PubMed · 8220744
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H G Griffin, A M Griffin. 1993. Plasmid sequencing.. https://doi.org/10.1385/0-89603-248-5%3A131
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The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 bp region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR I) showed some homology with other previously described SSOs of the SSOA type, as well as with the conversion signal of the Escherichia coli phage phi X174. This IR was shown to have a partial, RNA polymerase-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerase-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).
The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.
Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1 alpha and 1 beta) that encode different 5'-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue- or developmental-specific manner.