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Copyright 0 1996 by the Genetics Society of America

The Efficiency of Meiotic Recombination Between Dispersed Sequencesin Saccharomyces cerevkiae Depends Upon Their Chromosomal Location Alastair S . H. Goldman and Michael Lichten Laboratory of Biochemistry, Division of Basic Sciemes, National Cancer Institute, National Institutes of Health, Bethesda, Ma?yland 20892 Manuscript received March 11, 1996 Accepted for publication June 10, 1996

ABSTRACT To examine constraints imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARM gene containedin pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, butalso are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb)was significantly greater than thatobserved with inserts >50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.


HE segregation of homologous chromosomes during the reductional divisionofmeiosis requires that their position within the nucleus be fixed with respect to each other. Inmost organisms, this positioning is achieved through the processes of chromosome pairing and synapsis, withreciprocal recombination (crossovers) creating cytologically visible joints between homologues. Both homologue pairing and recombination are of fundamental importanceto the outcomeof meiosis, but it is not clear exactly how they relate to each other. For example, it remains unknown whether the relative position of homologues affects their ability to recombine, or on the other hand, if recombination effects the relative positioning of homologues. One of the difficulties in relating recombination to the homologous pairing of chromosomes during meiosis is that both functions involve multiple and continuous steps, at least some of which remain unknown. For the purposes of discussion we would like to define chromosomes as being in one of four states (Figure 1, a-d) : unpaired, when homologous chromosomes are distrih uted randomly throughout thenucleus; loosely associated, when one ormore regions of chromosomes are nearer to each other than expected by random distribution; paired, when chromosomes are in end to end alignment and close to each other over their entire length; spapsed, when chromosomes are separated, along their Curresponding authur: Alastair S. H. Goldman, Building 37 Room 4Dl1, National Institutes of Health, 37 Convent Dr. MSC 4255, Bethesda MD 208924255. E-mail: [email protected] Genetics 144: 43-55 (September, 1996)

plane of contact, only by the synaptonemal complex (SC) , which has a width of a 1 0 0 nm (see SYM et al. 1993). Some cytologicalstudies have suggested that homologous chromosomes first organize into pairs in somatic cells before the onsetof meiosis ( DLAZand LEWIS1975; THERMAN and SARTO 1977; BENNETT 1984; LOIDL 1990). In situ hybridization studies in fission yeast and budding yeast have lent support to theidea that homologues are atleast loosely associatedbefore the onset of meiosis ( SCHERTHAN et aZ. 1994; WEINERand KLECKNER 1994). This relationship appears to be transient. By the end of meiotic S-phase or at the onset of first meiotic prophase, homologous chromosomes are distant from each other in a large proportion of cells (GOLDMAN and HULTEN 1992; SCHERTHAN et ai. 1992; WElNER and KLECKNER 1994). Thus, for homologues to be paired and synapsed in all meiotic cells, a reassociation must take place during the first meiotic prophase. Molecular studies of meiotic recombination in yeast havefocusedprimarily on DNA double-strand breaks ( DSBs ) , the earliest identifiable molecular events required for recombination ( SUN et al. 1989; GAO et al. 1990) . DSB formation is shortly followedby the creation of joint DNA molecules (COLLINSand NEWLON1994; SCHWACHA and KLECKNER 1994,1995) and subsequently by the appearance of heteroduplex DNA and mature recombination products ( PADMORE et al. 1991; GOYON and LIGHTEN1993; NAGand PETES1993; COLLINSand NEWLON 1994). How do these recombination-associated

A. S. H. Goldman and M. Lichten


Loosely associated











Allelic recombination

Ectopic recombination between homologues -



Ectopic recombination between heterologues RCURE1.-We have considered four states of spatial relationship between homologues (a-d) andmeasured meiotic recombination between sequences in three configurations (eg ) . ( a ) Unpaired: before the onset of first prophase pairing, chromosomes could be randomlydispersed throughout the nucleus, there being no special spatial relationship between homologues. ( b ) Loose4 associated: homologous chromosomes are more likely to be closer to each other than any specific heterologue but are not aligned end to end. (c) Paired: homologous chromosomes have moved closer to each other and lie in pairs, aligned end to end. ( d ) Synapsed: the pairing process is complete and homologues are separated by "100 nm. ( e ) Allelic recombination was measured between sequences present at identical locations on homologues. ( f and g ) Ectopic recombination was measuredbetween sequences presentat nonidentical locations, either on homologous chromosomes ( f ) or on heterologous chromosomes ( g ) .

molecular events relate to the loose associationof homologous chromosomes, pairing and synapsis? There is good evidence that recombination is initiated before SCs are fully formed. Temporal studies of synchronized yeast cultures show that by the time synapsisis complete, DSBs are disappearing from the population ( PADMORE et al. 1991). In addition, studies of meiotic mutants indicate that substantial levels of meiotic recombination can take place in the absence of mature SC (ROCKMILL and ROEDER1990; SYM et al. 1993). The relationship between the intermediate stages of chromosome pairing and recombination is less clear. Before the time of DSB formation, and in some DSB-

deficient mutants, segments of homologous chromosomes can be seen close to each other (WEINER and KLECKNER 1994). Thusthe initiation of homologue pairing appears to be independent of the first known molecular steps of meiotic recombination. Finally, the mature products of recombination do not appear until after chromosomes are paired and mostlikely not until synapsis is complete ( PADMORE et al. 1991; GOYON and LICHTEN 1993). If homologous chromosomes are to find each other with accuracy and maintain a tight association throughout the DNA recombination process, then one problem that must be overcome is presented by dispersed homologous sequences. Recombination between dispersed repeats (ectopic recombination)can produce lethal rearrangements and has the potential to disrupt homologue association and segregation. Protection against ectopic recombination is probably achieved, in part, bymismatch repair functions that reduce recombination between diverged sequences ( SELVAet al. 1995; DATI'Aet al. 1996). However, there are numerous examples of meiotic recombination between nondiverged dispersed sequences in mammals (MURTI et al. 1994) and yeast ( ~ E etRal. 1991;JINKSROBERTSON and PETES 1986; LICHTEN et al. 1987; NAGand PETES1990), andit may even bea useful evolutionary strategyinPlasmodia UANSE and MONS 1992; DE BRUINet a/.1994; HINTERBERG et d . 1994). To address the issue of the importance of chromosome pairing formeiotic recombination, it is worth considering whetheror not ectopic recombination is mechanistically distinguishable from recombination between identically located sequences (allelic recombination ) . Ectopic recombination between artificial inserts in the yeast genome can occur at frequencies approaching those seen for allelic recombination, and asimilar proportion of ectopic and allelic events produce areciprocal exchange (JINKS-ROBERTSONand PETES 1986;LICHTEN et al. 1987; HABER et al. 1991) . In addition, both ectopic and allelic recombination involve the formation of heteroduplex DNA ( LICHTENet al. 1990; NAG and PETES1990). Finally, none of the meiotic mutants that have been suitably examined affect ectopic interchromasomal recombination differently from allelic recombination ( STEELE et al. 1991) . If there are truly no differences between allelic and ectopic interchromasomal recombination, then this wouldimply that the loose association, pairing and synapsis ofhomologues do not influence the ability of sequences to find each other and recombine. We have undertaken a study ofectopic recombination designed to examine the differences between locating dispersed sequences in the context of meioticallypairing chromosomes ( i e . , on homologues) us. locating them on chromosomes that are not expected to pair during meiosis ( ie., on heterologues) ( see Figure 1, e-g) . We find that ectopic recombination between sequences located on homologues is significantly more efficient


Ectopic Recombination in Meiosis

TABLE 1 Diploid yeast strains Diploid strain

MJLl654/55 MJL1808/09 MJL1729/31 MJL1762/63 MJL1758/59 MJL1604/05 THRl MJL1844/45 MJL1806/07 MJLl242 MJL1241/1374/75 MJL1239 MJL1770/71 MJLl747/48 MJL1610/11 MJL1703 MJL1836/37 MJL1240 MJL1812/13 MJL1652/1369/70 MJL1797/98 MJL1783/84 MJL1775/76 MJL1635/36 MJLl708 MJL1832/33 MJL1810/11 MJL1729/30 MJL1648/49 MJLl799/ 1800 MJLl787/88 MJLl779/80 MJL1637 MJL1840/41 MJL1510a/b MJL1760/61 MJLl768/69 MJL1781/82 MJL1785/86 MJL1737/38 MJL1739/40 MJL1764/65 MJL1793/94 MJL1819

Diploid strain

arg4-bgl arg4-nsp

CHAl CHAl CHAl CHAl CHAl CHAl CHAl CHAI HIS4 HIS4 HIS4 HIS4 HIS4 HIS4 HIS4 HIS4 HIS4 LEU2 LEU2 LEU2 LEU2 LEU2 LEU2 LEU2 LEU2 LEU2 MA Ta MA Ta LEU2 MA Ta MA Ta MA Ta MA Ta MA Ta MA Ta pH01 1 pH01 1 pH01 1 pH01 1 pH01 1 pH01 1 pH01 1 pH01 1 pH01 1

MJL1756/57 MJLl745/46 MJL1773/74 MJLl777/78 MJLl741/42 MJL1743/44 MJL1749/50 MJL1789/90 MJL1821 MJLI606/07 MJL1608/09 MJL1701 MJL1645 MJL1705 MJLl642 MJLl766/67 MJL1751/52 MJL1825/6 MJL1827/8 MJL1599/1600 MJL1713/1714 MJL1656/57 MJL1711 MJL1643/44 MJL1842/43 MJL1834/35 MJL1830/31 MJL1838/39 MJL1795/96 MJL1791/92 MJLl658/59 MJLl709 MJL1646/47 MJL1802/03 MJL1650/51 MJL1804/05 MJL1509a/b MJLl818 MJL1820 MJL1639/40 MJL1822/23

HIS4 LEU2 MA Ta pH01 1 pH012 > PUT2

URA3 CHAl LEU2 MA Ta pH01 1 pH01 2 < PUT2 > PUT2 THRl


CHA1 HIS4 MA Ta pH01 1 pH01 2 < PUT2 PUT2 THRl


CHAl HIS4 pH01 1 pH012 < PUT2 THRl


CHAl HIS4 LEU2 MA Ta pH01 1 pH01 2 > PUT2 THRl URA3

arg4-bgl arg4-nsp

pH012 pH01 2 pH01 2 pH01 2 pH01 2 pH01 2 pH01 2 pH012 pH012 > PUT2 < PUT2 > PUT2 < PUT2 > PUT2 < PUT2 > PUT2 > PUT2 < PUT2 > PUT2 < PUT2 > PUT2 < PUT2 > PUT2 > PUT2 THRl THRl THRl THRl THRl THRl THRl THRl THRl

CHAl hi54

1eu2 MA Ta pH01 1 pH01 2 > PUT2 THRl C’RA3 CHAl h154 h154

1eu2 1eu2

MA Ta pH01 1 pH01 2 > PUT2 < PUT2 < PUT2 > PUT2 THRl THRl URA3 CHAl h154 1bu2


MA Ta pH01 1 pH01 2 < PUT2 > PUT2 THRl CHAl



uRA3 uRA3 uRA3



1eu2 MA Ta pH01 1 pH01 2 < PUT2 THRl


The location of arg4-nspand arg4-bglplasmidinserts are indicated. Haploid parentsof these diploidswere made by transforming S95 (MATa ura3 lys2 ho::LYS2 leu2-K arg4-nsp, bgl) with arg4-bgkontaining plasmids and by transforming S105 (MATa ura3 lys2 ho::LYS2 leu2-R arg4-nsp, bgl) with arg4-nspcontaining plasmids. Open arrows indicate the orientation of inserts located at PUT2, with arg4 being transcribed either toward (>) or away from (8.5 kb of homology between otherwise unrelated loci. In all cases, the normal ARCA locus contained the nrg4-nsp,bgZdouble mutation and was thus prevented from contributing detectable ARG4 recombinants. ( b ) Loci used as insertion sites for the nrg4 mutant alleles. Arrows indicate insert orientation in terms of the direction of nrg4 transcription. For the purposes of analysis, inserts at least 50 kb from thenearest telomere were classified asinterstitial, and inserts 16 kb or less from the nearest telomere were classified as subtelomeric.

370 kb


- PHO11 12

10 kb





at a locus reflects the relative frequency at which lesions that initiate meiotic recombination occur within a locus (see W U and LICHTEN1995). Furthermore, we hypothesize that the frequency of ectopic recombination with arg4-nsp at locus 1 and arg-4-bgl at locus 2 can be expressed as

h . 1,2 (Arg+) =

E,,,(j,,,,,(Arg+)C,,+fne,o:!(Arg+)Cd, ( 2 )

and for arg4-nsp at locus 1 and arg4-bgl at locus 2 j , 2 , 1 i l ( A r g += )

El,djk,p.,dArg')Gt +j,l,61(Arg+)G7),). ( 3 )

referred to subsequently as the efficiency of ectopic recombination, is a term that reflects the propensity of locus 1 and locus 2 to encounter each other and recombine, relative to the case of allelic recombination. In the case of allelic recombination at locus 1 or locus 2 , El,,

= G.2 =



Summing ( 2 ) and (3) and solving for E,,,, we obtain E1.2




J t l . 6 > ( ~ g ++fn;),bl(Arg+) ) ( Arg + ) + fn2, ( Arg ) c,


+ [email protected]+)


+ hi.61 (Arg')




which reduces to G . 2 =

f?ll.b~~(ATg+) + J n . r , ~(Arg+) f,l.61(Arg+) +hP./,:!(kg+)'


This formula was used to calculate the efficiencies of ectopic recombination reported in Table 4. Statistical analysis comparing the mean values of efficiencies of ectopic recombination was carried out using the Mann-Whitney U-test.


To determine the importance of chromosome pairing context on the ability of sequences to recombine with each other during meiosis, we examined meiotic recombination between arg4-nsp and arg4-bgl mutant alleles inserted at various places in the yeast genome. The arg4 heteroalleles were inserted into target loci by integrating a pBR322-based plasmid that contained a 3.3-kb arg4 fragment, along with a 1.2-kb URA3 fragment for selection of transformants (Figure 2a; WU and LICHTEN1995). Nine insert loci were used, located on chromosomes Z, ZZZ, Vand VZZI (Figure 4 b ) . These were classified as being eitherinterstitial ( >50 kb from each telomere)or subtelomeric (within 16 kb of a telomere) . Recombination was measured in diploids containing two inserts positioned either identically on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination) . Recombination frequencies were calculated by measuring t h e frequency of Arg' colonies. Inall crosses, the normal ARC2 locus contained the nsp,bgl double mutation, thus preventing it from contributing detectable recombinants. For analysis, the ectopic combinations were divided into five groups. Two ofthese involved ectopic recombination between inserts located on homologous chromosomes: in the first group, only interstitial inserts were used (interstitial X interstitial) ; in the second group, one interstitial and one subtelomeric insert was used (interstitial X subtelomeric) . The three other groups


A. S. H. Goldman and M. Lichten

TABLE 2 Ectopic recombination frequencies

Chromosome and insert locus nsp + bgl I






> CHAl
























>pH012 >PUT2 THRl

0.68 0.37 ND 0.22

0.81 ND 0.43 0.24

ND 2.1 5.6

0.53 2.2 4.5



>pH011 I 111



0.91 1.33 1.2 0.61






1.0 0.98 0.86 0.85

0.69 0.81 0.60 0.49

0.27 ND 0.27 0.46

0.20 ND 0.21 0.34



0.44 ND 0.55




2.0 2.2

Mean frequencies of ARG4 recombinant spores(XlO’) are given for each combinationof inserts and markers.arg4-nsp insert locations (locus and chromosome) aregiven at the top of the table; arg4-bgl insert locations are given at the left. Open arrows indicate the orientationof each insert,with arg4 being transcribed either toward (>) or away from (50 kb from a telomere, sequences located at dispersed loci on homologous chromosomes are more likely to recombine during meiosis than sequences located on heterologous chromosomes. The efficiency of recombination between sequences dispersed on homologous chromosomes is dependent


Recombination Ectopic

(Int x Int) C Segmgatlon






. . e



(Tel x Tel)

FIGURE 4.-Detection of translocations amongst ARG4 recombinants by pulsed-field gelelectrophoresis. Chromosomes are displayed by ethidium bromide staining ( a and b ) or hybridization of blots with either PH012- or CHAl-specific DNA probes ( c ) . ( a ) The only two viable ARC4 karyotypes recovered from diploids with arg4 inserts at LEU2 and URA3 were either normal (no associated crossover) or balanced (with an associated crossover). The latter contained both III" and V" translocationchromosomes. In this diploid there were essential genes distal to both insert loci. Therefore all unbalanced translocation karytotypesare inviable. Out of 100 ARC4 recombinants examined, one contained 3:l nondisjunction segregation products, with normal chromosomes IIZ and Vas well as a translocation chromosome, III" ( b ) Three viable karyotypes were recovered from diploids with arg4 inserts at LEU2 and PH012. These were normal, balanced and unbalanced (both with an associated crossover). The latter contains a normal chromosome III and a translocation chromosome, WII". This unbalanced karyotype hasa duplication of chromosome III material distal to LEU2 and a deletion of chromosome WII material distal to PH012. There are no essential genes in the deleted segment. ( c ) Four viable karyotypes were recovered from diploids with [email protected] inserts at CHAl and PH012. These were normal, balanced and the two expected unbalanced karyotypes, containing either a normal chromosome WII and a translocation chromosome, III"'If or a translocation chromosome WII'" and a normal chromosome, HI. Both unbalanced karyotypes are viablebecause there are no essential genes distal toeither CHAl or PH012. Crossing over between inserts at CHAl or pH012 changed chromosomes sizesby only 6 kb, thus hybridization with pH012 and CHAl sequences was necessary to determine which chromosomes were normal or translocated. Because the probes used contained locus-specificsequences from both sides of the insert, 3:l nondisjunction segregation products would not be detected by this analysis.



upon the physical distance between insert loci: If homologous chromosomes are paired, i e . , in end to end alignment, the distance between a sequence on one homologue and a different sequence on the second homologue will be related to the linear physicaldistance between the two loci. If recombination occurs after homologues are aligned end to end,then sequences inserted on different homologues at nearby loci should be able to find each other and recombine more often than sequences inserted at distant loci. Consistent with this, the efficiencies of ectopic recombination between all inserts on homologues covered a broad range (from 0.99-0.16), with a general trend towards reducing the efficiency of ectopic recombination with increasing separation between insert loci (Figure 6 ) . The efficiency of ectopic recombination between the two closest loci used ( HZS4and LEU2, separated by -17 kb) was 0.95; at the other extreme, the efficiency of ectopic recombination for the two most distant loci used (pH012 and THRI; separated by -370 kb) was 0.17. These results are consistent with the suggestion that by the time most ectopic recombination takes place, homologous chromosomes are already paired, i e . , aligned end to end. Locating both inserts close to telomeres increases the efficiency of ectopic recombination: Ectopic recombination was also measured in three combinations of inserts at subtelomeric loci on heterologous chromosomes. The mean efficiency of ectopic recombination for these was 0.26, with a range of 0.24-0.28 (Figure 5; Table 4).This represents a modest but significant twofold increase over the efficiency of ectopic recombination calculated for both interstitial X interstitial and interstitial X subtelomeric inserts on heterologous chromosomes ( P < 0.01 ) . DISCUSSION

We have measured the frequencies of meiotic ectopic recombination between five different groups ofdispersed sequences. To facilitate comparison, these recombination frequencies were converted into ectopic recombination efficiencies. This conversion takes into account three potential sources of variation: position effects, marker configuration, and the loss of ectopic recombinants due to associated crossing over. This conversion reduces the apparent differences both within and between the five groups (compare the values in Table 2 with those of Table 4 ) , and thus leads to a conservative estimate ofhow the relative position of inserts influences their ability to recombine with each other. An examination of the efficiencies of ectopic recombination between all inserts located on heterologous chromosomes indicates that these dipersed sequences are three to 16 times less likely to recombine than sequences in an allelic arrangement. This is consistent with the finding OfJINKs-ROBERTSONand PETES (1986)


A. S. H. Goldman and M. Lichten

TABLE 4 Efficiencies of ectopic recombination ~~


Chromosome and insert locus _______





>pH01 1




> CHAl

















0.56 0.95

0.21 0.18 0.13 0.12

0.28 0.21 0.13

ND 0.18 0.11 ND0.15

0.14 0.17 0.10 0.087

0.068 0.12 0.067







0.17 0.47 0.99






> CHAl > HIS4 > LEU2 < MAT

0.27 0.17 0.11 0.062

0.62 0.42 0.20

0.95 0.27


0.40 0.55 0.48 0.15


> URA3



0.1 1




>pH012 > PUT2 < PUT2 > THRl

0.24 0.073 ND 0.049

0.28 ND 0.068 0.050 0.055

0.17 0.12 0.085 0.13

0.10 ND 0.068

0.074 ND 0.054 0.074


0.052 0.12 ND 0.13

0.029 0.053

These values were calculated as described in MATERIALSAND METHODS.Values below the diagonal represent ectopic recombination efficiencies for each insert pair are calculated from the uncorrected recombination frequencies reported in Table 2; values above the diagonal are corrected to account forloss of recombinants due to the unbalancedsegregation of translocations (see MATERIALS AND METHODS). ND, not done.

that ectopic recombination between uru3 sequences on heterologues is 17 times less frequent than allelic recombination for uru3 at its normal locus. By contrast, ectopic recombination between sequences located on homologous chromosomes can be as efficient as allelic recombination, or at worst six times less efficient. Furthermore, theefficiencies of ectopic recombination for inserts located on homologues is related to the physical distance between insert sites. Ectopic recombination between the closest loci used (separated by = 17 kb) is as efficient as allelic recombination, whereas for the two most distant loci used (separated by -370 kb) , the efficiency of ectopic recombination is n o greater than




5 0.25 G




M Int x Int Int x Int Homo Hetero Homo


Tel Int x Tel Tel Tel Hetero Hetero



0.80.6 -

Type of Cross

0.4 FIGURE5."Summary of the ectopic recombination efficiencies for the five different insert configurations. Int denotes interstitial inserts; Tel denotes subtelomeric inserts. In groups designated Homo theinserts were located on homologous chromosomes; in those designated Hetero inserts were located on heterologous chromosomes. Bars show the mean recombination efficiency for each group; thecircles represent ectopic recombination efficiencies obtained for each insert site combination. For both Int X Int and IntX Tel, the mean ectopic recombination efficiencies for the Homo group are significantly higher than therespective efficiencies in theHetero group ( P < 0.005 and P < 0.05, respectively). The mean ectopic recombination efficiency for the Tel X Tel group is significantly higher than those of both Int X Int (Hetero) ( P < 0.05) and the Int X Tel (Hetero) ( P < 0.05).



0 0



0 0 0

1 4 50 100 150 200 250 300 350 400 450 Distance between insert loa (kb)

FIGURE6.-The relationship between the efficiency of ectopic recombination between inserts on homologues and the distance, in trans, between insert sites. Circles represent the ectopic recombination efficiencies obtained for each insert site combination. The dotted line represents the mean efficiency of ectopic recombination seen forinserts on heterologous chromosomes inwhich at least one insert locus was interstitial.

Ectopic Recombination in Meiosis

that observed for some combinations of inserts on heterologous chromosomes (see Figures 5 and 6 ) . It has previously been reported that the location of sequences does not influence theirability to recombine with each other during meiosis ( HABER et al. 1991) . However, this and a similar study ( LICHTEN et al. 1987) used inserts located primarily on chromosome IIZ homologues. In the few crossesof these studies where ectopic recombination was measured between inserts at the URA3 locus on chromosome Vand chromosome ZIIloci, the frequency of recombination was lower than that foundfor crosses withboth inserts on chromosome ZZZ homologues ( LICHTENet al. 1987;J. E. HABER and W.-Y. LEUNG,personal communication ) . Analysisof their data shows that there was a trend of reducing ectopic recombination frequencies with increasing physical distance between insert loci on homologous chromsomes ( LICHTEN et al. 1987; HABER et dl. 1991) . The data we present here show that the efficiency of ectopic recombination between sequences located on homologues is consistently higher than that found between sequences located on heterologous chromsomes. This implies that, at the time ectopic recombination intermediatesform, homologous chromosomes are more likely to encountereach other thanany one heterologous chromosome. This, in turn, suggests thata large proportion of ectopic events happen after homologous chromosomes are atleast loosely associated. Such loose but specific associations would increase the local concentration of dispersed sequences relative to the low local concentration expectedfrom random nuclear distribution of chromosomes. Further conclusions can be drawn from the observation that the efficiency ofectopic recombination between sequences on homologues is reduced as the physical distance between insert loci increases.This implies that homologues are not just loosely associated,but that thereis a specific orientational relationship between them when most ectopic recombination takesplace. We interpret this to mean that homologous chromosomes are already in end to end alignment (i.e., fully paired as in Figure I C )when most ectopic joints first become irreversible. How do homologouschromosomescometobe paired by the time most ectopic recombination takes place? Pairing p-eceedsrecombination: It has been suggested that during early first prophase of meiosis, successive and reversible chance encounters between allelic sequences bring homologous chromosomes closer together ( KLECKNER and WEINER1993). Over time these interactions would colocalize and align homologues, their spatial relationship being fixed later by irreversible recombination events (Figure 7a). Such chance encounters couldalso arise between inserts on heterologous chromosomes, but theywould be relatively few in number compared to allelic interactions along the entire length of homologues. The forces of multiple interactions pulling homologues together might enhance the chances of ectopic interaction be-


Phase 1

Phase 2

Phase 3

Phase 1

Phase 2

Phase 3

FIGURE7.-Possible mechanisms of chromosome pairing (sister chromatids not shown). ( a ) Different unstable interactions (vertical dashes) between homologues gradually draw them together (phases 1 and 2). so that by the time recomhination takes place (crosses, phase 3) homologues are colocalized and aligned end to end. In this model the relative concentration of inserts (boxes)located on homologues is expected to be significantly higher than that of inserts located on homologous chromosomes. (b) Irreversible recomhination events (crosses) drive chromosomes together throughout the pairing process (phases 1 to 3). Initially all types ofrecombination events might be equally likely (phase 1). As more allelic interactions take place, and homologues are pulled together, nonrecombinant insert? on homologues might he more likely to make contact and recombine (solid cross hetween boxes, phase 2) than would inserts on heterologues (dashed crosses between boxes, phase 2). Finally, once homologues are fully paired, i . ~ aligned . end to end, the local concentration of nonrecombinant inserts on homologues, if not separated by too great a physical distance, would be significantly greater than between randomly dispersed heterologues.

tween inserts at close locion homologues. On the other hand, the same forces and the effects of chromosome condensation could pull apart both inserts on heterologues and inserts at distant loci on homologues. Most encounters between either inserts on heterologues, or distant inserts on homologues, would be lost by the time recombination fixes the spatial relationship between homologues. Such a model would predict both the higher efficiency ofectopic recombination between inserts on homologues and the inverse relationship found between the efficiency of ectopic recombination and distance between insert loci. However, the idea that all meiotic recombination takes place after homologues are paired is difficult to reconcile with certain aspects of our data. For example, the efficiency of ectopic recombination between inserts on heterologues can be higher than 20%.Also some loci, particularly his4::arg4, have a general tendency toward higher efficiencies of ectopic recombination regardless of where the recombining partner is (see Table 4 ) . These observations are consistent with a modification of the above model. Some ectopic


A. S. H. Goldman and M. Lichten

recombination could occur earlier in the pairing process, before homologues are finally fixed in specific spatial domains by allelic recombination. Recombination at different loci might be taking place during specific programmed times throughout first prophase ( PLOTKIN1978). If this is true, then higher ectopic recombinationfrequenciescould arise fromlonger overlaps between the time windows open for two given loci to recombine. Recombination and homologue pairing are contemporaneous ments: If all meiotic recombination was contemporaneous with the process of homologue pairing, then the effects we see can be explained without invoking either a mechanistic temporal difference between ectopic and allelic recombination or a programmedorder of recombination events. During the earliest moments of homologue pairing,when the first allelic recombination events would take place, both forms of ectopic recombination would be equally likely to occur (Figure 7b, phase 1 ) . The firstallelic recombination events would have the effect of increasing the concentration of other homologous sequences nearby. This, in turn, would improve the chances of the next allelic encounter taking place, producing anew recombination event, and so on until homologue pairing and end to end alignment are achieved (Figure 7b, phases 2 and 3 ) . Each successive allelic recombination event would increase the likelihood of dispersed inserts, located at nearby loci, on homologues finding each other. Conversely,as chromosome pairs become progressively more aligned, nonrecombinant inserts at distant loci on homologues would get less of a chance to find each other. The forces of recombination and chromosome condensation would tend to pull homologues together and separate heterologues, decreasing the chances of inserts on heterologues finding each other. By such a process, sequences dispersed on homologues would, on average, have more time to recombine than sequences dispersed on heterologues. The insert loci that display relatively high efficiencies of ectopic recombination between heterologues may be more able to recombine earlier than others. Distinguishing between the alternative models described above will require a more precise knowledge of both the nature and timing of the first contacts required for meiotic homologue pairing and recombination. In fact, the two pairing mechanisms are not mutually exclusive. It is possible that both transient unstable contacts and early recombination events play a role in the colocalization and end to end alignment of homologous chromosomes. A role for premeiotic pairing ofhomologues? WEINER and KLECKNER ( 1994) have provided evidence that before meiotic S-phase 50% of homologues appear close to each other, in at least one region along their length. Since this association is not seen in meiotic Sphase cells, it seems unlikely to play a direct role in the meiotic pairing of homologous chromosomes. HOW-

ever, it remains possible that contacts between homolcgous chromosomes are maintained during this period, but in a form that does not survive the chromosome spreading techniques used. Such contacts could serve as the initiators of homologue pairing, perhaps by providing the first sitesat which allelicrecombination takes place. Localization of telomeres to the nuclear periphery appears to increase the chances of nearby sequences interacting: It is generally accepted that mitotic and meiotic telomeres are located in the nuclear periphery and bundled together in groups ( BYERSand GOETSCH 1975; DRESSERand GIROUX 1988; KLEIN et al. 1992; PAL LADINO et al. 1993;WEINER and KLECKNER 1994).These two properties are expectedto bring regions of nonhomologous chromosomes into close proximity and may facilitate the successful search for homology between the dispersed inserts located close to telomeres. Consistent with this suggestion, we found that the mean efficiency of ectopic recombination between inserts located in three heterologous subtelomeric regions was twice the mean for other crosses between inserts on heterologues, even when one insert was subtelomeric. Others have foundthat locating both dispersed sequences within either telomere adjacent Y’ sequences or telomere repeat sequences of yeast leads to highly efficient ectopic recombination during meiosis (T. C. HUCKLEand E. J. LOUIS,personal communication). The relatively modest twofold effect we found may reflect the distance ( 10-16 kb) between the subtelomeric location and the true telomeres. However, the twofold increase in efficiency could simply reflect spatial constraints imposed by the localization of telomeres to the nuclearperiphery. If the nucleus is considereda sphere, then 50% of its volume resides within one-fifth of the radius fromthenuclear envelope. Thus,the chances of a random encounterbetween two sequences restricted to this outer fifth of the nuclear radius would be twice thechance of anencounter between unrestricted (interstitial) sequences. We are grateful to T.-C. Wu for constructing several of the strains used in this study. We thank H. BUSSEYand M. JOHNSTON for providing us with sequence informationfromchromosomes I and WII, respectively, before publication, M. C. BRANDRISSfor supplying US with the plasmid pKB8 and S. BURGESS, J. HABER,T. C. HUCKLEand E. J. LOUISfor sharing unpublished data with us. We are also grateful to S. M. GASSER for teaching us the geometry of a sphere as well as S. BURGESS, J. HABER, N. HOLLINGSWORTH, S. KEENEY, A. RATTRAY and J. SEKELSKY for comments that improved the manuscript.

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Communicating editor: S. JINKS-ROBERTSON

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