Diversification of bovine lambda-light chain genes.
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Animals↗
Biomedical subjects
B A Osborne
Publications and source records attributed to B A Osborne.
Cell death suffers a TKO.
The cytokine interferon-gamma (IFN-gamma), initiates both cell cycle arrest and cell death in certain cell lines. Through a novel strategy of cell transfection with episomal vectors expressing antisense cDNAs, Deiss et al. have demonstrated that it is possible to isolate genes that are required for the initiation of cell death by the cytokine IFN-gamma. This approach, referred to as TKO, for Technical Knock Out, has identified several genes whose activity appears to be essential for the induction of apoptosis by IFN-gamma in HeLa cells. Interestingly, these genes appear to mediate IFN-gamma-induced apoptosis in HeLa cells, but their inhibition by antisense does not ameliorate the antiproliferative effects of IFN-gamma in these cells. The clever strategy employed by these authors holds promise for others who wish to isolate genes required for other differentiative processes in cultured cell lines.
Antigens, Viral↗
Induction of genes during apoptosis: examples from the immune system.
The immune system provides many examples of cell populations that are susceptible to the induction of apoptosis. Self-reactive immature cells are deleted by triggering apoptosis. Additionally, mature peripheral lymphocytes are induced to undergo apoptosis, particularly when hyperactivated. In the past few years, several genes have been linked to cell death in lymphoid cells. However, only a handful of these genes has been shown to be required for cell death to occur in the immune system. This review focuses on signals known to mediate apoptosis in the immune system and those genes demonstrated to be required for the induction of cell death.
Animals↗
Molecular events in thymocyte apoptosis.
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Animals↗
Transient transfection assays to examine the requirement of putative cell death genes.
In conclusion, this chapter provides a convenient and efficient method for the detection and analysis of transiently transfected cells. Such strategies allow a fast and simple analysis of the requirement for particular genes that have been identified as being induced during apoptosis. Our experience has been that, when screening for "cell death genes," it is easy to isolate genes induced during apoptosis but far more difficult to determine the requirement for any given gene. These protocols have rendered such determinations much simpler to perform.
Antigens, CD↗
Quantitative analysis of the B cell repertoire by limiting dilution analysis and fluorescent in situ hybridization.
We have made a quantitative and concurrent analysis of B cell frequencies and VH gene family expression to study the influence of tissue type and age on the development and establishment of the primary B cell repertoire. Using LPS-mediated limiting dilution analysis and a panel of antigens we show that the newly generated B cell specificities from bone marrow get distributed without a bias to peripheral tissues such as spleen and Peyer's patches throughout the lifetime of the animal. Comparison of the B cell frequencies in animals of four different age groups (2-4 days old, 3, 12, and 18 months old) reveals that while the neonatal repertoire is comparable to that of adults, there was a selective twofold increase in the generation and distribution of B cells reactive with autologous mouse red blood cells in older mice compared to young ones. By means of a novel technique that employs fluorescent in situ hybridization and flow cytometry, we have also compared the VH gene family usage in large numbers of single B cells from these mice. Analysis of the same cell population (surface Ig+, functional B lineage cells) for expression of 7183, J558, and S107 VH families shows a preferential twofold increase in the use of VH 7183 in neonates compared to adults, while all three families show no significant difference in levels of expression during adult life or between primary and secondary lymphoid tissues. Taken together, our data indicate that specific and selective changes occur in both VH gene usage and antibody frequencies during murine ontogeny.
Age Factors↗
Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28.
CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.
Animals↗
Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77.
Engagement of the T-cell antigen receptor (TCR) on immature thymic T cells induces death by apoptosis. Although several lines of evidence indicate that apoptosis requires de novo gene expression, little is known about the molecular pathways that mediate this response. Here we show that nur77 (refs 4-7), a zinc-finger transcription factor, is expressed in response to TCR engagement in immature T cells and T-cell hybrids. Antisense inhibition of nur77 expression prevents apoptosis in TCR-stimulated cells. nur77 is also expressed in response to mitogens, but in this case transcription is regulated by 5' upstream elements that are distinct from those used for induction of apoptosis. In addition, polyadenylation is only observed on nur77 transcripts found in condemned cells. These data support a role for nur77 in cell death that may be distinct from that of activation.
Animals↗
Ced-3/ICE: evolutionarily conserved regulation of cell death.
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Animals↗
Identification of genes induced during apoptosis in T lymphocytes.
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Animals↗
Programmed cell death, apoptosis and killer genes.
A cursory examination of the literature reveals that the study of programmed cell death and apoptosis is increasing exponentially. Most contributors to this field have come either from developmental biology or immunology and view programmed cell death from different perspectives, leading both to confusion and an inability to fully appreciate the literature from other disciplines. Here, Lawrence Schwartz and Barbara Osborne define the terms and ideas relevant to the study of cell death in a way that will be accessible to investigators from all fields.
Animals↗
Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry.
We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.
Animals↗
Analysis of a novel VHS107 haplotype in CLA-2 and WSA mice. Evidence for gene conversion among IgVH genes in outbred populations.
Gene conversion has been suggested as the basis for many VH allelic differences, particularly in the murine VHS107 family. Whether conversion among IgVH genes is likely to have occurred in outbred populations has not been directly addressed. The CLA-2/Cn and WSA strains, which were recently and independently derived from a feral population exhibiting low responsiveness to PC, provide the opportunity to approach this question. In previous studies, the heavy chain cDNA sequence of a PC-specific hybridoma derived from CLA-2/Cn suggested gene conversion events within the VHS107 family. Accordingly, we have examined the germline VHS107 genes of CLA-2/Cn and WSA. The results indicate that: (a) The CLA-2 and WSA strains bear an identical but novel VHS107 family haplotype, which lacks a V3 element and contains a V1, a V13, and two V11 genes; (b) low PC responsiveness in these populations is unlikely due to an inability to express the V1 member of the VHS107 gene family; and (c) when compared with the other known VHS107 haplotypes, the proportion of differences consistent with gene conversion greatly exceeds that expected by random base substitution. Thus, gene conversion events appear to have occurred with considerable frequency in the evolution of the murine VHS107 family, especially among the V3, V13, and V11 members.
Alleles↗
Interaction and sequence diversity among T15 VH genes in CBA/J mice.
Nucleotide sequences of the four genes composing the T15 heavy chain variable region (VH) family of the CBA/J mouse have been determined. Comparison of these sequences with their published BALB/c and C57BL/10 homologues reveals that nucleotide differences found between given alleles of two strains, i.e., CBA/J and BALB/c, are observed in other family members of the same strain. We suggest that these patterns of sequence variation are most readily explained by gene interaction (conversion). Additionally, the sequence of a CBA/J hybridoma, 6G6, proposed to have been generated by gene conversion, is directly encoded by the CBA/J V11 gene indicating that the putative conversion has occurred meiotically in the germline. These results are consistent with the premise that gene correction is occurring frequently among members of this family and that such processes may contribute significantly to the evolution of Ig variable region genes even in the relatively short time frame of inbred strain derivation.
Alleles↗
Evolution of the IgA heavy chain gene in the genus Mus.
To examine questions of immunoglobulin gene evolution, the IgA alpha heavy chain gene from Mus pahari, an evolutionarily distant relative to Mus musculus domesticus, was cloned and sequenced. The sequence, when compared to the IgA gene of BALB/c or human, demonstrated that the IgA gene is evolving in a mosaic fashion with the hinge region accumulating mutations most rapidly and the third domain at a considerably lower frequency. In spite of this pronounced accumulation of mutations, the hinge region appears to maintain the conformation of a random coil. A marked propensity to accumulate replacement over silent site changes in the coding regions was noted, as was a definite codon bias. The possibility that these two phenomena are interrelated is discussed.
Amino Acid Sequence↗
Evolution of the mouse IgA gene: nucleotide sequence comparison of IgA in BALB/c and Mus pahari.
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Animals↗
Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochemical characterization of a human sarcoma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin-related determinants.
An immunoregulatory factor (IRF) that suppresses Con A-mediated peripheral blood mononuclear cell (PBM) proliferative responses was partially purified by DEAE anion exchange chromatography and affinity chromatography from a 3 M KCl extract of a human liposarcoma. The factor (m.w. = 70K) co-purified with albumin, monitored by two-dimensional gel electrophoresis, and demonstrated a heterogeneous isoelectric point (pI 7.6-7.8). Xenoantisera produced against the DEAE-purified fraction and coupled to Affigel 10 removed suppressive activity that could subsequently be eluted by glycine-HCl, pH 3.5. An anti-albumin column partially removed activity, but the unbound 70K factor could still be detected in the column effluent. A xenoantiserum to this 70K effluent coupled to acrylamide beads completely removed the immunosuppressive activity in immunodepletion experiments. Further direct binding enzyme-linked immunoassays (ELISA) and solid-phase immunoabsorption experiments with monoclonal antibodies to human anti-HLA-DR framework determinants and a constant region of the IgM mu-chain demonstrated determinants on the 70K factor recognized by these antibodies.
Cells, Cultured↗