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发布于:2019-5-14 03:33:16  访问:72 次 回复:0 篇
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37 for 4 h. Lane 1, cells grown in their very own cell-free spent culture
6, lane three) that was absent from the handle cells (Fig. six, lane 1). In equivalent experiments, induction of salA1 transcription wasFIG. 5. RT-PCR evaluation of sal locus transcripts from S. salivarius 20P3. cDNA was generated from mRNA by utilizing the oligonucleotide SalRterm. (A) Lanes 2 to five PCRs performed with primers SalAF and SalXR; lanes 7 to ten, PCRs performed with primers SalY2S and SalRterm; lanes two and 7, cDNA template generated by RT; lanes three and 8, RNA controls (no RT); lanes 4 and 9, chromosomal DNA template; lanes five and ten, no-template controls. (B) PCRs performed with primers SalAF and SalRR. Lane 2, cDNA template generated by RT; lane 3, RNA control; lane 4, chromosomal DNA; lane 5, no template. The molecular mass markers (panel A, lanes 1 and 6; panel B, lane 1) have been DNA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/21795619 HindIII fragments (23.1, 9.41, six.56, 4.36, 2.32, and two.02 kb).observed in S. pyogenes UB1308 salB cells when these cells have been grown in spent culture supernatant from S. pyogenes T11 cultures (SalA1 ) (Fig. 3, lane six) but not when they were grown in fresh broth or in spent culture supernatant from their own cultures (Fig. 3, lane 5). Moreover, S. pyogenes SF370 cells, which did not normally express salA1 mRNA (Fig. 3, lane 3), were induced to express the salA1 mRNA transcript after they were grown in T11 culture supernatant (Fig. three, lane four). These data strongly suggested that production of SalA and production of SalA1 have been autoregulated in S. salivarius 20P3 and S. pyogenes T11, respectively. To confirm this, S. salivarius 20P3 cells had been grown for the mid-exponential phase in M17CS medium, harvested, washed, and suspended in fresh medium containing 0 to five pmol of purified SalA peptide per ml. Immediately after incubation of your cultures at 37 for four h, mRNAs had been extracted and subjected to Northern evaluation.37 for four h. Lane 1, cells grown in their own cell-free spent culture medium; lane 2, cells grown in fresh M17CS medium containing five pmol of purified SalA/ml; lane 3, cells grown in cell-free S. salivarius 20P3 spent culture medium; lane 4, cells grown in cell-free S. pyogenes T11 spent culture medium.SalA as determined by an agar inhibition assay and didn‘t purchase3-Methylpyrazole considerably inhibit development of S. pyogenes (Table 2). The analogous salB gene inactivation experiment was then carried out with S. pyogenes T11 by integrating plasmid pMU1016 in the salB chromosomal gene (Fig. 1B). The resulting isogenic mutant, UB1308, did not produce a 0.2-kb salA1 transcript (Fig. three, lane five) and was also inhibitor production adverse (SalA1 ) as determined by the agar inhibition assay (Table 2). These final results confirmed that the salBCT gene merchandise were essential for production of SalA and SalA1 peptides and suggested that SalA and SalA1 lantibiotics modulated salA and salA1 gene transcription. Expression of SalA is autoregulated. To test the hypothesis that salA expression depended upon the presence of active SalA, S. salivarius 20P3 cells have been removed from a late-exponential-phase culture in M17CS medium. The spent medium was inoculated with S.
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