loss of the plasmid when it is present in hemizygous or homozygous configuration. Our results could support the long heteroduplex model for coincident recombination events, but only if novel as- sumptions regarding the formation and fate of mismatched DNA are made. Therefore, an alternative model that proposes multiple, concerted recombination events is discussed.
N yeast, mitotic recombination appears to differ from its meiotic counterpart I in several ways including the timing within the cell cycle (FABRE 1978; ESPOSITO 1978; ROMAN 1980; GOLIN and ESPOSITO 1981) and the structure of the intermediates (see ESPOSITO and WAGSTAFF 1981 for review). In addi- tion, it has recently been demonstrated that, although spontaneous mitotic recombination events are very infrequent, they can encompass a large region of a chromosome (ESPOSITO 1978; GOLIN and ESPOSITO 1981, 1984; MONTE- LONE, PRAKASH and PRAKASH 1981). Thus, in yeast, mitotic gene conversion events involving sites 16 cM apart occur 1000 times more frequently than is expected for two independent events. Such enhancement is not found among meiotic or X-ray-induced recombinants (ESPOSITO and WAGSTAFF 198 1 ; Ro- MAN and FABRE 1983; ROMAN 1984). Furthermore, the effect shows distance dependence (GOLIN and ESPOSITO 1984). Sites farther apart show less enhance- ment. It has been proposed that a major portion of the coincident events could be due to a long, continuous heteroduplex (ESPOSITO 1978; GOLIN and ESPOS- ITO 1984). The data, however, do not rule out alternative possibilities. For
' Present address: Department of Biology, Catholiq University, Washington, D.C. 20064. * Present address: Department of Genetics, Hebrew University, Jerusalem, Israel.
Genetics 114: 1081-1094 December, 1986
1082 J. E. COLIN, S . C. FALCO AND J. P. MARGOLSKEE
TABLE 1
JG 400
IG 302
HO a leul-c trp5-c cyh2 metl3c ade5 ade2 lys2-2 tyrl-2 his7-2 ura3-1
HO a leul-12 trp5-d CYH2 metl3d ADE5 ade2 lys2-I tyrl-1 hisl ura3- 313
JG33-18B X JG 34-38A JG34-38A with pJM53 (URA3) inte-
grated by transformation between leul-12 and trp5-d
JG 231 X JG 33-18B
HO a leul-c pJM53 (URA?) trp5-C CYH2 metl3d ADE5 ade2 LYS2 tyrl-1 hisl ura3-313 his7
JG 300RS 8-16 X JG 34-38A
IC 231 X IC 300-RS-8-16
COLIN and ESPOSITO (1 977)
COLIN and ESPOSITO (1 977)
GOLIN and ESPOSITO (1977)
Random Lys’ spore from JG300
pJM53 inserted in the leul-c, trp5- c homologue “
” ” ” Homozygous for pJM53
example, pairing of chromosomes in mitosis could be rate-limiting for the initiation of recombination. Once such rare pairing occurs, multiple recombi- nation events might take place in a distance-dependent fashion.
In this report, we describe experiments in which coincident gene conversion was analyzed at loci on either side of a large (1 1.6 kilobase pair (kbp)] insertion. The data presented in this paper could accommodate the long heteroduplex model, although a series of observations requires novel assumptions regarding the formation and fate or recombination intermediates. For this reason, an alternative to the long heteroduplex model is presented. This model proposes that coincident conversion is due to concerted, multiple recombination events.
MATERIALS AND METHODS
Strains: The pertinent strain genotypes used in this study are found in Table 1, and the linkage relationships of the markers used in this study are illustrated in Figure 1. The detailed genotype and geneology of JG44 are found elsewhere (GOLIN and ESPOS- ITO 1977, 1984).
Plasmid Plasmid pJM53 is a YIp5 derivative (BOTSTEIN et al. 1979) that has the yeast URA3 gene inserted into the AvaI site of pBR322 and a 6.1-kbp fragment of yeast DNA inserted into the Hind111 site. The yeast segment was obtained using a cloned LEUl gene to probe a bacteriophage lambda library 0. P. MARCOLSKEE and I. HERSKOWJTZ, unpublished results). The plasmid can integrate by homologous recom- bination into a site 8.8 kbp distal to LEUl (see Table 1).
Media: Media used in this study are identical to those reported earlier (GOLIN and ESPOSITO 1977), except that cycloheximide was used at a concentration of 1 mg/liter. E. coli harboring pJM53 were cultured in Luria broth plus 50 pg/ml of Ampicillin.
Growth and plating of vegetative cells: The diploids used in recombination exper- iments were cultured as described previously (GOLIN and ESPOSITO 1981), except that 12-ml cultures were employed.
Calculation of recombination rates: Recombination rates were calculated using the
COINCIDENT GENE CONVERSION IN YEAST 1083
pJM53 (UffA3I
LEU1 1 TRP5 MET13 + 12 d + CYHZ + d ADE5
3.04 7 10 0.3 79 15 86 0 ; ; I I I 1 I I I I I I
e : : I I I
1 1 I I I I
FIGURE 1.-The arrangement of genetic markers. The diploids used in this study carry a series of markers on chromosome Vll that are used to monitor recombination. The site of pJM53 integration is indicated by an arrow. In addition to the pJM53 insertion, there are three pairs of noncomplementing alleles at LEUl , TRl5 and MET13. The ADE5 and CYHZ loci are heterozygous for recessive mutations. Numerals indicate distance in centimorgans.
method of LEA and COULSON (1948). A description of this method's salient features is found elsewhere (COLIN and ESPOSITO 1984).
Dissection of asci: Ascal dissection was performed by conventional means. Sporulated diploids were treated with 0.2 ml of 1:50 dilution of glusulase (DuPont) for 5 min to dissolve the ascal walls. Following this, tetrads were spread on a YPD plate. The plate was placed on a microscope fitted with a micromanipulator, and ascospores were teased apart with a dissection needle. The ascosporal colonies were allowed to germinate and grow for 2-3 days at 30" before genotypic analysis.
Selection of independent recombinants: To select clonally unrelated Leu+ Trp' prototrophs, cultures of the diploids were plated on synthetic complete medium. Single colonies were transferred to 2-ml YPD liquid cultures which were incubated overnight and then plated on leucine, tryptophan omission medium. Since diploids are heteroal- lelic at the loci in question, prototrophs resulting from mitotic gene conversion are obtained. Only one prototroph was picked per original colony to ensure independent origin.
Genetic analysis of prototrophs: Because of its diploid nature, a prototroph resulting from conversion between heteroalleles contains a chromosome with a genotype (at that locus) that remains undetermined. To ascertain the full genotype at the convertant locus, random ascospores were collected and analyzed as follows. The Leu+ Trp+ pro- totrophs remain heteroallelic at the unlinked LYSZ locus. During sporulation, Lys+ colonies arise as a result of gene conversion events (which occur at rates ca. 200 times the mitotic levels). Random Lys' ascosporal colonies were recovered when the sporu- lated master plate was replicated to lysineless medium. On the average, half of these were Trp+, whereas the other half harbored the masked chromosome. For example, when a diploid was TRP5ltrp5-c, half of the Lys+ colonies were TRP5 and the other half were trp5-c. The complete genotype of each recombinant was determined by anal- ysis of 18 Lys+ meiotic segregants. These segregants were backcrossed to tester strains for the leul, trp5 and met13 heteroallelic pairs to establish their genotypes at these loci. The Lys+ segregants were also used to determine the genotypes of the colonies at loci distal to TRP5 (CYHZ, MET13 and ADE5).
DNA preparation: Plasmid DNA was prepared from bacterial strains as described by DAVIS, BOTSTEIN and ROTH (1980) and was purified by cesium chloride centrifugation as described by MANIATIS, FRITSCH and SAMBROOK (1982). Yeast chromosomal DNA was isolated using the method described by FALCO et al. (1982).
Yeast integrative transformation: The transformation of strain JG34-38A was car- ried out as described by HINNEN, HICKS and FINK (1978).
Nick translation of probes: Nick translation of pJM53 DNA was carried out using a nick-translation reagent kit (BRL, Bethesda). Labeled DNA was separated from un-
1084 J. E. GOLIN, S. C. FALCO AND J. P. MARGOLSKEE
incorporated nucleotides on a 0.9 X 15-cm column of Sephadex G-50 (Pharmacia) equilibrated with 10 mM Tris-HCI, pH 8.0, 50 mM NaCl and 0.1 M EDTA.
Hybridization analysis: DNA hybridization was carried out by the method of SOUTH- ERN ( 1 975). Total yeast DNA was digested with restriction enzymes (BRL Laboratories) of choice and separated on 0.8% agarose gels by electrophoresis (40 mA, 16 hr). DNA was transferred to nitrocellulose filters (SOUTHERN 1975); following this, the filters were treated for 30 min at 42" in a solution of 50% formamide, 5 X SSPE, and 1 % Sarkosyl before hybridization. The DNA was then hybridized to heat denatured, labeled probe pJM53 in the above solution for 15 hr before washing four times (30 min, 50") in 0.1 X SSPE, 0.1% SDS. The washed filter was dried and exposed to Kodak X-0-Mat R film with DuPont Cronex Hi-Plus intensifying screens at -70" to reveal hybridized fragments.
RESULTS
Coincident mitotic gene conversion in diploids hemizygous for a plasmid insertion: The purpose of this study was to determine whether the high fre- quency of coincident mitotic conversion events is the result of a continuous heteroduplex structure. Our experimental approach was based on observations from a number of organisms, including yeast, which demonstrated that the presence of nonhomologous DNA can sometimes reduce recombination in neighboring regions (LICHTEN and FOX 1983; HAMZA et al. 1981; SMOLIK- UTLAUT and PETES 1983). Thus, if coincident gene conversion events at LEUl and TRP5 were due to a continuous structure, placing a large insertion or deletion between the loci might reduce the observed frequency of double recombinants. To accomplish this end, we placed the plasmid pJM53 via in- tegrative transformation between the two loci to create a large insertion flanked by a 6.1-kb direct repeat. The yeast strains JG44 and JG300 are isogenic except for the hemizygous plasmid insertion present in the latter (Table 1). The JG302 and JG400 strains are closely related, although not isogenic to the others. The diploids contain heteroallelic, noncomplementing mutations at a number of loci including TRPS and LEUl on chromosome VIZ. Recombination is assayed by plating out cells on the appropriate omission media and counting the colonies which arise. The colonies are not due to back mutation at either of the sites (GOLIN and ESPOSITO 1977).
The linkage relationships of the markers in question were verified by analysis of more than 50 tetrads (data not shown). The diploids were tested for the stability of the plasmid insertion. Approximately 500 colonies were picked from nonselective media and tested; all were Ura+. To determine whether the plas- mid was present in single or multiple copy number, Southern hybridization analysis (SOUTHERN 1975) was performed. These results, shown and explained in Figure 2, indicate that a single plasmid copy is present in each strain. To determine whether the presence of the insertion had any effect on the rate of formation of Leu+ Trp+ recombinants, ten cultures of the JG300 diploid and the isogenic but non-plasmid-bearing strain JG44 were grown and plated on leucine, tryptophan and tryptophan-leucine omission media as described in MATERIALS AND METHODS. The rate of gene conversion at each locus (LEU1 and TRPS) and the rate of coincident conversion were determined. In addition, Leu+ Trpf colonies were picked and analyzed for their genotypes at the URA3
COINCIDENT GENE CONVERSION IN YEAST 1085
FIGURE 2.-Characterization o f strains by hybridization analysis. To determine whether pJM53 was present in single or multiple copy, yeast DNA was prepared and digested overnight with PvulI, a restriction enzyme with one site in pJM53 outside the 6.1-kb pair yeast fragment. The DNA was then run on a gel, blotted and probed using the procedures described in MATERIALS AND METHODS. The presence of pJM53 in more than one copy per chromosome would have been indicated by a fragment of plasmid unit length. Lane 1 , PuulI cut pJM53; lane 2, JG34-38A; lane 3, JG231; lane 4, JG300-RS-8-16.
insertion, the TRP5 locus and the LEUl locus. Since the Leu+ Trp+ recombi- nants remain diploid, these prototrophs contain chromosomes with complete genotypes that remain undetermined. To ascertain the full genotype, random spore analysis was used (see MATERIALS AND METHODS).
The results, which are found in Tables 2 and 3, can be summarized as follows: First, the rate at which Leu+ Trp+ events arise in JG300 was only slightly less than the rate of comparable events in JG44. Therefore, the pres- ence of a plasmid insertion has little, or no, effect on the rate of coincident gene conversion at these loci. Second, the genotypes of the coincident recom- binants recovered in JG300 were similar to JG44. For example, Leu+ Trp+ events are three to five times more likely to be associated with homozygosity of markers distal to TRP5 than are Trp+ events which did not involve LEUl. Of the 53 Leu+ Trp+ events analyzed, 17 (30%) were homozygous for the CYH2, MET13 and ADE5 loci, in contrast to only 6% of the total Trp+ popu- lation. There is also a marked polarity for recovery of the leul-c allele among coincident convertants that is not found among single locus (Leu+) recombi- nants (see PLOTKIN 1978; COLIN and ESPOSITO 1981). This phenomenon was also found previously for the non-plasmid control (COLIN and ESPOSITO 1984; J. COLIN, unpublished results). Similar results were obtained in the analysis of many independent Leu+ Trp+ events described later. Third, most of the Leu+ Trp+ recombinants (68%) were Ura- and must therefore have lost the URA3 gene present in the plasmic insertion. The rest were primarily URA3lura3. URA31URA3 recombinants were rarely (1 in 53) recovered.
To verify the results obtained from this analysis, 172 independent Leu+ Trp+ recombinants were collected. One hundred twenty-five (73%) were Ura-. Ninety of the 172 were tested for their genotype at the CYH2 and ADE5 loci. A total of 23 (26%) exhibited homozygosity for these markers. Therefore, the
1086 J. E. COLIN, S. C. FALCO AND J. P. MARGOLSKEE
TABLE 2
Total Trp' Diploid Culture no. x lo5
JG300
10
10
3.5 2.7 1.7 2.2 3.5 1.4 1.3 2.2 1.7 1.5
1.7 0.2
7.1 5.1 3.6 1.9 3.1 3.2 2.9 3.0 4.2 3.7
3.2 0.5
rota1 Leu+ Total Leu+ x I O 5 Trp+ X 10'
3.3 0.1 20 0.3
3.8 0.2 2.8 0.2 3.4 0.2 2.3 0.4 2.5 0.1 3.4 0.5 2.4 0.2 2.4 0.2
2.8 0.2 0.4 0.1
20 1.4 14 0.6 5.3 0.6 2.2 0.3 3.5 0.3 3.9 0.5 3.8 0.2 3.4 0.6
10 0.2 5.2 0.1
3.9 0.3 0.5 0.2
4.1 6.8 9.0
11 4.5
behavior of the independent recombinants and those derived from culture are very similar.
Coincident gene conversion was also examined in JG400. This strain is hem- izygous with respect to pJM53, but the insertion is in the other homologue (Eeul-c, pJM53, trp5-c). As was the case with JG300, the presence of pJM53 had little, if any, effect on the rate of coincident Leu+ TrpC recombinants (data not shown). The distribution of URA3 genotypes is, nevertheless, mark- edly different. Of 60 independent Leu+ Trp+ recombinants analyzed, only ten (-16%) were Ura-. Of the remainder, 38 (75%) were URA3/ura3, while 12 (25%) were URA3/URAJ. The basis for the difference between JG300 and JG400 remains unclear at the present time.
Analysis of recombination in strains homozygous for an insertion: The pJM53 insertion is lost during coincident gene conversion in homozygous (JG302) diploids. Coincident Leu+ Trp+ recombinants arise in JG302 at about the same frequency that they do in the hemizygous strains (data not shown). Tetrad analysis of 57 independently derived coincident convertants revealed that 35 (-61 %) were either LJRA3/ura3 or ura3/ura3 (Table 4) and had there- fore lost one or both copies of the plasmid.
COINCIDENT GENE CONVERSION IN YEAST
TABLE 3
1087
Genotype at pJM53
- ura3 URA3 uRA3 Total Allele recovery - - ura3 ura3 um3 observed (no.) Genotype at L e d , TRPS
1. LEUl leul-c
1 leu-I2 (5)
TABLE 4
URA3 genotypes of Leu' Trp+ events collected from a pJM53 homozygote
ura3 an a 1 y z e d URA3 ura3 ura3
- - - No. of events LIRA3 URA3
57 22 28 7
Analysis of putative Ura+ revertants: A striking feature of Leu+ Ura- re- combinants is the appearance of nearly confluent growth on uracil omission media after 48-72 hr of incubation. When such material was streaked out on YPD (nutrient) media and the subclones tested for their phenotype, all were Leu+ Ura+ Trp+. When the original Leu+ Trp+ Ura- colony was streaked and tested, however, less than 0.1% of the subclones were Ura+ (see rate deter- mination data described below). Southern hybridization analysis of DNA pre- pared from single colony subclones of the initial Ura- recombinant and sub- sequent revertants demonstrated that pJM53 was absent in the former, but
1088 J. E. COLIN, S. C. FALCO AND J. P. MARCOLSKEE
1 2 3 4 5 6
FIGURE 3.-Hybridization analysis of Ura+ revertants. To determine whether a Ura+ revertant is due to the presence of integrated pJM53, Southern blot hybridization was performed on Leu+ Trp+ recombinants. The initial Ura- Leu+ Trp+ colony and subsequent Ura+ revertants were streaked on YPD media to obtain single colony isolates. These were used as a source of DNA. The DNA was cut with Xhol. an enzyme making a unique cut within the 6.1-kbp yeast fragment. The presence of an intact plasmid in the original JG300 strain (lane 1) and two revertants (lanes 3 and 5 ) is shown. The initial Ura- Leu+ Trp+ recombinants (lanes 4 and 6) have a hybridization pattern identical to the isogenic, non-plasmid-bearing control strain JG44 (lane 2).
TABLE 5
Revertant PD NPD TT
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10
14 12 15 21 20
5 18 17 10 29
0 0 0 0 0 8 0 0 0 0
0 1 1 0 2 5 2 1 0 4
a PD = parental ditype, NPD = nonparental ditype, TT = tetratype. For any cross, an excess of PDs us. TTs and NPDs indicates linkage.
present in the latter (Figure 3). The hybridization patterns of the revertants were identical to those obtained from the initial JG300 (pJM53 hemizygote) diploid. The map position of the putative Ura+ revertants derived from ten independently isolated Leu+ Trp+ recombinants was determined. To do this, single colonies isolated from revertant patches were sporulated, and the re-
COINCIDENT GENE CONVERSION IN YEAST
TABLE 6
1089
Total UraC Cfu/culture Culture x 1 0 5 x 10-7
1 1 . 1 2.3 x 107 2 20 1 . 1 x 107 3 1.3 0.9 x io7 4 0.5 7.4 x 107 5 15 5.4 x 107 6 22 1.4 x 107 7 9.3 3.9 x 107 8 3.3 4.7 x 107 9 110 2.3 x io7
Median frequency 3.3 Median rate 0.5
sulting tetrads were dissected. The results, shown in Table 5, demonstrate tight linkage of URA3 to LEU1 in nine of ten cases analyzed.
The rate of reversion from Ura- to Ura+ was also determined. T o do this, Leu+ Trp' Ura- colonies were suspended in 1 ml of YPD media and were allowed to grow overnight before harvesting and plating. The results, presents in Table 6, indicate a rate of about one Ura+ revertant/105 cell-forming units. This suggests that reinsertion takes place after an initial lag of over ten gen- erations.
DISCUSSION
The data presented in this paper can be summarized as follows. First, the presence of a large region of nonhomology (the plasmid insertion pJM53) has little or no effect on the frequency of coincident gene conversion events in- volving loci flanking both sides of the insertion. Second, in hemizygous strains, the plasmid insertion is lost (resulting in a Ura- phenotype) at high frequency (70%) from Leuf Trp+ recombinants when it is between the leul-12 and trp5- d alleles, but at a fourfold lower frequency when it is inserted between leul-c and trp5-c. Third, the plasmid is also lost at high frequency from diploids which bear it in homozygous configuration. Finally, reversion from a Ura- to a Ura+ phenotype frequently occurs. Genetic mapping and DNA hybridization experiments indicate that the Ura+ revertants result from restoration of pJM53 at its original location.
Two models have been put forth to account for the high frequency, distance- dependent coincident mitotic gene conversion of widely separated genes in yeast (GOLIN and ESPOSITO 1984). The first proposes that co-conversion is due to a single long heteroduplex recombination intermediate, whereas the second proposes that it is due to multiple concerted recombination events following some rate-limiting step, such as pairing of homologous chromosomes. These models can now be considered in light of the observations presented above.
LICHTEN and Fox (1983) found that regions of nonhomology bracketing a
1090 J. E. GOLIN, S. C. FALCO AND J. P. MARGOLSKEE
0
1
'T-- 2
I - > FIGURE 4.-A model to explain the behavior of pJM53 in coincident recombination events.
The figure shows the first of two mechanisms proposed to account for the behavior of pJM53 in coincident recombination events involving the LEU1 and TRP5 loci. The figure illustrates plasmid loss by excision of a single-stranded loop from a symmetric heteroduplex at the two-strand stage of mitosis. In this diagram, the plasmid is drawn with the duplicated yeast chromosomal region indicated as - and the YIp5 sequences as - - -. Each line represents a single polynucleotide chain. The "+" indicates the URA3 gene present in YIp5. If the loop is excised with great preference, the -/- class could be found in excess, whereas the +/+ class would be comparatively rare.
genetic interval on bacteriophage X reduce recombination in that interval, particularly in replication-restricted crosses. This observation is consistent with the idea that the nonhomology prevents branch migration of a Holliday-type heteroduplex recombination intermediate into the interval. However, in the same study, evidence from multifactor crosses carried out under replication- permitted conditions suggested that nonhomologies could be included in re- gions of heteroduplex DNA. In yeast, most mitotic gene conversion occurs before DNA replication (ESPOSITO 1978; FABRE 1978; COLIN and ESPOSITO 1981; ROMAN 1980; ROMAN and FABRE 1983). Thus, it was of interest to investigate the effect of a large nonhomologous insertion on coincident gene conversion of flanking markers. The observation that the nonhomology had little or no effect on the frequency or character of coincident conversion events indicates either that a nonhomology can be included in a region of heterodu- plex DNA in yeast or that long heteroduplexes are not the source of the coincident conversions.
The frequent loss of the plasmid insertions from hemizygous strains can be accommodated by either the long heteroduplex model or multiple concerted recombination events model. The long heteroduplex model could account for the Ura- events because the creation of extensive heteroduplex DNA spanning the LEUl-TRP5 region would result in a single-stranded loop that might be preferentially excised from the JG300 diploids (Figure 4). BENZ and BURGER (1973) proposed such a mechanism to account for selective loss of the wild- type allele from crosses between wild-type and rZZ-deletion mutants of bacte-
COINCIDENT GENE CONVERSION IN YEAST
1
1091
1
tion by intrachromosomal recombination. figure illustrates loss of the plasmid inser-
riophage T4. No such process has been observed previously in yeast, however. The multiple events model could explain loss of the plasmid insertion by a separate, but associated, intrachromosomal event: homologous recombination between the direct repeats (Figure 5) . There is ample precedent for the latter mechanism in yeast (SCHERER and DAVIS 1979; ROEDER and FINK 1980), al- though association of intrachromosomal and interchromosomal events has not been reported.
The frequent loss of the plasmid insertion among Leu' Trp+ recombinants from an insertion homozygote is more difficult to explain within the context of the long heteroduplex model. Figure 6 illustrates how misalignment of the insertion could lead to a series of single-stranded loops. One or both of these could be cleaved to give recombinants. Misalignment of the insert on both chromosomes is required to produce a ura3/ura3 recombinant. The fact that the plasmid is lost from the homozygous diploid as frequently as from the hemizygous diploid requires that the misaligned structure drawn in Figure 6 occurs more frequently than the properly aligned structure. This seems un-
1092 J. E. COLIN, S. C. FALCO AND J. P. MARGOLSKEE
1 -
FIGURE 6.-Loss of plasmid in a homozygote by misalignment. The figure illustrates how pJM53 could be lost from a homozygote strain by a mechanism analogous to that proposed in A. Misa- lignment of direct repeats in a heteroduplex could create two single-stranded loops. Preferential excision would lead to cells which are either Ura-/Ura- or Ura+/Ura-.
likely. The multiple events model can explain loss of the plasmid from the homozygous diploid by the same intrachromosomal recombination mechanism as proposed for the hemizygous strain.
Reversion from Ura- to Ura+ by restoration of pJM53 to its original chro- mosomal location is also more difficult to explain by the long heteroduplex model. It must be proposed that a precisely excised single-stranded DNA re- gion either persists and reintegrates by single-strand assimilation (RADDING 1982) or replicates and reintegrates later through a double-stranded interme- diate. The multiple events model proposes that the excised double-stranded circle persists and is later reintegrated by a reversal of the excision reaction.
Thus far, we have assumed that most mitotic gene conversion occurs before DNA replication. Genetic analysis clearly indicates that this is the case (ESPOS- ITO 1978; FABRE 1978; GOLIN and ESPOSITO 1981; ROMAN 1980; ROMAN and FABRE 1983). This interpretation can be extended to coincident convertants with two important reservations. First, genotypic analysis of coincident recom- binants to date cannot rule out a GP contribution. Second, recent evidence suggests that although X-ray-induced gene conversion occurs at the two-strand stage, associated intergenic exchange may be completed following DNA repli- cation (ROMAN and FABRE 1983; ROMAN 1984). This could provide an alter- native explanation for the high frequency loss of the plasmid insertion from a long heteroduplex recombination intermediate. A major difference between the two- and four-strand stages is the presence of sister chromatids in the latter. If coincident conversion in long heteroduplexes was associated with high frequencies of unequal crossing over between the direct repeats of sister strands, Ura- recombinants could be produced. Although sister chromatid
COINCIDENT GENE CONVERSION IN YEAST 1093
exchanges of this type have been reported in yeast, they appear to be relatively rare (JACKSON and FINK 1981). In addition, such a mechanism for generating Ura- recombinants does not account for the frequent reversion to Ura+ ob- served.
In summary, the data presented in this paper do not eliminate the long heteroduplex model as an explanation for coincident mitotic gene conversion events. However, a model proposing multiple associated recombinant event appears to better fit the data. The analysis of recombination in strains bearing insertions which do not have direct repeats is a critical test of the models. The long heteroduplex model predicts that these will also be lost with high fre- quency during recombination events involving LEU1 and TRP5 when the in- sertion is present in hemizygous configuration. In contrast, the multiple events model predicts increased stability because intrachromosomal recombination be- tween direct repeats cannot occur.
We thank DEBBIE CHALEFF, IRA HERSKOWITZ, BOB LAROSSA, HERSCHEL ROMAN and especially FRANK STAHL for their incisive comments on several drafts of this manuscript. Work at the University of California, San Francisco, on the construction of pJM53 was supported by National Institutes of Health research grants to IRA HERSKOWITZ.
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