Showing papers by "David Baltimore published in 2000"

ATR disruption leads to chromosomal fragmentation and early embryonic lethality.

Abstract: Although a small decrease in survival and increase in tumor incidence was observed in ATR+/− mice, ATR−/− embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR−/− blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR−/− cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53.

Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages.

Abstract: A major challenge in the study of gene regulation by NF-kappaB/Rel transcription factors is to understand, at the biological and mechanistic levels, the selective functions of individual Rel family members. To study selectivity, we have examined the NF-kappaB/Rel protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 cell development. In nuclear extracts from lipopolysaccharide-activated macrophages, the predominant Rel dimers capable of binding the IL-12 p40 Rel site were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The two heterodimers bound the site with comparable affinities and exhibited comparable transactivation activities. In striking contrast, p40 mRNA and protein concentrations were reduced dramatically in c-Rel(-/-) macrophages and only modestly in p65(-/-) macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel(-/-) macrophages. These results reveal that a c-Rel-containing complex is an essential and selective activator of p40 transcription, which may reflect unique regulatory mechanisms or biological functions of IL-12. Furthermore, because selectivity was not observed in vitro or in transient transactivation experiments, these findings suggest that an understanding of the selectivity mechanism may require an analysis of the endogenous p40 locus.

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Abstract: Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Severe B cell deficiency in mice lacking the tec kinase family members Tec and Btk.

Abstract: The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220+CD43+ stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)MloIgDhi B cells. Although Tec/Btknull mice were able to form germinal centers, the response to T cell–dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.

Role of the rasGAP-associated docking protein p62 dok in negative regulation of B cell receptor-mediated signaling

Abstract: Antigenic stimulation of the B-cell receptor (BCR) is a central event in the immune response. In contrast, antigen bound to IgG negatively regulates signals from the BCR by cross-linking it to the inhibitory receptor FcgammaRIIB. Here we show that upon cross-linking of BCR or BCR with FcgammaRIIB, the rasGAP-associated protein p62(dok) is prominently tyrosine phosphorylated in a Lyn-dependent manner. Inactivation of the dok gene by homologous recombination has shown that upon BCR cross-linking, p62(dok) suppresses MAP kinase and is indispensable for FcgammaRIIB-mediated negative regulation of cell proliferation. We propose that p62(dok), a downstream target of many PTKs, plays a negative role in various signaling situations.

Signal transduction - A cellular rescue team

Abstract: A protein called glycogen synthase kinase-3β is already known to participate in early embryonic development and cell-fate determination. Now it seems that the protein is also involved in a cell's decision to live or die.

V(D)J recombination is not activated by demethylation of the kappa locus

Abstract: V(D)J recombination is thought to be regulated by changes in the accessibility of target sites, such as modulation of methylation. To test whether demethylation of the kappa locus can activate recombination, we generated two recombinationally active B cell lines in which the gene for maintenance of genomic DNA methylation, Dnmt1, was flanked with loxP sites. Transduction with a retrovirus expressing both the cre recombinase and green fluorescent protein allowed us to purify recombinationally active cells devoid of methylation. Loss of methylation of the kappa locus was not sufficient to activate recombination, although transcription was activated in one line. It appears that demethylation of the kappa locus is not the rate-limiting step for altering accessibility and thus regulated demethylation does not generate specificity of recombination.