Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.
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PMID:In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease. 993 Aug 78

Gene A40R from vaccinia virus (VV) strain Western Reserve has been characterized. The open reading frame (ORF) was predicted to encode a 159 amino acid, 18152 Da protein with amino acid similarity to C-type animal lectins and to the VV A34R protein, a component of extracellular enveloped virus (EEV). Northern blotting and S1 nuclease mapping showed that gene A40R is transcribed early during infection from a position 12 nucleotides upstream of the ORF, producing a transcript of approximately 600 nucleotides. Rabbit anti-sera were raised against bacterial fusion proteins containing parts of the A40R protein. These were used to identify an 18 kDa primary translation product and N- and O-glycosylated forms of 28, 35 and 38 kDa. The A40R proteins were detected early during infection, formed higher molecular mass complexes under non-reducing conditions and were present on the cell surface but absent from virions. The proteins partitioned with integral membrane proteins in Triton X-114. Canine pancreatic microsomal membranes protected in vitro-translated A40R from proteinase K digestion, suggesting the A40R protein has type II membrane topology. A mutant virus with the A40R gene disrupted after amino acid 50, so as to remove the entire lectin-like domain, and a revertant virus were constructed. Disruption of the A40R gene did not affect virus plaque size, in vitro growth rate and titre, EEV formation, or virus virulence in a murine intranasal model.
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PMID:The vaccinia virus A4OR gene product is a nonstructural, type II membrane glycoprotein that is expressed at the cell surface. 1046 13

Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5' end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.
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PMID:Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate. 3179 46


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