We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by seletion of Tra- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants. Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: tra--kam--ColE1--amp--tet... Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA(TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8--17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1--9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.

In this paper we propose a method of direct selection for hybrid DNA molecules in paromomycin-dependent E. coli cells.

Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN‐1 and PYN‐2, inhibit RNA polymerase at the stage of RNA chain elongation. the PYN‐1 antibodies react with the β′‐subunit of the enzyme. The PYN‐2 antibodies react with the β‐subunit and with its 130 kDa amber fragment.

No abstract

We have analyzed the interaction of monoclonal antibodies against Escherichia coli RNA polymerase with products of its limited proteolysis. Two major proteolytic fragments of molecular masses 107 and 43 kDa originate as a result of a single cleavage in the vicinity of the 980th amino acid residue. Anti-/3 subunit monoclonal antibody PYN-2 inhibiting RNA polymerase activity at the stage of RNA elongation reacts with an epitope located between the amino-terminus and the 50th amino acid residue of the /3 subunit. DNA sequencing has shown that the RNA polymerase mutation rpoB22 converts the Gln(l111) codon of the fi subunit gene into the amber codon. An epitope for the monoclonal antibody PYN-6 was located between the major site of proteolytic cleavage and Gln(lll1) of the b subunit. RNA polymerase Monoclonal antibody LimitedproteolysisEpitope mapping Amber mutation