Break-induced replication (BIR) is a nonreciprocal recombinationdependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5 end of the LEU2 gene at one end was transformed into a strain in which the 5 end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2 ؉ . Ade1 ؉ Leu2 ؉ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ. D uring the past several years, some old ideas about how recombination occurs have received strong experimental support. Meselson and Weigle (1) first proposed that crossingover could be explained by a break-copy mechanism in which one end of a double-strand break (DSB) could invade an intact linear template molecule and initiate new DNA synthesis that could proceed to the end of the chromosome template. In essence, a recombination event led to the establishment of a unidirectional replication fork. Skalka (2) provided a more molecular view of this idea (a replicator's view of recombination, as she called it) to explain phage recombination. Mosig (3, 4) made a similar proposal to account for late DNA replication in phage T4. Formosa and Alberts (5) provided a key in vitro experimental demonstration for the formation of a replication fork by recombination. More recent studies by George and Kreuzer (6) of DSB-induced recombination, controlled by phage T4 genes in Escherichia coli have supported the idea that recombination leads to extensive replication. Similarly, recent experiments by Motamedi et al. (7) and by Kuzminov and Stahl (8) with phage have provided strong evidence that a major pathway to generate crossing-over involves extensive replication during break-copy recombination.These ideas were applied by Kogoma (9, 10) to explain origin-independent, recombination-dependent replication of the E. ...

SummaryMurine mammary tumor viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor Vfl chains. MMTVs are transmitted to the suckling offspring through milk. Here we show that B cell-deficient mice foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice, and no deletion of MMTV superantigen-reactive T cells occurred. By contrast, T cell deletion and positive selection due to endogenous MMTV superantigens occurred in B cell-deficient mice. We conclude that B cells are essential for the completion of the viral life cycle in vivo, but that endogenous MMTV superantigens can be presented by cell types other than B cells.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS ®broblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS ®broblasts, but it is not clear if this is su cient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were analysed at various passages during the spontaneous immortalization of LFS skin ®broblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain speci®city may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

No abstract

Short-chain carboxylic acids (SCCA) are metabolic by-products of bacterial pathogens which can alter cytoplasmic pH and inhibit a variety of polymorphonuclear leukocyte (PMN) motile functions. Since cytoskeletal F-actin alterations are central to PMN mobility, in this study we examined the effects of SCCA on cytoskeletal F-actin. Initially, we tested nine SCCA (formate, acetate, propionate, butyrate, valerate, caproate, lactate, succinate, and isobutyrate). We document here that while eight altered cytoplasmic pH, only six altered cytoskeletal F-actin. We then selected one SCCA that altered both F-actin and cytoplasmic pH (propionate) and one SCCA that altered only cytoplasmic pH (lactate) for further study. Propionate, but not lactate, caused an irregular cell shape and F-actin distribution. Furthermore, propionate, but not lactate, inhibited formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated PMN polarization, F-actin localization, and cytoplasmic pH oscillation. Propionate-induced changes in cytoskeletal F-actin and cytoplasmic acidification were not affected by the fMLP receptor antagonist N-t-BOC-1-methionyl-l-leucyl-l-phenylalanine; however, alkalinization was affected. Pertussis toxin treatment completely inhibited propionate-induced changes in F-actin but had no effect on propionate-induced cytoplasmic pH oscillation. These results indicate that propionate (i) bypasses the fMLP receptor and G protein(s) to induce cytoplasmic pH oscillation, (ii) operates through G protein(s) to induce actin oscillation, cell shape changes (to irregular), and F-actin localization, and (iii) inhibits fMLP-stimulated cytoplasmic pH and actin oscillation, PMN polarization, and F-actin localization.Neutrophils provide the first line of host defense against bacterial pathogens. How and why the delicate balance between the polymorphonuclear leukocytes (PMNs) and bacteria is swayed toward the bacteria in disease is often not clear. It is clear that chemical mediators which activate or inactivate the PMNs initially interact with receptors, secondarily activate biochemical responses, and finally activate cellular responses (5, 37, 50). Bacterial mediators found at the sites of infection may use similar pathways to activate or inactivate PMN function (1,10,11,25,43,59).Short-chain carboxylic acids (SCCA) are metabolic byproducts from pathogenic anaerobic bacteria and appear in millimolar concentrations at the sites of infection (1,6,17,28,51,58). Studies from several laboratories indicate that SCCA inhibit stimulated PMN functions, such as chemotaxis, phagocytosis, degranulation, oxidative burst, and phagocytic killing of ingested bacteria (9, 4244,55,59).Despite considerable evidence regarding the effects of SCCA on PMNs, the mechanism(s) of SCCA-induced inhibition is unknown. Paradoxically, some SCCA can stimulate cytoskeletal F-actin polymerization, right-angle light scatter, and changes in cytoplasmic pH (25, 54, 59) yet inhibit motile functions (1). The purpose of this study was to examine the mechanism of SCCA-mediated PMN...