Hideo Ikeda

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1

SummaryTwo modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 by upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC, mutation located at 62 min of the E. coli chromosome. The herC, mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC, mutation alone conferred cold-sensitive growth, suggesting that the herC, gene product is essential for cell growth. The 1832 by E. coli DNA fragment, containing the wild-type allele of the herC, mutation, was cloned and an open reading frame for the HerC protein was determined.

Formation of deletion in Escherichia coli between direct repeats located in the long inverted repeats of a cellular slime mold plasmid: Participation of DNA gyrase

SummaryWe constructed a recombinant plasmid containing the 2.1 kb HindIII fragment of plasmid pDG1, isolated from the cellular slime mold (Dictyostelium sp. strain GA11), and using pAG60 as cloning vector. We found that deletions of the recombinant plasmid took place frequently in Escherichia coli wild-type cells. However, the deletion was not observed when the plasmid was introduced into a strain that was an isogenic temperature-sensitive mutant of the gyrA gene. These results suggest that E. coli DNA gyrase is involved in the mechanisms of the deletion formation. It was shown that the 1.0 kb deletant derived from the 2.1 kb HindIII insert was produced by elimination of a 1.1 kb region. Sequence analysis of the deletants showed that cutting and rejoining took place between two out of the six nearly perfect direct repeats [21 bp palindromic sequences; AAAAAA(T/C)GGC(G/C)GCC(A/G)TTTTTT], located near the distal ends of the inverted repeats, preserving one copy of the repeats. These sequences consist of local short inverted repeats, where cutting and rejoining occur at one of the two regions.

Cloning and characterization of a repetitive sequence from Pneumocystis carinii

Four repetitive sequence clones measuring 10.9–23.4 kb in length were isolated from the genomic library ofPneumocystis carinii. Restriction enzymes mapping and cross-hybridization studies revealed that these clones are interrelated and that they derive from the common repeat unit, which is specific forP. carinii. Dot-blot analysis suggested that the copy number of the repeat sequence is about 100, assuming that the genome size is 1.5×107 bp. Interestingly, the repetition unit extended over at least 23.4 kb and included long, 5.2-kb inverted repeats, for example, A-B-A′-C, in which A′ is the inversion of A.