A zinc finger protein-encoding gene expressed in the post-meiotic phase of spermatogenesis

Gerzr. 106 (1991) 221-227 0 1991 Elsevier Science Publishers

GENE

B.V. All rights reserved.

221

0378-l 119/91/$03.50

06048

A zinc finger protein-encoding (Recombinant

DNA;

transcription

gene expressed in the post-meiotic factor;

gene expression;

cDNA

cloning;

phase of spermatogenesis

mouse;

testis)

Paul Denny and Alan Ashworth Chester Beat& Laboratories,

The Institute of Cancer Research, London

Received by R.W. Davies: 13 November Revised/Accepted: 1 May/2 May 1991 Received at publishers: 16 July 1991

SW3 6JB (U.K.)

1990

SUMMARY

Spermatogenesis is the complex series of physiological and morphological changes that occur when spermatogonial stem cells differentiate into mature spermatozoa. Some of these changes are likely to be regulated at the level of transcription. To approach this problem, we have cloned a cDNA from mouse testis, encoding a protein (Zfp-29) with 14 copies of the zinc linger (Zf) motif commonly found in transcriptional regulatory proteins. The expression of this gene, Zfp-29, is restricted to the testis in adult mice, but also occurs during embryonic development. Within the testis, Zfp-29 mRNA is enriched in round spermatids, the earliest post-meiotic cells. Thus, the putative Zfp-2Pencoded protein may have a role in regulating the class of genes that are expressed in post-meiotic germ cells.

INTRODUCTION

The developmental process by which cells derived from the male germ line give rise to spermatozoa is known as spermatogenesis (Bellve, 1979). In the neonatal mouse, primordial germ cells differentiate to form type-A spermatogonia. These may cycle and remain as stem cells or differentiate into type-B spermatogonia, enter meiosis and give rise to primary spermatocytes. These cells are tetraploid and, following two rounds of reduction division, form haploid

Correspmdence to: Dr. A. Ashworth, tute of Cancer

Research,

Tel. (44-71)352-8133, Abbreviations: albumin;

dpc,

Fulham

Chester Road,

aa, amino acid(s); days

post coitum;

glyceraldehyde-3-phosphate nucleotide(s);

chain reaction;

Insti-

SW3 6JB (U.K.)

ext. 5200; Fax (44-71)352-3299. bp, base pair(s); EKRB,

KCl/0.0252 M NaHC0,/0.0012 M 0.0013 M CaC12/0.01 1 M D-ghCOSe/0.001 nt,

Beatty Laboratories,

London

oligo,

NaC1/0.0048

M

KH,P0,/0.0012 M MgSO,/ M glutamine pH 7.0; GAPDH,

dehydrogenase;

kb, kilobase

oligodeoxyribonucleotide;

RACE, rapid amplification

BSA, bovine serum

0.12 M

or 1000 bp;

PCR,

polymerase

of cDNA ends; SDS, sodium

dodecyl sulfate; SSC, 0.15 M NaCI/O.O15 M Na. citrate finger(s); Zfp, protein binding to Zf; Zfp, Zfp-encoding

pH 7.0; Zf, zinc gene.

spermatids. Spermatids are nondividing cells which undergo drastic morphological and functional differentiation to produce spermatozoa in a process called spermiogenesis. Spermatogenesis is a long and complex process (taking 35 days in the mouse) and is probably regulated by a correspondingly complex set of control mechanisms. Part of this regulation is mediated at the level of transcription (Willison and Ashworth, 1987) possibly through the presence of novel transcription regulatory proteins (transcription factors). Many eukaryotic transcription factors share characteristic structural motifs, some of which are involved in DNA binding (Jones, 1990). One of these, the Zf motif, is specilied by a characteristic arrangement of Cys and His residues, which are thought to bind Zn2 + ions (Miller et al., 1985; Brown et al., 1985). The co-ordination of Zn2 + ions by these residues maintains the Zf structure, which probably interacts with the major groove of the DNA double helix (Fairall et al., 1986; Diakun et al., 1986). Many genes have now been identified as members of the Zfb gene family including transcription factors such as SPl (Kadonaga et al., 1987), developmental regulatory genes in Drosophilu such as Krtippef (Rosenberg et al., 1986) and genes activated (Kinzler et al., 1988; Morishita et al., 1988) or

222 with the Drosophilu segmentation gene, Krtippel (Ashworth et al., 1989). One of these genes, Zfp-4, is expressed in adult

inactivated (Call et al., 1990) in neoplasia. Other _Z/j genes have been identified by low-stringency hybridisation (Chavrier et al., 1988; Chowdhury et al., 1987; Ashworth et al., 1989). It is likely that many of these genes encode sequence-specific nucleic acid binding proteins which may regulate the transcription of their target genes. In this paper we describe a Zfp gene, Zfp-29, which exhibits a high level of transcription in adult murine testis and may have a role in the development

RESULTS

AND

murine testis (A.A., unpublished observation). To obtain full-length cDNAs for this gene, we screened an adult testis cDNA library in the vector AZAP (Klemm et al., 1990) with the partial .Zfp-4 cDNA, pE7 (Ashworth et al., 1989). Five positive signals were obtained and following plaque purification and excision of pBluescript plasmids, the cDNAs were partially sequenced. This showed that the five clones were overlapping but were not derived from Zfp-4. These clones are derived from another Zfp gene which, in consultation with the mouse gene nomenclature committee (D. Doolittle, Jackson Laboratory), we have called Zfp-29. Despite the fact that high-stringency conditions were used for the hybridisation the overall sequence homology between Zlj-4 and Z/j-2Y is not high (approx. 76% ; data not shown). This cross-hybridisation was probably due to the

of spermatozoa.

DISCUSSION

Isolation of cDNA for a Zfp gene expressed in mouse testis We have previously described the isolation and chromosomal localisation of murine genes which cross-hybridise

(a)

Kpn I

5’

Smal

Narl

BamHl

\/

RACE

Hindlll

I

3a

I

clone

pD26

pD28 200 Fig. I. Schematic shown. plasmid

bp

diagram

of Zfp-29 cDNA

An adult mouse testis cDNA

clones.

library

Unique restriction

in lZap

DNAs were excised as recommended

(Klemm

enzyme

sites and the Stul used to clone the product

et al., 1990) was screened

by the manufacturer

(Stratagene,

according

to standard

La Jolla, CA). The probe (l.l-kb

et al., 1989) was labelled with “P as described by Feinberg and Vogelstein (1984) and following hybridisation, of 0.2 x SSCj0.I “, SDS at 65°C. Plasmids pD26 and pD28 were obtained in the initial screening. To obtain RNA, a modified of Moloney

RACE (Frohman

murine

manufacturer,

leukemia

in a 20 ~1 reaction

kit is based on the method and binding of the hybrids ethanol/Tris

volume;

(Bethesda hybrids

(‘Glassmilk’),

Research

Laboratories),

(1979) and involves

brief centrifugation

inactivation

to collect the Glassmilk,

in the kit). DNA was &ted

to the Glassmilk

transferase

for 10 min, then heat-inactivating

(Bethesda

the enzyme

by addition

then washing

from the Glassmilk

and so is lost in the washing Research

Laboratories)

by incubation

into cDNA with 200 units

in the buffer supplied

by use of the Geneclean

of enzymes

pBluescript

filters were washed to a tinal stringency sequence from the 5’ end of the Zfp-29 as primer,

removed

of RACE (see below) are Recombinant

EcoRI insert of plasmid pE7; Ashworth

(1 pg) was reverse transcribed using oligo(dT)

were purified and excess oligo(dT)

supplied

does not bind efficiently

using 15 units of terminal at 37’C

was used. Mouse testis poly(A)‘RNA

and Gillespie

(at the concentrations

Excess oligo(dT) incubating

by Vogelstein

to glass microbreads

to the purified cDNA hybrids, reaction

transcriptase

volume. cDNAjmRNA

described

pH 7,5/NaCI/EDTA

at 55 a C, in water.

et al., 1988) method

virus reverse

procedures.

of 3 ~01s. of 6 M Na

the pellet repeatedly

by two successive,

This iodide

with 50”,,

5-min incubations

steps. A ‘tail’ of A residues

and a dATP concentration

by the

kit (BiolOl).

was appended

of 0.2 mM, in a 30 ~1

at 65°C for 15 min. The hybrids

were denatured

at

95 ‘C for 5 min, together with 10 pmol of a (dT),,-ohgo adapter (5’-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT, containing XhoI, Sal1 and C/u1 recognition site sequences; Frohman et al., 1988) and then cooled to 72°C. The second cDNA strand was synthesized using 2.5 units of Tuq polymerase annealing adapter

(C&us),

in a 50 ~1 reaction

containing

at 50°C for 2 min, then incubating primer

(5’-GACTCGAGTCGACATCG)

60 mM KC1115 mM Tris

HCl pH 8.8 (at 2O”C)/2.25

at 72°C for 40 min. Zfp-29 sequences and primer B (right blackened

were then specifically box; sequence

shown

mM MgCl, amplified

in Fig. 2) were added

cDNA, which was subjected to 30 cycles of denaturation. annealing and DNA synthesis in a Techne PHC-I for the PCR were as follows: 30 s incubation at 94’C, 30 s at 50°C 60 s at 72°C with a 5-min final extension of amplification were digested with &II. further amplification of Zfp-29 sequences

and each dNTP

of this reaction

was digested

to the double-stranded

programmable thermal cycler. Conditions at 72°C. The products of this initial round

which cuts once in the adapter sequence and ligated to Sal1 + EcoRV-cut pBluescript (as described above), but using an additional, nested primer (primer A, left blackened

in Fig. 2), together with a vector-specific primer. The major 0.45-kb product the sequences of several independent isolates were determmcd.

at 0.25 mM. by

using the PCR; 10 pmol each of an

SKII. This allowed a box; sequence shown

with StuI + SalI, cloned into pBluescript,

and

223 GTTGTlTClTGTGAAAGA

CGGCCTTCTCAGAGAGCCT

TFXACAATGGCAGCCGAAGTGCC

lTAGGCTPCAAGCCTPCGTGACCATCCAGGAAG

120

MAAEVP AGCAGTGACCACTCCCCTC~~~G~C~~~~ AVSTPLSPLVQVPQEEDEQAEVTTMILEDDAWVQEAVLQE

CAGGCAGAGGTCAC CACTATGATCCTGGAGG&TGACGCGTGGGl?GCAGGAAGC&XGC!TGCAGGA

240

GGATGGCCCTGAGTCXAGC CCTTl?X!CCAGAGTGCTGGAAAAGGCAGCCCC CACCAGCAGCACCCACCCGACCCACCCCAGGCTCCTePTGGCTG DGPESEPFPQSAGKGSPQEEDAAEGPQGALVRFRELCRRW

360

GCTGAGGCCAGAGGTGCACCACACllAAGGAGCA GATGCTAACTGTGCTGCCAA GAGAAAmGGCCTGGC LRPEVHTKEQMLTVLPREIQAWLQEHRPESSEEAVALVED

480

CCTGACCCAGACaTCGGCACAGrGATTmGAGATACAGAGcGAGAA~ LTQTFRHSDFEIQSENGENSNEDMFEGVESHGMFLNISGG

TGCAACAACATCGGCCTGAGAGCAGTGFiGGAGGCAGTGGCCCTG~

TCAAATGAACACATGmCACCCTGPCGACTCACATGCCATGG

GGAAGGTGG!PZAGCAGTCTGATCCCCACAGTCAeP l"XAGFaGAGACTGTGGCTCTCCAGGGACA!lXC!CCCGGGTG&GGACCC~ EGGQQSDGDSDFERDCGSGGAQGHAPGEDPRVVPSEGREV TXJXAGCTAATAGGCCTCTACC~ GQLIGLQGTYLGEKPYECPQCGKTFSRKSHLITHERTHTG

CGTATGAATGTCCCCAGTGT

GT 720

mTAGCffiGAAATCCCACCTTATCACCCATGAGCGGACCCAG

CACGCACACAGGGGAAAAGC

960

GGCGAGAAGCCTlTCCAG!PXGCCG>GTGGCAAGAG~

CGTACTfXTCXCCCGAG~GC

B

840

AGAAAAATACTACAAATGXATGAATGTGGGAAGA GCmACPGACGGCTCGAACTPTAGTAGACACCAAACGACTCG EKYYKCDECGKSFSDGSNFSRHQTTHTGEKPYKCRDCGKS CTlTAGCCGGAGTGCGAACClTAlXACGCACCAGAGGA~CACACC FSRSANLITHQRIHTGEKPFQCAECGKSFSRSPNLIAHQR

A

600

-cc!GGTccAGccTTAATAcTcAccA-CACC

CCCCAACC!XATCGCCCATG

1080

GGAWUUULACCCTACGCGTGCAAGGA

1200

THTGEKPYSCPECGKSFGNRSSLNTHQGIHTGEKPYACKE ATGCGGCG.AAAGCTICATccAAccmTccGAcAc CGESFSYNSNLIRHQRIHTGEKPYKCTECGQKFSQSSALI

CAGAGCTCCGCGCTCAT

CAWLWUL’PCCACACCC~G~C~C~~G~

1320

TACGCACCGGAGAACGCACACCGGGGAGAAG CCCTATCAGTGCGGCGAGTGCGG CAAGAAcTrCAGclxCAGC TCCAACCPGGCCACTCACCCGCGCACCCACCTGGTGGAGA THRRTHTGEKPYQCGECGKNFSRSSNLATHRRTHLVEKPY

1440

CAAGTGCGGGCTGTGCGGCAAGA GCTPCAGCCAGAGCTCCAGCCTGATCCCGCACCAGGGCACG KCGLCGKSFSQS SSLIAHQGTHTGEKPYECLTCGESFSWS

1560

CTCCAACCTCATCAAGCACCAGCGGACGCACACCGGC GAGAAGCCCTACAGAlGCGGCGAC~TGGGAAGGGC~CCAGCGCT'ZGCAGCTCGTGGTGCACCAGCGGACGCACACCGG SNLIKHQRTHTGEKPYRCGDCGKGFSQRSQLVVHQRTHTG

1680

CGAGAAGCCCTACAAGTGCCTCATGTGTG GCAAGAGCTlCAGCCGGGGCTC~

TGGTGATGCACCAGCGAGCGCAC!lTGGGJVZACAAGCCl7!ACAGGTGCCCGGAGTGCGGGAAGGG 1800

EKPYKCLMCGKSFSRGSILVMHQRAHLGDKPYRCPECGKG

FSWNSVLIIHQRIHTGEKPYRCPECGKGFSNSSNFITHQR GACGCACCTGAAAGAGAAGC TIJTACTGAAGXGCA GAAAAGAGAAGGAAGTC!lGACTC THLKEKLY

TGCAGGGAGAACTCCCACAGTGTCCCTCCCCACAACCCTCCCCCCACAACCCCCCT

2040

CCCCCCCGCCCGTG'!XGTCCTTl!AAAAGAACCAC~TAAA~

Fig. 2. Zfp-29 cDNA sequence The nt sequence combination

of unidirectional

to complete

the sequence

and

Swiss-Prot

and deduced

was determined

databases

EMBL/GenBank/DDJB

aa sequence.

using the dideoxy

deletion derivatives on each strand. using

databases

2094

(Henikoff,

Sequences

the FASTA

The sequences

method

under accession

A and B, used in the RACE (described

et al. (1977)

1984) subcloning

were compiled

algorithm

ofprimers

of Sanger

of specific restriction

and analysed

of Pearson

and

with Sequenase

Lipman

fragments

using Microgenie (1988).

(version

in Fig. 1 legend) are underlined.

2.0) T7 DNA

and custom-synthesized

(Beckman

polymerase

(USB).

Instruments)

and compared

sequence

has been deposited

The Zfp-29 cDNA

A

primers were used to EMBL in the

No. X55 126.

1 S

G

K

S

F

G

K

S

F

R SD S R

G K SF S

Q

s

R

S

Q

s

w

S

Q R w

S s

S N -_-J TGEKPY-C-EC

GKSFS-

SSNLI-

H

Q

R

T

H

Zfp-29 cO"Se"S"S TGEKPY-C--C

-K-F---S-L--~-R-~

General

consensus

Fig. 3. The Zf domain (14 tandem repeats) of Zfp-29. Where a particular aa is identical in seven or more Zf, the position is boxed. These aa are included in the consensus sequence shown immediately below the Zf repeats, together with a general Zf consensus (Gibson et al., 1988).

224 repetitive

nature

Chowdhury

of the Zf motif

(Schuh

et al.,

approx. 500 bp shorter than Zfp-2Y mRNA (see section d) and has no potential start codons in sequences conforming to the Kozak (1987) consensus. We therefore re-screened the testis cDNA library with a fragment of pD28 nearest the 5’ end, but did not obtain any overlapping clones which

1986;

et al., 1987).

(b) Isolation of full-length The largest

cDNA

cDNA clones for Zfp-29

clone

that

we isolated

(pD28)

is

A 12345

67

28S-

-28s

18S-

-18s

kb 11

4

Fig. 5.

Fig. 4. Fig. 4. Southern-blot with restriction about

25 ng of the insert

1 x IO” dpm/pg.

outside

Fig. 5. Analysis

of pD28,

in 0.8% agarose-gels

which was labelled

described

of 40 mM Na

by Church phosphate

and Gilbert

to nylon filters (Genescreen,

by random-priming

of Zfp-29 gene expression

by Northern-blot

was used in post-hybridisation

Laboratory)

1984) to a specific

but omitting washes.

were digested

The filters were hybridised

with

activity

of about

bovine serum albumin

from the

Lanes:

bands (in kb) are shown on the left margin;

1, 3 and 5, C57BL/bJ; NheI digestion

2, 4

gives a band

hybridisation.

(Panel A) Tissue-specific

using the pH 4.0 guanidinium

6, testis; 7, 2 kg embryonic

is part of the same filter, but probed

poly(A) + RNA, 14.5 dpc. (Panel B) Cell-specific

by treatment

with collagenase

(6 mM), was used throughout (DuPont)

expression.

Tissues

from adult male or foetal mice

thiocyanate-phenol-chloroform

method

(Chomczynski

to nylon filters (Genescreen, Dupont). Hybridisation and in 2.2 M formaldehyde/l O,, agarose gels and transferred in Fig. 4 legend. The large, upper segment is an autoradiograph of a filter probed with the 5’ end of pD28 (nt 379-678

shown in Fig. 2). The lower segment

a CelSep chamber

and Vogelstein,

(1984) were used for the hybridisation,

pH 7.2/l 9, SDS, 68°C

of the RNAs. Total RNA (10 pg) was loaded in all lanes, with the exception

cells produced

(Feinberg

DuPont).

range of the gel.

and Scacchi, 1987), electrophoresed washing were performed as described for integrity

and transferred

with “P

were used as source of RNA. Total RNA was extracted

of the sequence

from mouse inbred strains C57BL/6J and DBA/ZJ (Jackson

1 and 2, Kpnl; 3 and 4, NcoI; 5 and 6, NheI. Sizes of detected

the resolvable

(Parkes)

electrophoresed

The conditions

buffer, A final stringency and 6, DBA/ZJ;

of the Zfp-29 gene. DNAs (5 pg)

analysis

enzymes,

the preparation.

and trypsin,

as described

expression.

with a cDNA

of lane 7. Lanes:

for ubiquitous

GAPDH

mRNA,

as a control

1, brain; 2, heart; 3, kidney; 4, liver; 5, spleen;

Testes from four adult mice were dissected

by Romrell et al. (1976). EKRB, supplemented

with pyruvate

and a suspension

of

(1 mM) and lactate

Cells in 100 ml of EKRB plus 0.5% BSA were layered on a 900-ml density gradient of 2-49, BSA, in as of 104, BSA at the bottom and sedimented under unit gravity for 90 mitt, at room temperature,

with a 50-ml cushion

described by Willison et al. (1990). Seventeen fractions of 50 ml each were collected from the bottom of the gradient (densest region; the first fraction, containing the cushion, was discarded) and cell purity and numbers analysed. RNA was prepared from suitable pooled fractions, electrophoresed, and transferred

to Genescreen

membrane

as described

in panel A. Total RNA (10 pg) was loaded in each lane. Loading

of samples

was checked

by staining

gels with ethidium bromide to visualise the ribosomal RNA bands and densitometric scanning of suitably exposed photographs. The upper segment is an autoradiograph of a filter probed with the 5’ end of pd28 as described in panel A. The middle and bottom segments are the same filter, hybridised with actin and protamine DNA probes, respectively. Lanes: 1, primary spermatocytes (80-907, pure); 2, round spermatids (85-95% pure); 3, elongating spermatids (40”~ pure; main contaminants were round spermatids and residual bodies); 4, residual bodies (75-90:~) and mature spermatozoa. panels, 28s and 18s indicate the positions of the corresponding murine ribosomal RNAs, which are 4869 nt and 1869 nt, respectively.

In both

225 extended

the existing

sequence

(data not shown).

To cir-

cumvent this problem, we made use of a modified RACE method (Frohman et al., 1988), as described in the legend to Fig. 1. The product of the RACE procedure was cloned into pBluescript and the sequence of four independent isolates was determined. This allowed the assembly of the merged sequence shown in Fig. 2. This sequence has a long open reading frame, terminating at a stop codon, followed by a short 3’-untranslated region. There are several potential start codons; the ATG triplet nearest to the 5’ end of the sequence is the most likely candidate, as it is surrounded by residues favourable for efficient translation (Kozak, 1987) and is preceded reading frame.

by two stop codons

in the same

(c) Sequence of the protein encoded by Zfp-29 cDNA The protein encoded by the Zfp-29 cDNA has a deduced size of 68.7 kDa and consists of two major domains: an N-terminal region, rich in acidic aa and a C-terminal block of 14 tandem repeats of the Zf motif. The N-terminal region of 216 aa with its high content of acidic aa, is similar in character to the transcription activating domains of a class of eukaryotic transcription factor (Jones, 1990). Secondary structure predictions based on the algorithm of Garnier et al. (1978) (data not shown) show that this region also has the potential to form a-helical secondary structures which is typical of this class of activating domain. Fourteen zinc fingers of the Cys*His’ type are located at the C terminus of the predicted Zfp-29 protein. Between each pair of fingers is a copy of the H/C link (Schuh et al., 1986) which is responsible for the cross-hybridisation between different Zfp genes. The sequences of the zinc fingers do not deviate significantly from a consensus finger motif assembled by Gibson et al. (1988), with the exception of fingers 9 and 13, in which the highly conserved Thr in the H/C link motif is replaced by Leu. Comparison of the sequence of the Zfp-29 zinc finger region with the contents of the EMBL/Swiss-Prot databases demonstrated that Zfp-29 is distinct from other characterised Zf proteins, including those known to be expressed in testis, such as Zfp-35 (Cunliffe et al., 1990), an unnamed gene (Nelki et al., 1990), and Zfi-1 and Zfy-2 (Mardon and Page, 1989). There appear to be no other genes closely related to Zfp-29 in the genome as a single hybridising band was seen on Southern blots probed under high-stringency conditions (Church and Gilbert, 1984) (Fig. 4). Although the Zf domain of Zfp-29 is distinct from that of other characterised proteins there is quite strong similarity between the individual fingers. This is shown in Fig. 3. Several residues in the central part of the finger, which are thought to be involved in determining specificity of interaction with DNA, are almost invariant. This may reflect an underlying repeat in the sequence to which the Zfp-29 protein binds.

(d) Tissue- and cell-specific Northern-blot

analysis

expression

of Zfp-29

showed that a probe derived from

the nonfinger region of the Zfp-29 cDNA hybridized to a single class of mRNA of approx. 2.2 kb (Fig. 5A). This mRNA is expressed in the mouse embryo at 14.5 dpc and in the adult testis. Expression was detectable in other adult tissues, but at approx. 50-fold lower levels than that in the testis. The filter was subsequently hybridised with a probe for the ubiquitous mRNA encoding GAPDH to ensure integrity of the RNA samples and to control for any variations in loading (Fig. 5A). Many genes which are highly expressed in the testis are expressed in a subset of the spermatogenic cell types (Willison and Ashworth, 1987). To investigate this possibility we fractionated germ cells by unit gravity sedimentation in gradients of bovine serum albumin as previously described (CelSep, DuPont; Willison et al., 1990). The purity of fractions from typical experiments is described in the legend to Fig. 5. RNA was prepared from the cells, then analysed by Northern-blot hybridisation and the effectiveness of the CelSep confirmed by control hybridisation with the protamine probe (Fig. 5B, lower panel). It is clear that protamine transcripts are not detected in the primary spermatocyte fraction, but accumulate to high levels in elongating spermatids (lane 3; Peschon et al., 1987; Willison et al., 1990). RNA hybridising to a Zfp-29 probe is present in primary spermatocytes, round spermatids and elongating spermatids, but not in residual bodies. The highest steady state level of Zfp-29 mRNA was detected in the early post-meiotic cells, round spermatids, where it was 2-4.5-fold enriched over the level seen in other spermatogenic cells, when normalised with respect to ribosomal RNA. This pattern of expression is distinct from that of another testis-specific Zfp mRNA, Zfi-35. Transcripts from this gene are detected mainly in the primary spermatocyte, immediately prior to the meiotic reduction divisions. We do not know the origin of the Zfp-29 transcripts detected in the 14.5 dpc embryo. We are currently investigating the possibility that Zfp-29 is transcribed in the foetal as well as the adult testis as has been described for Zfy-1 (Koopman et al., 1989). During spermatogenesis, one of the major changes in the cell is the re-packaging of DNA. The typical nucleosomal structure is altered by the replacement of histone proteins by other basic proteins, the protamines. These changes are accompanied by a general decline in transcriptional activity, but it is clear that many genes are transcribed de novo, at higher levels or from alternative promoters, following meiosis (Willison and Ashworth, 1987). Examples include the genes for protamine (Kleene et al., 1983), pre-proacrosin (Adham et al., 1989), smooth-muscle-type actin (Slaughter et al., 1989) and the Y-chromosome-linked Zfp genes, Zfy-1 and Zfy-2 (Mardon and Page, 1989). This implies the existence of haploid specific transcription me-

226 chanisms, such as the modification of pre-existing transcription factors or the synthesis of new factors. It is possible that Zfp-29 is such a novel factor, as suggested by its structure and restricted pattern of expression. In addition, by analogy with TFIIIA (Miller et al., 1985) Zfp-29 may be a bi-functional protein, able to bind both DNA and RNA. Therefore Zfp-29 could also play a role in regulation of gene expression at the level of translational control, a significant regulatory step in spermatogenesis (Willison and Ashworth, 1987). Antisera specific for Zfp-29 will be required, however, to demonstrate that this putative protein is produced from the Zfp-29 gene and to determine the cell-type(s) in which the protein is synthesised.

of zinc linger protein

Bellve, A.R.: The molecular Finn, CA. Oxford Brown,

University

scription

D.E.: Isolation

chromosome

P., Lemaire,

Characterization

its expression

pattern

in spermatogenesis.

Eur. J. Biochem.

of tran-

FEBS Lett. D.A.,

of a zinc finger polypeptide

I I Wilm’s tumour

of

182 (1989)

563-568. Ashworth, A., Williams, B.P., Buchberg, A.M., Goodfellow, P.N., Solomon, E., Potter, J. and Willison, K.R.: Chromosomal localisation

gene

locus. Cell 60 (1990)

by

P., Revelant,

O., Bravo,

R. and Charnay,

P.:

family that encodes zinc linger

Mol. Cell. Biol. 8 (1988) 1319-1326. P. and Sacchi,

acid

guanidinium

Anal. Biochem.

N.: Single-step

method

of RNA isolation

thiocyanate-phenol-chloroform

extraction.

162 (1987) 156-159.

Chowdhury, K., Deutsch, U. and Gruss, P.: A multigene family encoding several ‘linger’ structures is present and differentially active in mammalian Church,

genomes.

Cell 48 (1987) 771-778.

G. and Gilbert,

USA Sl(1984) Cunliffc,

W.: Genomic

V., Koopman,

zinc linger

sequencing.

Proc. Natl. Acad. Sci.

1991-1995. P., McLaren,

gene which

A. and Trowsdale,

is transiently

expressed

genesis. EMBO J. 9 (1990) 197-205. Diakun, G.P., Fairall, L. and Klug, A.: EXAFS sites in the protein

transcription

J.: A mouse

during

spermato-

study of the zinc-binding

factor IIIA.

Nature

324 (1986)

698-699. Fairall, L., Rhodes, D. and Klug, A.: Mapping of the sites of protection on a 5S RNA gene by the Xenopus transcription factor IIIA. J. Mol. Biol. 192 (1986) 577-591. Feinberg,

A.P. and Vogelstein,

restriction

endonuclease

Biochem. Frohman,

for radiolabelling

to high specific

DNA

activity.

Anal.

137 (1984) 266-267.

M.A., Dush,

structure

B.: A technique fragments

cDNAs

M.K. and Martin,

G.R.:

from rare transcripts:

oligonucleotide

primer.

of globular

methods

proteins.

production

of

using a single

Proc. Natl. Acad.

D.J. and Robson, of simple

Rapid

amplification

Sci. USA 85

R.: Analysis

of the accuracy

for predicting

the secondary

J. Mol. Biol. 120 (1978) 97-120.

Gibson, T., Postma, J.P.M., Brown, R.S. and Argos, P.: A model for the tertiary structure of the 28 residue DNA-binding motif (‘zinc linger’) common

to many

eukaryotic

transcriptional

Prot. Eng. 2 (1988) 209-218. Henikoff, S.: Unidirectional digestion targeted Jones,

breakpoints

N.: Structure

Cancer Kadonaga, cDNA

with

exonuclease

for DNA sequencing. and function

regulatory

proteins. III creates

Gene 28 (1984) 35 l-359.

of transcription

factors.

Seminars

Biol. I (1990) S-17. J.T., Carner, K.R., Masiarz, F.R. and Tjian, R.: Isolation encoding transcription factor Spl and functional analysis domain.

K.W., Ruppert,

B.: The GLI

of the Kriippel family of zinc linger proteins.

plasmic

poly(A) + RNAs

haploid

phases

which

first appear

of spermatogenesis

(1983) 453-464. Klemm, U., Maier, W.M., Tsaousidou, and

Engel,

W.: Mouse

of of

Cell 51 (1987) 1079-1090.

J.M., Bigner, S.H. and Vogelstein,

Nature

332 (1988) 371-374. Kleene, K.C., Distel, R.J. and Hecht, N.B.: cDNA clones encoding

S., Tsaousidou, and analysis

structure

repeats.

A.J., Pelletier, J., Haber,

ofa mouse multigene

gene is a member

cloning of preproacrosin

In: Vol. 1.

509-520. Chavrier,

the DNA binding

U., Maier, W.M., Hoyer-Fender,

has twelve consecutive

and characterization

at the human

Kinzler,

I.M., Klemm,

P.: The primary

T., Ito, C.Y., Buckler,

(1988) 8998-9002. Garnier. J., Osguthorpe,

S. and Engel, W.: Molecular

Biology,

Rose, E.A., Kral, A., Yeger, H., Lewis, W.H., Jones, C. and Housman,

and implications

Adham,

spermatogenesis.

of Reproductive

I86 (1985) 271-274. Call, K.M., Glaser,

gene-specific

REFERENCES

4 (1989)

1979, pp. 159-261.

C. and Argos,

factor TFIHA

full-length

This work was supported by the Cancer Research Campaign and the Medical Research Council. We thank Gillian Hynes for help with the germ-cell fractionation, Sally Leevers and Pamela Taylor for oligo synthesis, Keith Willison for the mouse testis cDNA library and Muriel Davisson and Don Doolittle for help with gene nomenclature. In addition, we would like to thank Andy Goldsborough, Val Macaulay, Sally Swift and Pamela Taylor for constructive criticism of the manuscript.

Reviews

Press, Oxford,

R.S., Sander,

structures.

ACKNOWLEDGEMENTS

Genomics

biology of mammalian

(Ed.), Oxford

Chomczynski,

(e) Conclusions (I) We have cloned a cDNA that codes for a putative protein (Zfp-29) that would have 14 zinc fingers of the Cys*Hi? type and an acidic potential tmns-activation domain. Zfp-29 is distinct from other characterised zinc linger proteins. The zinc fingers of Zfp-29 show considerable homology with each other possibly reflecting some repeat in the nucleic acid to which they bind. (2) Zfp-29 mRNA is expressed in the adult mouse testis at a level at least 50-fold higher than any other organ we have examined. It is also expressed in the developing embryo. (3) Within the testis Zfp-29 mRNA is most highly expressed in the earliest post-meiotic cells - the round spermatids. If levels of active Zfp-29 protein show similar variation, this would suggest a role for Zfp-29 in regulating events during the post-meiotic stages of spermatogenesis.

genes in man and mouse.

323-327.

at detectable

in the mouse. S., Adham,

preproacrosin:

cDNA

Develop.

Biol. 98

I.M., Willison, sequence,

cyto-

levels in

K.W.

primary

227 structure tiation

and postmeiotic

expression

in spermatogenesis.

Koopman,

P., Gubbay,

expression

J., Collignon,

patterns

messenger

RNAs.

J. and Lovell-Badge,

are not compatible

Nucleic

encodes

a protein

oocytes. Morishita,

in the

protein

D.S., Mucenski,

N.G. and Ihle, J.N.: Retroviral

Sanger,

region of the mouse Y and 13

sequencing

from

Xenopus

N.A., Copeland,

of a novel gene encoding

matid-specific Natl. Acad. Romrell,

Proc.

A.R.: DNA sequencing Natl.

Acad.

Sci.

with chain-

USA

74 (1977)

R., Aicher,

W., Gaul, U., Cot& S., Preiss, A., Maier, D., Seifert,

conserved

U.,

Schrbder,

family of nuclear encoded

C., Kemler, proteins

R. and

containing

JBckle,

structural

by Kriippel. a Drosophila

H.:

A

elements

segmentation

gene. Cell 47 (1986) 1025-1032. a

myeloid leukemia cell lines. Cell P. and Akhavan,

of a zinc finger cDNA expressed

Slaughter,

G.R., Meistrich,

for calmodulins,

M.: Cloning

and

in mouse testis. Nucleic

tools for biological

sequence

Sci. USA 85 (1988) 2444-2448.

R.R., Brinster,

expression

Vogelstein,

M.L. and Means,

actins

and tubulins

R.L. and Palmiter,

of protamine

1 in transgenic

A.R.: Expression

of RNAs

in rat testis cells. Biol. Reprod.

D.W.:

from agarose.

K. and Ashworth,

sion. Trends

and analytical

A.: Mammalian

spermatogenic

Expression

mice. Proc.

D 17Leh66, in purified murine spermatogenic

of mouse

of three

purification gene expres-

3 (1987) 351-355.

Willison, K.R., Hynes, G., Davies, P., Goldsborough,

Res., Camb. Separation

Genet.

D.: Preparative

Proc. Natl. Acad. Sci. USA 76 (1979)615-619.

R.D.: Sper-

Sci. USA 84 (1987) 5316-5319.

L.J., Bellve, A.R. and Fawcett,

B. and Gillespie,

ofDNA Willison.

D.J.: Improved

Proc. Nat]. Acad.

J.J., Behringer,

of the Droso-

factor IIIA. Nature 3 19

40 (1989) 394-405.

K., Cunningham,

W.R. and Lipman,

comparison. Peschon,

of the product

5463-5467.

of the finger protein

M.L., Jenkins,

Acids Res. 18 (1990) 3655. Pearson,

homology

S. and Coulson,

inhibitors.

E., Nauber,

zinc-binding

54 (1988) 831-840. Nelki, D., Dudley,

C., Preiss, A., Kienlin, A., Cote. S., Riede,

H.: Structural

F., Nicklen,

terminating Schuh,

A.: Repetitive factor IIIA

activation

zinc finger protein in IL-3-dependent

U.B., Schroeder,

(1986) 336-339.

from 699 vertebrate

EMBO J. 4 (1985) 1609-1614. K., Parker,

velocity. Dcvel. Biol. 49 (1976)

phila Kriippel gene with Xenopus transcription

with a highly acidic domain

transcription

Rosenberg,

1. and Jackie,

role in mouse

Acids Res. 15 (1987) 8125-8148.

zinc fingers. Cell 56 (1989) 765-770. Miller, J., McLachlan, A.D. and Klug, domains

R.: Zfi gene

with a primary

G. and Page, D.C.: The sex-determining

chromosome

cells by sedimentation

119-131.

sex determination. Nature 342 (1989) 940-942. Kozak, M.: An analysis of 5’ non-coding sequences Mardon,

spermatogenic

Differen-

42 (1990) 160-166.

t-complex

56 (1990) 193-201.

genes,

Tcp-1,

A. and Lewis, K.A.: DI7Lehl17c3

cell populations.

and Genet.