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F Rougeon

Publications and source records attributed to F Rougeon.

At least 19 recordsLinked to original sources
PubMed1997

The mouse Vcs2 gene is a composite structure which evolved by gene fusion and encodes five distinct salivary mRNA species.

Genes of the VCS (variable coding sequence) family are characterized by an extensive evolutionary divergence in the protein-coding sequence. The VCS family has been characterized by cDNA cloning from submandibular glands in the rat, mouse and humans. At the genomic level, the sequences of two members of this family are known in the rat Rattus norvegicus: the VCSA1 gene, encoding the prohormone-like polypeptide SMR1, and the VCSB1 gene, encoding a salivary Pro-rich polypeptide. No genomic data were available for the VCS genes of other species. To understand the evolution of the VCS gene family better, we have now sequenced 23 kilobases (kb) of the mouse Vcs2 gene. The Vcs2 sequence reveals numerous genomic reorganizations such as an inversion, insertions of short elements and an unusually high number of long interspersed repeated elements (LINEs), which make up 42% of this region. Interestingly, Vcs2 is composed of three different VCS-like regions. The first of these regions contains all the exons necessary to encode the previously described mouse submandibular gland polypeptide MSG2alpha. This region aligns with the entire genomic sequences of rat VCSA1 and VCSB1 genes. The two other regions align with fragments of these rat sequences. The three regions are arrayed in tandem and flanked by LINEs. In particular, the third region also contains exons that were found in mRNA species from the submandibular gland. In total, we have characterized five mRNAs from mouse submandibular glands which have in common their first exon, and are produced by alternative splicing. Vcs2 is thus a single gene that arose by the fusion of three genes (or pseudogenes) of the VCS multigene family.

Alternative Splicing↗
PubMed1997

Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation.

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.

Animals↗
PubMed1997

Targets for SMR1-pentapeptide suggest a link between the circulating peptide and mineral transport.

The submandibular rat 1 protein (SMR1) is selectively processed at pairs of basic amino acid residues in a tissue- and sex-specific manner. We have mapped peripheral targets for the final secretory maturation product of SMR1, the pentapeptide QHNPR, by examining in vivo the tissue distribution of the radiolabeled peptide using beta-radio imager whole body autoradiography. The characteristics of tissue uptake allowed specific binding sites at physiological peptide concentrations to be identified within the renal outer medulla, bone and dental tissue, glandular gastric mucosa, and pancreatic lobules. Direct evidence that pentapeptide binding sites are localized in selective portions of the male rat nephron, within the S3, S2, and S1 segments of the proximal tubules, was obtained. In bone tissue the pentapeptide exclusively accumulates within the trabecular bone remodeling unit, and in dental tissue it concentrates within the tubules of the dentinal rat incisor. In relation to male rat-specific behavioral characteristics, our data suggest that the circulating androgen-regulated SMR1-derived pentapeptide is primarily involved in the modulation of mineral balance between at least four systems: kidney, bone, tooth, and circulation.

Aging↗
PubMed1997

B lineage-restricted rearrangement of a human Ig kappa transgene.

To study the control of immunoglobulin kappa light chain gene rearrangement, we generated transgenic mice carrying a germ-line human kappa minilocus (HK) containing the J kappa-proximal V gene, V kappa IV, the V-J intergenic region, the five J kappa segments and the C kappa gene. This construct includes the intronic, but not the 3' kappa enhancer. Rearrangement of the HK transgene was found to be lymphoid specific and restricted to the B cell lineage. Quantification of kappa gene rearrangement in pre-B cell lines established from HK transgenic mice showed that, like endogenous kappa genes, rearrangement of the transgene is repressed in mu-negative early B cell precursors. These results indicate that rearrangement of the HK transgene is subjected to the same B/T cell and developmental regulation as V kappa-J kappa rearrangement at the endogenous locus. Comparison with an unrearranged kappa transgenic construct lacking the V-J intergenic region, suggests that this region, or elements associated with the proximal V gene, may act to restrict kappa gene rearrangement to the B cell lineage.

Animals↗
PubMed1996

The presence of direct repeats does not influence coding joint formation during V(D)J recombination.

During the recombination process that assembles immunoglobulin and T-cell receptor gene segments, the coding ends to be joined are extensively processed. Contradictory reports have been made in the past about the existence of homology directed mechanisms in V(D)J recombination. In this study we analyse coding end processing and the influence of the presence of homology stretches on coding joint formation using artificial substrates in which short sequence changes creating direct repeats have been introduced. These changes were monitored 3 bp away from the termini in order to avoid any differences due to the initiation steps of V(D)J recombination. Our results show that the sequence of the coding ends influences joint formation, but no evidence was found for a mechanistic bias due to the presence of direct repeats.

3T3 Cells↗
PubMed1996

Episodic evolution and rapid divergence of members of the rat multigene family encoding the salivary prohormone-like protein SMR1.

In rodents, the variable coding sequence (VCS) multigene family displays extensive evolutionary divergence in the protein-coding region. While certain VCS genes coding for proline-rich proteins (hPR-PB, mMSG1, rPR-VB1) are conserved in primates and rodents, others seem to be specific to certain genera. This appears to be the case for the Rattus genes forming the A-subclass. This subclass is composed of three genes in R. norvegicus and probably five genes in R. rattus. The first described VCSA gene (Rn. VCSA1) was found to encode a prohormone-like protein named SMR1 (-VA1), expressed mainly in the submandibular glands (SMG) of male rats. To further understand the evolution of this variable multigene family, we have cloned the two additional genes (Rn. VCSA2 and Rn. VCSA3) forming the R. norvegicus A-subclass and three VCSA genes (Rr. VCSA1a, b and Rr. VCSA2) of R. rattus. The putative SMR1 proteins encoded by all these genes display the same prohormone-like structure as Rn. SMR1-VA1. However, we observe a polymorphism in some internal cleavage sites which suggests that multiple processing of the SMR1 proteins could result in the liberation of peptides differing in structure and length. The phylogenetic analysis of the sequences reveals that the duplication events giving rise to the VCSA1, -A2, and -A3 progenitors were anterior to the R. norvegicus and R. rattus split, and that a VCSA1 duplication event likely occurred specifically in R. rattus. A striking observation is that the coding sequences of the VCSA genes have rapidly diverged from their ancestors. Along all branches of the phylogeny, the nonsynonymous divergence rate is identical or superior to the synonymous divergence rate. We suggest that frequent changes in functional requirements are mainly responsible for the episodic evolution and the rapid divergence of the VCSA genes.

Amino Acid Sequence↗
PubMed1996

Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties.

The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularly with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+, K(+)-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.

Animals↗
PubMed1995

Various transcripts are generated from the VCSA1 gene by alternative splicing and poly(A) processing in the rat submandibular gland.

The members of the VCS (variable coding sequence) multigene family display extensive evolutionary divergence in the protein-coding region. The first described gene (VCSA1) was found to encode a major 0.7-kb mRNA (VCSA1*1T1) coding for a prohormone-like preproprotein, SMR1-VA1, in the submandibular gland (SMG) of Rattus norvegicus. We report here the cloning of four other VCSA1 cDNAs corresponding to mRNAs (VCSA1*1T2 to *1T5) expressed in the SMG. VCSA1*1T1 to *1T4 mRNAs share the three exons previously described and differ in their 3' untranslated regions (UTR). Their differences originate from the alternative utilization of four polyadenylation sites. Comparison of the tissue levels of VCSA1*1T1 and VCSA1*1T4 during post-natal development of the male rat SMG suggests that the poly(A) addition sites are both used at each stage. The fifth RNA transcript (VCSA1*1T5) contains only the first two exons. The nucleotide sequence of the cDNA reveals that VCSA1 has an additional exon (exon 4) which is spliced to exon 2 in VCSA1*1T5. In addition to VCSA1*1T1, at least VCSA1*1T4 and VCSA1*1T5 are actively translated in vivo, as revealed by their association to the polysomal fractions. The protein, P2-VA1, coded by VCSA1*1T5 is 68 amino acids in length and it is likely to be a glycosylated secretory protein. The putative mature P2-VA1 protein completely differs from the SMR1-VA1 pro-protein and very likely has a different function. VCSA1*1T1 is accumulated in the male rat SMG 200-1000-fold more than the other transcripts. Run-on experiments reveal that almost all transcription proceeds several hundred bp downstream from the poly(A) site corresponding to VCSA1*1T1.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗
PubMed1995

The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization.

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.

Alternative Splicing↗
PubMed1995

Recent evolution of genes encoding the prohormone-like protein SMR1 in the rat submandibular gland.

The Variable Coding Sequence (VCS) multigene family of Rattus norvegicus, is composed of at least 10 members, and shows extensive evolutionary divergence in the protein-coding region. Three members of the VCSA subclass, have been characterized: one of them, the VCSA1 gene mainly expressed in the submandibular gland (SMG) encodes the prohormone-like protein, SMR1-VA1. As VCSA-related genes have not been detected in Mus musculus, the VCSA genes subclass is presumed to have recently emerged. To study the evolution of this subclass, we have looked for VCSA genes in a closely related species, Rattus rattus. By Northern analysis, we demonstrate that VCS-related mRNAs are present in the SMG, and that the level of VCSA mRNA accumulation is approximately equal in both sexes. By contrast, in R. norvegicus, males accumulate about 3,000 times more VCSA1 mRNA than females. Using total SMG mRNA, an almost full-length cDNA, homologous to the cDNA of the R. norvegicus VCSA1 gene, was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The putative corresponding SMR1-VA1 protein is 146 amino acids long and presents the features characteristic of a secreted protein, with a potential signal peptide of 22 amino acids in the amino-terminal portion. The presence of potential processing multibasic sites suggests that small peptides could be generated (particularly a hexapeptide: Arg-Gln-His-Asn-Leu-Arg), as in the case of the SMR1-VA1 protein of R. norvegicus. From Southern blot analysis there appears that species-species modifications of VCSA gene copy number have occurred; R. rattus contains a greater VCSA1 copy number than R. norvegicus (two or three and one, respectively).

Amino Acid Sequence↗
PubMed1994

Three novel SMR1-related cDNAs characterized in the submaxillary gland of mice show extensive evolutionary divergence in the protein coding region.

We have previously characterized an abundant male-specific mRNA from the submaxillary gland (SMG) of rats, encoding the SMR1 (androgen-regulated) protein, which has the structure of a prohormone and is processed by maturation enzymes to release a small peptide in the blood and saliva. We have now characterized three SMR1-related cDNAs in the SMG of Balb/c mice. These cDNAs encode three novel proteins, designated MSG1, MSG2 and MSG3. They are 639, 662 and 471 nucleotides (nt) long, respectively, and the corresponding mRNAs appear to be expressed only in the SMG. The putative polypeptides they encode carry an N-terminal secretory peptide sequence and are, therefore, presumably secreted into saliva. Although closely related, the three mRNAs show striking differences: a particularly different expression pattern and an extremely high degree of variability observed in the central part of the molecules. The MSG1 and MSG3 cDNAs are identical, except for a 173-bp insert found only in MSG1. This insert contains three Pro-rich repeats (GPGIGRPPPPPP), reminiscent of the most abundant multigenic family of the SMG, the Pro-rich proteins (PRP). Although MSG1 shares several common features with PRP, it is structurally related to SMR1. The unusually high ratio of replacement/silent nt changes provides a basis to address complex aspects concerning the molecular events leading to the emergence of new proteins in the SMG.

Amino Acid Sequence↗
PubMed1994

Selective processing of submandibular rat 1 protein at dibasic cleavage sites. Salivary and bloodstream secretion products.

The amino acid sequence of submandibular rat 1 (SMR1) protein, deduced from its cDNA sequence, led to the prediction that the SMR1 gene encodes a hormone-like precursor [Rosinski-Chupin, I., Tronik, D. & Rougeon, F. (1988) Proc. Natl Acad. Sci. USA 85, 8553-8557]. SMR1 contains an N-terminal putative secretory signal sequence and a tetrapeptide (QHNP), located between dibasic amino acids which constitute the most common signal for prohormone processing. We have isolated and characterized from the male rat submandibular gland and its secretions three structurally related peptides, namely an undecapeptide (VRGPRRQHNPR), a hexapeptide (RQHNPR) and a pentapeptide (QHNPR) generated from SMR1 by selective proteolytic cleavages at pairs of arginine residues. The biosynthesis of these peptides is subjected to distinct regulatory pathways depending on the organ, sex and age of the rat. Furthermore, the peptides are differentially distributed in the submandibular gland and in resting or epinephrine-elicited submandibular salivary secretions, suggesting distinct proteolytic pathways for their maturation. The undecapeptide is generated in the gland of both male and female rats, but under basal conditions it is only released into the saliva in male animals. The hexapeptide is produced in large amounts in the gland of adult male rats and released into the saliva in both resting and stimulated conditions. The pentapeptide appears only in the male saliva and is present mostly under stimulated conditions. In addition, administration of epinephrine induces the release of the hexapeptide from the submandibular gland into the bloodstream. The evidence indicates that the rat submandibular gland can function as a dual exocrine and endocrine organ for the SMR1-derived hexapeptide, as has been reported for nerve growth factor, epidermal growth factor, renin and kallikrein. Although the biological activities of the SMR1-derived peptides are not yet known, their high production and adrenergic-induced release only into the saliva and bloodstream of adult male rats, suggest a physiological involvement in some male-specific processes.

Amino Acid Sequence↗
PubMed1994

A new proline-rich protein precursor expressed in the salivary glands of the rat is encoded by a gene homologous to the gene coding for the prohormone-like protein SMR1.

A gene encoding a prohormone-like protein (SMR1) has previously been characterized and shown to be expressed in the rat submandibular glands under androgenic control (Rosinski-Chupin, I., Tronik, D., and Rougeon, F. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8553-8557). This gene, now named VCS-alpha 1, belongs to a multigene family (Rosinski-Chupin, I., and Rougeon, F. (1990) DNA Cell Biol. 9, 553-559). We now describe the structure and the expression of a second member (VCS-beta 1) of this family. The two genes differ principally in the protein-coding region, therefore we have named these related genes VCS (variable coding sequence). Genomic clones containing the VCS-beta 1 gene were obtained by screening a lambda EMBL3 library with a probe corresponding to the VCS-alpha 1 cDNA. The nucleotide sequence of VCS-beta 1 predicted a structure containing three exons. This structure, confirmed by sequencing a VCS-beta 1 cDNA obtained by reverse polymerase chain reaction, is identical to the organization of the VCS-alpha 1 gene. Comparison of the VCS-beta 1 and VCS-alpha 1 genomic sequences indicates regions of homology which are unevenly distributed, suggesting a differential evolution of some areas (particularly the third exon) of the VCS genes. The VCS-beta 1 cDNA codes for a proline-rich protein precursor named PR-V beta 1 (148 amino acids, 39.2% proline, 10.8% glycine) and characterized by a secretory signal-peptide and three repeats of a unit rich in proline residues surrounded by two clusters of potential endoprotease cleavage sites. mrNA coding for PR-V beta 1 was detected in the submandibular-sublingual gland complex of male and female rats. PR-V beta 1 is homologous to the proline-rich peptide B isolated from human saliva (Isemura, S., Saitoh, E., and Sanada, K. (1979) J. Biochem. (Tokyo) 86, 79-86) and to the submandibular proline-rich protein precursor MSG1 of the mouse (D. Tronik-Le Roux, M. Senorale-Pose, and F. Rougeon, manuscript in preparation). Our observations provide evidence that in addition to the known proline-rich protein genes, there is, in rodent and probably human genomes, another class of genes coding for salivary proline-rich proteins. The high conservation of various sites for bacterial collagenases localized in the repeat region of PR-V beta 1, MSG1, and PRP-B suggest a protective function of these proteins in the oral cavity.

Amino Acid Sequence↗
PubMed1993

Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase.

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.

3T3 Cells↗
PubMed1993

Methylation status of immunoglobulin kappa gene segments correlates with their recombination potential.

We have previously shown that unlike endogenous chi genes, unrearranged chi transgenes undergo V chi-J chi recombination in Tas well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous chi genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged chi transgenes was compared. The J chi-C chi locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous chi genes was tissue and developmentally regulated. Hypomethylation of the endogenous J chi-C chi region occurs only in cells of the B lineage undergoing, or having completed chi gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous chi genes. These results suggest that hypomethylation defines an accessible state of the chi locus and that methylation/demethylation could be involved in the control of chi gene rearrangement during lymphocyte differentiation.

Animals↗
PubMed1993

The intronic immunoglobulin kappa gene enhancer acts independently on rearrangement and on transcription.

It is not known which cis-acting elements regulate kappa gene rearrangement. However, the onset of rearrangement coincides with the onset of transcription suggesting that common regulatory elements are used. We have investigated the role of the intronic enhancer on rearrangement using mice transgenic for kappa minigenes. The rabbit kappa enhancer, which is defective in activation of transcription in mouse cells, allows a high level of rearrangement. However, transgenes which possess no enhancer region at all are rearranged a hundred times less compared to transgenes which possess the rabbit enhancer. Our results suggest that while the enhancer region can activate rearrangement as well as transcription, its action on both phenomena can be uncoupled.

Animals↗
PubMed1993

Rag-1: a topoisomerase?

Recombination activating genes Rag-1 and Rag-2 were isolated on the basis of their ability to confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the products of these genes is not known. Based on sequence comparison data, it was suggested that Rag-1 protein could act like a topoisomerase and that tyrosine in position 998 could be the active site tyrosine. We tested this hypothesis by introducing a point mutation on the Rag-1 cDNA, transforming the tyrosine codon into a phenylalanine codon. We show that the mutation has no effect on site specific recombination implying that Tyr-998 is not essential for the recombination reaction.

3T3 Cells↗