Genes induced by growth arrest in a pancreatic β cell line: identification by analysis of cDNA arrays

AbstractPancreatic β cell lines are a potentially attractive source of material for cell therapy of insulin-dependent diabetes mellitus. However, induction of proliferation in post-mitotic, differentiated β cells is likely to affect the expression of multiple genes associated with cell function, resulting in dedifferentiation. We have developed a murine β cell line by conditional transformation with the SV40 T antigen oncoprotein. These cells can undergo reversible induction of proliferation and growth arrest. Here we utilized this model to identify differences in gene expression between proliferating and quiescent β cells, by analyzing known β cell genes and differentially secreted proteins, as well as by a systematic survey of a mouse cDNA array. Our findings demonstrate that growth arrest stimulates expression of the insulin gene and genes encoding components of the insulin secretory vesicles. Screening of the cDNA array revealed the activation of multiple genes following growth arrest, many of them novel genes which may be related to β cell function. Characterization of these genes is likely to contribute to our understanding of β cell function and the ability to employ β cell lines in cell therapy of diabetes

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2−/− males, but not females, providing an explanation for the sexual dimorphism seen in Pms2−/− mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2−/− and Msh3−/− mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis

Distinct Functions of MLH3 at Recombination Hot Spots in the Mouse

The four mammalian MutL homologs (MLH1, MLH3, PMS1, and PMS2) participate in a variety of events, including postreplicative DNA repair, prevention of homeologous recombination, and crossover formation during meiosis. In this latter role, MLH1–MLH3 heterodimers predominate and are essential for prophase I progression. Previous studies demonstrated that mice lacking Mlh1 exhibit a 90% reduction in crossing over at the Psmb9 hot spot while noncrossovers, which do not result in exchange of flanking markers but arise from the same double-strand break event, are unaffected. Using a PCR-based strategy that allows for detailed analysis of crossovers and noncrossovers, we show here that Mlh3−/− exhibit a 85–94% reduction in the number of crossovers at the Psmb9 hot spot. Most of the remaining crossovers in Mlh3−/− meiocytes represent simple exchanges similar to those seen in wild-type mice, with a small fraction (6%) representing complex events that can extend far from the initiation zone. Interestingly, we detect an increase of noncrossovers in Mlh3−/− spermatocytes. These results suggest that MLH3 functions predominantly with MLH1 to promote crossovers, while noncrossover events do not require these activities. Furthermore, these results indicate that ∼10% of crossovers in the mouse are independent of MLH3, suggesting the existence of alternative crossover pathways in mammals

Adenovirus Early Region 3 Transgenes Expressed in β Cells Prevent Autoimmune Diabetes in Nonobese Diabetic Mice: Effects of Deleting the Adenovirus Death Protein 11.6K

The incidence of type 1 diabetes (T1D) is decreased in nonobese diabetic mice expressing the complete cassette of adenovirus early region 3 (E3) immunomodulating genes in pancreatic β cells. Embedded among the antiapoptotic E3 genes is one encoding an adenovirus death protein (ADP), which contributes to release of virion particles by promoting cell lysis. Because removal of this proapoptotic protein might have further enhanced the ability of E3 proteins to prevent T1D, an ADP-inactivated E3 construct was tested. Significantly, deletion of ADP did not improve the diabetes-protective effect of an E3 gene cassette

Global Gene Expression Profiling of Yersinia pestis Replicating inside Macrophages Reveals the Roles of a Putative Stress-Induced Operon in Regulating Type III Secretion and Intracellular Cell Division▿ †

Yersinia pestis, the causative agent of plague, is a facultative intracellular pathogen. Previous studies have indicated that the ability of Y. pestis to survive inside macrophages may be critical during the early stages of plague pathogenesis. To gain insights into the biology of intracellular Y. pestis and its environment following phagocytosis, we determined the genome-wide transcriptional profile of Y. pestis KIM5 replicating inside J774.1 macrophage-like cells using DNA microarrays. At 1.5, 4, and 8 h postinfection, a total of 801, 464, and 416 Y. pestis genes were differentially regulated, respectively, compared to the level of gene expression of control bacteria grown in tissue culture medium. A number of stress-response genes, including those involved in detoxification of reactive oxygen species, as well as several metabolic genes involved in macromolecule synthesis, were significantly induced in intracellular Y. pestis, consistent with the presence of oxidative stress and nutrient starvation inside Yersinia-containing vacuoles. A putative stress-induced operon consisting of y2313, y2315, and y2316 (y2313-y2316), and a previously unidentified open reading frame, orfX, was studied further on the basis of its high level of intracellular expression. Mutant strains harboring either deletion, Δy2313-y2316 or ΔorfX, exhibited diverse phenotypes, including reduced effector secretion by the type III secretion system, increased intracellular replication, and filamentous morphology of the bacteria growing inside macrophages. The results suggest a possible role for these genes in regulating cell envelope characteristics in the intracellular environment