DEVELOPMENTAL

BIOLOGY

142,147-154 (1990)

A New Rat Gene RT7 Is Specifically Expressed F. A. VANDERHOORN,'

H.A.

TARNASKY,AND~.

during Spermatogenesis K. NORDEEN*

Department of Medical Biochemistry, University of Calgary Health Sciences Centre, 3330 Hospital Drive N. If?, Calgary, Alberta, Canada T2N ~NI; and *Department of Pathology, University of Colorado Health Sciewzs Center, 4200 East 9th Avenue, Dewver, Colorado 80262 Accepted July 17, 1990 We report the isolation of a new rat male germ cell-specific gene, RT7, by differential cDNA cloning procedures. The RT7 cDNA nucleotide sequence is not homologous to any sequences present in the GenBank library. RT7 RNA is expressed at very high levels in rat early spermatids, while its expression is not detectable in any other organ or tissue examined. Mapping of the RT7 transcription start site by two independent procedures demonstrated that RT7 has two major and a number of upstream minor start sites for transcription. RT7 encodes a putative go-amino acid protein, of which the N-terminus is predicted to fold as an amphipathic a helix with features resembling the leucine zipper structure found in a family of transcription factors. However, unlike the leucine zipper proteins the RT7 alpha helix is not preceded by a basic region. Analysis of the RT7 promoter sequence indicates that it contains a putative testis-specific regulatory sequence found in protamine P, and P, promoters, as well as binding sites for several other transcription faCtOrS.

0 1990 Academic

Press, Inc.

confers haploid-specific expression (Stewart et al., 1988). Another example of this approach showed that testis-specific expression is regulated by a 323-bp fragment of the human phosphoglycerate kinase 2 promoter (Robinson et ah, 1989). Comparison of promoter sequences of genes expressed specifically in the testis resulted in a list of putative &s-acting, nuclear factor binding sites (Johnson et aZ., 1988; Krawetz and Dixon, 1988). The analysis of the importance of such sequences in transgenic animals is cumbersome and expensive. An alternative approach to the study of testis-specific transcription factors is in vitro transcription. Several studies using liver-specific transcription systems demonstrate the potential of this approach (Gorski et ab, 1986; Corthesy et ak, 1988; Lichtsteiner and Schibler, 1989). One recent study reported the use of a testis-specific in vitro transcription system to analyze the mouse protamine P, promoter (Bunick et at, 1990). We report the isolation and characterization of a new rat male germ cell-specific gene, which is expressed at a very high level in early spermatids. Its promoter has been sequenced and putative regulatory elements were identified. Also, analysis of the N-terminus of the putative RT7 protein predicts that it may fold as an amphipathic (Y helix bearing similarity to the transcription factor leucine zipper motif (Landschulz et al., 1988).

INTRODUCTION

Analysis of the tissue-specific expression of a large number of gene model systems has indicated that DNAprotein interactions between specific sequence elements and ubiquitous- as well as tissue-specific nuclear factors play a crucial role (Jones et aZ., 1988; Mitchell and Tjian, 1989). Studies on gene regulation in lymphocytes as well as in cells derived from liver, brain, and pituitary, to name just a few, demonstrate the existence of families of transcription factors (Jones, 1990). In spite of the considerable number of genes known to be expressed specifically in the testis (Hecht et al, 1984; Cate et ah, 1986; Boer et ah, 1987; Kistler et ab, 1987; Willison and Ashworth, 1987; Wolgemuth et al, 198’7; Allen et ah, 1988; Herzog et al., 1988; Krawczyk et al., 1988; Propst et ak, 1988; Affara et al, 1989; Wolfes et al., 1989), the study of mechanisms underlying male germ cell-specific transcription is lagging due to the lack of permanent spermatogenic cell lines or of an appropriate tissue culture system which supports spermatogenesis (Balhorn, 1989; Hecht, 1989). Most studies aimed at identification of cisacting elements which are involved in testis-specific transcription resort to the use of transgenic mice. Thus Peschon et ab, (1987,1989) used promoter fragments of the mouse protamine mP, gene to address the problem of haploid-specific expression. They report that an mP, promoter fragment from -465 to -40 confers early spermatid-specific expression of reporter genes. Similarly, an 859-bp promoter fragment of the mP, gene

MATERIALS

Isolation Sequence data from this article have been desposited with the EMBL/GenBank Data Libraries under Accession No. M37323. r To whom correspondence should be addressed at Department of Medical Biochemistry, University of Calgary Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4Nl.

AND METHODS

of Rat Male Germ Cells

Rat male germ cells were isolated from testes of 2month-old Wistar rats as described by Grootegoed et aZ. (1982). The germ cell suspension was separated on a nonlinear gradient of bovine serum albumin (Groote147

0012-1606190 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

148

DEVELOPMENTALBIOLOGY V0~~~~142,1990

goed et al., 19’7’7) and fractions containing pachytene spermatocytes or early spermatids were pooled for RNA isolation: we estimated these fractions to contain 90 and 80% of the respective germ cells. The late spermatid fraction contained 30% early spermatids and 30% residual bodies. In some cases entire seminiferous tubules were used for RNA isolation which were obtained after collagenase treatment of decapsulated rat testes. Differential

cDNA

Cloning

and Sequenke Analysis

RNA was isolated as described by Chomczynski and Sacchi (1987). Polyadenylated RNA was isolated by oligo-dT column fractionation (Maniatis et al., 1982). cDNA was synthesized using AMV reverse transcriptase (cDNA synthesis kit, Pharmacia) and cloned in the EcoRI site of bacteriophage lambda-ZAP II (Stratagene) using Gigapack Gold packaging extracts (Stratagene). Radiolabeled single strand cDNA was prepared as described by Krug and Berger (1987) using AMV reverse transcriptase (Boehringer-Mannheim) and [32P]dCTP (ICN). Differential cDNA screening was performed by the +/- screening method (Sargent, 1987) as follows: a cDNA library was prepared using RNA isolated from rat seminiferous tubules as source. Radiolabeled singlestrand cDNA probes were prepared using RNA from rat seminiferous tubules and from total rat testis. Three times 1000 plaques of the library were grown on three plates and duplicate filters from the three plates were screened with the two probes. After autoradiography, exposures of corresponding duplicate filters were examined for the presence of plaques that showed stronger hybridization signal with the probe prepared from seminiferous tubule RNA than with the total testis probe. Candidate plaques were purified in two further rounds of screening and the observed difference in hybridization signal in the original screening was confirmed. Clone pRT7-0.56, which demonstrated the largest difference in hybridization signal, was used to isolate near full-length cDNA clones from the cDNA library: one of these with the largest insert, clone pRT7, was used for DNA sequence analysis. Sequence analysis was performed on small-scale plasmid preparations of pRT7 or subclones derived from it (pRT7-0.4 and pRT7-0.6) using the ?sequencing kit (Pharmacia). Cosmid Cloning and Restriction

Mapping

The insert of clone pRT7-0.4 was radiolabeled by nicktranslation and used as probe to screen a cosmid library prepared from rat spleen DNA (Stratagene). A positive cosmid clone, cosRT-7, was purified and an 8-kb XbaI DNA fragment, which hybridized to the above mentioned probe, was subcloned in pBluescript SK- generating pRT7-cl5 Restriction analysis of the clone was

performed using Pharmacia scribed by the manufacturer. Primer

Extension.

restriction

and RNase

enzymes as de-

Mapping

Primer extension analysis of the mRNA cap site(s) of gene RT7 was carried out essentially as described by Calzone et al., (1987). In short, 10 ng (1.9 pmol) of the synthetic oligonucleotide oligoRT7 (5’ GAAATGGCACTCAGAG 3’) was radiolabeled with r’P]ATP and polynucleotide kinase (Pharmacia) in 10 ~1. Thirty-six fmol primer was annealed at 43°C for 30 min with 13.8 pg total RNA from rat muscle and pachytene spermatocytes and with 1.3 and 6.5 pg polyadenylated RNA from rat seminiferous tubules. After primer extension with AMV reverse transcriptase (AMV Super RT, Molecular Genetic Resources) the products were separated on 5% urea-polyacrylamide gels and visualized by autoradiography. For RNase mapping of the gene RT7 mRNA cap site(s) a clone pRT7-4 was constructed using PCR technology: pRT7-cl5 DNA was linearized with BamHI and subjected to 40 cycles (1 min 94°C; 2 min 42°C; 5 min 75°C) of PCR reaction using Taq DNA polymerase (Boehringer-Mannheim), primer RT7 and a T3 sequencing primer as described by the manufacturer. The 4-kb reaction product was made blunt-end with Klenow DNA polymerase (Pharmacia) and cloned in the SmaI site of pBluescript SK- generating clone pRT7-4. pRT7-4 DNA was digested with EcoRI, purified, and transcribed in vitro with T3 RNA polymerase (Pharmacia) in the presence of r2P]UTP (Amersham). After DNaseI digestion (Pharmacia), the labeled RNA probe (283 nucleotides) was purified and annealed to 4.6 pg total rat muscle RNA and to 0.65 and 5.2 pg polyadenylated rat seminiferous tubule RNA in 80% formamide, 40 mM Pipes (pH 6.5), 1 mMEDTA, 0.4 MNaCl for 16 hr at 50°C. Reaction products were digested with 30 pg RNase A and 120 units RNase T, in 300 ~1 of 0.3 MNaCl, 10 mMTris. HCl (pH 8), 5 mlM EDTA for 30 min at 30°C purified and separated on 5% urea-polyacrylamide gels. In Vitro Translation

Clone pRT7 was linearized with XbaI or with XhoI and the linear DNAs were transcribed in vitro with T7 RNA polymerase and T3 RNA polymerase, respectively, in the presence of m7GpppG using the TransProbe T kit (Pharmacia). Capped RNAs were purified and translated in vitro in the presence of [35S]cysteine (Amersham) using a rabbit reticulocyte lysate system (Promega). Protein products were separated on 12.5% SDSpolyacrylamide gels and analyzed by autoradiography. Other RNA/DNA

Techniques

All other RNA and DNA manipulations were carried out as described by Maniatis et ab, (1982).

VAN DER HOORN, TARNASKY, AND NORDEEN RESULTS

A ZG

cDNA Isolation of a Rat Male Germ Cell-Speci$c Gene RTY

To study transcription regulation of genes expressed specifically during mammalian spermatogenesis we set out to isolate cDNAs of abundantly expressed rat male germ cell-specific genes. The relatively simple +/- screening technique was used, because it allows one to isolate cDNA from a gene that is expressed as a very abundant RNA in one cell type (1% of total cellular mRNA) and is absent from another (Sargent, 1987). We choose to compare RNA from seminiferous tubules and total testis in the +/- screening approach to increase our chances of isolating a germ cell-specific gene rather than a testis-specific gene. A cDNA library in bacteriophage lambda-ZAP II prepared from rat seminiferous tubule mRNA was +/- screened using radiolabeled single-strand cDNA probes prepared from total testis mRNA (testis probe) and seminiferous tubule mRNA (tubule probe) (see Materials and Methods). Several cDNA clones were obtained of differentially expressed genes one of which, pRT7-0.56, contained an insert which when used as a probe detected a very abundantly expressed male germ cell-specific gene (see below). The insert of pRT7-0.56 was used to isolate a near full-length cDNA from the cDNA library, clone pRT7. The result of DNA sequence analysis of clone pRT7 and of subclones derived from it is presented in Fig. 1A. The sequence indicates the presence of a short open reading frame, which could encode a putative go-amino acid protein. Also, a long 3’ nontranslated region is present, in the middle of which a sequence 5’ CCCTGC( G/A) (G/A) 3’ is repeated 15 times. A comparative search between the nucleotide sequence of the open reading frame and all sequences entered in the GenBank library did not reveal any significant homology with published sequences. Interestingly, the putative RT7 protein is very eysteinerich (17% of all residues) and has a relatively lysine-arginine-rich N-terminus (23% of the N-terminal 34 residues). On the basis of the Chou-Fasman program for determination of secondary protein structure (Chou and Fasman, 1978) the 42 N-terminal amino acid residues of the putative RT7 protein are predicted to form an (Yhelix. A top projection of the 42 residues (Fig. 1B) indicates the possibility of formation of an amphipathic (Y helix; the hydrophobic face of the alpha helix contains predominantly leucine, isoleucine, and valine residues. RT7’ RNA Is only Expressed in Male Germ, Cells

To investigate the pattern of RNA expression of gene RT7, RNA was isolated from a large number of rat tissues and organs and analyzed by the Northern blotting technique. The results (Fig. 2A) indicate that gene RT7

149

RT7 Expression in Rat Male Germ Cells

PlAALGCLLDGVRRDIKK ATG GCC GCF\

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K &FIG

R CGR

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G TCC

C TGT

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L CTG

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D GQC

L CTC

Y TFIC

Y TX

P CCG

c TGC

c TGC

L CTG

c TGT

D G&C

Y T&C

K AAG

L CTG

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c TGC

L CTC

R CGC

P G 5 A A t cc0 TCG TCC GCA GCT T&G FiGAGACTCAGAFIGGCIcG~c~*~T~G~TGGccTccTcTTG Pstl CTtCAGC~GT~ClCFITTTT~GG~TCGGTG~~CGTCTGCGGCTTTG~~CCTG~TC~GGTC~~~GTGCGC

EcoAl

GamHl GTTCICIC\GFITGGALFIGGTCTGCGT~TCGGCCG~G~GG~~~~C~GGT~CG~CTGCCTCG~~~~~~

747 814 GGI

FIG. 1. Gene RT7 cDNA sequence and putative protein structure. (A) The nucleotide sequence of the largest RT7 cDNA clone, pRT7, was determined. RT7 cDNA contains a 270-bp open reading frame, which is not homologous to any published sequences. Indicated are enzyme restriction sites for BamHI, EcoRI, and PstI as well as the location of the synthetic oligonucleotide oligoRT7 and its orientation with respect to the cDNA RT7 coding strand. (B) On the basis of the Chou and Fasman (1978) method of determination of protein secondary structure the N-terminus of the putative RT7 protein is predicted to form an (Y helix. Shown is a top projection of the possible (Y helix, which indicates that the LYhelix is amphipathic. Charged amino acid residues are indicated with filled circles.

is exclusively expressed in rat testis as a LO- to l.l-kb RNA. After stripping the filter it was reprobed with a /3-actin probe to demonstrate that intact RNA was present in all lanes (Fig. 2A). To localize RT7 RNA expression within the testis, testicular cells were separated in the following fractions: pachytene spermatocytes (PS), early spermatids (ES), and late spermatids (LS) (see Materials and Methods). Northern blotting analysis was performed on RNA isolated from these fractions as well as from total testis, seminiferous tubules, and muscle.

150

DEVELOPMENTALBIOLOGY V0~~~~142,1990 A k

B A%

Ep

Li

Th

Ki

He

Lu

Mu

Bl

Ca

M

Te

TV PS ES

LS

_’

kb

geneRT7

FIG.2. RT7 RNA is expressed during spermatogenesis. (A) Total RNA was isolated from rat organs and analyzed for RT7 RNA expression by the Northern blotting technique using a radiolabeled insert from clone pRT7 as probe. After removal of the hybridizing RT7 probe the filters were rehybridized to a p-a&in probe to confirm the presence of intact RNA in all lanes. Te, total testis; A-Te, nonpolyadenylated testis RNA; Ep, epididymis; Li, liver; Th, thymus; Ki, kidney; He, heart; Lu, lung; Mu, muscle; Bl, bladder; and Ca, cartilage. (B) The expression of RT7 RNA in rat male germ cells was investigated by Northern blotting analysis of RNA isolated from male germ cells separated on unit-gravity bovine serum albumin gradients using the above described probe. i!4, muscle; Te, total testis; Tu, seminiferous tubules; PS, fraction enriched in pachytene spermatocytes; ES, fraction enriched in early spermatids, and LS, fraction enriched in late spermatids.

The results (Fig. 2B) demonstrate that gene RT7 is predominantly expressed in early spermatids, much less so in pachytene spermatocytes. The expression in the germ cells accounts for the observed expression in the testis. Reprobing of these filters with a rat protamine cDNA probe to detect the very abundantly expressed protamine 1 RNA (Hecht, 1989) showed (a) that the expression level of RT7 RNA in early spermatids is as high as that of protamine and (b) that protamine RNA is present in the pachytene cell RNA fraction (data not shown). As such cells do not express protamine RNA (Hecht, 1989), we conclude that the pachytene spermatocyte fraction must be contaminated with early spermatids. Thus we cannot rule out that the RT7 RNA signal observed in pachytene spermatocytes is due to contamination of that fraction with early spermatids. As expected no expression was observed in muscle. Mapping

of Gene RT7 Transcription

Start Sites

Gene RT7 is specifically expressed in rat male germ cells at very high levels. To localize the gene RT7 promoter we performed primer extension analysis using an end-labeled oligonucleotide (oligoRT7) which is indicated in Fig. 1A. OligoRT7 was annealed to muscle RNA, to total testis RNA, and to different amounts of seminiferous tubule polyadenylated RNA. After reverse transcription of the annealed material the products were analyzed by denaturing polyacrylamide gel electrophoresis. Two major transcription start sites (Fig. 3A, asterisk) separated by five nucleotides were detected using testis RNA (lane a) or seminiferous tubule RNA (lanes c and d). In addition, 5 minor upstream transcription start sites (Fig. 3A, arrowheads) were detectable when

using the largest amount of tubule mRNA (lane d). To confirm the presence of two rather than one major transcription start sites we analyzed the gene RT7 mRNA cap sites with RNase mapping. A labeled anti-sense RNA probe was prepared in vitro, which has a region of homology with gene RT7 sequences starting at and including the oligoRT7 sequence and extending upstream to the RT7 promoter EcoRI site at -166 (see Fig. 4A and Materials and Methods for details). Therefore, we expected to identify products protected from RNase A and T, digestion which are similar in size to those seen with the primer extension assay. As shown in Fig. 3B, when using seminiferous tubule mRNA (lanes b and c) we observed the same pattern of major and minor transcription start sites using the RNase mapping technique. No protection was observed using muscle RNA (lane a). However, although the doublet of minor start sites appear to be identical in size to the doublet observed using primer extension, RNase mapping localizes the major start sites four nucleotides downstream of the sites defined with primer extension. The location of the transcription start sites as defined by these two methods are indicated in Fig. 4B. The Gene RT7 Promoter

Sequence

Fragments of clone pRT7 were used to screen a rat genomic cosmid library, and a positive clone, cosRT7, was further analyzed. An 8-kbXba1 fragment of cosRT7 was subcloned into the SmaI site of pBluescript SKgenerating pRT7-cl5 (Fig. 4A), which has T3 and T7 RNA polymerase start sites outside the multiple cloning sequence as indicated. Southern blotting analysis of pRT7-cl5 had indicated that sequences homologous to the 5’ end of the RT7 cDNA are present on a 1.9-kb EcoRI

VAN DER HOORN,

TARNASKY,

AND NORDEEN

147-

67-

67l

l

I

l

abed

abc

FIG. 3. Gene RT7 has major and minor transcription start sites. (A) Primer extension was used to map the gene RT7 transcription start site(s). Radiolabeled oligoRT’7 DNA (see Fig. 1A) was annealed to total testis RNA (lane a), total muscle RNA (lane b), and two different amounts of polyadenylated seminiferous tubule RNA (lanes c and d). After transcription of the primer-templates with reverse transcriptase the products were analyzed by 5% urea-polyacrylamide gel electrophoresis. The sizes of the major products (indicated by asterisk) are 58/59 and 62/63 nucleotides. The minor products are indicated by arrowheads. (B) To confirm the presence of multiple transcription start sites RNase mapping was used. A radiolabeled, anti-sense RNA probe extending from the EcoRI site at position -166 down to the oligoRT7 sequence at +30 (Fig. 5B) was annealed to total muscle RNA (lane a) and to two different amounts of polyadenylated seminiferous tubule RNA (lanes b and c). After digestion of the hybrids with

pRT7-cl5 and to subclone the gene RT7 promoter at the same time we performed the polymerase chain reaction (PCR) technique on linearized pRT7-cl5 DNA using either the oligoRT7 and T3 sequencing primers or the oligoRT7 and T7 sequencing primers. The first combination of primers led to the synthesis of a 4-kb PCR product (data not shown), thus defining the location of oligoRT7 (which is anti-sense with respect to the RT7 coding region) as indicated in Fig. 4A. Also, the orientation of the gene RT7 sequences on clone pRT7-cl5 DNA is from the left to the right in Fig. 4A. No PCR product was generated using the oligoRT7/T7 combination. The 4-kb PCR product was subcloned to generate clone pRT7-4. Part of pRT7-4 as well as subclones derived from it were

RT7 Expression in Rat Male Germ Cells

151

sequenced. Figure 4B presents the sequence of the promoter region of gene RT7. Indicated are the major and minor transcription start sites as determined by primer extension (open circles) and RNase mapping (closed circles). Note that the major start sites lie in a very ATrich region. We assigned the major start site, which appeared strongest of all as determined by primer extension, as RT7 mRNA cap site +l. Also indicated is the start of gene RT7 sequences present in cDNA clone pRT7 as well as the location of oligoRT7. On the basis of sequence comparison, homologies were detected between gene RT7 promoter sequences and the consensus binding sites for the following nuclear factors: ATF/ CREB (Jones et ab, 1988), AP-1 (Jones et aZ., 1988) and Egr-1 (Cao et ah, 1990) as indicated in Fig. 4B. Importantly, we detected a region in this promoter which shares homology with an element called P,-box (Krawetz and Dixon, 1988) or Sequence D (Johnson et al, 1988), which is a putative protamine P, and P, transcription regulatory sequence. Finally, an RT7 promoter sequence at position -280 is homologous to the B-box sequence identified in the protamine 1 and 2 promoters (Johnson et ah, 1988), which may act as a positive regulatory element (Bunick et ah, 1990).

nP-l ATFKREB -447 GFITCCtTGGCCICCFIGGC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~

FIG. 4. The RT7 promoter contains transcription factor consensus binding sites. (A) The restriction map of the 8-kb XbaI fragment isolated from a rat genomic cosmid clone cosRT7 is presented (E, EcoRI; H, HindIII; P, P&I, and X, XbaI). The location and orientation of the T3 and T7 bacteriophage promoters on the vector and the location of the oligoRT7 sequence in the insert are shown. (B) The gene RT7 promoter sequence ranging from -515 to +59 is shown. Indicated are restriction sites for EcoRI and HindIII, the oligoRT7 sequence, the 5’ nucleotide present in cDNA clone pRT7, the +l start site A (underlined), sequences showing homology to the indicated transcription factor binding sites, and the major and minor transcription start sites as determined by primer extension (open circles) and RNase mapping (closed circles).

152

DEVELOPMENTALBIOLOGY kD

2

’

2009369-

3

4

5

.‘.,

46-

(Fig. 5, lane 5), identical in size to the protein translated from full-length RT7 RNA (Fig. 5, lane 4). No lo-kDa protein can be detected. Thus RT7 cDNA contains the open reading frame indicated in Fig. lA, not a larger one. DISCUSSION

30-

6-

FIG. 5. The putative RT’7 protein can be synthesized in vitro. To determine if the putative RT’7 protein can be produced, in vitrosynthesized sense- and anti-sense RT7 RNAs were translated in vitro in the presence of [BsS]cysteine, and the products were analyzed by polyacrylamide gel electrophoresis. Lane 1, no RNA added. Lanes 2 and 4, full length RT7 sense RNA was added. Lane 3, full-length RT7 anti-sense RNA was added. Lane 5, truncated sense RT7 RNA produced from an RT7 template linearized at the BumHI site was added.

In Vitro Translation

VOLUME 142,199O

of RT7 Protein

To confirm the potential of the open reading frame to encode a protein we prepared full-length sense- and anti-sense-capped RNAs in in vitro transcriptions using pRT7 DNA linearized with restriction enzymes which cleave outside the cDNA insert. Both RNAs were translated in vitro. Analysis of the in vitro translated proteins indicated that, as expected, anti-sense RT7 RNA does not encode a protein (Fig. 5, lane 3). Full-length sense RT7 RNA (Fig. 2, lane 2) directs translation of a protein. Labeled globin (15 kDa) can be detected in all cases. Surprisingly, the apparent molecular weight of the translated protein as analyzed by SDS-PAGE is 25 kDa instead of 10 kDa as we predicted on the basis of the 270 nucleotide open reading frame (Fig. 5). A 25-kDa protein may be encoded on an open reading frame of approximately 680 nucleotides. To rule out that sequencing errors may have led us to erroneously conclude that the open reading frame is 270 nucleotides in size rather than 680 nucleotides, we prepared truncated capped RT7 sense RNA by in vitro transcription of BamHI linearized pRT7 DNA (Fig. 1A). If RT7 cDNA would contain the longer open reading frame we expect to generate a smaller sized protein (approximately 16 kDa) translated from the truncated RNA compared to full-length sense RNA. The results in Fig. 5 show that truncated RNA directs synthesis of the 25kDa protein

We report the isolation and initial characterization of a new mammalian male germ cell-specific gene, RT7. This gene has several features, the study of which will increase our, at the present limited, knowledge concerning the role of specific proteins in mammalian spermatogenesis and the mechanism(s) underlying the regulation of gene expression during spermatogenesis (Hecht, 1989). RT7 is very abundantly expressed in male germ cells, at least as abundant as protamine 1 RNA, in particular in early spermatids. The detection of RT7 mRNA in the pachytene spermatocyte cell fraction could be due to it being contaminated with approximately 10% early spermatids. No RT7 RNA could be detected in the interstitial cell fraction or in any other rat tissue or organ examined. The very limited expression pattern, which is reminiscent of that of the protamine genes, suggests an important role for the product of gene RT7 during spermiogenesis. The nucleotide sequence of RT7 cDNA predicts that a go-amino acid protein can be encoded. Interestingly, in vitro translation of in vitro synthesized RT7 RNA directs the synthesis of a protein which has a higher apparent molecular weight than the one predicted for a go-amino acid protein. The cause of this phenomenon is unknown, but similar shifts in the mobilities of proteins on SDS-PAGE are known to occur, for instance, for the SV40 T-antigen (Levine, 1989) and several transcription factors. Possibly the very high cysteine content and the presence of an amphipathic (Yhelix confer a secondary structure leading to the observed effect. The predicted amphipathic helical structure is reminiscent of the leutine-zipper motif (Landschulz et al, 1988) found in a family of transcription factors as well as in microtubule-associated protein-2 (MAP-2) and tau (Lewis et al, 1989), which can homo- or heterooligomerize (Matus, 1990; Schuermann et al, 1989 and references therein). In the case of transcription factors the leucine-zipper is invariably preceded by a basic region involved in DNA binding. Interestingly, Lewis et aZ., (1989) demonstrated that the leucine-zipper of MAP-2 can be functionally replaced by the leucine-zipper of a yeast transcription factor. The putative RT7 amphipathic (Yhelix is N-terminal and not linked to a basic region. The likelihood of formation of an amphipathic helix suggests that RT7 protein may interact via its hydrophobic surface with

VAN DER HOORN, TARNASKY, AND NORDEEN

itself or structurally related proteins. Thus, the RT7 product may be an abundant structural protein the function of which may be dependent on the amphipathic cyhelix. Alternatively, the RT7 protein may act as a repressor of transcription by dimerizing and inactivating leucine-zipper containing transcription factors. Interestingly, recent publications describe two proteins, Id and emc (Benezra et al., 1990; Ellis et ak, 1990; Garrell and Modolell, 1990), that appear to be negative regulators of transcription which bind certain helix-loop-helix-containing transcription factors thus inactivating the DNA binding capacity of those transcription factors (reviewed by Jones, 1990). Id and emc contain amphipathic 01helices as part of a conserved helix-loop-helix motif found in transcription factors such as MyoD and the E12/E4’7 proteins (Kingston, 1989), but miss a basic DNA binding region. We are currently in the process of generating antisera against synthetic RT7 peptides to distinguish between the above mentioned possibilities for RT7 protein function. We will address questions such as the apparent size of the cellular RT7 product, its subcellular localization and its interaction with itself or other cellular proteins, possibly mediated by its putative IX helix. Mapping of the RT7 cap site led to the discovery of two major start sites for transcription as well as a number of minor start sites, a feature often encountered in TATA-less promoters of housekeeping genes. However, RT7 is not a housekeeping gene. Inspection of its promoter sequence reveals the presence of the sequence TTTAAA at the position normally occupied by the TATA-box. Possibly this TTTAAA motif acts to a certain extent as an imperfect TATA-box, allowing for multiple transcription start sites. Further analysis of the promoter and upstream sequences indicated regions with homology to consensus binding sites for the following transcription factors: (a) complex AP-1 (Jones et cd., 1988) which contains the c-fos and c-jun proteins and is involved in the transcription of genes responsive to a number of external stimuli, (b) ATF/CREB which may constitute a family of factors involved in transcription of genes that are induced by CAMP and in transcription of several Adenovirus early genes (Jones et aZ., 1988), and (c) Egr-1 which regulates transcription of early genes induced by growth factor stimuli (Cao et al., 1990). Interestingly, the gene RT7 region from position -117 to -94 is 70% homologous to an element found in the promoter of several protamine 1 genes as well as the mouse protamine 2 promoter. This element, designated the P, box or Sequence D (Krawetz and Dixon, 1988; Johnson et al., 1988), is quite well conserved among the analyzed protamine promoters. As the mouse protamine genes and RT7 display a similar or identical pattern of RNA expression, it can be speculated that a fac-

RT7 Expressim

in Rat Male Germ

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tor binding to the P,/D box may be involved in the regulation of male germ cell-specific expression. It was recently suggested that this &s-acting element may be involved in repression of protamine expression in cells other than haploid male germ cells (Bunick et ah, 1990). This possibility is currently being investigated by analysis of DNA-protein interactions and mutagenesis of the RT7 P, box-like sequence in in vitro transcription assays. Our knowledge concerning &s-acting elements that are involved in the regulation of male germ cell-specific transcription is rudimentary (Hecht, 1989): transgenic mouse experiments indicated that both the mouse protamine 1 promoter region of 465 bp and the mouse protamine 2 promoter fragment of 859 bp are capable of conferring haploid-specific expression (Stewart et al., 1988; Peschon et ah, 1989). Data for testis-specific transcription factors are virtually nonexistent. We will approach the identification and characterization of nuclear factors and their DNA binding sites, that regulate male germ cell-specific transcription, using a testis-specific in vitro transcription system that we recently developed. As more sequence data become available of promoters regulating expression of haploid-specific genes it will be possible to search for common protein binding sites, which can be tested for function both in in vitro transcription assays and in transgenic mice. The authors thank Dr. B. Kuhnel for critical reading of the manuscript. This work has been supported in part by a grant from the Council for Tobacco Research to S.K.N. and by a National Cancer Institute of Canada research Grant 1578 to F.A.vdH. REFERENCES AFFARA, N. A., CHAMBERS, D., O’BRIEN, J., HABEEBU, S. S., KALAITSIDAKI, M., BISHOP, C. E., and FERGUSON-SMITH, M. A. (1989). Evidence for distinguishable transcripts of the putative testis determining gene (ZFY) and mapping of homologous cDNA sequences to chromosomes X, Y and 9. Nucleic Acids Res. 17,2987-2999. ALLEN, R. L., O’BRIEN, D. A., JONES, C. C., ROCKETS, D. L., and EDDY, E. M. (1988). Expression of heat shock proteins by isolated mouse spermatogenic cells. Mol. Cell. Biol. 8, 3260-3266. BALHORN, R. (1989). In “Molecular Biology of Chromosome Function” (K. W. Adolph, Ed.), pp. 366-395. Springer, New York. BENEZRA, R., DAVIS, R. L., LOCKSHON, D., TURNER, D. L., and WEINTRAUB, H. (1990). The protein Id: A negative regulator of helix-loophelix DNA-binding proteins. Cell 61,49-59. BOER, P. H., ADRA, C. N., LAU, Y. F., and MCBURNEY, M. W. (1987). The testis-specific phosphoglycerate kinase gene pgk-2 is a recruited retroposon. Mol. Cell Biol. 7, 31073112. BUNICK, D., JOHNSON, P. A., JOHNSON,T. R., and HECHT, N. B. (1990). Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system. Proc. Natl. Acad. Sci. USA 87,891-895.

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