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J Stavnezer

Publications and source records attributed to J Stavnezer.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements.

Inasmuch as transcription of unrearranged, or germ-line, Ig CH genes appears to direct switch recombination, understanding the regulation of this transcription is essential for understanding the regulation of class switching. Transforming growth factor-beta 1 (TGF-beta 1) induces germ-line alpha transcripts and increases class switching to IgA in the I.29 mu B lymphoma and in Peyer's patch and splenic B cells. It has been previously demonstrated that induction of germ-line alpha transcripts by TGF-beta occurs at the transcriptional level in I.29 mu cells. We now demonstrate that the DNA segment located 5' to the initiation sites of germ-line alpha RNA drives expression of a luciferase reporter gene construct in transient transfection experiments. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. By creating a series of deletion and substitution mutations, we have demonstrated that an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line alpha promoter, but mutation of this motif does not diminish TGF-beta induction. Inducibility by TGF-beta requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemly repeated sequence 5' CA-CAG(G)CCAGAC 3' (termed Ig alpha TGF-beta-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats confers TGF-beta inducibility to a heterologous promoter. An additional copy of the TGF-beta-RE was identified at -41/-30 and its deletion reduces the TGF-beta response. Thus, we conclude that tandem repeats of a novel TGF-beta-RE are the positive regulatory element for the TGF-beta response. Our study provides further evidence that TGF-beta directs class switching to IgA through induction of transcription of the germ-line C alpha gene and demonstrates that TGF-beta can activate the promoter for the germ-line alpha gene.

Activating Transcription Factors↗
PubMed1992

Regulation of transcription of immunoglobulin germ-line gamma 1 RNA: analysis of the promoter/enhancer.

Antibody class switching is achieved by recombinations between switch (S) regions which consist of tandemly repeated sequences located 5' to Ig heavy chain constant (CH) region genes. RNA transcripts from specific unrearranged or germ-line Ig CH genes are induced in IgM+ B cells prior to their undergoing class switch recombination to the same CH genes. Thus, the antibody class switch appears to be directed by induction of accessibility, as assayed by transcription of germ line CH genes. For example, IL-4 induces transcripts from the mouse germ-line C gamma 1 and C epsilon genes to which it also directs switch recombination. We report here that the 150 bp region upstream of the first initiation site of RNA transcribed from the murine germ-line C gamma 1 gene, contains promoter and enhancer elements responsible for basal level transcription and inducibility by anti-Ig phorbol myristate acetate (PMA) and for synergy of these inducers with IL-4 in a surface IgM+ B cell line, L10A6.2 and a surface IgG2a+ B cell line, A20.3. Linker-scanning mutations demonstrated that multiple interdependent elements are required for inducibility by PMA and also for synergy with IL-4. Within the 150 bp region are several consensus sequences that bind known or putative transcription factors, including a C/EBP binding site--IL-4 responsive element, four CACCC boxes, a PU box, a TGF beta inhibitory element (TIE), an alpha beta-interferon response element (alpha beta-IRE) and an AP-3 site. The relationship between transcription regulated by these elements and the regulation of endogenous germ-line gamma 1 transcripts and switching to IgG1 is discussed.

Animals↗
PubMed1991

Effect of cytokines on switching to IgA and alpha germline transcripts in the B lymphoma I.29 mu. Transforming growth factor-beta activates transcription of the unrearranged C alpha gene.

H chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region that will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and whether the regulation of germline transcripts correlates with the regulation of switching, we are studying this process in the murine B lymphoma cell line I.29 mu, which switches, after treatment with bacterial LPS, primarily to IgA and less frequently to IgE. Levels of alpha germline transcripts initiating upstream of alpha switch (S alpha) sequences are elevated in clones of this line that switch well, compared with clones that switch less frequently. Transforming growth factor-beta (TGF-beta) has been shown to increase alpha germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B cells. We now demonstrate that TGF-beta increases LPS-induced switching to IgA by 10-fold at optimal doses and increases the level of alpha germline transcripts 5- to 9-fold in I.29 mu cells. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGF-beta appears to direct switching to IgA by inducing transcription from the unrearranged S alpha-C alpha DNA segment. Germline alpha RNA is quite stable in I.29 mu cells, having a half-life of about 5 h, and we find no evidence for further stabilization in the presence of TGF-beta. Levels of epsilon germline transcripts are decreased by TGF-beta treatment. IL-4, which modestly increases switching in I.29 mu cells, slightly increases transcription of alpha germline RNA. IFN-gamma, which reduces switching to IgA in these cells, also reduces the level of alpha germline transcripts. IFN-gamma also reduces the level of epsilon germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines regulates the specificity of recombination. We also present evidence that IL-4 may provide other signals, distinct from transcriptional targeting, that increase LPS-induced switching to IgA.

Animals↗
PubMed1990

Induction of germ-line immunoglobulin heavy chain transcripts by mitogens and interleukins prior to switch recombination.

It has recently been postulated that immunoglobulin class switching is preceded by transcription from unrearranged heavy chain genes. In this report, we have investigated the conditions under which RNA transcribed from unrearranged C gamma 3, C gamma 1, C gamma 2b, C gamma 2a, C epsilon and C alpha genes are induced in normal spleen cells by mitogens and/or interleukin (IL) 4, IL 5 and interferon-gamma. Lipopolysaccharide (LPS) plus IL 4 induced germ-line gamma 1 and epsilon transcripts. LPS induced gamma 2b and gamma 3 transcripts and high doses of IL 4 suppressed these LPS-induced transcripts. Interferon-gamma induced low levels of germ-line gamma 2a transcripts and profoundly suppressed the gamma 1 and epsilon transcripts induced by LPS and IL 4. IL 5 alone or in combination with IL 4 and/or LPS did not induce germ-line alpha transcripts. Spleen cells of the partially immunodeficient mice CBA/N and C3H/HeJ, which do not express IgG3 could be induced, however, by polyclonal activators to express germ-line gamma 3 and gamma 2b transcripts. The data indicate that the capacity of a ligand to induce/suppress transcription of a particular unrearranged heavy chain gene is a good indicator of its capacity to induce switching to the corresponding Ig isotype. However, it is also clear that control of switching can be carried out at other levels.

Animals↗
PubMed1990

Structure of germ line immunoglobulin alpha heavy-chain RNA and its location on polysomes.

We describe the structure of the major germ line RNA transcribed from unrearranged immunoglobulin alpha heavy-chain genes in immunoglobulin M-expressing cells of the I.29 mu B-cell lymphoma, a cell line capable of switching to immunoglobulin A expression upon lipopolysaccharide treatment. This germ line alpha RNA has a small open reading frame that does not include the C alpha domain, and this RNA appears to be present on polysomes in I.29 mu cells.

Animals↗
PubMed1990

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.

Animals↗
PubMed1990

Structure of germline immunoglobulin heavy-chain gamma 1 transcripts in interleukin 4 treated mouse spleen cells.

Antibody class switching is mediated by a DNA recombination event that replaces the C mu gene with one of the other heavy (H) chain constant region (CH) genes located 3' to the C mu gene. The regulation of this process is essential to the immune response because different CH regions provide different biological functions. Correlative evidence indicates that the isotype (class) specificity of the switch is determined by the accessibility of specific CH genes as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germline CH genes is unknown. In this report, we describe the structure of RNA transcribed from unrearranged gamma 1 genes in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4. The germline gamma 1 RNA is initiated at multiple start sites 5' to the tandem repeats of the gamma 1 switch (S gamma 1) region. As is true for analogous RNAs transcribed from unrearranged gamma 2b and alpha genes, the germline gamma 1 RNA has an I exon transcribed from the region 5' to S gamma 1 sequences, which is spliced at a unique site to the C gamma gene. The germline gamma 1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acid in length.

Amino Acid Sequence↗
PubMed1989

Mouse antibody response to group A streptococcal carbohydrate.

In an attempt to more fully understand the generation of antibody diversity to carbohydrate (CHO) Ag, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal CHO from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein Ag). We have analyzed the use of H and L chain V region genes in the early (day 7) and late response (hyperimmune) and have sequenced the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results indicate that the level of somatic mutation in the hyperimmune response is generally low in comparison with the response to haptens and protein Ag. These data also suggest that there is a positive selection for mutation in CDR 1 during the hyperimmune response to group A streptococcal CHO.

Amino Acid Sequence↗
PubMed1989

Reticulum cell sarcomas of SJL mice have rearranged immunoglobulin heavy and light chain genes.

The immunoglobulin (Ig) heavy (H) and light (L) chain gene rearrangements of the high incidence SJL lymphomas (reticulum cell sarcoma, RCS) have been analyzed. Both primary and transplanted RCS show rearrangements of H and kappa L chains, demonstrating that these tumors are of B cell origin. These data are consistent with previous results indicating that these tumors are a mouse model for follicular lymphoma. A long-term transplanted line and the in vitro line derived from it, cRCS-X, have a single rearranged JH-C gamma 2a fragment and one rearranged C alpha gene fragment which does not hybridize with a probe for the JH gene segments. These cell lines also have two rearranged J kappa-C kappa fragments. Primary tumors and early passages are more heterogeneous with respect to Ig gene rearrangements, possibly because more than one B cell clone is present. Although no synthesis of IgG2a, or of any Ig, could be detected by the in vitro cRCS-X cells, these cells contain abundant poly(A)+ RNA that hybridize with gamma 2a and kappa probes as well as lesser amounts of alpha and epsilon RNA. None of these H chain RNA hybridized with probes for the JH gene segments. The epsilon and alpha RNA are the same size as transcripts of germ-line CH genes which have been identified in other systems. However, the gamma 2a RNA are smaller than previously described germ-line C gamma 2a RNA and appear to be transcribed from aberrantly rearranged JH-C gamma 2a genes.

Animals↗
PubMed1989

A B-cell-specific nuclear protein that binds to DNA sites 5' to immunoglobulin S alpha tandem repeats is regulated during differentiation.

Immunoglobulin heavy-chain switching is effected by recombination events between sites associated with tandemly repeated switch sequences located 5' to immunoglobulin heavy-chain genes. Using the band mobility shift assay, we have identified two distinct sites 5' to the alpha heavy-chain switch sequence with affinity for a single B-cell-specific DNA-binding protein, S alpha-BP. S alpha-BP was present in nuclear extracts from pre-B and B cells but was not detected in extracts from plasmacytomas, B-cell hybridomas, T-cell lymphomas, or a macrophage cell line. It was also not detectable in other nonlymphoid cells tested. Evidence suggests there are S alpha-BP-binding sites near other immunoglobulin switch sequences. As with the S alpha sites, these sites appear to be distinct from the consensus tandem repeats characteristic of immunoglobulin switch sequences. The possible functions of S alpha-BP on contacting its binding sites are discussed in the context of immunoglobulin heavy-chain switch recombination.

Animals↗
PubMed1989

Mutations, duplication, and deletion of recombined switch regions suggest a role for DNA replication in the immunoglobulin heavy-chain switch.

The heavy-chain switch from immunoglobulin M (IgM) expression to IgA expression is mediated by a recombination event between segments of DNA called switch regions. The switch regions lie two to six kilobases upstream of the mu and alpha constant region coding segments. Switch recombination to IgA expression results in a recombinant mu-alpha switch region upstream of the expressed alpha constant region gene. We have characterized the products of switch recombination by a lymphoma cell line, I.29. Two sets of molecular clones represent the expected products of simple mu to alpha switches. Five members of a third set of molecular clones share the same recombination site in both the mu and the alpha switch regions, implying that the five molecular clones were derived from a single switch recombination event. Surprisingly, the five clones fall into two sets of sequences, which differ from each other by several point mutations and small deletions. Duplication of switch region sequences are also found in these five molecular clones. An explanation for these data is that switch recombination involves DNA synthesis, which results in nucleotide substitutions, small deletions, and duplications.

Animals↗
PubMed1988

I.29 lymphoma cells express a nonmutated VH gene before and after H chain switch.

The I.29 B cell lymphoma consists of IgM+ and IgA+ cells which express the same germ-line VH gene. IgA+ cells of the I.29 lymphoma were derived from the IgM+ cells by a typical H chain switch recombination event. The IgM+ cells can be induced with LPS to undergo H chain switching in culture. It has been proposed that the somatic hypermutation process is activated during H chain switch, since V genes expressed in IgG+ and IgA+ cells have more frequently undergone mutation than those expressed in IgM+ cells. We have investigated this question by sequencing VH genes expressed before and after H chain switch in the I.29 lymphoma. We have also sequenced the germ-line VH gene corresponding to the gene expressed by I.29 cells to determine whether the VH gene expressed in the IgM+ cells had already undergone somatic mutation. Our results indicate that somatic mutation was not activated in the precursor cell for the I.29 lymphoma, nor during isotype switch in I.29 cells. It is possible that cells of the I.29 lymphoma, or their precursor, have not received the signal which induces somatic mutation, or that I.29 cells belong to a subset of B cells that cannot be induced to undergo any (or much) somatic mutation.

Amino Acid Sequence↗
PubMed1988

The J kappa proximal region of the human K locus contains three uncommon V kappa genes which are arranged in opposite transcriptional polarities.

The structure of one of the V kappa gene-containing regions of the locus coding for the human immunoglobulin light chains of the kappa type is described. This so-called B region contains three genes: B1, B2 and B3. According to its sequence B1 is a pseudogene which does not fit well into the present subgroup classification. In lymphoid cell lines the B1 gene region is frequently deleted. B2 and B3 are the previously reported EV15 and V kappa IV genes. The transcriptional polarity of the B1 gene is found to be opposite to one of the B2 and B3 genes. This observation together with the fact that the B region is proximal to the J kappa C kappa gene segment leads to the conclusions to the mechanism of the V kappa-J kappa recombination and allows us to explain the formation of the recombination products in a particular cell line by two consecutive inversions.

Base Sequence↗