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Biomedical subjects

R M Gerstein

Publications and source records attributed to R M Gerstein.

12 recordsLinked to original sources
PubMed2001

Splenic and peritoneal B-1 cells differ in terms of transcriptional and proliferative features that separate peritoneal B-1 from splenic B-2 cells.

B-1 cells constitute a distinct B cell subset with characteristic phenotypic and functional features. B-1 cells are highly represented among peritoneal lymphocytes; substantial numbers of B-1 cells are also located within splenic tissue. Here a number of differences in transcription factor and gene expression were identified that separate peritoneal B-1 and splenic B-2 cells, and then splenic B-1 cells obtained from immunoglobulin transgenic mice were tested for these parameters. Splenic B-1 cells resembled splenic B-2 cells rather than peritoneal B-1 cells in terms of nuclear expression of DNA-binding STAT3, CREB, and PU.1, with respect to transcriptional activation of IL-10, and in the failure to enter cell cycle in response to PMA. Splenic B-1 cells (B-1S) appear to constitute a unique population of B-1 cells, which, while sharing with peritoneal B-1 cells (B-1P) certain phenotypic features, differ from them in transcription factor and gene expression and in signaling for cell cycle progression.

Animals↗
PubMed1999

The role of cytokine receptor signaling in lymphocyte development.

Cytokine receptor signaling plays an essential role in the early stages of lymphocyte development. Signals through various cytokine receptors - such as c-kit, flt3/flk2, CXCR4, the IL-7 receptor and the IL-15 receptor - are known to promote the expansion and survival of uncommitted progenitor cells as well as their migration to the appropriate microenvironment and subsequent differentiation into B, T or natural killer cells. The recent generation of mice deficient in one or more of these signaling pathways has revealed which cytokines play unique and/or redundant roles in each of these lymphocyte lineages during this developmental process.

Animals↗
PubMed1998

Pairs of violet-light-excited fluorochromes for flow cytometric analysis.

We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new fluorochrome that we have synthesized and is used for the first time in the present study. The two emission classes are sufficiently different that Cascade Blue can be paired with Cascade Yellow, Lucifer Yellow, or GFP-Vex in flow cytometric analysis. Furthermore, with proper detection filters, these fluorochromes can be combined with all of the currently used fluorochromes in a three-laser FACS system. With these data, the total number of fluorochromes that can be used as antibody labels for simultaneous detection in combined FACS analysis increases to nine. This study demonstrates the sensitivity and power of the combined use of these reagents in a single eight-color analysis by identifying murine T-lymphocyte subsets that could not otherwise be readily distinguished.

Animals↗
PubMed1997

Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.

Animals↗
PubMed1995

Defective B cell development and function in Btk-deficient mice.

Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1993

Extent to which homology can constrain coding exon junctional diversity in V(D)J recombination.

Among site-directed DNA recombination systems, V(D)J recombination is noteworthy in that identical reactants yield different recombination products at the junction of joined segments. This variation is the basis for diversity at the base of antigen receptor binding pockets and corresponds to V-(D)-J DNA junctions. An abundance of certain junctions has been noted. It has been proposed that these junctions are favoured because they occur where short regions of homology in participating coding ends might align preferentially. Here we use a system that is entirely free from cellular selection to show that the diversity of coding joints can be severely restricted when the coding ends participating in the reaction have short regions of homology. This constraint on diversity is diminished but not eliminated by terminal deoxynucleotidyl transferase, a mechanistic feature that has implications for the establishment of the immune repertoire.

Animals↗
PubMed1993

Somatic hypermutation of an immunoglobulin mu heavy chain transgene.

We have analyzed somatic hypermutation of an immunoglobulin (Ig) heavy chain transgene. Hybridomas expressing the transgene were produced from immunized transgenic mice and transgene copies were sequenced to assay for mutation. In two IgM-producing hybridomas, as well as in several IgG-producing hybridomas, mutations were found in the VDJ region of the transgene. In the IgM-producing hybridomas, both mutated and unmutated transgene copies were present and expressed as mRNA. Several mutated transgene copies were present in a single cell and these showed different patterns of mutation. Two IgG-producing hybridomas isolated from a single animal also showed a hierarchical pattern of mutation indicating that transgene mutations can accumulate during B cell proliferation, similar to the mutational process for endogenous antibody genes. Among hybridomas that expressed both IgG and IgM molecules derived from the transgene, the isotype-switched gamma transgene copy exhibited a higher level of mutation than the mu transgene copies. Our results indicate that the 15-kb ARSmu transgene contains all the sequence information required to target the Ig-specific hypermutational machinery, and raise the possibility that sequences associated with the endogenous CH locus might enhance somatic mutation.

Amino Acid Sequence↗
PubMed1993

Coding end sequence can markedly affect the initiation of V(D)J recombination.

In V(D)J recombination, two site-specific cuts are made adjacent to V, D, and J subexons to create four DNA ends, two of which (the coding ends) are joined to generate the exon that encodes the variable domain of the antigen receptor. Although deviations from consensus signal sequences have been reported previously to have a large impact on the efficiency of V(D)J recombination, coding end sequence has been assumed to be neutral with respect to the efficiency of recombination. We have used extrachromosomal V(D)J recombination substrates to undertake a systematic comparison of coding end sequences. Substrates were constructed that contain identical consensus recombination signal sequences, where only the coding ends were varied. Surprisingly, we found that nucleotide sequence at the coding end can affect the efficiency of V(D)J recombination > 250-fold. Variable initiation of recombination appears to account for most of the effect. This finding has mechanistic implications because it indicates that signal-binding proteins involved in V(D)J recombination may have different levels of activity when confronted with coding ends of different sequence. Our results also indicate that coding end sequence must be considered to be among the major factors that shape the antigen receptor repertoire.

Animals↗
PubMed1990

Isotype switching of an immunoglobulin heavy chain transgene occurs by DNA recombination between different chromosomes.

Transgenic mice carrying an immunoglobulin mu heavy chain transgene exhibit isotype switching of the transgene. We have now characterized the mechanism of transgene switching in these mice. The site of mu transgene insertion in one transgenic line has been localized to chromosome 5 using a series of polymorphic endogenous retroviruses as genetic markers in backcross mice. The endogenous immunoglobulin heavy chain locus resides on mouse chromosome 12, which shows that transgene isotype switching can occur between two different chromosomes even though normal antibody gene switching has generally been thought to occur within one chromosome. We find that transgene isotype switching involves interchromosomal DNA recombination, and our data suggest that the same enzymatic mechanisms mediate both normal isotype switch recombination and interchromosomal transgene switching. Our findings also support the notion that the isotype switching mechanism can induce chromosomal translocations such as observed for the c-myc gene in some B cell tumors.

Animals↗
PubMed1989

Quantitative analysis of idiotypic mimicry and allelic exclusion in mice with a mu Ig transgene.

Analysis of C57BL/6 mice (IgM allotype, Igh-6b or mu b) that carry an Ig H chain transgene of a different allotype (mu a) shows that IgM molecules of mixed allotype (mu a mu b) are present among serum antibodies. The finding was extended to hybridomas prepared from nonimmune transgenic mice, many of which also failed to exhibit allelic exclusion. The proportions of mu a and mu b secreted by individual hybridomas varied markedly, and the product of an individual hybridoma was found to be heterogeneous with respect to the allotype content of individual molecules. The ratio of mu a:mu b chains secreted by individual hybridomas was found to correlate with the number of transgene copies remaining in each hybridoma, and several hybridomas that secrete only mu b-positive molecules had apparently lost all but one copy of the transgene. An idiotype characteristic of the transgene was found to be present only in association with the transgenic (mu a) allotype, and indirect evidence strongly suggests that the idiotype was present only on mu a polypeptide chains. Thus, there is no evidence in this system for the induction of idiotypically cross-reactive endogenous molecules.

Alleles↗
PubMed1989

Isotype switching by a microinjected mu immunoglobulin heavy chain gene in transgenic mice.

Immunization of transgenic mice carrying an immunoglobulin mu heavy chain resulted in a response dominated by expression of the transgene variable region. Unexpectedly, in a large proportion of the antibody produced by immunized mice, the transgene variable region was associated with IgG rather than IgM. This demonstrates that the transgene can undergo an isotype switch. Four transgenic founder lines all exhibited transgene isotype switching despite the likelihood of random chromosomal integration of the transgene. In addition one of the lines was analyzed by breeding studies and the transgene was found to be genetically unlinked to the immunoglobulin heavy chain (Igh) locus. These results indicate that a precise chromosomal location is not required for isotype switching and suggest the possibility that the isotype switching process can occur interchromosomally.

Animals↗