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Random amplified polymorphic DNA (RAPD) was tested for the identification and classification of Leishmania. RAPD was found to be useful for the identification of species of L. (Leishmania) and L. (Yiannia) and for the classification of L. (Yiannia) species. The polymerase chain reaction (PCR) was tested for the identification of Leishmania from mammals and lizards, using both published primers and new primers which amplify kinetoplast minicircle DNA. The size of the PCR product was found to be useful for discriminating between some sympatric pairs of species such as L. braziliensis and L. mexicana. Isotopically labelled probes prepared from the variable region of the kinetoplast minicircle were tested for specificity for the identification of New and Old World species of Leishmania. The specificity was dependent on the concentration of target DNA and was manipulated to investigate relationships between Leishmania species. Restriction digests of kinetoplast DNA (schizodemes) prepared by PCR and by centrifugation through 20% sucrose were compared for the identification of strains of L. infantum and L. chagasi. Twenty three strains of L. chagasi from cases of visceral and cutaneous leishmaniasis in Honduras were examined by RAPD, schizodemes, differential display, isoenzyrnes, RFLPs and PFGE to discover whether genetic differences existed between parasites causing the two different pathologies. The parasites were found to be unusually homogeneous and no differences were found which correlated with pathology by any of these methods. Restriction digests of PCR amplified small subunit ribosomal DNA (SSU rDNA) (ribodemes) were tested to find markers specific for the genus Leishmania. A classification of the Leishmania based on the restriction fragments indicated that L. hertigi and L. herreri were more closely related to Endotrypanum than to Leishmania, and that the lizard Leishmania could not be placed in separate genus from the Leishmania. Ribodemes were used to identify two strains of parasites supplied by colleagues in Central America that could not be identified by existing methods for the identification of Leishmania. One of these strains appeared to be identical to a C. luciliae reference strain. The other strain produced a fingerprint unlike any of the available reference strains. A variable region of the SSU rRNA gene was identified that was suitable for classifying trypanosomatids and the sequence of this region was used to classify the strain that could not be identified by fingerprinting.

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The bacteriophage T4 utilizes at least three modes of initiation of replication, termed primary, secondary and tertiary (Mosig, 1983; Kreuzer and Alberts, 1985). Two origins of replication have been isolated that utilize the tertiary mode of initiation. The DNA sequence requirements of the two tertiary origins have been characterized at the nucleotide level. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 basepairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.

Actinomycin D in low concentrations was suggested to inhibit ribosomal RNA (rRNA) transcription via an extranucleolar mechanism. Actinomycin D was proposed to inhibit unique messenger RNAs (mRNAs) coding for proteins needed for the maintenance of rRNA transcription. According to this hypothesis actinomycin D would bind to specific nonribosomal DNA with high affinity. This hypothesis was investigated by isolating high molecular weight rat liver DNA, digesting it with restriction endonuclease EcoRI, adding [³H] actinomycin D in low concentration, performing RPC-5 chromatography to separate the restriction fragments and subsequent hybridization to rRNA. It was observed that actinomycin D bound to nonribosomal DNA with high affinity. The same experiment was performed with nucleolar DNA. High affinity actinomycin D binding was not observed in nucleolar DNA. Discrete high affinity binding DNA for actinomycin in rat liver DNA was also observed when another restriction endonuclease BamHI was used to cleave rat liver DNA. However, with rat liver DNA digested with restriction endonuclease HindIII, such a high affinity actinomycin D binding DNA was not observed. Actinomycin D was also demonstrated to bind to discrete site(s) in at least four more eukaryotic species (salmon, calf, herring and human) after DNA from these species were digested by EcoRI, labeled actinomycin D added, and RPC-5 chromatography performed. Labeled actinomycin D bound to its high affinity binding DNA was displaced by unlabeled actinomycin D in a concentration range of biological significance. However, six other antitumor agents, (doxorubicin, aclacinomycin, carminomycin, marcellomycin, musettamycin and pyrromycin) which also intercalate into DNA, did not significantly displace labeled actinomycin D from its high affinity binding DNA. Since this high affinity actinomycin D binding DNA is hypothesized to be involved in the inhibition of rRNA transcription, the actinomycin D binding DNA could have a role in the regulation of rRNA transcription. To date this is the first time that a probable regulatory DNA has been characterized by selective drug binding.

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