PATTERNS

Candidate Testis-Determining Gene, Maestro(Mro), Encodes a Novel HEAT Repeat ProteinLee Smith,1 Nick Van Hateren,1 John Willan,1 Rosario Romero,1 Gonzalo Blanco,1 Pam Siggers,1

James Walsh,1 Ruby Banerjee,1 Paul Denny,1 Chris Ponting,2 and Andy Greenfield1*

Mammalian sex determination depends on the presence or absence of SRY transcripts in the embryonic gonad.Expression of SRY initiates a pathway of gene expression resulting in testis development. Here, we describe a novelgene potentially functioning in this pathway using a cDNA microarray screen for genes exhibiting sexually dimorphicexpression during murine gonad development. Maestro (Mro) transcripts are first detected in the developing malegonad before overt testis differentiation. By 12.5 days postcoitus (dpc), Mro transcription is restricted to thedeveloping testis cords and its expression is not germ cell-dependent. No expression is observed in female gonadsbetween 10.5 and 14.5 dpc. Maestro encodes a protein containing HEAT-like repeats that localizes to the nucleolusin cell transfection assays. Maestro maps to a region of mouse chromosome 18 containing a genetic modifier of XXsex reversal. We discuss the possible function of Maestro in light of these data. Developmental Dynamics 227:600–607, 2003. © 2003 Wiley-Liss, Inc.

Key words: sex determination; gonad development; Maestro; HEAT repeat; nucleolus

Received 13 March 2003; Accepted 7 May 2003

INTRODUCTION

The mammalian gonads are derivedfrom the sexually dimorphic devel-opment of a bipotential gonadalprimordium, the genital ridge. Thefate of the developing genital ridgeis under the control of the Y-linkedsex determining gene SRY (Koop-man et al., 1991). Expression of SRY,a putative transcriptional regulator,in the supporting cell lineage of thebipotential gonad is responsible forthe commitment of those cells to theSertoli cell (i.e., testicular) fate(Koopman et al., 1990; Palmer andBurgoyne, 1991; Hacker et al., 1995;Jeske et al., 1995). In the absence ofSRY expression, the indifferent go-

nad becomes committed to theovarian fate. The details of the tran-scriptional cascade initiated by SRYare still unknown, although it is likelythat SOX9 is a key target gene dur-ing gonadogenesis given that ex-pression of this SRY-related gene inthe embryonic gonad is both neces-sary (Foster et al., 1994) and suffi-cient (Vidal et al., 2001) for develop-ment of the testis. How thesupporting cell lineage exerts a mas-culinizing effect on other gonadalsomatic lineages is unclear, al-though the production of a che-moattractant responsible for the mi-gration of mesonephric cells into theXY gonad appears to be a key

event (Martineau et al., 1997; Capelet al., 1999; Tilmann and Capel,1999). Much less is known about thetranscriptional events responsible forovary development.

In addition to SRY and SOX9, a sur-vey of other key genes functioningduring sex determination, such asWNT4 (Vainio et al., 1999), DAX1(Swain et al., 1996, 1998), WT1 (Pelle-tier et al., 1991), DMRT1 (Raymond etal., 1999, 2000), SF1 (Sadovsky et al.,1995; Ikeda et al., 1996), AMH (Mun-sterberg and Lovell-Badge, 1991; Be-hringer et al., 1994), DHH (Bitgood etal., 1996), M33 (Katoh-Fukui et al.,1998), and FGF9 (Colvin et al., 2001),reveals that transcriptional regula-

1MRC Mammalian Genetics Unit, Harwell, Didcot, United Kingdom2MRC Functional Genetics Unit, Department of Human Anatomy & Genetics, University of Oxford, South Parks Road, Oxford, United KingdomGrant sponsor: EC; Grant number: HPRN CT 2000 0087.Drs. Smith and Van Hateren contributed equally to this work.*Correspondence to: Dr. Andy Greenfield, MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK. E-mail:a.greenfield@har.mrc.ac.uk

DOI 10.1002/dvdy.10342

DEVELOPMENTAL DYNAMICS 227:600–607, 2003

© 2003 Wiley-Liss, Inc.

tors are represented at unusuallyhigh frequency. The putative missinggenes from this pathway, such asthose encoding additional mem-brane-bound or secreted signalingmolecules, their receptors, signaltransduction elements, protein traf-ficking mediators, nuclear import/export regulators, growth factors,enzymes, and structural proteins re-main to be identified. We have de-scribed recently a DNA microarray-based screen for genes exhibitingsexually dimorphic transcription dur-ing mouse gonad development thatidentified the molecules Vanin-1and Pn-1 as potential elements insexual development (Grimmond etal., 2000). By using the same strat-egy, we describe here the identifi-cation of a novel gene, Maestro(Mro), which exhibits male-specifictranscription both before and afterovert differentiation of the testis. Wepresent the preliminary character-ization of this novel gene and discussits possible role in gonadogenesis.

RESULTS

Microarrays were constructed by us-ing randomly picked polymerasechain reaction (PCR)-amplifiedcDNA clones from the developingurogenital ridge library NMUR (Grim-mond et al., 2000). To overcome thelimitations imposed by the smallamounts of RNA available from em-bryonic tissue, a Taq polymerase-based amplification procedure wasused before labeling of RNA (see Ex-perimental Procedures section andSmith et al., 2003). This procedure al-lowed hybridizations to be per-formed with as little as 50 ng of totalRNA without significant loss of theability to reliably detect differentialexpression. Clone 10p06 was identi-fied by its exhibiting a male-en-hanced signal after dual hybridiza-tion with two amplified cDNAsamples derived from RNA ex-tracted from 13.5 days postcoitus(dpc) embryonic male and femalegonads (Fig. 1a). Routine validationof the observed microarray expres-sion ratios was performed by usingwhole-mount in situ hybridization(WMISH) of explanted gonad–meso-nephros pairs at 13.5 and 12.5 dpcwith a 10p06 probe. Prominent ex-

pression was observed in the testiscords at 13.5 and 12.5 dpc, but noexpression was observed in the de-veloping ovary or mesonephros (Fig.1b,c). Expression analysis of embryosat 11.5 dpc, before overt testis differ-entiation, revealed transcripts in themale (XY) gonad at the 18 and 13tail-somite stages (Fig. 1d,e). No ex-pression was observed in male go-nads before 13 tail somites, and noexpression was observed in femalegonads. No transcripts were de-tected in other embryonic organs at10.5 or 11.5 dpc (data not shown).This gene was named Maestro (Mro)due to its male-specific transcriptionin the developing reproductive or-gans. The gene name “Maestro”and symbol “Mro” have been ap-proved by the mouse nomenclaturecommittee.

To determine the cellular specific-ity of Mro transcription, 13.5 dpc em-bryonic gonads were sectioned af-ter WMISH. Transcripts were found tobe present throughout the testiscord (Fig. 2a), consistent with expres-sion in Sertoli cells and germ cells. Toconfirm that Mro transcription wasnot germ-cell dependent, expres-sion was examined in 12.5 and 13.5dpc male gonads dissected fromembryos homozygous for the ex-treme allele of dominant white spot-ting (We/We), which lack germ cells(Cattanach, 1978). Reverse tran-scriptase (RT)-PCR demonstratedthat transcripts were present in bothwild-type and We/We homozygousembryonic gonads at 12.5 dpc (Fig.2b), confirming that Maestro tran-scripts are present in the Sertoli cellsof the testis cord.

The length of its embryonic tran-script was determined by hybridizinga Maestro probe to a Northern blotcontaining polyA� RNA from maleand female embryonic gonads (13.5and 14.5 dpc). A single major tran-script of approximately 2.0 kb wasobserved in male samples alongwith a much less abundant tran-script of approximately 4.5 kb (Fig.3a). Northern analysis of unsexedadult RNA samples revealed tran-scripts in several tissues includingbrain, heart, liver, and testis (Fig. 3b).

Sequence analysis of clone10p06revealed a 1072-bp cDNA clonecontaining an open reading frame

of 249 bp that remained open at the5� end. To extend the Mro cDNAclone in the 5� direction, 5� rapid am-plification of cDNA ends (RACE) PCRwas used (for details see Experimen-tal Procedures section). RACE PCRgenerated an 800-bp product over-lapping clone 10p06. RT-PCR, usingprimers from the novel RACE se-quence and clone 10p06, was usedto establish the validity of the com-posite 1891-bp cDNA sequencegenerated. The Maestro cDNA, pre-sumably representing the major2.0-kb transcript detected by North-ern blotting, encodes a predictedprotein of 248 amino acids. To deter-mine whether Maestro is a homo-logue of any known protein, its pre-dicted peptide sequence wascompared with current databasesusing PSI-BLAST (Altschul et al., 1997),with an E-value inclusion threshold of5 � 10�3. Maestro was found to bemost similar to human B29 (E � 2�10�94). B29 is a predicted proteinof unknown function that was iden-tified by sequencing of the genomicregion associated with a tumor sup-pressor locus on human chromo-some 18q21 (Yanaihara et al., 2001).B29 cDNA has also been identified inthe monkey (Macaca fascicularis)testis (Osada et al., 2002). Homolo-gous regions were found in the C-termini of Drosophila melanogasterLD28902p and Caenorhabditis el-egans C34G6.1. A second round ofPSI-BLAST searches revealed signifi-cant similarity (E � 5 � 10�3) be-tween these homologues and theSchizosaccharomyces pombe im-portin-� subunit, within a region con-taining multiple HEAT repeats (Fig. 4).HEAT repeats were originally identi-fied in a functionally diverse group ofproteins, huntingtin, elongation fac-tor 3, the PR65/A subunit of proteinphosphatase 2A (PP2A), and thelipid kinase TOR1 (Andrade andBork, 1995), and are also evolution-arily related to armadillo repeats,such as those in importin-� subunits(Malik et al., 1997). Maestro containsfour HEAT repeats (Fig. 4). A nucleo-tide BLAST search of the mouse ex-pressed sequence tag databasealso revealed identity betweenMaestro and a shorter cDNA,4933435E20Rik, identified in an adultmouse testis library.

MAESTRO: A CANDIDATE TESTIS DETERMINING GENE 601

Fig. 1. Expression analysis of NMUR clone 10p6 (Maestro) during mousegonadogenesis using DNA microarrays and whole-mount in situ hybrid-ization (WMISH). a: False-color overlay of a region of an NMUR microar-ray after hybridization with female (green) and male (red) fluorescentlylabeled embryonic gonadal cDNA (13.5 dpc). Clone 10p6 is indicatedwith a white arrow. The male-enhanced expression results in the redsignal, in contrast to the yellow (equivalent female : male signal) of allother clones. b: WMISH of a Maestro probe to male and female gonad–mesonephros explants at 13.5 days postcoitus (dpc) and (c) 12.5 dpc,confirming the microarray data. Maestro transcription is male-specificand restricted to the testis cords. d: WMISH of 11.5 dpc (18 tail-somitesstage) male and female gonad–mesonephros explants showing thatmale-specific expression in the gonad precedes overt differentiation.e: Male-specific gonadal expression of Maestro at 15 tail-somites stage(approximately 11.25 dpc).

Fig. 2. Maestro (Mro) expression in the developingmale gonad is not germ cell-dependent. a: Longitudinalsection of a 13.5 days postcoitus (dpc) male gonadafter whole-mount in situ hybridization with a Maestroprobe. Signal is observed throughout each testis cord,consistent with expression in both germ cells in the cen-tre and Sertoli cells at the periphery of the cord. b: Reversetranscription-polymerase chain reaction (RT-PCR) ex-pression analysis indicates that Maestro transcripts arepresent in gonads from 12.5 dpc We/We homozygotes.These mutant gonads are devoid of germ cells, as evi-denced by the absence of the germ cell marker Oct4.Expression in We/We homozygotes confirms that Maestrotranscription is not germ cell-dependent and, thus, ispresent in the supporting (Sertoli) cell lineage of thetestis. In contrast, gonads from We/We homozygoteslack Oct4 expression. Expression of Hprt in all samplesacts as a control of RNA integrity and RT-PCR. R�, reversetranscribed sample; R-, RNA sample without reverse tran-scription; nt, water blank; G, genomic DNA amplifica-tion; M, size markers.

602 SMITH ET AL.

To determine the subcellular local-ization of the Maestro protein, weused a GFP-Maestro fusion con-struct. Transfection of this constructinto COS-7, Swiss 3T3, and HeLa cellsrevealed the great majority of fluo-rescent signal to be restricted to thenucleolus, with occasional signalfound in other smaller, discrete bod-ies in the nucleus (Fig. 5a–c).

Genome mapping studies wereperformed by using fluorescent insitu hybridization (FISH) on mousechromosome spreads with a BACclone containing Mro. The identity ofthis BAC clone was confirmed by se-quencing, which showed that all ex-ons identified were identical in se-quence to clone 10p06. The FISHrevealed a single strong hybridiza-tion signal on mouse chromosome18 (Fig. 6a,b), a region syntenic withHSA 18q21. To position Maestro withrespect to known chromosome 18genetic markers, we used the T31radiation hybrid panel (McCarthy etal., 1997). Radiation hybrid mappingplaced Mro on distal chromosome18, with pair-wise logarithm of odds�6 with anchor markers D18Mit189and D18Mit142 and with a most likelylocation between D18Mit153 andD18Mit189. This map location is inagreement with that predicted byinterrogation of the Ensembl mousegenome sequence database withthe cDNA sequence of Maestro(Fig. 6c).

DISCUSSION

We describe here the identificationand characterization of a gene,Maestro (Mro), encoding a novelHEAT-repeat containing protein ex-pressed in a male-specific mannerduring mouse gonad development.The transcription of Mro in the go-nadal somatic cells and its onset be-fore sexual differentiation of the go-nad suggests that it may be regulatedby male-determining transcriptionfactors such as SRY and SOX9 andplay an early role in testis differentia-tion.

The HEAT-repeat protein motif com-prises approximately 37–43 amino ac-ids and normally appears in tandemarrays of at least three in length, witheach unit consisting of two interactinganti-parallel �-helical domains sepa-

rated by a nonhelical region (An-drade et al., 2001; Fig. 4). Crystal struc-ture studies of the PP2A PR65/Asubunit and �-importin reveal that theHEAT repeats of these proteins formflexible solenoid-like structures thatsupport multiple protein–protein inter-action (Groves et al., 1999; Vetter etal., 1999). It is likely, therefore, thatMRO interacts with other proteins, per-haps within a complex. However, thefamily of HEAT-repeat containing pro-teins is functionally diverse, includingmolecules with roles in modulation oftranslation, protein trafficking, and nu-clear transport. Notwithstanding thisdiversity, it is noteworthy, given the nu-clear import function of the HEAT re-peat-containing protein �-importin,that failure of nuclear import of thesex-determining protein SOX9 is be-lieved to underlie at least one case ofhuman XY sex reversal (Preiss et al.,2001).

Transfection of GFP-Maestro fusionconstructs into three different mam-

malian cell lines suggests a nuclearlocalization for Maestro protein, withthe majority of the signal restrictedto the nucleolus. The significance ofthese data is unclear, given therapid development of our under-standing of the function of the nu-cleolus in recent years. In addition toacting as a “ribosome factory,”data suggest that the nucleolus canfunction as a storehouse for titrationor sequestration of specific proteinsand, thereby, act to modulate mo-lecular pathways (reviewed in Olsonet al., 2000; Bernardi and Pandolfi,2003). An example of this phenome-non is the nucleolar retention ofMDM2, a negative regulator of p53,which results in stabilization of nucle-oplasmic p53 (Weber et al., 1999).The putative nucleolar localizationof Maestro awaits confirmation byimmunohistochemical studies of thedeveloping testis with an anti-Mae-stro antibody.

The chromosomal region to which

Fig. 3. Embryonic and adult Maestro transcription. a: Northern analysis of Maestro tran-scription during mouse gonad development. A prominent transcript of approximately 2.0kb is detected in male gonadal polyA� RNA at 13.5 and 14.5 dpc (lower arrow). No signalis detected in female RNA tracks. A much fainter transcript of approximately 4.5 kb is alsovisible in both male samples (upper arrow). A �-actin loading control is also shown.b: Northern blot of unsexed adult mouse tissues revealing Maestro transcription in severalorgans. Expression is most prominent in testis (T), brain (B), liver (Li), and heart (H), withweaker expression in kidney (K). No expression is detected in spleen (Sp), lung (Lu), orskeletal muscle (Sk). Variation in transcript size between tissues and the occasional pres-ence of more than one transcript in a tissue suggest some alternate splicing may occur. A�-actin loading control is also shown.

MAESTRO: A CANDIDATE TESTIS DETERMINING GENE 603

Fig. 4. Multiple sequence alignments of theHEAT repeats of Maestro, human B29, andother proteins. Predicted HEAT repeats frommouse Maestro and human B29 are alignedwith those from a B29-like protein (B29b), afruit fly hypothetical protein (LD28902p), andthe human protein phosphatase 2A PR65/Asubunit (Groves et al., 1999; Protein Data-bank Code: 1B3U). The two HEAT repeat �-he-lices seen in crystal structures are repre-sented above the alignment. The alignmentwas prepared by comparing a hiddenMarkov model (Eddy, 1998), generated byusing repeats from 1B3U, against Maestroand its homologues. Consensus coloring ofalignment columns used CHROMA (Good-stadt and Ponting, 2001). Amino acids, orgroups of amino acids, are colored if theyoccur in more than 75% of these 26 se-quence repeats: D, aspartic acid (in red ongray); l, aliphatic residues (I, L, V; in gray onyellow); s, small residues (A, C, D, G, N, P, S,T, V; in green); p, polar residues (C, D, E, H, K,N, Q, R, S, T; in blue); and h, hydrophobicresidues (A, C, F, G, H, I, L, M, T, V, W, Y; inblack on yellow). Species abbreviations:Dm, Drosophila melanogaster; Hs, Homo sa-piens; Mm, Mus musculus. Numbers repre-sent the sequences' amino acids shown.

Fig. 5. Subcellular localization of a Maestro–green fluorescent protein (GFP) fusion pro-tein. All images shown are after transfection with a construct having full-length Maestro atthe N-terminus of the fusion protein. The same results were observed with the reverseorientation construct. a: Representative image of a transfected COS-7 cell is shown, withGFP signal most intense in the nucleolus. COS-7 cells have a small number of prominent,large nucleoli, as revealed by 4�,6-diamidine-2-phenylidole-dihydrochloride (DAPI) stain-ing (data not shown). b: Occasionally, GFP signal was observed in small, discrete nuclearbodies outside of the nucleolus. c: Transfected 3T3 cells, in contrast to COS-7, have largernumbers of small nucleoli. Weak signal is also observed throughout the nucleus in mosttransfectants. Transfected HeLa cells also showed nucleolar localization of Maestro–GFP(data not shown).

Fig. 6. Chromosomal localization of Mae-stro. a: Representative chromosomal spreadafter fluorescent in situ hybridization analy-sis of a mouse BAC clone containing Mae-stro. b: The identity of the hybridizing chro-mosome was confirmed by G-banding.c: Composite radiation hybrid (RH) andphysical map of distal mouse chromosome18. RH mapping places Maestro betweenD18Mit141 and D18Mit210, in agreement withthe position predicted by SSAHA alignmentof the Mro cDNA with the Ensembl annotatedmouse genome sequence. Distances be-tween microsatellite markers and Mro are inmegabases (Mb). Mro–RH anchor markerpair-wise logarithm of odds (LOD) scoreswere calculated by The Jackson LaboratoryMouse Radiation Hybrid Database.

604 SMITH ET AL.

Maestro maps in humans, 18q21,contains no loci associated with ab-normalities in sexual development.However, a recent report has identi-fied a locus on mouse chromosome18 (Odsm1) that controls XX sex re-versal in mutant Odd Sex (Ods) mice(Qin et al., 2003). The Ods mutantmouse was originally identified whenrandom insertion of a transgene up-stream of the sex-determining geneSox9 resulted in XX transgenics de-veloping as males (Bishop et al.,2000). When alleles of Odsm1 de-rived from the A/J strain of mouseare bred to homozygosity on theOds transgenic background, XXtransgenics develop normally as fe-males. Thus, the Odsm1 locus onmouse chromosome 18 is a geneticmodifier of the Ods sex reversal phe-notype. The mechanistic basis of thisgenetic interaction is unclear, al-though it is possible that the A/J-de-rived Odsm1 protein acts as a“strong” female-promoting mole-cule, allowing repression of inappro-priate Sox9 expression in XX Odstransgenics (Qin et al., 2003). How-ever, an alternative interpretation isthat the A/J Odsm1 allele acts as a“weak” promoter of testis develop-ment in the same context, failing tosufficiently block repression of Sox9transcription. The radiation hybridmapping data presented hereplace Maestro in the Odsm1 criticalregion (Qin et al., 2003) betweenD18Mit184 and D18Mit25, close toD18Mit210. Despite the absence of aknown function for Maestro in go-nad development, it is still formally acandidate gene for the Odsm1 lo-cus in light of these considerations.However, it will be necessary to per-form direct genetic linkage studiesbetween Maestro and Odsm1 be-fore this candidature can be ex-plored further. Our current studiesare aimed at determining a role forMaestro in mouse gonad develop-ment using gene targeting.

EXPERIMENTAL PROCEDURES

DNA Microarray Analysis

A normalized cDNA library (NMUR)from urogenital ridges (UGRs) dis-sected from 11.5 and 12.5 dpc em-bryos was used as a source of cDNAclones for microarray construction,

as previously described (Grimmondet al., 2000). Total RNA was preparedfrom embryonic gonads at 13.5 dpcas previously described (Grimmondet al., 2000). Due to the limitingamounts of RNA available, we usedan amplification strategy based onPCR (for details, see Smith et al.,2003). Briefly, first-strand cDNA wasgenerated by using a modifiedoligo-dT primer (Van Gelder et al.,1990). Double-stranded cDNA wasgenerated by using a cDNA synthe-sis system (Roche). Multiple cycles ofamplification (up to 40) were per-formed using a heel primer comple-mentary to the modified oligo-dTprimer and Taq DNA polymerase(ABgene). Amplified cDNA was fluo-rescently labeled by incorporationof Cy5/Cy3-dCTP (Amersham) usingrandom priming and extension withKlenow fragment (Invitrogen).

Microarray hybridization andscanning were performed as previ-ously described (Grimmond et al.,2000). ImaGene (Biodiscovery) andGenespring (Silicon Genetics) wereused for data capture and subse-quent bioinformatic analysis.

Expression Analysis

Embryo collection, genotyping,whole-mount in situ hybridization, andsectioning were performed as previ-ously described (Grimmond et al.,2000). RT-PCR using gonadal RNA ex-tracted from We/We embryos wasperformed using Superscript II (Invitro-gen). Mro primers were 5�-ATGACT-GTTCGAGCCTGTGT-3� and 5�-AG-CAAGCTCCAAGCCAGTTA-3�; Oct4primers were 5�-CTCAGCCTTAAGAA-CATGTG-3� and 5�-TTCTCTTGTCTAC-CTCCCTT-3�; Hprt primers were 5�-CCTGCTGGATTACATTAAAGCACTG-3�and 5�-GTCAAGGGCATATCCAACAA-CAAAC-3�.

Cloning of Maestro cDNA

The 1072-bp insert of NMUR cDNAclone 10p06 was sequenced by us-ing vector primers. 5� RACE PCR wasperformed with RNA from 13.5 dpcembryonic male gonads by using aSMART RACE cDNA amplification kit(Clontech). Briefly, we generatedfirst-strand cDNA by oligo-dT priming.The first round of PCR was performed

by using the SMART universal primermix and Mro primer 5�-GCCTGTGGT-TCTTCTGTTCT-3�. A second roundwas performed on a dilution of first-round products using the nestedSMART universal primer mix and Mroprimer 5�-CACAGGCTCGAACAGT-CATT-3�. Products were cloned intopGEM-T�easy vector (Promega) andsequenced by using vector primers.The sequence of the composite MrocDNA generated by addition of the5� RACE product was subsequentlyverified by RT-PCR experiments andhas been assigned GenBank acces-sion no. AY243876.

Isolation of Mro BAC Clone

Mro primers 5�-GTACTGACTAGGC-CATCTCC-3� and 5�-TCCTGTAGAC-GGTGTAGAGC-3� were used toscreen superpools of the mouse BACII library (Research Genetics). Onepositive clone was identified.

Subcellular Localization

Green fluorescent protein (GFP)–Maestro fusion constructs weregenerated by inserting mouseMaestro cDNA both upstream anddownstream of GFP in thepcDNA3.1/CT-GFP-TOPO vector(Invitrogen). Full-length Maestrowas amplified by RT-PCR usingprimers: 5�ACCATGGAACAAACTG-GGAAAATC-3� and 5�-CCAGG-ATTTTATTCGCATAGAAG-3�. NIH3T3, Cos-7, and HeLa cells weremaintained in DMEM-glutamaxsupplemented with 10% fetal bo-vine serum and penicillin/strepto-mycin. Cells were seeded ontoeight-well chamber slides at 50%confluence 24 – 48 hr before trans-fection. Control pcDNA3.1/CT-GFP-TOPO vector or Maestro–GFP con-struct was transfected into cells byusing Fugene 6 reagent (Roche)according to the manufacturer’sinstructions. After incubation for 48hr, cells were fixed with 3.7% para-formaldehyde/PBS for 10 min atroom temperature, washed in PBS,and mounted with Vectashield(Vector Laboratories, Inc.). GFPwas visualized by confocal micros-copy.

MAESTRO: A CANDIDATE TESTIS DETERMINING GENE 605

Chromosomal Localization

FISH.

Chromosome spreads from mousespleens were prepared by usingstandard procedures. Chromosomeswere denatured in 70% formamide in2� SSC at 72°C for 2 min. The Mae-stro BAC probe was labeled with Bi-otin-16dUTP (Boehringer Mannheim)by using the Bio-Nick Kit (Gibco-BRL)at 16°C for 90 min. Probe was dena-tured for 8 min at 75°C followed bypreannealing at 37°C for 30 min. Af-ter applying probes to slides, hybrid-ization was performed overnight in ahumid chamber at 37°C. Posthybrid-ization washes were in 40% form-amide at 42°C the following morn-ing. The probe was detected withAvidin fluorescein isothiocyanate(FITC) and chromosomes counter-stained with 4�,6-diamidine-2-pheny-lidole-dihydrochloride (DAPI). Slideswere mounted with Vectashieldmounting medium (Vector Labs)containing 1 �g/ml DAPI. FISH im-ages were generated with an epif-luorescent microscope equippedwith a cooled CCD camera (Photo-metrics, Sensys) and computer-con-trolled filter wheels with excitationand emission filters for visualization inDAPI and FITC. Chromosomes werepseudo-G–banded, and the BACwas chromosomally localized by us-ing the ideograms installed in theSmartCapture karyotyper.

Radiation Hybrid Mapping

For mapping of Maestro we used theT31 panel of whole-genome radia-tion hybrids from Research Genetics(McCarthy et al., 1997). This panelconsists of 100 mouse/hamsterclones with an average mouse ge-nome retention frequency of 30%.We performed mapping by analyz-ing a subset of 94 clones. PCR anal-ysis of each clone was used to de-termine the presence or absence ofMro. PCR primers were 5� GTACT-GACTAGGCCATCTCC 3� and 5� TC-CTGTAGACGGTGTAGAGC 3�. Theseprimers amplify a 340-bp Maestroproduct from mouse genomic DNAand give no amplification productswith hamster genomic DNA. DNAsamples (25 ng) from 94 of the hybridclones, plus mouse and hamster

genomic DNA controls were used forPCR amplification in 96-well microti-ter plates. Cycling conditions wereinitial denaturation at 94°C for 5 min,followed by 39 cycles of 94°C for 1min, 58°C for 1 min, and 72°C for 1min. PCR analysis was performed induplicate and no more than threediscrepancies between duplicatedata sets were tolerated. Any dis-crepancies were scored as “not de-termined,” and a single compositedata set was obtained. These datawere analyzed by using Mapman-ager QT and The Jackson LaboratoryMouse Radiation Hybrid Databaseautomated mapping system foundat: http://lena.jax.org/resources/documents/cmdata/rhmap/rhsubmit.html.

ACKNOWLEDGMENTSWe thank Elaine Whitehill for animalhusbandry, Terry Hacker for assis-tance with histology, and the GASCore at Harwell for DNA sequenc-ing.

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