GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE RETROPOSON

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The proposed research is directed towards gaining a more complete understanding of the biochemical and genetic behavior of the L1 (or LINE1) element in mice. The L1 family is ubiquitous in mammals and shares some characteristics with known transposable elements. Genetic considerations and DNA sequence analysis suggest that L1 is an active transposable element in mice and other mammals; its movement provides a major source of genomic flux in evolutionary time, and expression and movement of L1 is likely to occur in the germline. Characterization of the control of L1 expression and movement in the animal may lead to important insights into the general mechanisms of genome rearrangement and developmental control of gene expression. One result of these processes going awry in adult tissue may be cancer. Hypotheses based on DNA sequence analysis suggest that L1 is a retroposon-like transposable element. There is, however, no direct knowledge about the intermediates involved in the process of L1 insertion, or about the expression and control of L1 in the organsim. The proposed experiments address these unknowns. A collection of rabbit anti-L1 antisera will be made against unidentified open reading frames found in the DNA sequence of mouse L1. These antisera will be used to characterize the pattern of expression of L1 proteins. Preliminary experiments suggest that F9 teratocarcinoma cells will be a fruitful source for further study of the intermediates involved in L1 expression and movement, because a "genomic" L1 RNA is made in these cells and appears to be associated with a non-polyribosomal particle. Other cell lines will be examined as well with a goal of identifying a cell source of L1 expression products to aid further study. Antibodies will be used to identify the L1 proteins expressed in F9 and other cells, and to characterize the particle by immunoelectron microscopy. The antibodies will also be used to screen mouse tissues by immunocytochemistry, focusing particularly on expression in embryos and germ cells. One of the open reading frames found in L1 is related to reverse transcriptases; enzyme assays will be done on extracts from both mouse cells and E. coli that are known to be expressing this region of L1. Finally, the ability of L1 to move into new locations in DNA will be tested using a selection for insertional inactivation of the thymidine kinase gene of herpes simplex virus.