Analysis of a genetically unstable region in Streptomyces lividans 66–TK64.

Genetic and molecular analyses of an unstable region encompassing the gene loci cml arg and a 5.7 kb amplifiable unit of DNA were done. Spontaneous mutants from Cm1R→CmlS and the revertants from CmlS→CmlR were analysed for mutations at arg locus and amplification of amplifiable unit of DNA. Twenty-one revertants were analysed. Two of these had large-scale amplification and one of these was also Arg–. Nine of the revertants which were Arg had low-level or intermediate-level amplification of the 5.7 kb DNA sequence but no deletions of the flanking sequences were detected. Five of the CmIR' revertants, which were also Arg, had lost one of the two copies from the doublet of amplifiable unit of DNA. The remaining five revertants did not show any other change. The amplifiable unit of DNA, therefore, not only undergoes amplification but can also suffer specific deletion of one copy. Thus, this region as a whole is characterized by instability and the events appear to take place at more than one locus concomitantly with a high frequency.

An estimate of the physical distance between two linked markers in Haemophilus influenzae.

Using DNA clones, the physical distance between the linked genes nov and str in Haemophilus influenzae was estimated. Although none of the cloned inserts contained both the markers, pJ1-8StrR 13 (insert of 18·7 kb) included str gene at one end and part of nov gene at the other end of the insert. By EcoRI restriction analysis and by Southern hybridization, the distance between the two EcoRI sites, cutting at which inactivates the two genes, was estimated to be 17·7 kb. A single continuous EcoRI fragment (containing 4 EcoRI sites within it) carrying both the genes intact would need to be 20·4 kb in size. These estimates were confirmed independently using different clones of novr and strr alleles as probes for hybridization with BamHI-digested chromosomal DNA.

Cloning and characterization of a DNA repair gene in Haemophilus influenzae.

Using a high-efficiency DNA cloning vector pJ1-8, a DNA repair gene uvr1 has been self-cloned in bacterium Haemophilus influenzae. Chimeric plasmid pKuvrl, carrying wild type allele of uvr1 gene and flanking DNA sequences, specifically complements a uvrl gene mutation in the bacterial chromosome. A uvr1– mutation could be transferred from chromosome by in vivo recombination to pKuvr1 and isolated and designated as plasmid pKuvrl –. Plasmid pKuvrl carries a 11·3 kb chromosomal DNA insert which was scanned for the presence of any other DNA repair genes by a novel method of directed mutagenesis. Preliminary analysis of the 3 new mutants isolated by this method supports the notion that the insert contains more than one gene concerned with ultraviolet radiation-sensitivity.

Genetic transformation in bacteria.

Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria like biplococcus pneumoniae and Gram-negative bacteria represented by Haemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low in Haemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. In Haemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.

A new DNA-cloning vector for Haemophilus influenzae Rd.

A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF 0885 (ampr) and Haemophilus influenzae chromosomal DNA. pJl-8 has only one EcoRI site and a molecular weight of only 2.5 × 106. No detectable ampr transformation was obtained with pJl-8 DNA. However, ampr transformation increases markedly if Haemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts.

Genetic and molecular events in transformation of Haemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA.

In Haemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ in Haemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. The rec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen in rec 1– strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency on rec 1+ strain.