Biomedical subjects

B K Birshtein

Publications and source records attributed to B K Birshtein.

At least 37 records · Page 2Linked to original sources

PubMed1991

DNA sequences 3' of the Ig H chain cluster rearrange in mouse B cell lines.

A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to study DNA rearrangements that occur at the Ig H chain locus. One variant, F5.5, has acquired both VH gene and C epsilon gene rearrangements. Through genomic Southern blot analysis initially directed to mapping the C epsilon gene rearrangement, we observed that the VH region rearrangement was linked, through an inversion event, to sequences that originate 3' of the CH cluster, i.e., 3' of the C alpha gene. Subsequent studies have shown that DNA rearrangements within the region 3' of the C alpha gene are detected in several other mouse myeloma and hybridoma cell lines and are not associated with the expression of specific isotypes.

Animals↗

PubMed1990

Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange.

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' regions of the C gamma 2a and C gamma 2b heavy chain genes results in duplication of the C gamma 2a heavy chain gene and generation of a novel recombination joint. The USCE occurs between (TC)n tracts adjacent to alternating purine-pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical probing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influence of negative supercoiling the alternating purine-pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine.oligopyrimidine (pur.pyr) regions of these regions can adopt intramolecular triplexes at low pH (less than or equal to pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z-DNA segments adjacent to pur.pyr tract prevents intramolecular triplex formation. In summary, we conclude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC)n tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z-DNA at a site of USCE suggests that formation of this structure may enhance recombination between adjacent pur.pyr tracts.

Animals↗

PubMed1990

Intravascular metabolism of normal and mutant mouse immunoglobulin molecules.

The metabolism of IgG immunoglobulins in the body is tightly regulated in order to maintain their intravascular concentration. Different subclasses may have different intravascular half-lives, and in the mouse, passively administered IgG2b disappears from the circulation more rapidly than IgG2a. We have attempted to localize the sequences in the constant region which are responsible for this difference by examining the intravascular metabolism of mutant immunoglobulins that were generated in tissue culture and have undergone deletions of individual constant region domains or contain different combinations of gamma 2b and gamma 2a CH2 and CH3 domains. Our results suggest that the regulation of intravascular metabolism is complex but indicate that sequences in the CH3 domain are important in determining the different intravascular half-lives of IgG2b and IgG2a antibodies in the mouse.

Animals↗

PubMed1989

Nucleotide sequence of an unequal sister chromatid exchange site in a mouse myeloma cell line.

The mouse myeloma cell line MPC 11 carries two C gamma 2a immunoglobulin heavy-chain genes on the expressed chromosome, a duplication shown to have occurred through unequal sister chromatid exchange (USCE). In the present report, we present the nucleotide sequence of the USCE joint and show that both breaks occurred within tracts of repeated TC dinucleotides. Additional TC dinucleotide tracts and two oligonucleotide segments (N sequences) were inserted at the USCE site.

Animals↗

PubMed1988

DNA rearrangements affecting both variable and constant regions of Ig H chain genes in MPC11 mouse myeloma variants.

We have examined the mechanisms that account for short Ig H chain production in two variants of the mouse myeloma cell line MPC11 (IgG2b, kappa) by mRNA sequencing and restriction enzyme mapping. One variant, F5.5, has a thymidine residue inserted into the (CH3) domain, of the Ig H chain, resulting in premature termination and translation of a gamma 2b H chain of 50,000 m.w. A second variant, E5.7A12, contains gamma 2a-derived sequences that extend from near the 3' end of the CH2 domain to the intervening sequence between the CH2 and CH3 domains, consistent with a microrecombination event (defined as either a double cross-over or gene conversion event). In this variant, the 5' end of the CH3 domain has been deleted, but the remainder of the gamma 2b(CH3) domain is present, resulting in the translation of a gamma 2b-gamma 2a-gamma 2b H chain of 52,000 m.w. Additional rearrangements affecting sequences in or adjacent to the variable region accompany H chain constant region alterations in these cell lines and subclones of these cell lines. In F5.5, novel sequences have recombined within one of two duplicated copies of the VH gene. In a sister clone of E5.7A12 that has ceased H chain production (E5.7A14), new sequences have recombined within 300 bp 5' of the enhancer element. Both F5.5 and E5.7A12, like their immediate unstable precursor cells, fail to assemble H-H dimers, halting the Ig assembly process at the heavy-light stage, and do not secrete H chains. We speculate that defects in H chain assembly and secretion, as exemplified by the parents of these variants (i.e., intermediates of these secondary variants), reactivate the Ig gene rearrangement machinery and result in the formation of these putatively equally unstable secondary variants.

Amino Acid Sequence↗

PubMed1988

Sites of switch recombination in IgG2b- and IgG2a-producing hybridomas.

The technique of genomic Southern blot analysis was employed to map the sites of class switch rearrangement in five hybridoma cell lines, four of which produce gamma 2b, whereas the fifth secretes gamma 2a. Two of the class switch recombinations we have mapped represent secondary switches to gamma 2b with a gamma 1 intermediate. We also observe isotype specificity in class switch recombination, with both allele rearranging to the same isotype in the three lines where an unexpressed chromosome is retained. Every 5' break occurred outside of and immediately 5' to the tandem repeats associated with Cmu, a result consistent with earlier studies on myelomas but in contrast to the pattern seen in lymphomas. Seven of the eight 3' switch sites, however, were within tandem repeats. This disparity in the distribution of 5' and 3' break points may reflect a unique role for the mu-associated tandem repeats in the switch process.

Genes, Immunoglobulin↗

PubMed1988

Site of unequal sister chromatid exchange contains a potential Z-DNA-forming tract.

Previous studies from this laboratory have provided evidence that the duplication of the IgG2a heavy chain constant region gene (C gamma 2a) in the murine myeloma cell line MPC-11 occurred via unequal sister chromatid exchange. We now report the determination of the DNA sequences of the germ-line regions in which this exchange has occurred. The two donor sequences have long regions of virtual identity. Furthermore, both chromatids contain stretches of T-C and T-G dinucleotides; the latter has the potential to form Z-DNA. Localization of the actual breakpoints via genomic Southern blot analysis suggests a novel mechanism for the recombination and strongly implicates the simple sequence in the process.

Animals↗

PubMed1988

Mechanism of class switching.

We have documented the action of unequal sister chromatid exchange on antibody genes, and our studies provide the first molecular description of an unequal sister chromatid exchange event. We delineated the sequences that mediate class switch recombination in normal B cells through genomic Southern analysis of five IgG2-producing hybridomas. Successive subclass switching was documented in two of eight maps and may possibly account for interposed DNA in three additional maps, implying that subclass switching may be a relatively frequent occurrence in normal B cells. As in most other myelomas and hybridomas, the 5' breakpoint of switch recombination in our panel of hybridomas falls 5' to Cmu-associated tandem repeats, whereas the 3' breakpoint falls within C gamma-associated tandem repeats. We consider various hypotheses in the text, the most interesting of which is that deletion of Cmu-associated tandem repeats is a necessary first step before class switch recombination in mature B cells. This hypothesis suggests that Cmu-associated tandem repeats may be a separately regulated DNA element.

Animals↗

PubMed1987

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice.

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.

Animals↗

PubMed1987

Absence of somatic mutation in the variable region of MPC 11 variants expressing a different heavy chain isotype.

Somatic mutation of immunoglobulin variable regions contributes to the diversity of the immune response. Some investigators have postulated that somatic mutation is coupled to isotype switching; others have presented evidence that the two processes are not linked. By using in vitro variants of the MPC 11 mouse myeloma cell line that produce heavy chains of different isotypes, we have examined several VH regions at the nucleotide level using a variety of different sequencing procedures. We have found no evidence of somatic mutation and conclude that the processes of somatic mutation and isotype switching may be independent.

Animals↗

PubMed1986

T cell clones specific for IgG2a of the a allotype: direct evidence for presentation of endogenous antigen.

T cell clones specific for IgG2a of the alpha allotype have been isolated from C57BL/6J mice. Antigenic determinants recognized by these clones were localized by using a panel of hybrid IgG2b-IgG2a myeloma proteins. These experiments provide evidence for two distinct antigenic sites, one located in a segment encompassing the hinge region and most of the CH2 domain, and the other in a segment spanning the CH3 domain and the C-terminal eight residues of the CH2 domain. As judged by their failure to respond in the presence of B6.C-H-2bm12 spleen cells, all the clones recognize determinants created, in part, by the I-A beta chain. A strong proliferative response was observed in the presence of spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP, and BALB.B. Experiments testing reactivity directed toward spleen cells from appropriate allotype-congenic mouse strains demonstrated that this response was controlled by Igh-linked genes. These results clearly indicated 1) that Igh-1a-specific T cell clones are stimulated by endogenously synthesized IgG2a, and 2) these T cells recognize shared determinants expressed on IgG2a molecules of various strains. These experiments thus provide strong evidence for presentation of self antigens under normal physiological conditions.

Animals↗

PubMed1985

Unequal sister chromatid exchange. A mechanism affecting Ig gene arrangement and expression.

Two gamma 2a gene forms in the MPC11 mouse myeloma cell line (gamma 2b, K) have been localized to the expressed H chain chromosome, where they exist in tandem downstream of the expressed gamma 2b gene. The gamma 2a gene duplication has apparently occurred by spontaneous, unequal sister chromatid exchange (SCE) in a B lymphoid precursor of MPC11 or in MPC11 itself. It is especially significant that either of the tandem gamma 2a gene forms may be used in the class switch from gamma 2b to gamma 2a production occurring in the MPC11 line, indicating that heavy chain constant region (CH) gene duplication (or deletion) may be a mechanism normally impinging on CH gene expression. The gamma 2a genes of MPC11 undergo further copy number variation via unequal SCE in variants of this line derived by mutagenesis and selection for altered H chain production. The remarkable frequency with which copy number variation occurs in these cells suggests that unequal SCE may play a physiological role in CH gene arrangement and expression.

Animals↗

PubMed1985

Independent immunoglobulin class-switch events occurring in a single myeloma cell line.

Five gamma 2a-producing cell lines, all derived from the gamma 2b-producing mouse myeloma MPC11, were analyzed for changes in gene structure. Also examined was a cell line (ICR9.7.1) that acts as an intermediate to some of these class switches. All six of the MPC11 variants have undergone DNA rearrangement. Rearrangement sites in the gamma 2a-producing cells are different in each case and generally do not involve tandem repeat sequences. An enhancer for heavy chain gene transcription was deleted in at least one of these cell lines as a result of its class-switch rearrangement. DNA sequence analysis of cloned genes from two of the MPC11 variants revealed shared sequences at their rearrangement breakpoints. The same sequences are found at the breakpoints of two additional immunoglobulin gene rearrangements in MPC11.

Animals↗

PubMed1985

Site of binding of IgG2b and IgG2a by mouse macrophage Fc receptors by using cyanogen bromide fragments.

Cyanogen bromide fragments of murine IgG2b and IgG2a immunoglobulins were used to localize the sequences that are bound by specific IgG2b and IgG2a Fc receptors on murine macrophages. One fragment from the CH2 domain of IgG2b bound to the gamma 2b Fc receptor. Two fragments from IgG2a--one one from the CH2 domain, differing by only four amino acids from the homologous IgG2b fragment, and the other from the CH3 domain--specifically bound to the gamma 2a Fc receptor. In both a rosetting assay and a radioactive binding assay, these two fragments from IgG2a competed with intact IgG2a: however, they did not compete with each other. Rather, binding of the fragment from the CH3 domain of IgG2a augmented the binding of the fragment from the CH2 domain of IgG2a but not that of the homologous fragment from IgG2b. The binding of both IgG2a fragments was abolished by trypsin treatment of macrophages. These data suggest that 1) a sequence in the CH2 domain of IgG2b is sufficient for binding to the gamma 2b Fc receptor, 2) sequences from both the CH2 and CH3 domains of IgG2a bind to the gamma 2a Fc receptor, and 3) the binding of sequences from the CH3 domain of IgG2a may induce a conformational change in the gamma 2a Fc receptor that leads to enhanced binding of sequences from the CH2 domain.

Animals↗

PubMed1985

Structural requirements of immunoglobulin G for binding to the Fc gamma receptors of the human tumor cell lines U937, HL-60, ML-1, and K562.

Fc receptors (FcR) on U937, HL-60, ML-1, and K562 cells were characterized by using competitive binding assays and EA rosette inhibition studies. Like human monocytes, U937, HL-60, and ML-1 cells bound monomeric human IgG Fc with high affinity and mildly reduced and alkylated human IgG and Fc with a somewhat diminished affinity. In contrast, K562 cells had a much lower affinity for monomeric human IgG or Fc. Concentrations of these proteins as high as 10(-5) M were needed to completely block EA rosette formation. There was no binding of reduced and alkylated IgG and Fc. We assessed the influence of various segments of IgG on FcR interactions by using human pFc', rabbit Facb, mouse IgG2b-IgG2a hybrid Ig, and also studied the effect of reduction of the interchain disulfide bonds. The FcR on all four cell types bound rabbit IgG but not rabbit Facb or human pFc', suggesting that rabbit C gamma 3 domains are required for FcR interaction and that isolated human C gamma 3 domains do not have a human FcR binding site. Murine IgG2a, but not IgG2b, was cytophilic for U937, HL-60, and ML-1 cells. Binding studies with the use of several mouse myeloma variant Ig molecules having hybrid gamma 2b-gamma 2a heavy chains showed that variants with a complete gamma 2a Fc region bound to these FcR-like IgG2a, whereas those having gamma 2a sequences only in the C gamma 3 region and in a short adjacent segment of the C gamma 2 region behaved like IgG2b and did not bind. These results suggested that additional murine gamma 2a sequences are required for FcR binding. Interestingly, the Fc fragments from murine proteins with a complete gamma 2a Fc region bound only to a limited extent. These fragments are shorter than the human IgG1 Fc fragments, and they lack that segment of the hinge region that includes the interchain disulfide bonds. Cleavage of the interchain disulfide bonds of murine Ig having a complete gamma 2a Fc region diminished binding to a similar extent as that observed with human IgG. Together, these findings provide additional insight into the roles of the hinge, C gamma 2, and C gamma 3 regions of human, rabbit, and mouse IgG in their interaction with the FcR of human tumor cells.

Animals↗

PubMed1984

Products of a reciprocal chromosome translocation involving the c-myc gene in a murine plasmacytoma.

The structures of the rearranged c-myc gene products derived from a rcp T(12;15) have been investigated in the MPC-11 (IgG2b kappa) plasmacytoma. The rcp T(12;15) in MPC-11 is a reciprocal exchange between the c-myc gene on chromosome 15 and the immunoglobulin gamma 2a switch region (S gamma 2a) on chromosome 12. The c-myc gene is broken within a 5'-nontranslated exon, thereby separating the promoter region of the normal c-myc gene from its protein coding sequences. This reciprocal rearrangement results in the loss of 11 base pairs of c-myc sequence and 300 base pairs of S gamma 2a sequence at the point of recombination. Sequences that represent the promoter region of the normal c-myc gene are present in the 5'-myc reciprocal fragment. A comparison of the nucleotide sequences at the recombination site of a number of c-myc rearrangements reveals a common feature that may have mechanistic importance for these translocation events.

Animals↗

PubMed1984

Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly.

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.

Amino Acid Sequence↗

PubMed1984

Loss of a consensus splice signal in a mutant immunoglobulin gene eliminates the CH1 domain exon from the mRNA.

A series of mouse myeloma mutants, derived from a cell line of the murine MPC-11 tumor (gamma 2b, kappa), resemble human heavy-chain disease in their loss of an internal domain (exon). In these mutants, most of the gamma 2b CH1 exon was present in the nuclear RNA but was removed during splicing to form the mature cytoplasmic RNA. Amino acid sequence studies of one mutant (10.1) are consistent with the loss of the complete CH1 domain. A second mutant cell line (I17) derived from 10.1 and containing the same CH1 alteration was shown by S1 nuclease protection experiments to have an additional mRNA deletion spanning the CH2-CH3 domain boundary. This second deletion was shown to result from a genomic alteration that provided a marker for the isolation of the expressed H-chain allele. To determine the basis of the CH1 splicing defect, the 117 genome-expressed gamma 2b constant region DNA was cloned. Sequence studies showed a deletion of 99 nucleotides around the 3' end of the CH1 domain, which removed the splice site and flanking DNA, apparently causing the aberrant splicing of the RNA transcript. The sequence deleted in the mutant is flanked by short repeats of the octameric sequence CCAGCCAG in the wild-type gene. In the mutant, one copy of the repeat, in addition to the sequences between the repeats, has been lost.

Amino Acid Sequence↗