Papers by Barbara Birshtein
Birth defects original article series
Society of General Physiologists series
The Journal of Immunology
We report here the primary structure of an immunoglobulin heavy chain synthesized by ICR 16, a va... more We report here the primary structure of an immunoglobulin heavy chain synthesized by ICR 16, a variant of the MPC 11 mouse myeloma cell line. The ICR 16 heavy chain is a gamma 2b-gamma 2a hybrid, consisting of the CH1 domain of gamma 2b and the hinge, CH2 and CH3 domains of gamma 2a subclasses. The genetic mechanism by which ICR 16 occurred may be recombination, based on homologies in both coding and intervening sequences in gamma 2b and gamma 2a constant region genes. Although the Fc fragment of ICR 16 is completely gamma 2a-like and has been shown to bind to gamma 2a Fc receptors on mouse macrophages, the intact H2L2 molecules is unable to do so. Such an observation underscores the crucial role that conformation may play in the ability of immunoglobulins to carry out biologic functions.
Journal of Experimental Medicine
The Journal of Immunology
A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobu... more A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.
The Journal of Immunology
Fc receptors (FcR) on U937, HL-60, ML-1, and K562 cells were characterized by using competitive b... more 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.
The Journal of Immunology
ABSTRACT
The Journal of Immunology
We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nucle... more We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.
Nucleic Acids Research
In addition to E,u, several elements downstream of the IgH cluster, i.e. 3' of the Ca gene, are i... more In addition to E,u, several elements downstream of the IgH cluster, i.e. 3' of the Ca gene, are involved in regulating IgH gene rearrangement and expression. This entire downstream regulatory region was shown to be deleted in the mutant myeloma cell line, LP1.2. The deletion encompasses -34 kb and is presumably responsible for the reduced levels of IgH expression in this cell line. An additional regulatory element, included in the LP1.2 deletion, was identified by investigation of a DNase I hypersensitivity site located -33 kb downstream of the a gene and present in pre-B and plasma cells. This novel IgH gene enhancer element, termed 3'a-hs4, Is capable of activity throughout B cell development. Transient transfection of 3'a-hs4 in a CAT reporter gene construct shows transcriptional enhancement activity approximating that of E,u in S194 plasmacytoma and M12.4.1 and A-20 B cell lines; while in a pre-B cell line, 18-81, the average activity is 25% that of El. Enhancer activity was localized to an 800 bp fragment. The activity of 3'a-hs4 is orientation independent and appears to be B cell specific. Tight regulation of 3'a-hs4 is inferred from its variable activity in different plasmacytoma cell lines and within the pre B cell line, 18-81.
The Journal of Immunology
A mouse myeloma cell line MPC11 (IgG2b, kappa) and variants derived from it have been used to stu... more 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.
The Journal of Immunology
ABSTRACT
The Journal of Immunology
Somatic mutation of immunoglobulin variable regions contributes to the diversity of the immune re... more 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.
Journal of Biological Chemistry
An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' ... more 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 Journal of Immunology
Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived fr... more 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 assigne...
The Journal of Immunology
The technique of genomic Southern blot analysis was employed to map the sites of class switch rea... more 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.
Molecular and Cellular Biology
We have identified a nuclear factor expressed in pro-B-, pre-B-, and B-cell lines that binds to t... more We have identified a nuclear factor expressed in pro-B-, pre-B-, and B-cell lines that binds to two sites within the murine immunoglobulin heavy-chain (IgH) 3' alpha enhancer (3' alpha E). These sites were defined by oligonucleotide competition in an electrophoretic mobility shift assay (EMSA) and methylation interference footprinting. The 3' alpha E-binding factor is indistinguishable from NF-HB (B-lineage-specific nuclear factor that binds to the IgH gene) and the B-lineage-specific transcription factor BSAP by several criteria, including similar cell type distribution of binding activity, cross-competition of binding sites in EMSA, similar protein size as demonstrated by UV cross-linking, and sequence identity of one of the 3' alpha E-binding sites with a BSAP-binding site within the promoter of the sea urchin late histone gene H2A-2.2. These observations indicate that 3' alpha E is one of the mammalian targets for NF-HB (BSAP). Transient-transfection assays w...
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Papers by Barbara Birshtein