Detection and characterization of Leishmania species and strains from mammals and vectors by hybridization and restriction endonuclease digestion of kinetoplast DNA.

Leishmania parasites from animals, man or insect vectors were characterized by the gel electrophoresis of restriction endonuclease enzyme-produced mitochondrial (kinetoplast) DNA (kDNA) fragments and/or by DNA-DNA hybridization with 32P-labelled cloned, or uncloned, kDNA fragment probes from type isolates. The electrophoretic separation of kDNA fragments is a sensitive method for detecting genetic similarities and differences among Leishmania. Parasites with similar kDNA restriction fragment patterns belong to the same schizodeme and schizodeme analysis is useful for studying Leishmania populations. Cloned, species-specific kDNA probes detected Leishmania in sandflies and in liver, spleen or blood preparations from infected animals. Cloned DNA probes also hybridized to immobilized kDNA from in vitro cultivated promastigotes and detected as few as 100 parasites in a species-specific manner. Sensitive DNA hybridization probes should be useful in research on the immunology, chemotherapy or epidemiology of animal and human leishmaniasis.

Identification of pathogenic Leishmania promastigotes by DNA: DNA hybridization with kinetoplast DNA cloned into E. coli plasmids.

We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restriction endonuclease-produced DNA fragments vary from species to species. However, kDNA isolates from different species and strains cross-reacted to a great extent in Southern hybridization experiments. Only kDNA isolated from L. infantum and L. major had little homology during hybridization reactions. To prepare DNA probes that would differentiate between species of Leishmania, minicircle kDNA was digested with restriction enzymes and ligated to an E. coli plasmid. Several plasmids were isolated that specifically detect in hybridization experiments as few as 5 X 10(3) L. donovani or L. infantum promastigotes lysed on nitrocellulose filters.

Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection.

The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie.

DNA strand specificity of temporal RNA classes produced during infection of Bacillus subtilis by SP82.

The DNA of the Bacillus subtilis bacteriophage SP82 has been separated into heavy (H) and light (L) fractions by centrifugation in buoyant density gradients in the presence of polyguanylic acid. Competition-hybridization experiments were performed with these separated fractions using RNAs isolated from cells labeled at intervals which account for 80% of the lytic cycle and unlabeled competitor RNAs isolated from phage-infected cells at 2-min intervals throughout infection. The analysis of temporal RNA classes were facilitated by use of a double reciprocal plot of the data. Five temporal classes binding to the H fraction and three binding to the L fraction were detected; the possible existence of an additional class transcribed from the H fraction is discussed. RNA synthesized in the presence of chloramphenicol contains two of the three classes produced from L-DNA and two of the five classes transcribed from H-DNA.

DNA strand specificity of transcripts produced in vivo and in vitro by RNA polymerase from SP82-infected Bacillus subtilis.

Phage-specific RNA synthesized early in the infection of Bacillus subtilis with SP82 hybridizes to both heavy (H) and light (L) strands of SP82 DNA nearly equally. Phage RNA synthesized during the middle of the infection hybridizes preferentially to the H strand. The ratio of H/L strand binding of RNAs synthesized in vitro by RNA polymerases isolated from uninfected and infected cells resembles the ratios of early and middle phage RNA classes, respectively. This supports the conclusion that a modified RNA polymerase is required for the transcription of middle RNA classes.