The long-term goal of this proposal is to investigate the organization of the subtelomeric regions of human chromosomes, and their relationship to the evolution of the olfactory receptor (OLFR) family. Evidence from several sources suggests that these regions are made up of large blocks of sequence which are present in various combinations at the telomeres of multiple chromosomes. The presence and number of these regions varies among chromosome and among individuals. The applicant hypothesizes that these subtelomeric regions have evolved by duplications, deletions and exchanges among homologous and nonhomologous chromosomes, that this process has been important for the generation of diversity in gene families as the OLFR, which are preferentially associated with these regions, and that they may form a source of clinically important chromosomal rearrangements.
The specific aims of the project are to characterize one such subtelomeric region, that on 3q, by (1) constructing a physical map of the region (2) identifying the distribution of the various repetitive elements on other chromosomes, and locating the boundaries of differentially distributed segments (3) sequencing 200 kb of 3q to identify genes in the region, and look for motifs that may predispose to recombination (4) assay normal variation at the cytogenetic and sequence levels and use this to describe the recent evolution of these regions and the genes they contain and (5) to test if genes duplicated near telomeres are expressed, and mapping additional OLFR genes to determine if association with subtelomeric regions is common in this family. The reference physical map of 3q will be constructed by walking using a chromosome 3 cosmid library arrayed on dense filters.
The aim i s to walk to the telomeric repeat sequence at one end, and to unique chromosomal DNA on the other. Walks will be confirmed by typing the clones for STS defined from the region, by FISH and by restriction fragment mapping. 10 cosmid clones distributed across the tiling path in the reference map will by used for FISH in 50 individuals representing 10 different human populations, as well as in 6 different primate species, dog and mouse. This will define the relative frequency of each block on each chromosome in different populations, and will be used to construct phylogenetic trees based on allele frequencies. 25 unrelated individuals from other populations will also studied using one cosmid showing particularly striking variation in chromosomal distribution. In addition STS mapping on flow sorted chromosomes and southern blotting on zoo-blots will be used to study the extent of sequence homology detected or undetected by FISH. The evolution of the subtelomeric regions will also be studied by sequencing the OLFRA and OLFRB genes from multiple chromosomes in 3 different populations chosen to maximize interpopulational differences. The chromosomal distribution of other OLFR genes in the human genome will be assessed using FISH with a pool of OLFR sequences obtained by PCR using degenerate primers, and known OLFR-containing clones. Assay for expression of subtelomeric OLFR genes will be attempted using RT-PCR on olfactory neuroepithelium mRNA, and hybridization or PCR on olfactory cDNA libraries, as well as sequence analysis to reveal pseudogenes.
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