This application continues to pursue the major goals of the prior proposal. Historically, the three projects being pursued through this proposal developed simultaneously from the investigators interest in the association between viral sequences and histocompatibility (H) genes. Studies of the H-30 locus kindled the investigators interest in the nearby Ly-6 locus. During the next funding period Dr. Meruelo and coworkers seek to accomplish three major specific aims.
The first aim i s to complete the physical map of the 1200 kb Ly6 region of C57BL/6 mice. Also inherent within this aim is the identity and position, within the locus, all Ly-6 coding sequences/genes and the identity of products of Ly-6 genes using transfection analysis and monoclonal antibodies. An attempt will be made to understand the regulation of expression of these genes and their functional role(s). The last endeavor of this specific aim will be an attempt to identify the human counterpart of the Ly-6 locus. The second specific aim is to continue efforts to identify and characterize endogenous retroviral sequences within the MHC and to explore the possible role of these sequences on class I expression. The last specific aim entails the use of field inversion gel electrophoresis as well as existing and new probes to develop a fine molecular map of the Ly-6 --H-30 region. To generate new probes, three approaches will be taken. First, the investigators will screen subtracted cDNA libraries by subtractive hybridization. Second, these workers will use insertional mutagenesis to obtain markers from this region of DNA. Finally, Dr. Meruelo and coworkers will use the phenol emulsion reassociation (PERT) technique which promotes the very rapid reassociation of DNA permitting the specific cloning of DNA fragments absent in one mouse strain and present in another using very small amounts of DNA. After establishing RFLP boundaries close enough to H-30, the investigators will use cosmid walking methodologies and chromosome hopping techniques to clone H-30 candidate genes. Potential H-30 candidates will be assayed by transfection analysis and construction of transgenic mice. When these workers demonstrate that a cloned gene from this region is recognized by specific anti-H-30 CTLs, and transgenic mice carrying this gene reject H-30 incompatible skin grafts, then the investigators will refer to that gene as H-30, and molecularly define the H-30 gene and serologically and biochemically characterize its product.
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