THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 18, Issue of June 25, pp. 10709-10713.1990 Cc: 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Prrnted ,n US A.

A New Member of the Glutamine-rich Protein Gene Family Is Characterized by the Absence of Internal Repeats and the Androgen Control of Its Expression in the Submandibular Gland of Rats*

(Received for pubhcation, November 6, 1989)

Isabelle Rosinski-Chuping and Frangois Rougeon@ From the i&it& de G&&ique et Biochimie du D&eloppement, Labor&o&e associti Centre National de la Recherche Scientifique 361, Ins&t Pasteur, 75722 Paris Cedex 15, France

A cDNA, corresponding to a rat submandibular mRNA which is accumulated at a 20-fold higher level in males than females, has been isolated. The predicted protein, SMRB, has a calculated molecular mass of 15.4 kDa and is rich in glutamine/glutamic acid, proline, and asparagine/aspartic acid, a characteristic of the so-called salivary glutamine-rich proteins (GRPs) of the submandibular gland of rats. Nucleotide sequence comparisons indeed revealed strong similarities be- tween the sequences of the SMR2 mRNA and that of GRPs, except in the region encoding the carboxyl- terminal part of the proteins. In particular, the SMR2 mRNA contains the 5’-untranslated region and the signal peptide region shared by both groups of GRPs and proline-rich proteins (PRPs). A major difference is that, in SMRS, the peptidic motif which is repeated four or five times in GRPs, is only found once. The SMR2 gene is about 3.5 kilobases in length and con- tains 4 exons. The second intron, which does not exist in characterized GRP genes, splits the “transition” re- gion which separates the repetitive sequences from the signal peptide. This structure is reminiscent of that found in most PRP genes, strengthening the hypothesis that GRP and PRP genes have the same ancestral origin.

Two different functions are frequently assigned to the submandibular gland (SMG)’ of rodents. One is an exocrine function consisting in the constitution of the salivary fluids. The other is an endocrine function which leads to the release into the blood of certain growth factors and hormones.

Exocrine secretions mainly involve the acinar cells of the SMG. Among the secreted proteins are families of tissue- specific proteins characterized by highly repetitive contiguous peptide sequences. According to their predominant amino acids, these proteins have been classified into proline-rich

* This work was supported by grants from the Association pour la Recherche sur le Cancer and the Institut Pasteur de Paris. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBnnkTM/EMBL Data Bank with accession number(s) JO5490 and 505491.

§ To whom correspondence and reprint requests should be ad- dressed.

’ The abbreviations used are: SMG, submandibular gland; GRP, glutamine-rich protein; PRP, proline-rich protein; bp, base pair(s); NaDodSO,, sodium dodecyl sulfate; PAGE, polyacrylamide gel elec- trophoresis; kb, kilobase(

proteins (PRPs) or glutamine-rich proteins (GRPs). Their role in the salivary fluids has not been elucidated. On account of their high affinity for calcium phosphate (1, 2), it has been proposed that they could be involved in the protective pro- teinaceous structure of teeth surfaces. In addition, a role in the detoxification of certain substances, such as tannins, has been postulated for PRPs (3, 4).

The cDNAs corresponding to several mouse (5), rat (5, 6), and human (7) PRPs and rat GRPs (2, 8) have been cloned, and their sequences have been determined. In addition, the structure of some PRP and GRP genes (8-12) has been established. The peptide sequence of GRPs and PRPs can similarly be divided into four regions: a signal peptide, a “transition” region (which separates the repetitive region from the signal peptide), a repetitive region, and a carboxyl-termi- nal region. The organization of GRP and PRP genes is very similar and, in particular, the sequence of the first exon (corresponding to 5’-untranslated region and signal peptide) is highly conserved among these genes (8, 9, 11, 12). This suggests that GRP and PRP genes may derive from a common ancestor.

PRPs are encoded by a multigenic family mapped on chro- mosome 12 in man (13). In mouse, the PRP genes were firstly assigned to chromosome 8 (14) on the basis of results with mouse x hamster somatic cell hybrids but new linkage data indicate that they are on chromosome 6 (15). Evolutionary models for this gene family include a series of internal dupli- cations of a 42-bp unit (9). Diversity would have been gener- ated by recruitment or deletion of three bases from the ances- tral unit during the duplication events, leading to a final length which varies between 42 and 63 bp. Finally, gene conversion would have homogenized the divergence between the internal repeats.

GRP genes differ from PRP genes, in particular, by the length (69 bp) and the sequence of the repeats. They are also part of a multigenic family; more than 10 GRP genes have been detected by Southern blot analysis in rats (8). The sequences of the two characterized GRP mRNAs are identical except for the number of repetitive motifs and the carboxyl- terminal part of the proteins, probably due to recent gene conversion events (8).

We are interested in the androgen regulation of genes expressed in the SMG of rodents. A number of growth factors, hormones, and other proteins with biologically defined prop- erties are synthesized in large amounts in the SMG of rodents under androgen control (16, 17). It is the case, for instance, for renin, epidermal growth factor, and nerve growth factor in the SMG of mice. The role of these peptides in the saliva is unclear. Curiously, the pattern of proteins expressed at a higher level in the SMG of males than females appears to be

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10710 Evolutionary Aspects of the Glutamine-rich Protein Family

species-specific. In an attempt to characterize some of the peptides whose expression is regulated by androgens in the SMG of rats, we have compared the patterns of in vitro translation products directed by the SMG mRNAs prepared from males and females (17). We have shown that several polypeptides are translated in higher amounts from male than female SMG mRNAs. One of them, SMRl, was shown to have the structure of a hormonal precursor, which could potentially give rise to a thyrotropin releasing hormone-like peptide after processing (18).

Here we report the characterization of another SMG mRNA, accumulated under androgen control. This mRNA encodes a protein, SMR2, which is related to the GRPs but contains only once the repetitive unit present in GRPs. The structure of the corresponding gene has been studied and is very similar to that of GRP except for the presence of an additional intron in the transition region coding sequence, reminiscent of that found in most PRP genes.

EXPERIMENTAL PROCEDURES AND RESULTS’

DISCUSSION

In this paper, we report the characterization of a gene which is expressed under androgen control in the SMG of rats. The product of this gene, SMR2, belongs to the family of salivary glutamine-rich proteins. The nucleotidic sequence of the SMR2 mRNA is about 75% homologous to that of GRP mRNAs (except in the regions encoding the carboxyl-terminal part of the proteins). A similar intron-exon structure is found in GRP and SMRP genes.

Like the GRPs, SMR2 contains a relatively high proportion of glutamine + glutamic acid (20%), proline (12%), and as- paragine + aspartic acid (13%). In addition, SMR2 and GRPs share a certain number of structural properties. They display an overall negative charge and a similar distribution of charges along the sequence with an excess of negative charges in the central part of the proteins and an excess of positive charges in the carboxyl-terminal region. Analysis of SMRP and GRP primary structure by the method of Hopp and Woods (32) (data not shown and Ref. 2) reveals that they are hydrophilic, except in the amino-terminal (signal peptide) and in the carboxyl-terminal regions which are more hydro- phobic. Curiously, both GRPs and SMR2 have the same aberrant behavior on NaDodSO,-PAGE. Their predicted mo- lecular masses (including signal peptide) are, respectively, 26.5 kDa and 15.4 kDa while the molecular masses determined from the electrophoretic mobility of the in vitro translation products are about 70 kDa (2) and 35 kDa. By analogy with the aberrant mobility observed for collagen (33), PRP (34), and chromogranins (35), Mirels et al. (2) have suggested that GRP aberrant behavior may be due to the high proline content and to the anionic net charge.

However, a major difference between SMRP and GRPs concerns the number of internal repeats. The peptidic motif which is found perfectly repeated four or five times in GRPs only occurs once in SMR2. One hypothesis is that the dupli- cation leading to the separation of the GRP and SMR2 genes could have occurred before the process of internal duplication in GRPs. Alternatively, most of the repeats could have been deleted in SMR2 gene (for instance, during an unequal cross- ing-over event).

’ Portions of this paper (including “Experimental Procedures,” “Results,” and Figs. 1-5 and 8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

The structure of the SMR2 gene is very similar to that described for the GRP genes (8). In both cases, there is one intron at the end of the signal peptide sequence and one immediately downstream from the stop codon. However, in addition to the two introns found in GRP genes, the SMR2 gene contains a third intron splitting the transition region. Interestingly, the other family of salivary contiguous repeat proteins (proline-rich proteins) shares the same gene organi- zation and can also be divided into two groups on the basis of the presence or absence of a third intron inside the transition region. The human PRHl and PRH, genes (10) and the hamster HZ9 gene (12) have the same structure as the SMR2 gene in that exon II is divided into two parts (exon IIa and exon IIb) by an intron in the transition region (Fig. 6). On the other hand, the mouse Ml4 and MP, genes (9), as GRP genes, contain a unique exon (exon II) encoding the entire transition region, the repetitive segments, and the carboxyl- terminal region. However, Ann et al. (11) have proposed that combination of exon IIa and IIb in the mouse PRP genes could represent a species-specific difference in PRP gene structure.

Curiously, in the region where intron II occurs in SMR2 gene, GRP mRNAs contain a 15-bp sequence which does not align with the SMR2 mRNA sequence (see dot matrix of homology on Fig. 4). In an attempt to optimize these align- ments, we found that the sequence present in GRP mRNA and absent in SMR2 mRNA is highly homologous to the 3’ end of the SMR2 intron II. As shown on Fig. 7, one possibility is that this sequence was originally a part of the exonic sequence. We propose that, after insertion of intron II into a GRP-like gene, this sequence has been released inside intron II by the use of a new more 3’ splicing site, leading to the present structure of the SMR2 gene. Since the sequences of PRP genes have too much diverged in this region, they cannot

“ “ “an PRP PRH,

Rot S”RZ - -

FIG. 6. Comparison of PRP and GRP gene organizations. Exons are indicated by thick bars. The mouse Ml4 and MP2 PRP genes (9), as well as the rat CRP2 gene (8), have 3 exons. The hamster H29 (12) and human PRHl (10) PRPs and the rat SMR2 genes are characterized by a 4-exon structure due to an additional intron splitting the transition region.

FIG. 7. Alignment of SMRP and GRP gene sequences inside the transition region: proposed model for the loss of an exonic sequence in SMR2. SMRZ and CRPP sequences have been aligned in the region surrounding intron II in SMRZ. Numbering refers to the mRNA sequences. Exon and intron sequences are written, re- spectively, in capital and small letters. A striking observation is that the sequence 130-153 of CRP2 may rather well be aligned with the 3’ end of SMRL intron II. We propose that the ancestral gene of CRP2 and SMR2 did not contain intron II (“GRP-like structure”). The structure found in the SMRL gene was created by the insertion (marked by an arrow) of intron II and the subsequent displacement of the 3’ splice site (after mutation events), leading to the integration of an originally exonic sequence into the intron. The sequence boxed in the figure may have diverged after intron insertion by replicating errors or by unequal crossing-over, due to its repetitive nature.

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Evolutionary Aspects of the Glutamine-rich Protein Family

be used to verify the validity of such a model. One should therefore also consider the possibility that this region of the ancestral gene had the same structure as in the SMR2 gene and that the GRP structure was created by the imprecise excision of the intron II.

Exons I of SMRP and GRP genes, are 92% homologous and are also highly homologous to that of PRP genes (Fig. 8). The potential significance of this surprising conservation of exon I in PRPs has been discussed by Ann et al. (9). This sequence could be critical for the synthesis and/or secretion of these proteins.

It has been proposed that the transition region and the carboxyl-terminal region of the PRPs could have emerged, like the repeats, during the internal duplication events (9). A surprising finding is that SMRP mRNA is about 75% homol- ogous to GRPs mRNAs (including the transition region and the 3’-untranslated region) except in the carboxyl-terminal region, where no significant homology can be found. A similar observation has also been reported for the two characterized GRP mRNAs. The sequences of these mRNAs are identical except in the carboxyl-terminal regions which are only about 60% homologous in terms of nucleotidic sequences. Heinrich and Habener (8) have therefore proposed that the two genes have undergone multiple conversion events in the recent evolutionary past. Since more than 10 GRP genes have been detected by Southern blot analysis of rat DNA (8), we cannot exclude that some other GRP genes share the same carboxyl- terminal region as SMRB. The high level of sequence diver- gence in the carboxyl-terminal region, together with differ- ences in repeat number, could contribute to give functional specificity to the different GRPs and SMRB.

Although SMR2 and GRP genes obviously have a common evolutionary origin, their expression is submitted to different regulation. No sexual dimorphism has been reported for GRP gene expression. In contrast, SMR2 mRNA accumulation is about 20 times higher in the SMG of male than female rats. Whether the androgen regulation is transcriptional or not, direct or indirect is under investigation. However, since the 5’- and 3’-untranslated regions which are the most often involved in mRNA stability (36) are relatively well conserved between SMRP and GRP genes, a mechanism of mRNA stabilization by androgens seems rather improbable. Due to the high level of sequence homology between the GRP and SMR2 genes, this family could therefore provide an interest- ing model to study the mechanisms involved in differential regulation of the genes expressed in the SMG, and particularly in the regulation by androgens.

Acknoruledgments-We especially thank Dr. M. Tosi for helpful discussions. We also thank C. Gachet for her skillful typing of the manuscript and Drs. M. Goodhardt and D. Tronik for critical reading of this manuscript.

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Natl. Acad. Sci. U. S. A. 65, 8553-8557 Tronik, D., Dreyfus, M., Babinet, C., and Rougeon, F. (1987)

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10712 Evolutionary Aspects of the Glutamine-rich Protein Family

Supphxnenta, Material 10 : A new member of the glulamine rxn protein family is characterized by the absence of internal repeats and the androgen control of its expression in the submandibulsr gland of ra,s lsabelle Rosinski-Chupin and Franpois Rougeon

EXPERIMENTAL PROCEDURES

/\nimds and hormone ~reaua~& Ten week old m”le and female Wistar rats were purchased from lffa Credo (Lyon. France). Androgens were withdrawn by c~slralion and. where indicated 35 mg of ~eslos~erone (Swrandryl Retard. Hoechsl- Roussel) was injected s”be”laneo”sly IO days later. Where indicated the same dose of tesms!erone was “dminislered to female rats. Rats were killed one week after androgcn treatmen,.

n and 8” w,r” ,rW RNA was prepared from ,a, ,,SI”CF ds dcscr,bed (19). In v,lro wanslauon of RNAs WPI performed w,,h ,he mRh’A- dependent reuculocyte lysate lra”sb,,,o” syswm (20). The products were snrlyzed by d~ml”rw N*D”dSQ polyacrylamtde gel elec~rophoresa

Nord~ern blot analysis (figure 2 Al “sing the full length cDNA insert as a probe revealed that il corresponds IO a short mRNA of about 600 nucleolides. accumulated in higher amo”ms in the SMG of male rats than in female rals. In addition. there was some cross.hybridiralion with a mRNA of 950 ““cleoddes present al the same level in m”,es and females. This cross-hybridizadon can be ahmost complclcly abolished by “se of a shorter probe corresponding to the 3’ end of the cDNA ioscrl. indicadng that only the 600 nucleotides mRNA corresponds to SMRI. Since SMR2 and GRP mRNAs have highly homologous 5’ ends (see below). the 950 ““cleoddes mRNA probably corresponds IO one of the GRP mRNAs.

In order 10 verify whether SMR2 mRNA acc”m”la~io” in male rats was due IO a regulation by androgens. castration experiments and androgen ~reaooenl of carlrazed males and of females were performed as described in the ezpcrimenml procedures. As shown in figure 2B. SMR2 mRN.4 accumulates 10 abo”l *O-fold higher levels in male than in female rats. The amount of SMR2 mRNA in males is reduced about IO- fold 20 days after castration. Testosterone treament of these castrated males or of females induces SMR2 mRNA acc”m”lalion to a level similar IO that of males.

The tissue distribution in rats of SMR2 mRNA was invesligaled by Northern blot analysis. Among the different ral organs slvdied : SMG. proswe. scmmal vesicles. kidney. liver. guts. SMR2 mRNA was dewcled only in Ihc SMG (not shown,.

&m~g and chmtlon of ,he SMR2 cDNA The s”bmand,b”lsr gland cDNA hbrary was cons,r”c,ed and screened as descr,bed I” ,181 The rccombmn”, clones werf idcnrified by DNA-mRNA hybrid arreswd ‘celi free tr”ns,aio” cxperimenrs (21). h cDNA insert was subcloned in Ml3 mp9 vector and sequenced by Ihc dideoxynvclootide chain-termination method (22). The starch for sequence homologies bewecn SMR2 and Ihe proteins of the PSEQUlP library (23) or bewee” SMR2 mRNA and Ihc nucleotidic sequences of Ihe EMRL data bank was done with. respectively. Ihc FASTP and the FASTN program according 10 Lipman and Pearson (24).

‘.” ran eenomic lihrart DNA of high molecular weight was prcparcd from ral spleens. parlially digested with So” 3A and loaded on a 5-25 I NaC, gradient in buffer 10 mM Tris.HCI. 3 mM EDTA. pH8. After 4 h 30 of ccnlrifugation in a SW 41 (Beckman) rotor al 37 000 rpm. the grndienls were collected and fractions were analysed by clectrophorcsis in a 0.5 % agarose gel. Fragments of 16-20 kb were inserted in a XEMBL3 vector (Gcnofil) obtained by double digestion with EcoRl and BomHl according 10 the inslr”ctio”s of lhe manufacwrcr. Packaging “sing Ihc “Cigapack Gold exwacts” (S~ralagcnc) and infection of LE392 bacteria were realized according to the iowucliow of manufacturer. Approximately 106 rccombinanl phages were screened by hybridization with a 32P labeled cDNA insert afler lransferl on ni~rocell”lose fihcr. Miniprcprralions of DNA from posh&e cIo”cs were realized according $0 Crossbergcr (25) and analyscd after Soulher” blotting. ADN from one clone XEB228 was prepared in higher ~mo”ms

according 10 Manialis (26). Different overlapping fragments were subcloned in Bluscript vccnor (Strrlagcne) and sequenced. either directly or after progressive deletions of Ihe insert by the Exonucleaselll Mung Bean syswm.

BN&x&& Total RNA was elecwophoresed in an agarasc/formaldchyde gel (27). lransfcrrcd 10 a nylon membrane and hybridized with the SMR2 cDNA probes. The probe was the full length insert or the 3’ Toaql restriclion fragment of the insen.

RESULTS

gkd of rats under andraen contra To ISO,.,~ mRNAs accumulated under androgen control I” the submrndtbular

gland of rats, a cDNA bbrary was prepared and screened as dcscrlbed (18) The positive clones were characlermed by DNA-mRNA hybrtd arresled cell free lr.mslaboo (19). As shown on figure I, one of the cDNA inserts abohshed the m rtwn synlhcsls of a polyppdde wfh an apparem Mr 35 kDa on NaDodSO4 PAGE. SMR2

O’Q uoc - - + + 56 plK9

92-

- - - - - - 69-

46-

f

--- _ -.WRZ

30-

- - - -

--s r r ( --A

ABCD Fig. 1. -arrestcds of SMRZ EDNA C,O~ Fwo m~rograms of total RNA prepared from SMG of male rats ( lane A. C. D).

female rals ( lane B) wcrc Iranslated I” a reuculocyle cell free system eather dmecfly (lanes A and B) or after hybr,d,zaoon wwh 500 nanograms of the purified SMR2 cDN.4 ,“serl S6 (lane C) o, 600 nanograms of p”C9 DNA (lane D) The I” v,,ro lranslat~on products were elecwophoresed ,n a NaDodSO4 12 5% po,yacry,am,de ge, and rutoradiographed.

1 2 345 67

Fig. 2. (A) Characterization of SMR2 mRNA bv RNA b,o# analvsis. Two microsram~ nf IO,P, RNA from male (I) or female (2) raw were

elcctrophoresed 1; -a I 7% agarose, formaldehyde gel, transferred to a nylon membrane and hybrtd,zed wrh the full-,eng,h SMR2 cDNA probe

(B) wreevlatlon of SMR2 mRNA accymula(lon. One mmogram of total RNA from male (3). castrated male (5). castrated and

testosterone treated male (6). testosterone [rexed female (7) raps and wemy mtcrograms of total RNA from female rats (4, were electrophoresed I” a I 5 agaroselformaldehydc gel. transferred to a nylon membrane and hybrtdzed wh #ha 3’ fragment of the SMR2 cDNA msen obtamed after Taoql resw~cuo”

Nucleotide seo”ence of SMR2 cDNA and homoloeies with the GRP mRNAs The SMR2 cDNA insert (527 bp long excluding Ihe polyA watt) was sequenced

(hgure 3) The po,yA tract II preceded by an “operfec, polyadenylsuon r,gnal AGUAA (28) Only one open rsadmg frame. begmnmg after 15 bp of 5’ ““wanslated sequences can be found. Thw opn readmg frame II 411 bp long and corresponds zo a prole!” of 137 armno-actds The pred,c,ed prolet” 1s r,ch I” glu,amme,gl”!am,c ac,d (17%). prolme (12%) and asparagme,aspar,~ acld (13%) The molecular mass of the protean calculated from Ihe sequence IS about 15 4 kDa. which IS wkmgly lower than the molecular mass (35 kDa) esumared by NaDodSO4 PAGE elecwophorew

Fig. 3. Nuclcolide of SMR2 cDNA clone and ~rcdicrad

The protein sequence is numbered from the first methianine residue. The putative signal peptide is underlined. Broken underline indicates the polyadenylarion signal. The position of the inlrons is marked by arrows. The sequence which is repeated in CRPs is overlined.

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Cornpurer search for sequence hamologxr in the data banks revealed d agnificant degree of homology between SMR2 and mRNAr encoding the glummine- rich proteins (GRPs) of ,a,% These prorems are cba,ac,~,iz~d by the presence of contiguous idcmical peplidcs, rich in glunmine/glulamic acid. Figure 4A shows a dot matrix of homology between SMRZ and CRPZ. the longer GRP mRNA sequence rtporwd by Hcinricb and Habcnc, (8). Parallel diagonals are mdieativc of the presence of the interna, repeats in the sequences of GRPs.

By analogy with GRPs, the sequcncc of SMR2 mRNA can bc divided into fwe rcgionr (Pi~urcs 3 and 4). The firs, 70 nuclcotides, conespondmg 10 ,be 5 untranslated region and a highly hydrophobic segmenr cba,acte,is,ic of s,gnal peplides for recreuon are rrrongly conrewed (92% homologous) between SMRZ and ORPI. The next ssgmenr. 147 bp long in GRPs and 132 bp long m SMR2 defines the “t**“Si,lO”- region which displays approximately 75% nuclco,idc sequence conrervllion between hofh mRNAs. The diffcrcnt length of ,he two sequences m rhrs region is due to a IS bp gap in the SMR2 sequence. The ,hi,d segmenr canespandr 10 rhc region of contiguous repearr in GRPs. Each repeated motif is 69 bp long and the number of repents is variable in ,be two characterized GRP mRNAs. In SMRZ mRNA. the motif is only found once (Figure 48) and is 75 % homologous LO rha, of GRPr in nucleoddc sequence, but only SO ‘h homologous wab rc~pcct ,o ammo-aezd sequence. Regions corresponding 10 ,hc carboxy-,c,mmal part of Ihe pratein~ are highly dlvcrgcnt both in rams of nncleotide and pcptidc sequencer. This region IE aI10 the mml divergem between bmb characterized GRPs. CRP2 and CRPJ (8). Surprirmgly. a high degree of similsnly is again found in the 3’ untranslated regions of SMRZ and GRP quenees.

Fig. 4. (A) ~MRotGRP(PC m RPZ RNA

Stnngcncy of comparison II the ,a,io of required marcher 10 ,hc length of comparison is 28/U The differen, regions defined in each mRNA sequence are indicacd. S’c S : 5’ untranslated ,&on and signal pepride; Tram : ,,ansr,,on rcgwn Rep reperirivc mEion; CT : carbarv-renninal meion: 3’ 3’ unrranslawd ,e~ion.

(BiSehcmatic CRP2 and CRP3 correspond IO the two GRP mRNAs described by Hemnch and

Habenc, (8). S is rhc sir& peprtdc ; T is the vans~dan region : R corrcrponds IO Ihe pepride motif pcrfeedy repeated five ttmes in CRp2 and four ,,mes in CRB ; R is only found one rime in SMRZ : R’ and R” are the less conrencd repeats found in GRPr : CT Indicates the carboxy-terminal x&on ( rhadded on the fi&) which IP rhe least conserved belween the differen, GRPr and SMRZ. 3’ indicaw ,be )’ sequence.

In order to characrerize SMR2 genamnc svuc,u,c. the cDNA mscn was used as a probe to screen a gcnomic library of Wirrar ,a,. prepared ,n a XEMBl.3 vec,o, after partial digestion of the DNA by Sou3A. The clone EB228 conrains a 18 kb msen m&ding a full lenglh SMRZ gene. A regmn of 4.5 Lb corresponding to ,he SMRZ gene and adjacent sequences was sequenced (Fagwe 5). The imron-cron junctions we,e determmed by comparison wilh the wqucnce of rhc cDNA. The gene IE 3S kb long wth four exons separated by m,,ons of tcrpectwcly 900. 970 and 1140 nucleoudcr. A,, ~pliec sites obey the GTfAG rule (29).

A sequence reremblmg ,he YGTGTTYY sequence (31,. wh,ch has been shown to be ~mponanr for efficient formarion of the 3’ terminus of mRNAs occu,~ 8 bp after the polyadenylation rite characccrned I” rbc cDNA sequence.

Ar shown on fiE”,U 3 and 5, exon I corresponds ,,I ,he 5’ “ntranrlatcd region and ,o rhe region encoding rbe signal Fqttde ; Elton 2 conesponds 10 ,be 14.15 f,,r, amino-acids of the ,ransidon region : exon 3 encoder the end of ,be ,,anri,ion region. rht “repeat modf” and the carboxy-rerminrl ponion of SMR2 ; exon 4 comains only the 3’ vnrranslatcd regian. This or~=nitatmn IS similar 10 that described for GRP genes (8). cxcepr for the prerencc d ,he rcfond inlron which dots no, OCCU, m GRP

a E ept K egli E E ego Bg11 E c

Exl Ex2 EX3 Ex4

Ikb

Fig. 5. (A) yscaucnce The exonr are underl ined by wuavy liner. Splice sites are mdtcdled by wianglcr.

Tranrcriplion miriatton sires are rrancd. TATA box is Yndellined by a Ihick bar. Polyadenyladon signal and YGTG’ITYY resembling sequence are underl ined by thm bars. The lranslalion mlriarian codan is boxed.

(B) Rcsnicrionmao of rhe 4.5 kh w&w&&&~ The rcsricdon mrymcs are: E Em RI. Bgf : Bgll. Bgll Egl I;. K : Kppn 1.

Fig. 8. ms ar the 5’ end of difIrrcnr, Nucleoodc sequences of different PRP cDNAs from human (cP3 .cPS. cPI) (7).

mouse (pUMP40. pMP1) (5). rat (pRP25. pRP33) (5.6). PRP gene, from hamster (pH29) (12). GRP cDNA from rat (pCRP2) (8) and SMRZ cDNA are aligned ID show sequence homologies. The ATG inidanon codon is ahgned and underl ined T& mitmlron codonr are aligned with the A dcsngnated as nucleodde I

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I Rosinski-Chupin and F Rougeonsubmandibular gland of rats.

absence of internal repeats and the androgen control of its expression in the A new member of the glutamine-rich protein gene family is characterized by the

1990, 265:10709-10713.J. Biol. Chem. 

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