9806033 Martin This POWRE project will support a one year Visiting Researcher Fellowship for Sandra L. Martin. The planned research is ideally suited for a POWRE award because the PI will gain experience with a wide range of new biochemical techniques while on sabbatical leave at the Salk Institute. The main goals of PI are to acquire new biochemical skills and then apply them to studies of LINE-1 retrotransposition. LINE-1 (long interspersed repeated sequence one, or L1 ) is a major dynamic force in the mammalian genome. It is a transposable element that has amplified to high copy number numerous times during evolution. Retrotransposition deposits the progeny of L1 throughout the genome, sometimes leading to gene disruption, modified expression of adjacent genes, and/or transduction of neighboring DNA. In addition, L1, as interspersed, repetitive DNA, provides a substrate for homologous recombination of mispaired sequences, leading to gene duplication, deletion, chromosome translocation, and potentially, exon shuffling. LINE-1 retrotransposition begins with transcription of a full-length, sense-strand L1 RNA and requires two L1-encoded polypeptides which are the products of open reading frames one and two, ORFI and ORF2. The two L1-encoded proteins likely also catalyze the reverse transcription and integration of SINEs (short interspersed repeated sequences) and processed pseudogenes, thereby amplifying the effects of LINE-1 in mammalian genome dynamics. The long-range goal of research in the PI's laboratory is to understand the retrotransposition process in detail, including the biochemical intermediates involved, as well as its control in genetic and evolutionary time. The sabbatical project is designed to develop the recipient's skills as a biochemist while devising purification and assay methods for L1 retrotransposition intermediates. Experimental effort will be directed along two fronts, development of: l) assays for detection of steps in the L1 retrotransposition cycle, and 2) metho ds for purification of L1 ribonucleoprotein particles which are hypothesized to be intermediates in the retrotransposition reaction. Because of their complementary nature, work on these two objectives will proceed simultaneously. This effort will be greatly facilitated by working in Dr. Frederic Bushman's laboratory at the Salk Institute because of its expertise in using similar approaches to study the integration reaction of retroviral pre-integration complexes. Between 10 and 20% of the genome in all mammals is comprised of the long interspersed repetitive element, LINE-1. LINEs have replicated to this high copy number by a process of duplicative transposition that uses an RNA intermediate, also known as retrotransposition. When LINE elements replicate, a new copy is inserted into a new site within the genome. Sometimes the insertion disrupts an essential gene, which can lead to loss of function of that gene, but most LINE insertions do not cause mutations. Little is known about the replication cycle of LINE elements, but it is clear that the two proteins encoded by LINEs are essential for the retrotransposition process. The goal of this work is to develop an in vitro system for the retrotransposition reaction. This requires development of novel assays to detect various steps in the process of retrotransposition as well as methods for purification of transposition intermediates, including the two proteins made by the LINE elements themselves. Once these assays and materials are available, they will be used to dissect the pathway of LINE retrotransposition in detail. This information may be used in the future to prevent mutations caused by LINE insertions, and/or to harness LINE retrotransposition for use as a tool in biotechnology.
