RESUMO
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.
Assuntos
Impressões Digitais de DNA , DNA/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase , TatoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Saccharomyces cerevisiae/metabolismo , Telômero/ultraestrutura , Transcrição Gênica , Northern Blotting , Cromatina/metabolismo , Proteínas de Ligação a DNA , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Cinética , Nuclease do Micrococo/metabolismo , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , Fenótipo , Retroelementos , Proteínas de Schizosaccharomyces pombe , Temperatura , Fatores de Tempo , Ativação Transcricional , Regulação para CimaRESUMO
MCM (minichromosome maintenance) proteins function as a replication licensing factor (RLF-M), which contributes to limiting initiation of DNA replication to once per cell cycle. In the present study we show that a truncation of the pol II CTD in a S. cerevisiae strain harboring a mutation in mcm5 partially reverses its ts phenotype and improves maintenance of CEN/ARS minichromosomes. We correlate this phenotype to effects on DNA replication rather than to effects on transcription or specific gene expression. We also demonstrate that a similar truncation of the CTD reduces minichromosome stability and impairs stimulation of DNA replication by trans-activators and that tethering of recombinant pol II CTD to an origin of replication has a significant stimulatory effect on minichromosome stability. Furthermore, we show that pol II is recruited to ARS1. We propose that in S. cerevisiae a mechanism of coordinating pol II transcription and DNA replication is mediated by the CTD of pol II.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , RNA Polimerase II/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Mutação , Estrutura Terciária de Proteína , RNA Polimerase II/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.