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F Rougeon

Publications and source records attributed to F Rougeon.

At least 55 records · Page 3Linked to original sources

Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes.

The somatic diversity immunoglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Rag1 and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Rag1 and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types of junctional diversity observed during lymphoid development.

3T3 Cells↗

Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OF1 mice.

Two cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OF1) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown by in vitro transcription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females. In vitro translation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only one kap gene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.

Alleles↗

Demonstration of a divergent transcript from the bidirectional heavy chain immunoglobulin promoter VH441 in B-cells.

The mouse heavy chain immunoglobulin promoter VH441 can lead in vitro to bidirectional transcription, due to a symmetrical organization of immunoglobulin heavy chain promoters with two TATA-like sequences bracketing the upstream promoter element ATGCAAAT (the so called octamer). We demonstrate here that divergent transcription also occurs in vivo in mature B cells from a myeloma which expresses the VH441 gene and even from the spleen of BALB/c mice. The level of VH441 divergent transcript increases in the spleen of BALB/c mice after immunisation by beta-(1,6)-galactan, showing that it is expressed in B cells which actively transcribe the VH441 gene. The divergent transcript has been characterized: its major transcription start site was mapped within 33 base pairs from the divergent TATA-like region, it is unspliced and not polyadenylated. In the light of these results, the functions of the divergent transcript and the bidirectional promoter are discussed.

Animals↗

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells.

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.

Amino Acid Sequence↗

Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.

Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages.

Animals↗

One amino acid change in rat SMR1 polypeptide induces a 1 kDa difference in its apparent molecular mass determined by electrophoretic analysis.

SMR1 is a male-specific, 19 kDa, in vitro translation product of Wistar rat submaxillary glands, which may be the precursor of a small hormone resembling the TRH. In Sprague-Dawley and Fischer rats, instead of SMR1, a male-specific 18 kDa polypeptide may be found. We have cloned the cDNA encoding the 18 kDa polypeptide. We show that the 19 and the 18 kDa polypeptides have the same sequence except for one amino and change.

Animals↗

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.

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, SMR2, 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 between 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 SMR2, 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 contains 4 exons. The second intron, which does not exist in characterized GRP genes, splits the "transition" region 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.

Amino Acid Sequence↗

Nucleotide sequence of the VH, VL regions of an anti-idiotopic antibody reacting with a private idiotope of the anti-lysozyme D1.3 antibody.

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.

Amino Acid Sequence↗

The gene encoding SMR1, a precursor-like polypeptide of the male rat submaxillary gland, has the same organization as the preprothyrotropin-releasing hormone gene.

SMR1 is a precursor-like polypeptide of the submaxillary glands of rats. Sequence analysis predicts that it could be processed by maturation enzymes to release a small peptide resembling the thyrotropin-releasing hormone. The SMR1 gene was isolated from a rat genomic library and sequenced. The SMR1 gene spans 4.7 kb and consists of three exons. The two introns occur a few nucleotides before the initiation codon in the 5' untranslated region, and a few nucleotides before the first predicted processing site, respectively. Such a structure is reminiscent of that of the preprothyrotropin-releasing hormone gene. The site of transcriptional initiation of the SMR1 gene was determined and 1.4 kb of 5'-flanking sequence was sequenced. The sequence analysis revealed the presence of alternating purine-pyrimidine tracts and of purine-rich sequences. In addition, some sequences which could be involved in the regulation of SMR1 gene expression were identified.

Amino Acid Sequence↗

The activity of the mouse renin promoter in cells that do not normally produce renin is dependent upon the presence of a functional enhancer.

The expression of a hybrid gene containing the promoter region of the mouse Ren-1 or Ren-2 genes and the chloramphenicol acetyl transferase (CAT) gene coding region was analysed in five cell lines that do not normally express the renin gene. The renin promoter is inactive in each of these cell lines unless the SV40 enhancer is also present in the construct. In the latter case, transcription initiates at the normal renin start site. This is in contrast to the situation observed in lymphoid cells where the renin promoter is inactive even when coupled to a functional enhancer [(1987) EMBO J. 6, 1685-1690]. Furthermore, 5'-flanking sequences of Ren-2, placed upstream of the thymidine kinase or SV40 early region promoters do not alter the activity of these promoters in three different cell lines. The results suggest that, except for B cells, the renin promoter is not tissue-specific but that its lack of activity in cells that do not express the gene is not the result of repression but may be due to the absence in these cells of one or several trans-acting positive factors.

Animals↗

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.

Animals↗

Determinant differences between the rabbit and mouse immunoglobulin kappa enhancers impair the activity of the rabbit enhancer in mouse myeloma cells.

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

Animals↗

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways.

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.

8-Bromo Cyclic Adenosine Monophosphate↗

Regulatory elements involved in the bidirectional activity of an immunoglobulin promoter.

We show that the promoter from the mouse VH441 heavy-chain immunoglobulin gene, when present on plasmids transiently introduced into myeloma cells, promotes transcription bidirectionally, due to the presence on both strands of TATA-like sequences bracketing the highly conserved decanucleotide element. The two divergent promoters compete for the transcriptional machinery, their relative strength ultimately reflecting the likeness of the two TATA boxes to the consensus sequence. Moreover, their relative activity is also strongly influenced by certain point mutations within the distally located heavy-chain enhancer. The bearing of these results on current concepts of promoter function is discussed.

Animals↗

Rearrangement of the immunoglobulin kappa light chain genes in a b4 rabbit and a Basilea rabbit.

The immunoglobulin chi light chain gene family of the rabbit is characterized by the presence of two constant region exons, C chi 1 and C chi 2 encoded at the chi 1 and chi 2 loci, and linked to their own cluster of joining pieces (J chi). The gene segments at the two loci are very unequally expressed. Thus, in domestic rabbits, the immunoglobulin light chains are essentially of the chi 1 type, even though the gene segments at the chi 2 locus are structurally functional. We have investigated the origin of the weak expression of the genes at the chi 2 locus by analysing the pattern of rearrangement of the chi 1 and chi 2 J chi segments in rabbit B-cell populations. Southern blot analysis of B cells isolated from a rabbit expressing chi 1 light chains suggests that the genes at the chi 2 locus underwent very few, if any, rearrangements. However, using more sensitive approaches, it was possible to detect transcripts originating from the rearranged chi 2 locus. In contrast, in B cells isolated from a Basilea rabbit, which cannot express chi 1 chains, Southern blots revealed the rearrangement of the chi 2 genes, whereas the chi 1 rearranged fragments were barely detectable. These results could be explained either by preferential rearrangement of genes at the chi 1 locus or by clonal amplification of only cells producing chi 1. Furthermore, results of Southern blot analysis provide evidence that V-J recombination may be accompanied by an inversion of the intervening DNA region.

Alleles↗