Biomedical subjects
S M Mirkin
Publications and source records attributed to S M Mirkin.
[Primary structure of two natural IS1-flanking transposons Tn9* and Tn9' coding for stability to chloramphenicol].
The DNA nucleotide sequence from the central region of the composite transposons Tn9* and Tn9' at the junction with the right copy of IS1 was determined. From the data obtained it follows that both transposons are members of the Tn9 family, although they contain additional DNA segments with regard to Tn9 of the length about 320 and 290 b.p. respectively lying distal to the cat gene. It was proposed that all the transposons of the Tn9 family have been formed as a result of IS1-mediated deletions of the plasmid R100 r-determinant. It was revealed from the data of computer analysis that in the sequenced DNA there are two potential promotors with transcription directed opposite in relation to the transcription of the cat gene.
[Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression].
We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.
[Effect of DNA supercoiling on transcription performed by normal and mutant Escherichia coli RNA-polymerases].
A specific DNA-gyrase, inhibitor, coumermycin A1, causes differential changes in the spectrum of proteins synthesized in E. coli wild types cells, while protein spectrum in the mutant cells with coumermycin-resistant DNA-gyrase is left unaffected. The mutation of RNA-polymerase RpoB265 lowers the sensitivity of bacteria to coumermycin A1, whereas the RpoC3 enhances it. The differences between the normal and mutant RpoB265 RNA polymerases on interaction in vitro with ColE1 DNA plasmid depend on its supercoiling. No dependences of this kind were detected when comparing the normal and RpoC3-RNA polymerase. The obtained data indicate that the template supercoiling may significantly affect the transcription in vivo and that the properties of RNA polymerase may in some cases define this influence.
[Effect of superhelical DNA on the transcription of cloned genes of the T4 phage].
The effect of DNA superhelicity on the transcription of T4 DNA fragments containing early genes uvs W, Y and late genes 25-29 was studied. RNA polymerase transcribes both early and late phage genes within the supercoiled recombinant plasmid. Late genes relative transcription increases essentially when T4-modified RNA polymerase is used. DNA relaxation causes a sharp decrease of modified RNA polymerase activity, especially on the late genes. The same effect is obtained in intact cells with recombinant plasmid, which superhelicity is lowered by temperature-sensitive mutation in DNA gyrase. The data obtained prove that DNA superhelicity is required for the T4 late transcription by phage-modified RNA polymerase. It is suggested that the dependence of late transcription on the phage DNA replication in infected cells is connected with phage DNA superspiralization throughout its replication.
[Interactions of subunits of DNA gyrase].
Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied. For this purpose a E. coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated. It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain. Hence, the mutation affecting the B-subunit is capable to change A-subunit properties. That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.