Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

The human genome contains multiple sequences of varying homology to mouse mammary tumour virus DNA

Sequences related to the mouse mammary tumour virus (MuMTV) DNA were isolated from a genomic library of human DNA by screening under conditions of relaxed stringency. It is estimated that there are in the order of 50 MuMTV-like sequences per haploid genome and that the homology between the different human sequences and MuMTV varies by 15%.     

Recombination between satellite phage P4 and its helper P2. I. In vivo and in vitro construction of P4: :P2 hybrid satellite phage

P4::P2 hybrid satellite phages which carry a portion (including the P2 head gene Q and the cohesive end) of the left end of the P2 chromosome linked to the essential part of the P4 chromosome have been isolated by in vivo as well as in vitro recombination. These hybrids express gene Q and grow in the presence of a P2 helper even if defective in gene Q.