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Genomics 61, 170–182 (1999)Article ID geno.1999.5956, available online at http://www.idealibrary.com on

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A Mouse Homolog of the Saccharomyces cerevisiae MeioticRecombination DNA Transesterase Spo11p

Scott Keeney,*,1 Frederic Baudat,† Michael Angeles,* Zhi-Hong Zhou,† Neal G. Copeland,‡Nancy A. Jenkins,‡ Katia Manova,* and Maria Jasin†,2

*Molecular Biology Program and †Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,New York, New York 10021; and ‡Mammalian Genetics Laboratory, ABL-Basic Research Program,

NCI–Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Received July 8, 1999; accepted August 5, 1999

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The Saccharomyces cerevisiae Spo11 protein ishought to catalyze formation of the DNA double-strandreaks that initiate meiotic recombination. We haveloned cDNA and genomic DNA for a mouse gene encod-ng a protein with significant sequence similarity to con-erved domains found in proteins of the Spo11p family.his putative mouse Spo11 gene maps to the distal re-ion of chromosome 2 (homologous to human chromo-ome 20q13.2–q13.3) and comprises at least 12 exons,panning approximately 15–18 kb. Strong expression ofhe Spo11 message is seen in juvenile and adult testis byNA in situ hybridization, RT-PCR, and Northern blot,ith much weaker expression in thymus and brain. In

itu hybridization detects expression in oocytes of em-ryonic ovary, but not of adult ovary. RT-PCR and initu hybridization analyses of a time course of juvenileestis development indicate that Spo11 expression be-ins in early meiotic Prophase I, prior to the pachytenetage, with increasing accumulation of mRNA throughhe pachytene stage. Taken together, these resultstrongly suggest that this gene encodes the functionalomolog of yeast Spo11p, which in turn suggests that theechanism of meiotic recombination initiation is con-

erved between yeast and mammals. © 1999 Academic Press

INTRODUCTION

During meiosis, homologous recombination providespotent source of genetic variation in most sexually

eproducing organisms. Recombination also plays anssential mechanical role in chromosome segregationy forming physical connections between homologoushromosomes that allow them to orient properly on theeiotic spindle and to segregate accurately at the first

Sequence data from this article have been deposited with theMBL/GenBank Data Libraries under Accession Nos. AF163049–F163054.

1 To whom correspondence may be addressed. Telephone: (212)39-5182. Fax: (212) 717-3627. E-mail: s-keeney@ski.mskcc.org.

2 To whom correspondence may be addressed. Telephone: (212)39-7438. Fax: (212) 717-3317. E-mail: m-jasin@ski.mskcc.org.

170888-7543/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

ivision (see Roeder, 1997; Smith and Nicolas, 1998;ickler and Kleckner, 1998 for recent reviews).To date, the mechanism of meiotic recombination

as been best characterized in the budding yeast, Sac-haromyces cerevisiae. As originally suggested by Szos-ak et al. (1983), recombination is initiated by DNAouble-strand breaks (DSBs) (Cao et al., 1990; Sun etl., 1989). Normally, these breaks are exonucleolyti-ally resected at their 59 strand termini to yield mole-ules with variable-length 39 single-stranded tailsBishop et al., 1992; Sun et al., 1991), which are actedpon further by homologous recombination proteinsreviewed in Smith and Nicolas, 1998). However, inertain mutant backgrounds, DSBs accumulate in annresected form (Alani et al., 1990; Cao et al., 1990;cKee and Kleckner, 1996; Nairz and Klein, 1997;rinz et al., 1996), consisting of a covalent complexetween the Spo11p protein and the 59 strand terminin either side of the break (de Massy et al., 1995;eeney et al., 1997; Keeney and Kleckner, 1995; Liu etl., 1995). Spo11p is required for DSB formation (Caot al., 1990) and has sequence similarity to the DNA-leaving “A” subunit of topoisomerase VI, a new familyf type II topoisomerases recently discovered in archae-acteria (Bergerat et al., 1997). Taken together, theseesults strongly support a model in which Spo11p cat-lyzes formation of DSBs via a topoisomerase-likeransesterase reaction (Bergerat et al., 1997; Keeney etl., 1997).Homologs of Sa. cerevisiae Spo11p have been identified

n several other eukaryotes. One of these homologs, theroduct of the rec12 gene of Schizosaccharomyces pombe,s required for meiotic recombination in that organismLin and Smith, 1994). Based on this conservation oftructure and function between these widely divergedeasts, it was argued that the mechanism of meioticecombination initiation was evolutionarily conservedBergerat et al., 1997; Keeney et al., 1997). This argumentas subsequently extended to metazoans in an elegant

tudy of a Spo11p homolog in Caenorhabditis elegansDernburg et al., 1998) and by the observation that the

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171MOUSE HOMOLOG OF YEAST Spo11p

rosophila melanogaster meiW-68 mutation, whichauses a defect in meiotic recombination (McKim et al.,998), inactivates a Spo11p homolog (McKim and Ha-ashi-Hagihara, 1998).From these studies, it seemed likely that the mecha-

ism of meiotic recombination initiation would be con-erved in all sexually reproducing organisms. However, aammalian member of the Spo11p family of proteins hadot yet been described. We report here the cloning andharacterization of a mouse gene encoding a protein withignificant similarity to members of the Spo11p family.he tissue-specific and developmental expression pat-

erns of this gene are consistent with the idea that itncodes a functional homolog of yeast Spo11p.

MATERIALS AND METHODS

Oligonucleotides. Oligonucleotides for RACE and RT-PCR ofpo11 were as follows: Spo11-for1 (59-AATAGTCGAGAAGGATG-AACA), Spo11-rev1 (59-TAGATGCACATTATCTCGATGC), andP1R (59-GATCTGCATCGATCGACCAGTGTGAA). The positions ofhese primers are indicated in Fig. 1. Primers for PCR amplificationf intronic sequences at the 59 end of lambda clone 4 were as follows:4T3-for1 (59-ACATTTTGCCCATCTTCTTTG) and l4T3-rev1 (59-CAGACTTGGCTTGAAATACT). Other RT-PCR primers weremc.3 (59-TGAAGGAGGATCAAGTTGTGC) and Dmc.4 (59-TCATG-TCTACGTTGAAGCG) for Dmc1 (Pittman et al., 1998) and bactin1

59-ATCTACGAGGGCTATGCTCTC) and bactin2 (59-AGGTCTT-ACGGATGTCAACG) for the b-actin gene.

cDNA cloning. Independent RACE reactions were performed toenerate overlapping 39 and 59 portions of the Spo11 cDNA. PCRrimers for 39- and 59-RACE were designed based on the EST se-uence in IMAGE Consortium cDNA Clone 1446797. The 39 end ofhe cDNA was amplified according to the manufacturer’s instruc-ions from a Marathon mouse testis cDNA library (Clontech) usinghe Clontech Adapter Primer (AP1) and the gene-specific forwardrimer Spo11-for1. The amplification reaction generated two prod-cts of ca. 1.0 and 1.9 kb. These products were purified by agarose gellectrophoresis and GeneClean (Bio 101) and were used separately torogram PCR using primer Spo11-for1 and the reverse primerpo11-rev1. The 1.0-kb 39-RACE product generated a 204-bp PCRroduct, as expected based on the EST sequence. In contrast, the.9-kb RACE product generated a ca. 550-bp PCR product. We as-ribe this larger PCR product to the presence of an unspliced intron337 bp) between exons 8 and 9; the additional sequences in the.9-kb RACE product may derive from another unspliced intronurther downstream. We did not characterize this RACE producturther. The 1.0-kb RACE product was cloned into plasmid pCR2.1sing the topo-TA cloning kit (Invitrogen) and sequenced. Two suchlones were designated pSK62 and pSK63 and were used as theource of probe sequences in Northern blotting and in Southernlotting for genomic mapping, respectively.The 59 portion of the cDNA was amplified from the same Marathonouse testis library using the gene-specific reverse primer Spo11-

ev1. A more complex mixture of amplification products was obtainedn this case, with prominent species of ca. 0.8, 1.0, and 1.5 kb. Theseroducts were gel-purified and used to program PCR using primerspo11-for1 and Spo11-rev1. The 0.8-kb RACE product generated aCR product of 204 bp as expected, whereas the 1.0- and 1.5-kbACE products generated a PCR product of ca. 550 bp, which wegain attributed to the presence of an unspliced intron. The 0.8-kbACE product was cloned into pCR2.1 and sequenced as above.ecause we were unsure that we had cloned the 59-most end of theDNA (see Results), we tried to isolate longer 59-RACE clones usinghe Marathon cDNA library as well as oligo(dA)-tailed first-strandDNA prepared according to the manufacturer’s instructions (Boehr-nger Mannheim) from fresh testis poly(A)1 RNA. However, no ad-

itional 59 sequences were obtained through these attempts. Instead,onger clones of two distinct classes were obtained: those in which thepo11 message still contained intronic sequences, and those in whichhe target cDNA sequences had been artifactually fused to cDNAerived from other gene products.To control for PCR errors, a consensus sequence was derived by

omparison of multiple independent clones and by comparison of theDNA sequence to the genomic DNA sequence (below). In total,equence from three independent 39-RACE clones and eight indepen-ent 59-RACE clones was used to generate the consensus hypothet-cal cDNA sequence outlined in Fig. 1 (GenBank Accession Nos.F163053 for splice variant A and AF163054 for splice variant B).ll sequencing was performed by the DNA sequencing facility of theornell University BioResource Center (Ithaca, NY).Genomic DNA cloning. A 204-bp PCR product was generated

rom the 39-RACE clones using primers Spo11-for1 and Spo11-rev1.his DNA fragment was 32P-labeled by nick-translation and used tocreen a 129SvJ mouse genomic DNA library in the Lambda FIX IIector (Stratagene). All hybridizations and phage manipulationsere according to standard protocols (Sambrook et al., 1989). Of a

otal of 2.1 3 106 plaque-forming units screened, 15 hybridization-ositive clones were isolated and purified. Of these, clone 4 extendedurthest to the 59 side of the gene, but did not include sequencesorresponding to the 59-most cDNA sequences. A ca. 500-bp PCRroduct corresponding to intronic sequences at the 59 end of clone 4as generated using the primers l4T3-for1 and l4T3-rev1. Thisroduct was used to rescreen the 129SvJ library, and four new clonesere isolated.We selected three overlapping clones (4, 6, and 19) that cover the

ntire Spo11 coding sequence for detailed analysis. The genomictructure of Spo11 was deduced from PCR analysis and direct DNAequencing of these clones. In total, we determined 11,260 bp ofenomic sequence in four noncontiguous segments (GenBank Acces-ion Nos. AF163049 through AF163052), with the three gaps be-ween these sections (indicated by parentheses in Fig. 3) all fallingithin introns.Interspecific mouse backcross mapping. Interspecific backcross

rogeny were generated by mating (C57BL/6J 3 Mus spretus) F1

emales and C57BL/6J males as described (Copeland and Jenkins,991). A total of 205 N2 mice were used to map the Spo11 locus (seeext for details). DNA isolation, restriction enzyme digestion, agaroseel electrophoresis, Southern blot transfer, and hybridization wereerformed essentially as described (Jenkins et al., 1982). All blotsere prepared with Hybond-N1 nylon membrane (Amersham). Therobe, a 663-bp StuI/NotI fragment from pSK63, was labeled witha-32P]dCTP using a random-primed labeling kit (Stratagene); wash-ng was performed to a final stringency of 1.03 SSCP, 0.1% SDS at5°C. A fragment of 11.0 kb was detected in HincII-digested57BL/6J DNA, and a fragment of 7.0 kb was detected in HincII-igested M. spretus DNA. The presence or absence of the 7.0-kbincII M. spretus-specific fragment was followed in backcross mice.A description of the probes and restriction fragment length poly-orphisms (RFLPs) for the loci linked to Spo11 including Mc3r,ck1, and Lama5 has been reported previously (Miner et al., 1997;iracusa et al., 1989). Recombination distances were calculated us-

ng Map Manager, version 2.6.5. Gene order was determined byinimizing the number of recombination events required to explain

he allele distribution patterns.Northern blot analysis. Poly(A)1 RNA was extracted from vari-

us tissues of a 26-day-old mouse using a PolyATract System 1000it (Promega) according to the manufacturer’s recommendations.NA samples (2 mg each) were denatured, electrophoresed on a 1%garose–formaldehyde gel, transferred to a charged nylon membraneNEN), and then hybridized with 32P-labeled double-strand DNArobes. All manipulations were according to standard protocols (Aus-bel, 1988). The b-actin probe was a 2.0-kb fragment of human-actin cDNA (Clontech). The Spo11 probe was a 1.0-kb EcoRI/SpeIragment from plasmid pSK62.

RT-PCR analysis. Poly(A)1 RNA was extracted from various tis-ues of mice using a QuickPrep Micro mRNA Purification Kit (Am-

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172 KEENEY ET AL.

rsham/Pharmacia Biotech). RNA (75 ng) was reverse-transcribedith a poly(dT) primer, using the ProSTAR First-Strand RT-PCR kit

Stratagene). For the time course experiment with prepubertal mice,oly(A)1 RNA was extracted from four testes (6- and 7-day-old mice),wo testes (8, 9, and 11 days old), or one testis (10 days and older) andecovered in 400 ml, from which 10 ml was used for the reverse-ranscription reactions. cDNA was PCR-amplified using primer pairspecific for Spo11, Dmc1, and the b-actin gene.

RNA in situ hybridization. Procedures for tissue preparation andn situ hybridization were as described (Tomihara-Newberger et al.,

FIG. 1. Mouse Spo11 cDNA sequence and open reading frameverlapping 59- and 39-RACE clones. Features of the sequence are hlternative splice junctions between exons 6 and 7; shaded box, theutative polyadenylation signal; asterisk, putative catalytic tyrosiomain; horizontal arrows, positions of PCR primers used in this st

998). Briefly, tissues were fixed in 4% paraformaldehyde in PBS at°C, and then 5-mm paraffin sections were prepared. To generaterobes for hybridization, a 204-bp portion of the Spo11 cDNA wasmplified by PCR using the primers Spo11-for1 and Spo11-rev1. ThisCR product was cloned into plasmid pCR2.1 using the TA cloningit according to the manufacturer’s recommendations (Invitrogen).eparate clones with the Spo11 cDNA insert in opposite orientationsere selected to provide both sense and antisense probes. These weresed to prepare labeled RNA by in vitro transcription with bacterio-hage T7 RNA polymerase in the presence of [a-33P]UTP.

anslation. Sequence information was compiled from independentlighted as follows: filled triangles, splice junctions; open triangles,-bp region that differs between the two splice variants; open box,

residue; vertical arrows, conserved acidic residues in the Toprim.

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RESULTS

dentification of a Putative Mouse Homolog of YeastSpo11p

Comparison of Sa. cerevisiae Spo11p to translatedequences in an EST database identified a singleouse EST with significant similarity to a highly con-

erved region found in the C-terminal halves of Spo11pamily members. This sequence similarity raised theossibility that the EST (IMAGE Consortium Clone ID446797) was derived from a gene for the mouse ho-olog of yeast Spo11p. However, this conclusion was

omewhat tentative because the EST had been isolatedrom a thymus cDNA library, which was not expectedor a meiosis-specific gene product. Moreover, thislone could not have encoded a full-length functionalpo11p because the sequences 59 of the conserved re-ions were substantially diverged and contained sev-ral in-frame stop codons. To resolve these discrepan-ies, and to determine whether this sequence waserived from a bona fide Spo11p homolog, we clonedhe corresponding cDNA from mouse testis.

Based on the conserved portion of the EST sequence,CR primers were designed to separately amplify over-

apping fragments of the cDNA by 59- and 39-RACE ofoly(A)1 RNA from mouse testis. Three independentlones isolated by 39-RACE from a Marathon cDNAibrary derived from testes of BALB/c males (Clontech)greed with one another to within 61 bp at their 39nds. RACE reactions to amplify the 59 end were per-ormed on Marathon cDNA and on reverse-transcribedoly(A)1 testis RNA isolated from (C57BL/6 3 CBA/a)F1 mice. We obtained eight independent clones thatatched one another, varying only in their lengths at

he 59 end and in the presence or absence of 12 bp ofequence derived from an alternative splicing eventsee below).

A conceptual fusion of the longest cDNA sequences

FIG. 2. Sequence conservation of members of the Spo11p/Top6Aop6A proteins are shown. Residues that are identical in at least 50haded gray. Dashes represent gaps introduced into the sequences toasterisk) and conserved Toprim acidic residues (arrows; see Dis-amino-acid region encoded in mouse Spo11 splice variant A but nchizosaccharomyces pombe; D-me, Drosophila melanogaster; C-el,

ulgidus; M-th, Methanobacterium thermoautotrophicum; M-ja, Mehibatae.

btained is presented in Fig. 1. The cDNA for theongest splice variant (A) is 1599 bp long, not includinghe poly(A) tail, and comprises 52 bp of 59 untranslatedequence, an open reading frame of 1077 bp, and 470p of 39 untranslated sequence containing a potentialolyadenylation signal sequence 16 nucleotides fromhe end. A probe derived from the cDNA recognizes a.6- to 2.0-kb message on Northern blots of poly(A)1

NA from testis (see below). Assuming a poly(A) tailength of ca. 200 nt (Colgan and Manley, 1997), itould appear that the assembled cDNA represents theajority of the mRNA sequence.The first ATG triplet in the cDNA sequence is in-

rame and occurs within a good match to the Kozakonsensus sequence (gccA/GccATGG, in which theost critical residues are in uppercase letters (Kozak,

991)). The predicted amino acid sequence is very sim-lar in length to the sequences of the other knownukaryotic Spo11p homologs, and several attempts toxtend the 59 sequences further were unsuccessful (seeaterials and Methods). Based on these criteria, it is

ikely that the cDNA sequence includes the entirety ofhe coding sequence. However, it should be noted thathe reading frame continues upstream of the first ATGodon, past the end of the assembled sequence, raisinghe possibility that the reading frame continues fur-her upstream. Unfortunately, the alignment ofpo11p homologs is uninformative in this regard, be-ause the amino-terminal sequences of the familyembers are highly diverged (Keeney et al., 1997).The predicted translation product is 358 amino ac-

ds, with a molecular mass of 40.4 kDa. The sequence islearly similar to members of the Spo11p/Top6A fam-ly, showing 21–31% identity in pairwise comparisons.he sequence similarities are even more strikingithin several conserved domains previously identified

n the Spo11p/Top6A family (Bergerat et al., 1997;eeney et al., 1997). As an example, alignment of por-

mily. Portions of eukaryotic Spo11p homologs and archaebacterialf the family members are highlighted in black; similar residues areerate the alignment. The positions of the putative catalytic tyrosine

sion for details) are indicated. The horizontal line indicates thein splice variant B. S-ce, S. cerevisiae; M-mu, Mus musculus; S-po,enorhabditis elegans; C-ci, Coprinus cinereus; A-fu, Archaeoglobusnococcus jannaschi; P-ho, Pyrococcus horikoshii; S-sh, Sulfolobus

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174 KEENEY ET AL.

ions of two of these domains is presented in Fig. 2.ased on this homology, the new mouse gene has beenamed Spo11.Spo11 was independently cloned by another group

see Romanienko and Camerini-Otero, 1999). Our datare largely in agreement with theirs, except for theirbservation of an optional exon between exons 1 and 2.his exon was in a fraction of cDNAs and encoded 38dditional amino acids within a poorly conserved re-ion of the protein.

enomic Structure of the Spo11 Gene

We used a 204-bp probe derived from PCR amplifi-ation of mouse testis cDNA to screen a mouse genomicNA library in bacteriophage lambda. Fifteen hybrid-

zation-positive clones were isolated, all of whichroved to be overlapping clones containing the 39 por-ion of the Spo11 cDNA sequences. Clones spanninghe 59 portion of the gene were isolated using a secondrobe, derived from intronic sequences within the 59-ost end of one of the original lambda clones (Clone 4,ig. 3). Exon boundaries were identified by PCR and byirect sequencing of a subset of the lambda clones. Theene contains at least 12 exons and spans ca. 15–18 kb

FIG. 3. Overview of the mouse Spo11 genomic locus. Exons arendicated by solid boxes, and introns and upstream sequences arendicated by thin lines. Parentheses denote gaps for which sequenceas not been determined. Positions of the start codon (ATG), stopodon, putative catalytic tyrosine (Tyr), and Toprim domain are ashown. The asterisk marks the variantly spliced exon 7. The posi-ions of the partially sequenced bacteriophage l clones are indicated.estriction sites: H, HindIII; RV, EcoRV; Xb, XbaI.

TAB

Intron/Exon Bounda

Exons 59 Splice donor

1–2 TGGCCTCTAGz gtacagcagc2–3 AAGAAATTCGz gtaagttact3–4 CAACCAAGAGz gtaaatatat4–5 TCTGCACGTGz gtgagccact5–6 TAGTGCCACGz gtgagcatcc6–7Aa GGAATGCAGCz gtatccttta6–7Ba GGAATGCAGCz gtatccttta7–8 CATGGTTACGz gtagggttac8–9 GATCCCTACGz gtaatccttc9–10 CGGATCCATGz gtgagtacag

10–11 ATATCCAGAGz gtaagatggt11–12 GAAAAAAGAGz gtagatggtg

Note. Exon sequences are in uppercase; intron sequences are inderlined.

a Alternative splice acceptor sites at the 39 end of the intron betw

Fig. 3). The 11 introns follow the usual GT/AG ruleTable 1). Because we are not certain that we havesolated cDNA sequences corresponding to the 59-mostnd of the mRNA, we are unable to define the 59 bound-ry of the gene, and the exon and intron numberingust therefore be considered provisional. The exons

ary in length from 37 to 592 bp. The last exon is theongest and contains the last 40 codons and the 39 UTR.

The seventh exon consists of two alternative splicingariants. Removal of the intron between exons 6 and 7tilizes either of two 39 splice acceptors (Table 1 andig. 1), which are separated by 12 bp. Both spliceariants preserve the reading frame, differing by onlyhe presence or the absence of four codons (see shadedequence in Fig. 1). To determine how frequently theifferent splice acceptors were used, we took advantagef restriction site polymorphisms between the twopliced forms of the message. Usage of the first accep-or (designated “A” in Table 1) results in inclusion of aclI restriction enzyme site in the mature message.sage of the second acceptor (B) removes the BclI site,ut creates a new SphI site at the splice junction.irst-strand cDNA was reverse-transcribed fromoly(A)1 testis RNA and then PCR-amplified usingrimers flanking the exon 6/7 boundary. The resultingCR product was then digested with BclI or SphI andnalyzed by agarose gel electrophoresis. By this assay,qual amounts of BclI-sensitive and SphI-sensitiveCR product were obtained (data not shown), indicat-

ng that the two splice sites are used at approximatelyqual frequencies.It is not clear whether there is any functional differ-

nce between the two splice variants. The alignment ofhe Spo11p/Top6A family members is not informativen this regard because this portion of the sequence isot absolutely conserved. Inclusion of the four extramino acids in variant A matches the conserved spac-ng in this region and includes three moderately con-

1

s of the Spo11 Gene

Intron size 39 Splice acceptor

3.5–6 kb aaaactgcag z GTTTGATGAT766 bp tattttccag z CTCTGATTCT458 bp atctgcatag z AGACATATAC398 bp ttttttccag z CTATCTACTT808 bp tttaattcag z GCTACTGCTG299 bp aacactgcag z ATCTGATCAC311 bp ctgatcacag z ATGCGAAGTT1693 bp ctactttcag z GGAAAGGGCG337 bp ctctcaacag z GCATCGAGAT518 bp tttatttcag z TCCATGTCTTca. 2.2 kb ttttttccag z GTTAAATATA825 bp cttctggcag z CTGGAGATGA

owercase. Splice donor and acceptor consensus dinucleotides are

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175MOUSE HOMOLOG OF YEAST Spo11p

erved residues (Fig. 2). However, although deletion ofhese residues in variant B necessitates inclusion of aap in the alignment at this position, it should be notedhat gaps of one to three residues are seen here in theethanococcus jannaschi Top6A, Coprinus cinereus

po11p, and Sc. pombe rec12p sequences.Derivation of the intron/exon structure of Spo11 al-

ows us to account for the discrepancies noted earlieror the thymus cDNA first identified in the EST data-ase, namely, that sequences upstream of the con-erved region were divergent and contained in-frametop codons. The EST sequence contained a single baseeletion (presumably due to a frameshift mutation orequencing error) near the 59 end of exon 7 and re-ained intronic sequence upstream of exon 5, indicatinghat the RNA from which this cDNA clone was derivedas incompletely or improperly spliced. Inefficient

plicing may be a general property of the Spo11 tran-cript. Several independent clones that retained in-ronic sequences were isolated by 59-RACE from aarathon testis cDNA library (see Materials andethods). Moreover, RT-PCR of any of several differ-

nt regions of the Spo11 coding sequence from poly(A)1

NA from adult testis routinely yielded additional am-lification products of a size consistent with the reten-ion of one or more introns (data not shown). Thisattern of intron retention did not appear to vary overtime course of juvenile testis development, suggest-

ng that this property is not due to developmentallyrogrammed regulation of Spo11 splicing.

enetic Mapping

The chromosomal location of Spo11 was determinedy interspecific backcross analysis using progeny de-ived from matings of [(C57BL/6J 3 M. spretus)F1 357BL/6J] mice. This interspecific backcross mappinganel has been typed for over 2900 loci that are wellistributed among all the autosomes as well as the Xhromosome (Copeland and Jenkins, 1991). C57BL/6Jnd M. spretus DNAs were digested with several en-ymes and analyzed by Southern blot hybridization fornformative RFLPs using a mouse cDNA Spo11 probe.he 7.0-kb HincII M. spretus RFLP (see Materials andethods) was used to follow the segregation of thepo11 locus in backcross mice. The mapping results

ndicated that Spo11 is located in the distal region ofouse chromosome 2 linked to Mc3r, Pck1, andama5. Although 124 mice were analyzed for everyarker and are shown in the segregation analysis (Fig.

), up to 184 mice were typed for some pairs of mark-rs. Each locus was analyzed in pairwise combinationsor recombination frequencies using the additionalata. The ratios of the total number of mice exhibitingecombinant chromosomes to the total number of micenalyzed for each pair of loci and the most likelyene order are centromere–Mc3r–3/134–Pck1–0/137–po11–5/184–Lama5. The recombination frequencies

expressed as genetic distances in centimorgans 6 the

tandard error) are Mc3r–2.2 6 1.3–(Pck1, Spo11)–.7 6 1.2–Lama5. No recombinants were detected be-ween Pck1 and Spo11 in 137 animals typed in com-on, suggesting that the two loci are within 2.2 cM of

ach other (upper 95% confidence limit).We have compared our interspecific map of chromo-

ome 2 with a composite mouse linkage map that re-orts the map location of many uncloned mouse muta-ions (provided from Mouse Genome Database, aomputerized database maintained at The Jacksonaboratory, Bar Harbor, ME). Spo11 mapped in a re-ion of the composite map that lacks mouse mutationsith a phenotype that might be expected for an alter-tion in this locus (data not shown).The distal region of mouse chromosome 2 shares a

FIG. 4. Spo11 maps in the distal region of mouse chromosome 2.po11 was placed on mouse chromosome 2 by interspecific backcrossnalysis. The segregation patterns of Spo11 and flanking genes in24 backcross animals that were typed for all loci are shown at theop of the figure. For individual pairs of loci, more than 124 animalsere typed (see text). Each column represents the chromosome iden-

ified in the backcross progeny that was inherited from the (C57BL/J 3 M. spretus)F1 parent. The shaded boxes represent the presencef a C57BL/6J allele, and white boxes represent the presence of a M.pretus allele. The number of offspring inheriting each type of chro-osome is listed at the bottom of each column. A partial chromosomelinkage map showing the location of Spo11 in relation to linked

enes is shown at the bottom of the figure. Recombination distancesetween loci in centimorgans are shown to the left of the chromo-ome, and the positions of loci in human chromosomes, where known,re shown to the right. References for the human map positions ofoci cited in this study can be obtained from GDB (Genome Dataase), a computerized database of human linkage information main-

ained by The William H. Welch Medical Library of The Johnsopkins University (Baltimore, MD).

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176 KEENEY ET AL.

egion of homology with human chromosome 20q (sum-arized in Fig. 4). In particular, Pck1 has beenapped to 20q13.2–q13.3. The close linkage betweenck1 and Spo11 in mouse suggests that the humanomolog of Spo11 will map to 20q13, as well.

po11 Is Expressed Primarily in Germline Tissue

A functional homolog of the yeast SPO11 gene isxpected to be expressed primarily (if not exclusively)n meiotic cells. We therefore examined the tissue dis-ribution of Spo11 expression by Northern blot andT-PCR. Poly(A)1 RNA was isolated from various tis-ues and subjected to Northern blotting using as arobe 1.0 kb from the 39 end of the cDNA sequence. Aingle prominent message of 1.6–2.0 kb was easilyetected in testis, but not in brain or kidney, even withonger autoradiographic exposure times (Fig. 5A).herefore, the Spo11 message is much more abundant

n testis than in the other tissues examined.These results were confirmed and extended usingT-PCR analysis. Oligonucleotide primers were used

o amplify a portion of the Spo11 message spanningxons 7–8 from poly(A)1 RNA isolated from variousissues (Fig. 5B). An abundant RT-PCR product wasbtained from testis RNA, with much smaller amountsmplified from thymus and even smaller amounts fromrain. No products were detected with RNA frompleen, kidney, adult ovary, or lung. The lack of expres-ion in the adult ovary is as expected for an earlyeiotic gene, because mammalian oocytes progress

hrough the early stages of meiosis (including the pe-

FIG. 5. Tissue distribution of Spo11 expression. (A) Northern blolectrophoresed in duplicate on a formaldehyde/agarose gel, transferr a probe derived from the 39 end of Spo11. Positions of size markerlot was exposed for 11 h (short exposure) and 22 h (long exposure)issues was subjected to PCR amplification with (1) or without (2)mc1, or b-actin.

iod during which homologous recombination occurs)n embryonic development. The low level of Spo11 ex-ression in thymus accounts for the original isolationf a Spo11 EST from a thymus cDNA library. As aontrol, the meiosis-specific Dmc1 message was alsomplified. The Dmc1 message was detectable in RNArom testis, as expected (Habu et al., 1996). Interest-ngly, a weak signal for Dmc1 was also detected withNA from adult ovary.

po11 Is Expressed in Early Meiotic Cells within theAdult Testis

The mouse spermatogenesis cycle is well defined ands subdivided into 12 stages, with each stage consistingf a specific complement of male germ cells (Leblondnd Clermont, 1952; Oakberg, 1956a, b; Russell, 1990).ithin the adult testis, different seminiferous tubules

re in different stages of spermatogenesis at any oneime. Because a single histological testis section con-ains numerous seminiferous tubules, each sectionherefore contains spermatocytes that vary with re-pect to their stages of progression through meiosis,nd these stages can be assigned with precision. Weherefore turned to RNA in situ hybridization analysiso determine the spatial distribution of Spo11 expres-ion within the testis and to determine whether Spo11xpression is restricted to particular stages of sper-atocyte development.Gonads were dissected from adult males (2–5onths) fixed, sectioned, and hybridized to strand-spe-

ific RNA probes derived from 204 bp near the middle

nalysis. Poly(A)1 RNA (2 mg) isolated from the indicated organs wasto a nylon membrane, and probed in parallel with a b-actin probe

e shown on the left. The b-actin blot was exposed for 5 h; the Spo11) RT-PCR analysis. Poly(A)1 RNA (75 ng each) from the indicatedr reverse transcription (RT) using gene-specific primers for Spo11,

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f the Spo11 cDNA. Under low-magnification dark-eld illumination (Fig. 6A), a strong hybridization sig-al from the antisense probe was visible over the cellsetween the basal layer and the lumen of the seminif-rous tubules, whereas only background levels of sig-al were observed over interstitial cells (between theubules), over spermatogonia in the basal layer of theubules, and over the spermatids near the lumen. Thisesult indicates that Spo11 expression within the testiss restricted primarily to meiotic cells. The hybridiza-ion signal was specific for the Spo11 message, becausenly background signal was detected when a senserobe was used (Fig. 6B).The intensity of the Spo11 hybridization signal var-

ed substantially from tubule to tubule within the sameection, and this variation correlated with the stages ofhe tubules. Preleptotene/leptotene spermatocytesere not labeled (Fig. 6C). Scattered autoradiographicrains were found on zygotene primary spermatocytesn stage XI–XII tubules (Fig. 6D). Higher levels ofpo11 mRNA were detected in pachytene spermato-ytes, reaching a peak around stage VII (Fig. 6C)hrough stage X (not shown). Strong hybridization sig-al persisted in diplotene spermatocytes (Fig. 6D) buteclined continually in dividing and secondary sper-atocytes and in the round spermatids. Expression ofpo11 mRNA was essentially undetectable in step 4–5permatids.

he Timing of Spo11 Expression in Spermatocytes

Based on studies in budding yeast, DSB formationediated by Spo11p is expected to occur relatively

arly in meiotic prophase (discussed further below),o the expression of the Spo11 gene is likewise ex-ected to occur early in meiosis. The in situ hybrid-zation results for adult testis were consistent withhis expectation, but we wished to determine morerecisely the time of onset of Spo11 expression. To dohis, we used RNA in situ hybridization and RT-PCRo follow Spo11 expression during the course of pu-ertal testis development. Whereas meiosis occurssynchronously throughout the adult testis, the firstave of developing reproductive cells in the juvenile

estis progresses through meiosis in a synchronousnd highly stereotyped fashion.At embryonic day (E) 13.5, the male germ cells enteritotic arrest, where they remain during the entire

etal period until approximately the end of the firstostnatal week. Meiosis begins in the postnatal testis–3 days after resumption of mitotic activity, approxi-ately around day 9–10 (Bellve et al., 1977). No Spo11

xpression was detected in the male gonad by RNA initu hybridization in the fetus or in the early postnatalestis. Weak expression was first noticed in a few sem-niferous tubules at day 11 (not shown).

By day 12, the number of positive tubules and the

ntensity of the signal had increased (Fig. 6F). Fourategories of tubules present at this age have beenescribed by McKinney and Desjardins (1973) for theouse and by Clermont and Perey (1957) for the rat,

ased on the association of germ cells in specific stages.he first two types of tubules contained either sper-atogonia or spermatogonia along with resting/lepto-

ene spermatocytes, respectively. By in situ hybridiza-ion, no Spo11 expression was detected in tubules ofhese types (Figs. 6F and 6G). The third category ofubules contained zygotene spermatocytes. By in situybridization, tubules of this type showed discrete au-oradiographic grains distributed over cells a littlearger than the resting/early leptotene spermatocytesi.e., zygotene cells; Figs. 6F and 6H). The fourth cat-gory of tubules contained cells with large nuclei in theore of the tubules, interpreted as early pachytenepermatocytes. Significantly higher levels of Spo11essage were observed in this type of tubule (Figs. 6F

nd 6I).Between day 14 and day 20, the steady-state levels of

po11 message continued to increase, accompanyinghe progression of spermatocytes through pachytenenot shown). We found that Spo11 mRNA was presenthrough the end of the first division and persisteduring the short second meiotic division. A decline inessage levels was found thereafter, and only a low

evel remained in the round spermatids (data nothown), which occur around day 20 (Bellve et al., 1977).Spo11 expression was also examined by RT-PCR

nalysis of poly(A)1 RNA isolated from juvenile testest various days after birth (Fig. 7). RT-PCR of theeiosis-specific Dmc1 message and the ubiquitously

xpressed b-actin message served as controls. Expres-ion of the Spo11 gene was detectable by day 7 post-artum, and steady-state levels appeared to accumu-ate over the next several days. A similar pattern waseen for Dmc1, but expression of this gene was detectedt day 6, earlier than for Spo11. This may reflect ear-ier expression timing for Dmc1, higher expression lev-ls (and thus greater sensitivity for detection), or aombination of these factors.By cytological analysis (Goetz et al., 1984), cells clas-

ified as preleptotene occurred sporadically in day 8estes, whereas 98% of spermatocytes were in thereleptotene stage by day 9. A very small number ofachytene cells were seen as early as day 10, but ap-reciable levels (20%) did not accumulate until day1–13. Similar timing was reported in an independenttudy (Bellve et al., 1977). Comparing our RT-PCResults with these earlier studies, it appears thatpo11 expression begins very early in meiosis, appar-ntly before the preleptotene stage, although maximalevels are not reached until pachytene. The earlierpo11 expression detected in Fig. 7 compared to the initu hybridization analysis most likely reflects thereater sensitivity of the PCR-based assay.

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178 KEENEY ET AL.

xpression of Spo11 in Developing Oocytes within theEmbryonic Ovary

The germ cells in male and female mammals takeery different routes in their developmental progres-ion toward gamete formation. In contrast to the situ-tion in the testis, female germ cells enter meiosis at13.5 and progress through the leptotene, zygotene,nd pachytene stages. Around birth, oocytes withinrimordial follicles arrest in diplotene and resume mei-sis only when recruited to ovulate (McLaren andouthee, 1997).To determine whether Spo11 might play a role in

emale meiosis as well, RNA in situ hybridization anal-sis was carried out on sections of embryonic ovaries.e found that Spo11 mRNA was detectable in female

erm cells, although at a lower intensity than in theirale counterparts. Signal was first detected at E14.5

nd clearly corresponded to the meiotic cells (Figs. 6End 6J). Autoradiographic grains overlaid oocytes withval nuclei, localized in clusters (arrows in Fig. 6J),hile only background grains were present over thevarian somatic cells. Some oocyte clusters, however,ere devoid of signal (indicated by the stars in Fig. 6J).ost likely the positive and negative groups of oocytesere in different stages of meiotic prophase. Due to the

ow intensity of expression and to the specifics of fixa-ion and preparation of the specimens for radioactiven situ hybridization, we were unable to stage the oo-ytes more precisely. The pattern of expression per-isted in ovaries from late stage fetuses, but the num-er of labeled oocyte clusters gradually decreased (dataot shown). No expression was found in newborn andostnatal ovaries (data not shown), consistent with theT-PCR results described above.

DISCUSSION

Mouse Homolog of the Budding Yeast SPO11 Gene

Several lines of evidence support the idea that theew mouse gene isolated in this study is a functionalomolog of the Sa. cerevisiae SPO11 gene, whose prod-ct apparently catalyzes formation of the DSBs that

nitiate meiotic homologous recombination. First, theouse gene is expressed primarily in organs (testis

nd embryonic ovary) that contain meiotic cells. Sec-nd, within these organs, the gene is expressed in cells

FIG. 6. RNA in situ hybridization analysis of Spo11 expressiondult testis sections, hybridized with antisense (A) or sense (B) RNAarying densities of autoradiographic grains; the section in B shows oeminiferous tubules in stage VII and stage XI, respectively, hybridin preleptotene/leptotene (PL/L), zygotene (Z), pachytene (P), and dipeen, toward the tubule center in C. (F–I) Digital micrographs of jurobe. A dark-field image (magnification, 203) is presented in F. Aro the Spo11 probe. These tubules are shown in higher magnificationicrographs of fetal ovary at embryonic day 14.5, hybridized to the an

J; magnification, 1003) images are shown. The arrows in J indicutoradiographic grains in E. The stars in J indicate clusters of ooc

arly in meiotic prophase I, coincident with the timehen homologous recombination is thought to begin.Third, the predicted translation product of theouse gene contains sequences similar to regions that

re highly conserved in Spo11p homologs in eukaryotesnd archaebacteria. Two such domains are highlightedn Fig. 2. The first of these domains contains the con-erved tyrosine residue (asterisk in Fig. 2) that ishought to attack the DNA phosphodiester backboneuring DNA cleavage. Mutation of this tyrosine to phe-ylalanine in Sa. cerevisiae Spo11p confers a null phe-otype for DSB formation and meiotic recombinationBergerat et al., 1997). The second domain was definedhrough iterative sequence comparisons as being con-erved in topoisomerases, primases, and certain nucle-ses (Aravind et al., 1998). Based on this similarity,his motif has been termed the “Toprim” domain. Thisomain was independently identified based on struc-ural conservation between topoisomerase VI, classicalype II topisomerases, and type IA topoisomerasesBerger et al., 1998) (M. Nichols and J. Berger, pers.omm., University of California, Berkeley, 1999). A keyeature of the Toprim domain is a triad of acidic resi-ues (arrows in Fig. 2) that are important for SPO11unction: mutation of these acidic residues in Sa. cer-visiae Spo11p diminishes or abolishes its activity inivo (R. Diaz and S. Keeney, unpublished results).iven the strong conservation of these two domainsnd the critical role that residues within them play ina. cerevisiae Spo11p activity in vivo, sequence conser-ation within these domains can be considered a defin-ng hallmark of the Spo11p/Top6A family.

iming of Spo11 Expression during Meiosis

Despite the presence of Spo11p homologs in otherrganisms, meiotic DSBs have been detected only ina. cerevisiae. In budding yeast, Spo11p-mediatedSBs are known to occur at the leptotene stage (Pad-ore et al., 1991; Zickler and Kleckner, 1998), but the

iming of recombination initiation in other organismsas been the subject of debate. For example, synap-onemal complex (SC) formation is dependent uponpo11p activity in Sa. cerevisiae (Giroux et al., 1989;oidl et al., 1994) and Coprinus cinereus (M. Celerin,. S. Merino, and M. Zolan, pers. comm., Indiana Uni-ersity, 1999), but is independent of Spo11p activity in. melanogaster and C. elegans (Dernburg et al., 1998;

estis and embryonic ovary. (A, B) Dark-field digital micrographs ofobes; magnification, 103. Different seminiferous tubules in A showbackground grains. (C, D) Bright-field digital micrographs of adultto the antisense Spo11 probe; magnification, 1003. Spermatocytesne (D) are indicated. A layer of step 7 round spermatids can also beile testis at Day 12 postpartum, hybridized to the antisense Spo11s point to three tubules that show varying degrees of hybridizationght-field images in G, H, and I (magnification, 1003). (E, J) Digitalnse Spo11 probe. Dark-field (E; magnification, 203) and bright-fieldclusters of labeled oocytes that correspond to the patchy areas ofs devoid of hybridization signal.

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179MOUSE HOMOLOG OF YEAST Spo11p

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180 KEENEY ET AL.

cKim et al., 1998; McKim and Hayashi-Hagihara,998). Based in part on this difference, it has beenrgued that recombination initiates after, and re-uires, SC formation in metazoans and thus occursater than in fungi (McKim et al., 1998). However, ithould be stressed that the genetic independence of SCormation from Spo11p activity indicates nothingbout the relative timing of these events in normaleiosis. Moreover, mouse Rad51p foci (which are

hought to represent the sites of ongoing recombinationnd, thus, the sites where DSBs have formed) haveeen observed on chromosomes at the leptotene stagei.e., prior to the onset of synapsis), where they aressociated with short patches of axial cores (Moens etl., 1997).The results presented here cannot resolve this issue,

ecause the timing of Spo11 mRNA accumulation can-ot be used to infer the timing of Spo11p protein accu-ulation, much less the timing of Spo11p activity.owever, our results can put a limit on the earliest

ime at which Spo11p might act, namely, the earliestime at which the gene is expressed. In situ hybridiza-ion analysis suggests that maximal steady-state levelsf Spo11 mRNA do not accumulate until the pachytenetage, but the RT-PCR analysis of pubertal testis de-elopment indicates that Spo11 mRNA is first ex-ressed much earlier in meiosis, most likely at or be-ore the preleptotene stage. Thus, these results areonsistent with the possibility that Spo11p is present,nd active, prior to SC formation.

issue Distribution of Spo11 Expression

The major sites for expression of Spo11 are the adultestis and oocytes of the fetal ovary. However, Spo11essage is also detectable in the adult thymus and

very weakly) in the brain, and we cannot rule outxpression in other tissues or at other times duringevelopment. It is not clear whether there is any func-ional significance to Spo11 expression in nongermlineissues. Although the thymus is the site of significant

FIG. 7. Spo11 is expressed early during juvenile testis develop-ent. Poly(A)1 RNA isolated from juvenile testis at the indicated

ays postpartum was amplified by PCR with Spo11, Dmc1, or b-actinene-specific primers with (1) or without (2) prior reverse transcrip-ion (RT).

evels of DSB-mediated recombination in the form of(D)J rearrangements, there is no obvious role forpo11p in this process. On the other hand, mutation ofhe D. melanogaster Spo11p homolog, MEI-W68, gen-rates a mild mitotic hyperrecombination phenotype,uggesting that MEI-W68 may play a role in somaticissues as well (McKim and Hayashi-Hagihara, 1998).

volutionary Conservation of Meiotic Processes

Overall, it appears that the key processes unique tohe meiotic cell division pathway are conserved in mostexually reproducing organisms. At the ultrastructuralevel, examples of this conservation include the synap-onemal complex (reviewed in Moens et al., 1998) andhe stereotyped clustering of telomeres known as theouquet (reviewed in Zickler and Kleckner, 1998). Athe molecular level, several of the genes involved ineiotic recombination, such as the RecA homologMC1 (Bishop et al., 1992; Pittman et al., 1998; Yo-

hida et al., 1998), are also now known to be conservedoth structurally and functionally. One of the mosttriking conclusions to be drawn from the work pre-ented here, in conjunction with other recent studiesBergerat et al., 1997; Dernburg et al., 1998; Keeney etl., 1997; McKim and Hayashi-Hagihara, 1998), is thathe mechanism by which meiotic recombination ini-iates is also likely to be conserved.

Further delineation of the similarities between mei-tic events in mammals and other organisms, as wells identification of important differences, will comerom generation and characterization of mutant miceefective for meiotic recombination initiation. Thesetudies are now possible with the identification of theouse homolog of Spo11p described here.

ACKNOWLEDGMENTS

We thank Rosemary Bachvarova for providing advice and insightsegarding the histological interpretation of the gonad sections. Welso thank James Berger, Mimi Zolan, and Martina Celerin forharing data prior to publication; Tom Nguyen and Andrew Koff forroviding the mouse genomic DNA library; and Deborah J. Gilbertor excellent technical assistance. This work was supported, in part,y a grant from the New York City Council Speaker’s Fund foriomedical Research (to S.K.), by NIH Grants GM58673-01 (to S.K.)nd HG01740 (to M.J.), and by the National Cancer Institute,HHS, under contract with ABL.

Note added in proof. We have sequenced the optional exon betweenxons 1 and 2 (see Results) and it is in agreement with the sequencebtained by Romanienko and Camerini-Otero (Genomics 61: 171–83). Our EMBL/GenBank submission has been updated to reflecthis information.

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