9407082 Kinsey The long term goals of this project are to determine the mechanisms and control of transposition of the LINE-like element, Tad from Neurospora crassa. Tad is a 7 kilobase retrotransposon. It is present in active form in the Adiopodume strain of Neurospora crassa, but only inactive, multiply mutated form in other strains of Neurospora. The active Tad elements lacks terminal repeats, but contain two long non-overlapping ORFs on the DNA strand with the same polarity as the full length message. ORF1 shows homology with a small part of the gag genes of other LINE-like elements. ORF2 contains blocks of homology that are considered diagnostic for reverse transcriptase, including one block that is characteristic of the reverse transcriptase domains of other LINE-like elements. Tad is capable of transposing between nuclei in a heterokaryon, and an intron inserted into ORF has been used to demonstrate that TAD is a bona fide retrotransposon. In this project the focus is on the control of transcription and transposition. A combination of approaches are being taken to include: 1) in vitro mutagenesis of the 5' promoter region. 2) analysis of proteins that bind to the 5' end of TAD and the sequences to which they bind. 3) cloning and disruption of genes coding for proteins that bind to sites that are required for transcription and transposition. 4) analysis of sites near the 5' end where cytosine methylation is associated with transcriptional inactivity of Tad. 5) Isolation and characterization of chromosomal mutants that effect TAD expression and transposition. 6) analysis of mutations of minus strand ORFs and promoters. In addition experiments will be carried out to begin to analyze the relationship TAD and the RIP process. %%% Neurospora has one of the few genetic systems where this type of jumping gene can be studied. A similar jumping gene is present in tens of thousands of copies in human chromosomes. ***
