Search PubMed⌕ Search

Biomedical subjects

M Kohiyama

Publications and source records attributed to M Kohiyama.

76 records · Page 5Linked to original sources
PubMedDate not supplied

Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli.

The hemimethylated oriC binding activity of the E. coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting. Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle. Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Initiation of DNA replication in delta cya mutants of Escherichia coli K12.

The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K(A) value for cAMP as 0.819 muM(-1). The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competitive in nature with an inhibition constant (K(i)) of about 8.25 muM. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta++ cya, but also that DnaA protein is not degraded. The Delta cya mutants of E. coli are unable to continue DNA synthesis in the absence of de novo protein synthesis and the initiation of DNA replication in these mutants takes place from oriC.

Adenylyl Cyclases↗