GENOMICS

8,331-339

(1990)

Genomic Analysis of a Mouse Zinc Finger Gene, Zfp-35, That is Up-Regulated during Spermatogenesis VINCENT CUNLIFFE,’

SARAH WILLIAMS,

Imperial Cancer Research Fund Laboratories, Received

January

44 Lincoln’s inn Fields, London 16, 1990;

Academic

Press.

Inc.

INTRODUCTION

The zinc finger domain is a sequence-specific nucleic acid-binding motif that was first recognized in the Xenopus transcription factor IIIA (TFIIIA; Miller et al., 1985) and subsequently identified in the open reading frames of two Drosophila segmentation genes, Kruppel and Hunchback (Rosenberg et al., 1986; Tautz et al., 1987). The ADRl gene of Saccharomyces cerevisiae encodes a trans-activator which contains tandem repeating zinc finger units (Hartshorne et al., 1986), indicating that the zinc finger motif is an evolutionarily conserved DNA-binding domain. Several of the zinc finger domains of TFIIIA, Kruppel, and ADRl contain a short repeat located between the last histidine of one finger and the first cysteine of the next, similar or Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under Accession No. 504770. 1 To whom correspondence should he addressed.

revised

April

WCZA 3PX, United Kingdom

13, 1990

identical to the sequence TGEKPYE. This sequence is also found in the coding regions of other Drosophila and vertebrate zinc finger genes (Schuh et al., 1986; Chowdhury et al., 1987; Kadonaga et al., 1987; Ruiz i Altaba et al., 1987; Morishita et al., 1988; Parkhurst et al., 1988; Chavrier et al., 1989). To identify zinc finger genes with regulatory roles in mammalian germ cell development, we isolated cDNA clones from mouse embryonic gonad and adult testis libraries, using an oligonucleotide probe encoding the TGEKPYE sequence. In the course of our studies we identified a mouse zinc finger gene, Zfp-35, that gives rise to a 2.4-kb mRNA with an open reading frame that encodes a protein with a large block of 18 contiguous zinc finger domains (Cunliffe et al., 1990). The N-terminal region of this polypeptide contains an abundance of acidic residues that is characteristic of some transcription activators (Sigler, 1988). The 2.4kb mRNA is selectively expressed in testis and is specifically upregulated within the pachytene spermatocyte stage of male germ cell differentiation. To provide a structural basis for comparative studies on the organization, regulation, and function of zinc finger genes, we have elucidated the genomic architecture of the Zfp-35 gene, determined its chromosomal location, and investigated its conservation in the animal kingdom.

Zinc finger genes are a class of eukaryotic regulatory genes that encode sequence-specific nucleic acidbinding proteins. Members of this large gene family are required for growth and development in a wide range of organisms. We previously identified a mouse zinc finger gene, Zfp35, that was up-regulated during spermatogenesis at the pachytene spermatoeyte stage of development. We now describe the genomic organization of this gene, including its intron-exon structure, the sequence of its flanking regions, and its assignment to a region encompassing bands B3 to C of chromosome 18. The transcription unit has three exons. Intron 1 is within the 5’ untranslated region and exon 3 contains the block of all 18 zinc fingers. These two features are common to a number of zinc finger genes. We also show that Zfp-35 is conserved in some placental mammals and that it is a member of a subfamily of related mammalian zinc finger genes. 0 1990

AND JOHN TROWSDALE

MATERIALS

AND

METHODS

Screening of a Mouse Genomic DNA Library A mouse strain 129 library was constructed in pcos2EMBL from genomic DNA that was partially digested with Mb01 (Erich et al., 1987; kindly provided by Dr. Lisa Stubbs). The library was screened with probe Zfp35-0.6 (Cunliffe et al., 1990), and four distinct, overlapping cosmids were isolated. Restriction maps were compiled using the method of Rackwitz et al. (1985), and the approximate positions of exons were located by hybridization of cDNA probes to complete restriction enzyme digests of the cosmids.

the

331 Copyright 0 1990 All rights of reproduction

0888-7543/90 $3.00 by Academic Press, Inc. in any form reserved.

332 DNA

CUNLIFFE,

WILLIAMS,

Sequence Determination

Restriction fragments of cosmid Zfp35-1 were subcloned into the Bluescript plasmid (Stratagene) and sequenced by the primed synthesis chain termination method (Bankier et al., 1987). Primers included a family of oligonucleotides of sequences distributed throughout the Zfp-35 gene, as well as the forward and reverse Ml3 sequencing primers whose sequences were located in the vector polylinker. Primer

Extension

Total RNA (30 pg) was mixed with 2 ng [T-~~P]ATP end-labeled Zfp-35-specific oligonucleotide 5’-GGAAGCAACTGGAGAATT-3’ (complementary to positions +538 to +521 in Fig. 2) in 20 r&4 Tris * Cl (pH 8.5), 100 mM NaAc, 16 mM MgC12, in a final volume of 10 ~1. After heating at 90°C for 2 min, the mixture was cooled at 37°C for 15 min; then 10 ~120 mM DTT, 1 ~1 10 n&f dNTPs, 20 U RNasin, and 10 U AMV Reverse Transcriptase were added and the reaction was incubated for 45 min at 43°C. The reaction was terminated by adding EDTA to 20 mlW, and after phenol extraction nucleic acids were precipitated with ammonium acetate. cDNA products were analysed on 6% acrylamide-7 M urea denaturing gels. Southern

Blot Hybridization

The preparation of Southern blots, random primed probes, and the hybridization analyses were as previously described (Trowsdale et aZ., 1989). Briefly, DNA was digested with BamHI and electrophoresed in an 0.8% agarose gel in 1X Tris-acetate/EDTA buffer (Sambrook et al., 1989). DNA was transferred to Hybond N-plus (Amersham, UK), according to the manufacturer’s instructions, and hybridized in 6X standard sodium citrate (SSC), 5X Denhardt’s solution, 0.5% SDS, 10% dextran sulfate. Probes were labeled by random hexamer priming and were added to the hybridization mix at a concentration of 5 X lo5 dpm/ml (Feinberg and Vogelstein, 1983). Stringent posthybridization washes of filters were performed in 0.1X SSC, 0.1% SDS, at 65°C for 30-60 min, prior to autoradiography. Nonstringent posthybridization washes were performed in 2X or 3X SSC, 0.1% SDS at 65°C for 20 min. In Situ Hybridization Mouse bone marrow cultures were incubated for 60 min at 37“C in RPM1 1640 medium containing 10% fetal calf serum and 0.05 pg/ml colcemid prior to the preparation of metaphase chromosome spreads. Following G-banding and photography, in situ hybridization of the biotinylated cosmid Zfp35-1 to selected metaphases was performed essentially as described by

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TROWSDALE

Edwards et al. (1990). To prevent repeated sequences in the cosmid from hybridizing to the chromosomes, 80 ng of biotinylated probe was competed with 2.5 fig sonicated genomic mouse DNA at 37’C for 3 h before hybridization to the metaphase spreads. Zfp-35-specific signals were then detected with FITC-conjugated avidin and amplified by successive incubations with biotinylated anti-avidin and FITC-conjugated avidin. After staining with propidium iodide, metaphases were rephotographed using Fujicolor HG400 film with a Zeiss filter set 9. Only paired signals located on both chromatids of the same chromosome were scored from these photographs, and the distance from centromere to signal was expressed as a proportion of the distance from centromere to terminus. The location of the gene was inferred by comparison of the signal position with the prebanded photographs and the standard karyotype (Evans, 1989). RESULTS

Isolation of Cosmids Containing the Zfp-35 Gene To elucidate the genomic organization of the Zfp-35 gene and to determine the sequence of its 5’ flanking region, clones containing this gene were isolated from a cosmid library of mouse strain 129 genomic DNA, using the probe Zfp35-0.6 at high stringency (Cunliffe et oz., 1990; 0.1X SSC, 65’C, 1 h). Four distinct, overlapping cosmids were isolated and mapped for restriction enzyme sites using a partial restriction mapping procedure (Rackwitz et al, 1985). These clones covered 50 kb of the region containing Zfp-35 (Fig. 1). The approximate positions of the exons were located by hybridizing restriction enzyme digests of cosmids with Zfp-35 cDNA probes, prior to subcloning each BamHI fragment and sequencing. The resultant hybridization patterns were similar to the patterns obtained when Zfp-35 cDNAs were hybridized to restriction digests of genomic DNA (0.1X SSC, 65”C, 1 h), indicating that the cosmids had not been rearranged during cloning (data not shown). Intron-Exon Structure of the 2.4kb Zfp-35 Transcription Unit The transcribed regions of the gene were sequenced in the genomic DNA by priming subclones from cosmid Zfp35-1 with a battery of oligonucleotides encoding short stretches of the Zfp-35 cDNA sequence. In this way the positions of the intron-exon junctions that were used to generate the 2.4-kb transcript were located, and the sequences at each end of the introns were determined. This analysis revealed two introns in the transcription unit, the first located only 49 nucleotides into the 5’ untranslated region and the second interrupting co-

GENOMIC

ANALYSIS

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ZfP35-5

I

I 2fp35-2 I

I Zfp35-8 1

I Zfp35-1 I

I lkb

au

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BB

B

I I

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n Zfp35-PCR

FIG. 1. Genomic organization of the mouse Zfp-35 gene. (a) A series of overlapping cosmids that were ordered by restriction mapping. A composite map of the region of the genome from which they are derived, locating the position of BumHI sites, B, is shown beneath. (b) The intron-exon structure of Zfp-35 is represented by black boxes (exons) and lines (introns), and lies underneath the BanHI restriction map to indicate the location of exons within the cosmid contig. (c) Location of structural domains in the open reading frame for the Zfp-35 transcription unit. Lines represent 5’ and 3’ untranscribed regions and introns, open boxes represent 5’ and 3’ untranslated regions, black boxes represent nonfinger domains including the acidic region, and the gray box indicates the position of the zinc finger domain. The exons identified by cDNA probes Zfp35-PCR and Zfp35-2.0 are bracketed underneath.

don 17 of the open reading frame, in the nonfinger region (Fig. 2). Thus exon 1 contained 49 nucleotides of 5’ untranslated region, exon 2 contained 155 nucleotides of 5’ untranslated region plus the first 17 codons of the open reading frame, and exon 3 contained the remainder of the nonfinger region including the acidic region plus the whole of the zinc finger domain. All the transcribed portions of the gene were sequenced and no sequence differences from that of the cDNA were found. Intron 1 was approximately 500-700 bp in length, by restriction mapping, whereas intron 2 was much larger, spanning 12 kb. Comparison of the two pairs of splice donor and splice acceptor sequences showed that each corresponded to the canonical splice consensus sequences (Padgett et al., 1986). Definition of the Site of Transcription Primer Extension and Identification Flanking Sequences

Initiation by of 5’ and 3’

A 32P-end-labeled 18-mer oligonucleotide complementary to sequences between nucleotides +174 and +193 in the published cDNA sequence was used to prime cDNA synthesis on RNA from adult male spleen, thymus, and testis, and the products were analyzed on a denaturing acrylamide gel (Fig. 3). Yeast tRNA was used as a negative control for primer extension specificity, and a dideoxy sequencing ladder was used as marker. A testis-specific product 197 nucleotides in length was generated (Fig. 3, lane 2), and allowing for the

post-transcriptional addition of a single nucleotide cap, the site of transcription initiation was thus localized to a single position four nucleotides 5’ to the first nucleotide in the cDNA sequence previously described. The position of transcription initiation thus defined is consistent with the position of the 5’ ends of other cDNA clones recently isolated, which are also 4 nucleotides longer than the previously reported sequence. An additional extension product approximately 500 nucleotides long was also observed in all three mouse tissues and is likely to be either an alternative transcript from this gene or an experimental artifact. A second primer complementary to the known Zfp-35 cDNA sequence detected only the testis-specific RNA (data not shown). In view of the fact that Northern analysis of spleen and thymus reveals a low level of Zfp-35 mRNA (Cunliffe et al., 1990), the absence of extension products 197 nucleotides long in the samples from these tissues in Fig. 3 may reflect the lower sensitivity of the primer extension assay compared to that of the Northern analysis. Primers complementary to the 5’ end of the cDNA were used to sequence the region 5’ to the initiation site, up to position -232 (Fig. 2). This DNA sequence was compared with that of other promoters, and it bore only one obvious homology to known transcription factor binding sites: an APl binding site (TGAGTC; Jones et al, 1988) starting at position -196. The site of transcription initiation as defined by primer extension was shown to be precise and limited to a single nucleotide, yet no TATA box motifs, commonly as-

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WILLIAMS,

sociated with directing accurate initiation to a single nucleotide, were observed. Sequencing the B’end of the 1.3-kb BamHI fragment containing exon 3 identified two variant polyadenylation sequences (Fig. 2), both of which were located 5’ to the known position of poly(A) addition from previous cDNA cloning. Between these two sequenceswas the pentanucleotide CACTG, which is frequently found in close proximity to polyadenylation signals and may interact with the U4 snRNP particle (Berget, 1984). A substantially T-rich stretch on the sense strand was located close to the site of polyadenylation on its 3’ side, and such sequences have been associated with transcription termination. The gene thus encompasses 19 kb of DNA, of which only 2.4 kb encode the final transcription product. The Zfp-35 Gene Is Conserved in Some Mammals and Is a Member of a Large Mammalian Zinc Finger Gene Family Many features of mammalian spermatogenesis are conserved among mammals (Bellve, 1979), and it might be expected that some genes required for this process are similarly conserved. Previous work had established Zfp-35 as a potentially important regulatory gene that was up-regulated during the pachytene stage of spermatogenesis; therefore, using Southern blotting we analyzed genomic DNA from a variety of eukaryotes for the presence of Zfp-35related sequences(Fig. 4). Using the cDNA probe Zfp35PCR (Fig. lc), containing nonfinger coding sequencesand the 5’ untranslated region, only three BamHI fragments previously identified by cosmid cloning were visualized in mouse genomic DNA, even under low-stringency hybridization conditions (Fig. 4b). In addition, between one and four high-molecular-weight bands corresponding to the cognate Zfp3.5 gene could be identified in rat, human, whale, and horse DNA. This subset of eutherians represents four mammalian orders with relatively distinct evolutionary histories and dissimilar biological adaptations. As Zfp35 is a marker for the mouse pachytene spermatocyte, the observed sequence conservation may reflect a function during spermatogenesis that is common to these mammals. When the cDNA probe Zfp352.0 (Fig. lc), containing the acidic region and all 18 zinc finger domains, was hybridized at low stringency to the same samples of genomic DNA, a ladder of bands could be identified

AND

TROWSDALE

in all mammals (Fig. 4a). It is unlikely that these related sequencesare processed pseudogenes from the homologous Zfp-35 loci because none were identified by the Zfp35-PCR probe in Fig. 4b. This indicates that a substantial number of these sequencesare either functional genes or nonfunctional relics of ancestral gene duplication events. The complex patterns of Zfp-35-related hybridization signals observed in Fig. 4a do not persist after high-stringency washes (0.1X SSC, 65°C 1 h). Under these conditions, the Zfp35-2.0 probe nevertheless identifies the two BamHI fragments of approximately 1.5 and 5 kb identified at low stringency in Fig. 4a (data not shown) that correspond to the cognate fragments in the set of overlapping cosmid clones in Fig. 1. The organization of these related sequencesis similar in some closely related mammals. Note, for example, the similarity in the restriction fragment patterns on the three primate DNA samples (Fig. 4a, tracks 15,16, and 17) and the near identical restriction fragment patterns that hybridization to goat and sheep genomic DNA produces (Fig. 4a, tracks 6 and 8). Zfp-35 Is Located on Mouse Chromosome 18 Cosmid Zfp35-1 was labeled with biotin and used as a probe for in situ hybridization to mouse metaphase chromosome spreads. Hybridization was visualized with FITC-conjugated avidin (Fig. 5). Five metaphase spreads were analyzed and signals were detected on 18 of the 20 chromatids from chromosomes 18. There were no paired signals on any chromosome other than chromosome 18. Comparison with banded karyotypes showed that 72% of all paired signals were localized to band B3 or C of chromosome 18, indicating that Zfp35 maps to this region. A histogram of the distribution of signals is shown in Fig. 6. Although there is a relative paucity of both genetic and molecular markers for this autosome compared to those that exist for other chromosomes, the zinc finger gene Krox-24 has recently been localized to a region encompassing bands C and D of chromosome 18 (Jannsen-Timmen et al., 1989). DISCUSSION

The Genomic Organization of Zfp-35 and That of Other Members of the Zinc Finger Gene Superfamily Are Similar By means of cosmid cloning and sequencing, the organization of Zfp-35 has been determined. A small 47-

FIG. 2. Nucleotide sequence of the Zfp-35 gene including 5’ and 3’ flanking regions. The position of transcription initiation is indicated by the right-angled arrow, and the locations of the intron+rxon junctions are indicated as right-angled lines. The positions of two putative polyadenylation signals are underlined, and the point of poly(A) addition is shown by the vertical arrow. The sequence of the open reading frame is given beneath the nucleotide sequence. Numbering of the DNA sequence is on the left and follows from the designation of the first transcribed nucleotide as +l. This numbering is arbitrary, as the complete sequences of introns 1 and 2 were not determined. Numbering of the amino acid sequence of the open reading frame is on the right.

583 17

30

797 70

917 110

1037 150

i157

~GAGTCCACACTGGT-CCTTA~GTGTWITtA QRVHTGEKPYKCDECGKAFSQSSDLHIHPRlHTCEKDyQC

-CCcTACCMIGT 190

1277 230

270

,517

CCATATCCGTGTGCTUGM;T~C~GT~TAGT-TTCA~C~TATT~CACAGMG~T~A~C~GffiA~CC~AT~TGTAGTGMTG~~ffiC~TTMc i Y P c ., Q c NKSFSQNSDLIKHRRIHTCEKPYKCSECGKAFN

310

1637 350

1757 390

1877 430

1997 470

2117 510

2237 550

2357 580

2477

2597

2717

2837

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1

2

3

WILLIA

4GATC

: FIG. 3. Definition of the site of transcription initiation by primer extension. Gel electrophoresis and autoradiography of cDNA synthesis products, using a 32P-labeled primer complementary to positions +174 to +193 in Fig. 2 and total RNA from mouse organs as template. Lanes: 1,30 pg yeast tRNA control; 2,30 cg testis RNA; 3,30 pg spleen RNA; 4, 30 pg thymus RNA. Right: A dideoxy sequencing ladder size marker.

nt exon. 1 in the 5’ untranslated region is followed by a second small exon of 204 nt containing the remaining 152 nt of 5’ untranslated sequence and the first 17 codons of the open reading frame. The third exon contains the remainder of the 2.4-kb transcription unit. A noncoding first exon is a property conserved among several human, mouse, and Drosophila zinc finger genes (Tautz et al., 1987; Chowdhury et al., 1988a; Morishita et al., 1988; Parkhurst et al., 1988, Chavrier et al., 1989; Schneider-Gadicke et al., 1989) and may indicate a functional requirement for intron 1 sequences to be close to the promoter. Although six of the nine zinc finger domains in Xenopus TFIIIA each reside on individual exons (Tso et al., 1986), it has been argued that introns were inserted after the TFIIIA coding region was assembled (Rogers, 1986). Indeed, many of the zinc finger genes that have been well characterized possesszinc finger regions unperturbed by introns. The zinc finger regions of the mouse Krox-20 and Krox-24 genes are not perturbed by introns (Chavrier et al., 1989; Jannsen-Timmen et al., 1989), and the human ZFY and ZFX genes both contain a block of 13 zinc finger domains that is wholly located on a single exon (Page et al., 1987; SchneiderGadicke et al., 1989). To date, the block of 18 zinc finger domains in Zfp-35 constitutes the largest uninterrupted zinc finger-containing exon described for any gene. Such genomic integrity of the finger region indicates that these genes may have evolved by sequence duplication and domain shuffling by unequal crossing over. In this way recombination events between similar

MS.

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TROWSDALE

TGEKPYE repeats would either permit the exchange of zinc finger domains between genes or facilitate the insertion of one or more zinc finger domains within a gene. Such mechanisms could produce genes encoding proteins with diverse and overlapping DNA-binding specificities, and thereby engender the rapid evolution of new regulatory networks. The general absence of introns within the zinc finger region and the general presence of an intron in the 5’ untranslated region of zinc finger genes together suggest that their genomic organization may be conserved for a reason. The precise distribution of intron and exon sequences might confer important regulatory properties to such genes. Given the small size of the first two exons and the large size of the introns, it is conceivable that there are

a

b

1 2 3 4

3.7-

5 6 7 6 9 1011121314~51617

’

FIG. 4. Low-stringency hybridization of cDNA probes from the Zfp-35 2.4kb transcription unit to BamHI restriction digests of genomic DNA from a range of eukaryotes. (a) Hybridization was with probe Zfp35-2.0 [Fig. lc and Ref. (12)]. (b) Hybridization was with probe Zfp35-PCR [Fig. lc and Ref. (12)]. Lanes: 1, moth, 4 pg; 2, Drosophila mdamgaster, 4 pg; 3, Xenopus, 15 pg; 4, chicken, 15 gg; 5, pig, 15 pg; 6, goat, 15 pg; 7, horse, 15 gg; 8, sheep, 15 pg; 9, whale, 15 s 10, dog, 15 pg; 11, cat, 15 gg; 12, rat, 15 gg; 13, mouse, 15 pgg; 14, guinea pig, 15 gg; 15, cytomegalus monkey, 15 pg; 16, rhesus monkey, 15 pg; 17, human, 15 pg. Sizes of DNA markers are on the left (kb). In (a) the membrane was washed at 65“C for 20 min in 2X SSC, 0.1% SDS, and then exposed to fiIm for 18 h. In (b) the membrane was washed at 65°C for 20 min in 3X SSC, 0.1% SDS, and then exposed to film for 18 h.

GENOMIC

ANALYSIS

OF

other exons and/or promoters that could be used in conjunction with exon 3 to produce alternative mRNAs encoding a similar protein in different regulatory circumstances. In support of this, probes from the Zfp35 zinc finger region detect both the 2.4-kb mRNA and a second 3.5-kb mRNA of much lower abundance (Cunliffe et cd., 1990) which may represent an alternative transcript or partially processed transcription intermediate from the Zfp-35 gene. The use of alternative promoters and exons has been documented for vertebrate homeobox genes (Cho et al., 1988, Acampora et al., 1989), as well as for the Drosophila Kruppel, hunchback, and Serendipity zinc finger genes (Rosenberg et al, 1986; Tautz et al., 1987; Vincent et al., 1985).

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The 5’ Flanking Regions of Genes Selectively Expressed in Testis Have a Limited Number of Common Features The 5’ flanking regions of a number of genes selectively expressed in testis were compared to that of Zfp35. Analysis of the promoters from the human PGK2, mouse Hox 1.4, mouse protamine 1, and human germ cell alkaline phosphatase genes revealed no extensive sequence identities to Zfp-35 (Peschon et al., 1987; Millan and Manes, 1988; Galliot et al., 1989; Robinson et al., 1989). Transcription initiation is precise for Hox 1.4, PGK2, and Zfp-35, even though their 5’ flanking regions each lack an obvious TATA box (Bucher and Trifonov, 1986). Moreover, all three genes are coordinately up-regulated in the pachytene spermatocyte, and it is therefore tempting to speculate that such activation may involve a spermatocyte-specific, TATAindependent mechanism. A region of the PGK2 promoter that has been shown to direct pachytene spermatocyte-specific expression of a reporter transgene contains a sequence of 12 nucleotides that matches a sequence in the 5’ flanking region of Zfp-35. The sequence, AGGTTTTTACAT, is located at positions -512 to -501 in the PGK2 promoter (Robinson et al., 1989) and is identical at 10 of 12 positions with the sequence GGGTTTTGACAT located at positions -226 to -215 in the Zfp-35 5’ flanking region (Fig. 2). It will therefore be interesting to determine whether this homology has any functional significance. Several mouse zinc finger genes are now known to be expressed in the testis. The expression of the Zfy1 and Zfy-2 genes in the testis requires the presence of germ cells (Koopman et al., 1989), and an increase in the steady-state levels of mRNA from both of these genes coincides with the onset of meiotic prophase in prepuberal mice (Nagamine et al., 1990). Such patterns of expression parallel that of Zfp-35 (Cunliffe et al., 1990), suggesting that the three genes may share some common germ cell-specific regulatory elements. The mouse genes mKr4 and A4OK2 are also preferentially

FIG. 5. In situ hybridization of cosmid Zfp35-1 to mouse metaphase chromosomes. (a) Giemsa-banded chromosomes. (b) In situ hybridization of biotinylated Zfp35-1 cosmid to the same set of chromosomes was visualized with FITC-avidin. Arrows in (a) and (b) indicate position of Zfp-35 by this method.

expressed in adult testis (Chowdhury et al., 1988b; Ernoult-Lange et al., 1990) and represent further candidate genes that could be analyzed for regulatory elements that target expression to the testis.

Zfp-35-Specific Sequences Are Detected in a Variety of Placental Mammals Cell-type-specific regulatory controls limit Zfp-35 up-regulation to the pachytene spermatocyte, and so it is reasonable to postulate a role for this gene in mammalian spermatogenesis. Using a gene-specific probe, Zfp-35 could be identified in the genomes of a diverse group of mammals, which suggests that the function of its encoded protein may also be conserved in these organisms. To obtain further information about the evolutionary origin of the mouse Zfp-35, pairwise comparisons of each Zfp-35 zinc finger domain with every one of the 17 others were performed, and the number of nucleotide substitutions between each

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AND TROWSDALE 4. BERGET, S. M. (1984). Are U4 small nuclear ribonucleoproteins involved in polyadenylation? Nature (London) 309: 179-182. 5. BUCHER, P., AND TRIFONOV, E. N. (1986). Compilation and analysis of eukaryotic POL II promoter sequences. Nucleic Acids. Res. 14: 10009-10026. 6. CHAVRIER, P., JANNSEN-TIMMEN, U., MATTEI, M-G., ZERIAL, M., BRAVO, R., AND CHARNAY, P. (1989). Structure, chromosomal location, and expression of the mouse zinc finger gene Krox-20: Multiple gene products and coregulation with the gene c-fos. Mol.

18 FIG. 6. Histogram of the distribution of signals along chromosome 18 following in situ hybridization with cosmid Zfp35-1. Units on the vertical axis represent fractions of the total length of the chromosome, and units on the horizontal axis represent the number of signals in each fraction.

pair was computed (Nei, 1987). An estimate of the divergence time of the two most similar domains, based on the number of synonymous nucleotide differences, was approximately 200 MY (data not shown). By comparison, mammalian radiation is believed to have occurred only 80 MY ago. Such estimates suggest that the assembly of Zfpi-35 into a gene encoding a block of 18 zinc fingers may have predated the evolution of many mammalian species. The characterization of cDNA and genomic clones derived from the human homolog of .@I-35 should facilitate a more detailed analysis of the evolution and function of this gene. ACKNOWLEDGMENTS We are grateful to Dr. Lisa Stubbs (ICRF, London, UK) for providing the mouse genomic library from which Zfp-35 cosmids were isolated and to Dr. Takashi Gojobori (National Institute of Genetics, Mishima, Japan) for molecular evolutionary analysis of the Zfp-35 zinc finger domains.

REFERENCES ACAMPORA, D., D’ESPOSITO, M., FAIELLA, A., PANNESE, M., MIGLIACCIO, E., MORELLI, F., STORNAIUOLO, A., NIGRO, V., SIMEONE, A., AND BONCINELLI, E. (1989). The human HOX gene family. Nucleic Acids Res. 17: 10385-10426. BANKIER, A. T., WESTON, K. M., AND BARRELL, B. G. (1987). Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. In “Methods in Enzymology” (R. Wu, Ed.), Vol. 155, pp. 51-93, Academic Press, San Diego. BELLVE, A. R. (1979). The molecular biology of mammalian spermatogenesis. Oxford Rev. Reprod. Biol. 1: 159-261.

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