The long term goals of this project are to determine the mechanisms and control of transposition of the LINE-like (Long INterspersed Element) genetic element Tad from Neurospora crassa. Tad is a 7 kilobase retrotransposon. It is present in active form in the Adiopodoume strain of Neurospora crassa, but only in inactive, multiply mutated form in other strains of Neurospora. The active Tad elements lack terminal repeats, but contain two long non-overlapping open reading frames (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 ORF1 has been used to demonstrate that Tad is a bona fide retrotransposon. All LINE-like elements that have been studied appear to have an internal 5' promoter that is responsible for transcription of the full length message; however, none of these promoters have been thoroughly characterized. The Tad promoter is more than 30 bp internal to Tad, and the region that contains the promoter is approximately 100 bp. A variety of techniques are being used to identify critical promoter elements and the genes whose products are responsible for Tad expression. In addition there are a number of unique and interesting features of Tad: 1) Tad contains a unique two component silencer system. One component, which is in a 1.2 kb fragment contained within the 3' end of ORF2, acts as a silencer of an adjacent gene. The silencer itself is controlled by another element that is located within the 5' end of tad. This element is responsible for a reversible methvlation of the 5' end of Tad. When the sequences are heavily methylated the silencer is off. When the sequences are lightly methylated, not methylated, or deleted, the silencer is functional. The components of both elements of the silencer system are being characterized. 2) Tad contains an unusual 3' tail beyond the end of ORF2. Certain sequences near, but not at, the 3' end of this tail are essential for transposition. These essential sequences are being characterized. 3) Tad exists in a genus that has a very efficient mechanism for destroying active transposons. Active Tad has been found in only one strain of hundreds of strains collected from Africa; however all stains examined have Tad elements that appear to have been inactivated by the RIP process. The Tad elements from the African strain are susceptible to RIP and that strain is not RIP deficient. Soil-based PCR will be used to look for active Tad elements in order to try to determine the origin of Tad. Mobil genetic elements have been found in organisms as diverse as bacteria and humans and are responsible for mutations when they insert into active genes. These studies should lead to an understanding of how these elements arise and how some organisms protect their genomes from being damaged by potentially large numbers of them.
