Mycobacterium avium-intracellulare complex organisms are the most common cause of mycobacterial lung disease other than tuberculosis and are the leading cause of morbidity and mortality in the HIV-infected AIDS patients. Infections caused by M. avium patients with AIDS often go untreated as existing antibiotics are ineffective in adequately controlling these infections. This necessitates the need for novel molecular targets for chemotherapy. Replication of the bacterium leading to its multiplication is one of the necessary steps to establish an infection. Initiation is the first committed step in the replication, and the replication process is believed to be regulated at the level of initiation. Thus, understanding the basic steps involved in initiation of DNA replication in M. avium will help define important molecular targets against which new generation drugs can be developed. Initiation of replication occurs once per cell cycle at a specific site on the chromosome called oriC. Initiation is believed to be triggered by the interactions of dnaA with its recognition sequences present in oriC called the dnaA-boxes. These interactions are thought to facilitate recruiting of other proteins resulting in the completion of initiation. This research proposal focuses on identification and characterization of the oriC and Dna protein of M. avium. To obtain oriC, chromosomal DNA fragments of M. avium that support autonomous replication when present in nonreplicative plasmids will be identified, cloned and their nucleotide sequence will be determined. Sequential deletions from both the 5' and 3' regions will be carried out to identify the minimal DNA region that is essential for oriC activity. Site directed mutagenesis will be carried out to identify the putative DnaA boxes that are essential for oriC activity. The ability of M. avium oriC to function as autonomously replicating sequences in other bacteria will be determined. The dnaA gene will be over-expressed, the gene product will be purified and its interaction with the wild type and mutant sequences will be investigated. Antisense dnaA oligonucleotides that target to the dnaA mRNA to affect the expression of M. avium dnaA gene will be explored in an effort to evaluate the role of the M. avium gene. Using oriC plasmids and cell free extracts, an in vitro replication system will be established. The ultimate long-term goal of these experiments is to utilize the knowledge thus gained to develop rational drugs that affect the initiation of replication thereby preventing growth and resulting M. avium infections.
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