Structure and function of the adeno-associated virus (AAV) genome will be studied at the molecular level. Use will be made of a recombinant plasmid in which the AAV genome is inserted into pBR322. When this plasmid is transfected into human cells with helper, the viral genome is rescued and replicated. With this system alterations can be made at any place in the AAV genome and the effects on replication directly assessed. Problems to be studied include: 1) structural and sequence requirements in the inverted terminal repetition for DNA replication and rescue of the integrated genome, 2) identification of AAV sequences that regulate transcription, 3) identification of helper functions required for AAV transcription, 4) identification of additional spliced species of AAV transcripts, and 5) identification of nonstructural proteins. Additional projects include isolation of nonstructural proteins, and characterization of AAV sequences integrated into cellular DNA and in AAV-SV40 recombinants by cloning, restriction analysis, and sequence determination. Finally, computer analysis will be used to search for interesting sequence arrangements and potential secondary structures that might affect regulation of transcription and translation. Sequences that are so identified will be modified and the effect on transcription and translation determined using the biologically active clone described above.
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