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S Gerondakis

Publications and source records attributed to S Gerondakis.

53 records · Page 3Linked to original sources
PubMed1990

RNA splicing generates alternate forms of germline immunoglobulin alpha heavy chain transcripts.

Emerging evidence implicates germline immunoglobulin heavy chain gene transcription in the targeting of heavy chain genes for switch recombination. In this study, cloned cDNA copies of the major germline alpha heavy chain transcript expressed in the murine B cell lymphoma 1.29 mu, a cell line that switches to IgA in culture, have been used to characterize the germline alpha transcription unit. The 5' end of these transcripts are heterogeneous, being derived from an exon denoted I alpha located approximately 2.2 kb 5' of the alpha switch region. Sequence analysis of cDNA and genomic clones reveals that alternate splice donor sites generate I alpha exons of varying length. While the two smaller spliced forms of I alpha contain stop codons in the open reading frame of the C alpha gene, transcripts utilizing the 3' most splice donor signal may encode a protein in which amino acids derived from the 3' end of the I alpha exon are fused to the C alpha domain.

Animals↗
PubMed1990

The murine c-rel proto-oncogene encodes two mRNAs the expression of which is modulated by lymphoid stimuli.

Here we report a survey of c-rel proto-oncogene transcription in murine tissues, cell lines and lymphoid cells. In addition to the previously described 7.5-kb mRNA, we have identified a mRNA of 2.5-kb. As DNA hybridization indicates that there is only one gene with significant homology to c-rel in the mouse genome, it appears that multiple mRNAs are transcribed from c-rel. The nucleotide sequence of a cDNA clone derived from the 2.5-kb c-rel mRNA demonstrates that the 7.5- and 2.5-kb mRNAs encode identical proteins. The different size of the two mRNAs is due to variation in the length of the 3' untranslated region, which arises from the use of alternate polyadenylation signals. These mRNAs are present at low levels in organs tested, and in cell lines representing a wide variety of lineages. Fibroblasts are the only cells in which expression was not detectable. In B-cell lines representing different stages of differentiation, the highest levels of mRNA are seen in B-lymphomas, and this level drops markedly in plasmacytomas. There is a transient increase of 10- to 20-fold in the level of c-rel mRNAs in T-cells treated with concanavalin A, while lipopolysaccharide-stimulated B-cells exhibit a transient 5-fold elevation of c-rel expression. This study indicates that the control of c-rel expression can vary between and within different cell lineages, and the widespread expression of this gene points to a fundamental cellular function, rather than one restricted to hematopoietic cells as previously suggested.

Amino Acid Sequence↗
PubMed1990

Murine c-rel transcription is rapidly induced in T-cells and fibroblasts by mitogenic agents and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

The c-rel protooncogene is here shown to be a member of the early response gene family. Expression induced by different agents is regulated by both transcriptional and posttranscriptional mechanisms. In quiescent fibroblasts, c-rel expression is maximally induced by serum or 12-O-tetradecanoylphorbol-13-acetate within 60 min and is superinduced in serum-stimulated fibroblasts by cycloheximide. In T-cells, although 12-O-tetradecanoylphorbol-13-acetate and concanavalin A both rapidly activate c-rel expression, the kinetics of induction mediated by these agents differs markedly. Nuclear run-on analysis demonstrates that induced c-rel expression is due primarily to increased transcription, and the rapid decrease in expression observed in serum- and 12-O-tetradecanoylphorbol-13-acetate-stimulated cells results from mRNA turnover. In the B-lymphoid lineage, c-rel is constitutively transcribed, with the differentiation stage-specific decrease in c-rel expression seen in plasmacytomas reflecting posttranscriptional regulation.

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PubMed1989

Structure of a mammalian c-rel protein deduced from the nucleotide sequence of murine cDNA clones.

The avian retrovirus, rev-T, which carries the viral oncogene v-rel, causes an acute leukemia in birds and transforms immature lymphoid cells in vitro. Although the role of c-rel in normal cells is unknown, homology with the Drosophila gene dorsal, which is involved in determining embryonic dorsal-ventral polarity, raises the possibility that c-rel in vertebrates may play a role in differentiation. As a step towards understanding its role in mammalian cells, we have characterized the coding domain of the 7.5 kb murine c-rel mRNA by isolating cDNA clones that span its entire coding domain and part of the 5' and 3' untranslated regions. The nucleotide sequence reported here indicates that murine c-rel encodes a 588 amino acid polypeptide with a predicted molecular weight of 66 kd. The murine protein shares homology with avian v-rel and dorsal over a 300 amino acid stretch within the amino terminus, while the carboxyl terminal regions of these proteins diverge completely. This suggests that the conserved domain of the rel related family of proteins performs a common function that is modulated by the carboxyl terminal domain.

Amino Acid Sequence↗
PubMed1988

Immunoglobulin JH rearrangement in a T-cell line reflects fusion to the DH locus at a sequence lacking the nonamer recognition signal.

Rearrangements of the immunoglobulin heavy chain joining region (JH) genes occur in some T lymphocytes, probably because the mechanism for assembly of T-cell receptor encoded genes is very similar to that for immunoglobulins. Two such rearrangements described previously represented proper fusion of a DH and JH gene. We have cloned and analyzed the JH rearrangements found on both alleles in the T lymphoma ST4. One represents conventional recombination between a member of the SP2 DH family and JH3. On the other allele, JH4 has recombined with a sequence within the DH locus but not at a DH gene. This recombination involved the heptamer but not the nonamer of the bipartite recognition signal required for DH-JH joining. This result suggests that the heptamer may be the primary determinant of the specificity in V-gene assembly and that the DH locus as a whole may be preferred target for recombination.

Alleles↗
PubMed1986

Structure of the protein encoded by the chicken proto-oncogene c-myb.

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.

Amino Acid Sequence↗
PubMed1984

Activation of immunoglobulin mu gene expression involves stepwise demethylation.

In an attempt to identify stages of mu gene activation subsequent to VHDHJH assembly, we investigated two pre-B cell lines (A1 and A8) that have both alleles of the JH locus rearranged but do not make mu polypeptides. The block in A1 reflects incorrect VHDHJH assembly on both alleles. In A8, although an out-of-phase VHDHJH-C mu allele is transcribed, the properly assembled allele is silent, despite having a normal VH promoter and mu enhancer. Thus, transcription can be restricted to a single mu allele. Low level mu transcription in both lines was associated with demethylation of the VHDHJH and enhancer regions but not of the C mu gene. Markedly elevated mu transcription ensued on lipopolysaccharide stimulation or fusion to a plasmacytoma, but only fusion induced C mu demethylation. Hence stepwise demethylation is implicated in mu gene regulation, but enhanced expression can also occur independently of, or prior to, demethylation.

Alleles↗
PubMed1984

Translocation of the myc cellular oncogene to the immunoglobulin heavy chain locus in murine plasmacytomas is an imprecise reciprocal exchange.

The 15;12 translocations in murine plasmacytomas represent recombination of the myc oncogene with the immunoglobulin CH locus, often within CH switch recombination (SH) regions. Chromosome junctions cloned from four plasmacytomas confirmed that the translocation generates reciprocal CHSH-myc and 5'myc-SH structures. H locus targets included S alpha fused to Smu, Smu fused to S gamma 2b, and a germline S alpha region. The nature of two H locus targets suggests that the target need not be highly active transcriptionally. Switch recombination machinery is implicated in the translocation by the SH targets and by homology of certain c-myc breakpoints with normal switch recombination sites. Fusion regions revealed deletions, extraneous nucleotides, and one duplication. These results prompt a translocation model in which staggered single-stranded breaks on each chromosome are followed by single-strand excision or polymerization prior to ligation to the other chromosome.

Alleles↗
PubMed1983

Interchromosomal recombination of the cellular oncogene c-myc with the immunoglobulin heavy chain locus in murine plasmacytomas is a reciprocal exchange.

The 15:12 chromosome translocations found in most murine plasmacytomas involve the cellular gene (c-myc) homologous to the oncogene (v-myc) of avian retrovirus MC29, Translocation links the c-myc gene of chromosome 15 to the immunoglobulin heavy (H) chain locus of chromosome 12, often within the switch recombination (S) region 5' to the alpha constant region (C alpha) gene. We have investigated c-myc rearrangements in 21 BALB/c plasmacytomas and three B lymphomas by Southern blot analysis. We show that the t(15;12) is a reciprocal chromosome exchange since most tumours contain not only a c-myc gene linked to the S alpha C alpha region but also a separate structure with S mu or S alpha linked to the c-myc 5'-flanking region. Analysis of the two rearrangement products cloned from plasmacytoma J558 suggests that one type of H locus target for translocation is an S alpha region recombined with S mu; two other targets appear to be other switched heavy chain genes and an unrearranged C alpha gene. Nearly all the chromosome 15 breakpoints fall within a 1.1-kb region spanning a 5' c-myc exon; hence scission of the transcriptional unit by translocation can account for the altered c-myc transcription in plasmacytomas. The c-myc breakpoint region lacks substantial homology with S mu or S alpha, arguing against homologous recombination as the translocation mechanism.

Animals↗
PubMed1983

Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumours.

The 15;12 chromosome translocation in murine plasmacytomas and the 8;14 in human Burkitt lymphomas often link the cellular myc oncogene to the locus for constant regions of immunoglobulin heavy chains (CH locus). To clarify how and why c-myc translocation occurs, we have sequenced the mouse and human c-myc genes and correlated c-myc transcription with c-myc rearrangement. Both genes comprise three exons; the second and third encode the myc polypeptide, which is conserved between mammals and birds, particularly in its more basic C-terminal half. Southern blots showed that four of 12 Burkitt lines have c-myc linked near CH switch regions and two near the joining region (JH) locus. Hence, immunoglobulin recombination machinery may participate in translocation, although the common myc breakpoint region around exon 1 does not resemble a switch region. Tumours with breakpoints just 5' to exon 1, or distant from c-myc, had normal c-myc mRNAs of 2.25 and 2.4 kb, which differ at their 5' ends, while tumours with breakpoints within exon 1 or intron 1 had altered c-myc mRNAs (2.1-2.7 kb in Burkitt lines), initiated within intron 1. Both types of mRNAs probably yield the same polypeptide. Since the untranslocated c-myc allele was generally silent, translocation to the CH locus must induce constitutive c-myc expression. The presence of c-myc mRNA in immortal but non-tumorigenic lymphoblastoid cell lines may implicate c-myc in an immortalization step.

Amino Acid Sequence↗
PubMed1983

Cellular myc oncogene is altered by chromosome translocation to an immunoglobulin locus in murine plasmacytomas and is rearranged similarly in human Burkitt lymphomas.

Molecular cloning has recently established that the 15;12 chromosome translocations in murine plasmacytomas fuse DNA from chromosome 15 to the immunoglobulin heavy (H) chain locus, usually within the switch recombination region near the alpha constant region gene. We show here that the incoming DNA bears the cellular gene (c-myc) homologous to the oncogene (v-myc) of avian retrovirus MC29. In human Burkitt lymphomas bearing an 8;14 translocation, c-myc was also rearranged, apparently (in at least two cases) to an H chain switch recombination region (mu or alpha), and both products of a reciprocal chromosome exchange were detectable. Both the murine and human c-myc genes contain two exons homologous to v-myc, and additional 5' and 3' murine genomic segments (apparent exons) were defined by hybridization to c-myc mRNAs. In plasmacytomas, chromosome breakpoints fall near or within the 5' exon and apparently disrupt the normal c-myc transcriptional unit, because plasmacytoma c-myc mRNAs differ from the mRNA in lines without c-myc rearrangement. The translocated gene presumably has lost its normal 5' regulatory sequences and may well encode an altered myc polypeptide. We propose that altered expression of the c-myc gene, induced by translocation to an immunoglobulin locus, is a critical oncogenic event for these B lymphoid tumors. Two events may be required, because the plasmacytoma oncogene capable of transforming fibroblasts is not c-myc.

Animals↗
PubMed1982

Transcriptionally active DNA region that rearranges frequently in murine lymphoid tumors.

A DNA region not associated with conventional immunoglobulin gene rearrangement is rearranged in many lymphoid tumors. This region, designated here as lymphoid rearranging (LyR) DNA, was cloned from plasmacytoma J558 in which it had recombined 5' to a constant (C) region of the alpha heavy (H) chain gene, C alpha, within a switch (S) region, S alpha, involved in the switching of CH genes. Sequence determination established that LyR DNA had recombined within a S alpha recombination unit. LyR DNA does not originate from the H chain locus, and discordance between LyR DNA and CH copy number in certain lines suggests that LyR DNA probably derives from another chromosome. LyR DNA rearrangement is a characteristic of tumors of mature B cells; it was detected in 24 of 28 plasmacytomas and B-cell lymphomas, usually as LyR-S alpha, but not in 11 Abelson retrovirus-induced lymphomas of B-cell precursors nor detectably in normal B cells. In contrast, rearrangement was observed in only 3 of 18 T-cell lymphomas, and none of seven nonlymphoid lines. Most tumor lines (49 of 52), whether lymphoid or not, contained a low level of polyadenylylated LyR transcript(s), but several new RNA species with differences in their 5' regions appeared in B-cell lines in which LyR DNA was rearranged, suggesting that rearrangement may activate a new promoter or mode of splicing. The results suggest that the LyR-S alpha rearrangement represents a translocation to chromosome 12 that alters expression of LyR-encoded genes; hence, it may have participated in lymphoid tumor oncogenesis.

Animals↗
PubMed1981

Organization of genes and spacers within the mouse immunoglobulin VH locus.

The germline organization of mouse immunoglobulin VH genes has been investigated using cloned VH sequences. Hybridization studies with VH probes from plasmacytomas HPC76 (H76), S107, HOPC1 (H1), and lymphoma ABLS-8 (A8) demonstrated that the VH locus contains at least three distinct VH gene families. Comparison with other results suggests a total of about 10 such families, and of the order of 160 germline VH genes. Nucleotide sequencing revealed that the H76 family includes anti-inulin VH sequences, and the S107 family is known to encode antiphosphorylcholine sequences. The three VH gene families studied were mapped in the order H76-S107-A8/H1-CH by determining which VH genes had been deleted from several plasmacytomas by VH rearrangement events. Nine genomic clones from athe H76 family, and one each from the S107 and A8/H1 families, were characterized; collectively they span 103 kilobases (kb). Two clones from the H76 family and one from the S107 family each bore a pair of VH genes separated by approximately 14 kb, suggesting that related VH genes in these families are clustered with a typical spacing of approximately 14 kb. No other VH genes were detected within the spacers, arguing against intermingling of different families. Within the VH76 family cluster, however, two closely homologous VH genes were shown not to be adjacent. While spacer sequences were strongly conserved in the A8/H1 family, the H76 family had minimal conservation and that was restricted to regions immediately surrounding the genes. Hence conservation of spacer sequences cannot be essential for VH gene function, nor for maintenance of a VN family. Spacers in both the H76 and S108 family contained small repeat elements, some of which behaved like mobile DNA sequences.

Animals↗
PubMed1980

Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes.

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.

Animals↗
PubMedDate not supplied

Simultaneous expression of germline gamma1 and epsilon immunoglobulin heavy chain transcripts in single murine splenic B-cells.

While successive isotype switching can occur from IgM to IgE via IgG1, little is known about the pattern of germline transcript expression in normal B cells committed to switching to multiple isotypes. In this study we define the relationship between germline transcript expression and immunoglobulin isotype expression and secretion in murine splenic B cells. Following 7 days stimulation with LPS and IL-4, 10% of single cells secrete IgE and of these only 1 in 10 secrete IgE alone. In contrast, when cells were stimulated with LPS and IL-4 for 48 hr prior to sorting into clonal cultures, 71% secreted IgE alone. In an attempt to isolate switch intermediates, IgG1+IgM- cells were sorted and upon re-culture secreted predominantly IgG1 alone or IgG1 and IgE. The frequency of cells expressing germline epsilon transcripts was 34% for surface IgG1+IgM- expressing cells and 14% for surface IgG1-IgM+ negative cells. Furthermore, analysis of single surface IgG1+IgM- cells demonstrated that these cells can co-express germline gamma1 and epsilon transcripts. Sorting of IgG1-IgM+ cells according to surface expression of the integral membrane proteoglycan, Syndecan, demonstrated that IgM+Syndecan+ cells had a lower frequency of germline transcript expression and a lower frequency of isotype switching (32%) compared to IgM+Syndecan cells (83%). Collectively, these finding show that murine B cells can switch successively from IgM to IgE via IgG1 and that IgG1 expressing intermediates express germline transcripts. Furthermore, Syndecan expression appears to be linked to germline CH transcription and isotype switch commitment.

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