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PubMed2005

[Detection of methicillin resistance and identification of Staphylococcus spp. from positive blood culture bottles using the mecA and nucA genes with the LightCycler System].

INTRODUCTION: The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system. METHODS: One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene. RESULTS: All the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively. CONCLUSION: This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.

Automation↗
PubMed1991

Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.

The E1A pre-mRNA of adenovirus is spliced into three mRNA species (13S, 12S, and 9S mRNAs) by the use of three alternative 5'-splice sites. The 13S and 9S mRNAs predominate during the early and late periods of infection, respectively. With HeLa nuclear extracts isolated in early and late periods of infection, we were able to reproduce a 13S-9S modulation that resembles that occurring in infected cells. An in vitro analysis of the cis-acting parameters involved in the 13S-9S switch indicates that the 13S mRNA splicing inhibition is one of the first events of the late period and leads to the subsequent stimulation of the 9S mRNA reaction. The new abilities of the late nuclear extract for the 9S mRNA reaction were also confirmed by analyzing splicing of a major late transcript containing leaders 1 and 2 separated by the wild-type intervening sequence (IVS) of 1021 nucleotides. Complementation experiments show that the trans-acting factor(s) are micrococcal nuclease sensitive. They were partially characterized by induction experiments, and we show that the primary factors responsible for the 13S-9S modulation in vitro are viral RNAs of high molecular weight that accumulate late in infection. We postulate that the splicing modulation of E1A pre-mRNA results from an indirect mode of action for these viral RNAs, based on a sequestration of common splicing factors that are not present in vast excess in HeLa cells.

Adenoviridae↗
PubMed1981

Structure of the filamentous bacteriophage fl. Location of the A, C, and D minor coat proteins.

The location within the virion of the A, C, and D minor coat proteins of the filamentous bacteriophage fl has been analyzed. The A protein is present in approximately 5 copies/particle and is located at the tip of normal length phage, miniphage, and fl/pBR322 chimeric phage, a longer than normal length phage. The mole ratios of the A, C, and D proteins are the same for each type of particle, consistent with a model of phage organization in which the minor coat proteins are clustered near or at the ends of the phage. Normal length phage were fragmented by passing them through a French press, and those fragments that contained the A protein were separated from those that did not by treating the mixture with anti-A protein antibody. Analysis of the protein compositions of the two populations of fragments showed that the A and D proteins were found together in one population of fragments and that most, it not all, of the C protein was found in the other. These results show that the D protein is located near or at the A protein end of the phage and that the C protein is located in a region near or at the opposite end. Treatment of the virion with proteases which lowered the infectivity of the phage resulted in particles in which only the A protein was cleaved to any detectable extent. These particles remained resistant to the action of micrococcal nuclease.

Capsid↗
PubMed1980

Electrophoretic properties of the scrapie agent in agarose gels.

The molecular properties of the scrapie agent were investigated by subjecting partially purified preparations to electrophoresis on agarose gels. When electrophoresis was performed at room temperature in the presence of sodium dodecyl sulfate (NaDodSO4), most of the recoverable agent was found at the top of the gel, consistent with previous studies indicating aggregation of the agent upon exposure to elevated temperatures. In addition, less than 5% of the agent applied to the gel was found after electrophoresis, even though the study was performed with a low concentration of NaDodSO4 (0.1%). Further studies on the inactivation of the agent by NaDodSO4 suggest that this may be, in part, a function of the NaDodSO4: protein ratio in the sample. In contrast, sodium N-lauroyl sarcosinate (Sarkosyl) did not inactivate the agent in concentrations as high as 5% (wt/vol). Virtually all of the infectivity could be recovered after electrophoresis of the agent into 0.6% agarose gels at 4 degrees C in the presence of 0.2% Sarkosyl. Digestion of the preparations with micrococcal nuclease and proteinase K prior to Sarkosyl electrophoresis caused a substantial portion of the agent to migrate ahead of DNA fragments of 1 x 10(6) daltons. The behavior of the scrapie agent in electrophoretic gels is consistent with earlier studies showing that the monomeric form of the agent has a sedimentation coefficient of less than or equal to 40 S. Thus, the smallest or monomeric form of the agent is smaller than any known animal virus.

Detergents↗
PubMed1990

Potential retroviral RNAs in Creutzfeldt-Jakob disease.

The molecular nature of the related infectious agents that cause Creutzfeldt-Jakob disease (CJD) and scrapie is poorly understood, and an agent-specific nucleic acid genome has not yet been identified. Several biological manifestations of these agents resemble those seen in retrovirus-induced diseases. We therefore attempted to identify an agent-specific retrovirus-like RNA transcript in CJD infectious fractions. A series of synthetic oligonucleotides complementary to known mammalian retroviral primer binding sites were used in a primer extension assay. Substrate nucleic acids isolated from partially purified hamster brain CJD infectious fractions and from parallel normal brain fractions were compared with total starting brain RNA. This sensitive exogenous strong-stop reaction revealed that CJD infectious fractions contained a series of potential retroviral RNAs including apparent transcripts of endogenous hamster IAP genes. Most transcripts selectively recovered in the fractions were substantially protected from micrococcal nuclease digestion, and at least one substrate RNA, consistent with an intracisternal A particle, was packaged in a form that had the same buoyant density as CJD infectivity. Although a completely CJD-specific transcript was not identified, the copurification of potential retroviral transcripts with CJD infectivity suggests that models of disease involving retrovirus-like nucleic acid elements deserve further consideration.

Animals↗
PubMed1994

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.

Bacillus Phages↗