The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons. MCM1 (mini-chromosome maintenance) genes had been identified in screens for Saccharomyces cerevisiae mutants, which displayed high rates of minichromosome loss (1). MCM2-MCM7 homologues were subsequently found in all eukaryotes. It is believed that MCM proteins act as a component of the licensing machinery, which limits the occurrence of DNA replication to once per cell cycle (2, 3). MCM proteins form prereplicative complexes at active as well as silent origins in S. cerevisiae or on un-replicated DNA in higher eukaryotes in a manner dependent on origin recognition complex (ORC) and Cdc6 (2, 3). Activation of these pre-replicative complexes by protein kinases is required for initiation of replication (2, 3). Removal of the MCM complex from origins at the time of initiation is believed to limit origin firing until the next cell cycle when the pre-replicative complex is re-established (2, 3). MCM proteins also seem to have a post-initiation role in DNA replication as indicated by their association with moving replication forks (4) and by the requirement for their uninterrupted function for fork progression in S. cerevisiae (5). In vitro assembled MCM complexes have also been shown to have DNA helicase activity consistent with a role in both melting of origin DNA and unwinding during fork elongation (3).Some of the properties of MCM proteins suggest that they may have functions that extend beyond the regulation of DNA replication. They are 100-to 1000-fold more abundant than the estimated number of origins in S. cerevisiae (6, 7) and mammals (8, 9). Mammalian MCMs have been reported to associate with large complexes containing or contacting RNA polymerase II and general pol II transcription factors (10, 11), with the MAT1 component of CAK/TFIIH (12), with the transcriptional activator STAT1 (13,14), and with the tumor suppressor pRB (15, 16) thus suggesting a function of MCMs in pol II transcription and gene expression. Other studies have linked MCM proteins to chromatin remodeling based on the interaction between the MCM proteins and histone H3/H4 dimers (17,18) and between MCM2 and the histone acetyltransferase HBO1 (19).Genome-wid...
Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from "handler" and "wearer" areas of 764 items commonly submitted for examination. One hundred and fifty-five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T-shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison.
D-(؊)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(؉) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.Poly-3-hydroxybutyrate (PHB), a bacterial intracellular reserve of carbon and reducing energy, accumulates when a nutrient other than carbon is limiting for growth, as reviewed by Madison and Huisman (18). Based on biochemical evidence, the metabolism of PHB has been proposed to be a cyclical process, comprising the pathways for synthesis and degradation of PHB. Synthesis of PHB occurs when excess carbon, in the form of acetyl coenzyme A (acetyl-CoA), is condensed via a ketothiolase (EC 2.3
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