Two related projects: RDA and microarray analysis summarized in abstract form below:RDA: Gene isolation methods used during positional cloning rely on physical contigs consisting of bacterial artificial chromosomes, P1 or cosmid clones. However, in most instances, the initial framework for physical mapping consists of contigs of yeast artificial chromosome (YACs), large vectors which are suboptimal substrates for gene isolation. Here we report a strategy to identify gene sequences contained within a YAC by using cDNA representational difference analysis (RDA) to directly isolate transcripts expressed from the YAC in mammalian cells. The RDA tester cDNAs were generated from a previously reported hamster cell line derived by stable transfer of a 590 kb YAC (911D5) which expressed NPC1, the human gene responsible for Niemann-Pick type C (NP-C). The driver cDNAs were generated from a control hamster cell line which did not contain the YAC that expressed NPC1. Among the gene fragments obtained by RDA, NPC1 was the most abundant product. In addition, two non-NPC1 fragments were isolated which were mapped to and expressed from 911D5. One of these RDA gene fragments (7-R) spans more than one exon and has 98% sequence identity with a human cDNA clone previously reported as an expressed sequence tag (EST), but not mapped to a chromosomal region. The other fragment (2-R) which had no significant sequence similarities with known mammalian genes or ESTs, was further localized to the region of overlap between YACs 911D5 and 844E3. The latter YAC is part of a contig across the NP-C candidate region, but does not contain NPC1. This two part approach in which stable YAC transfer is followed by cDNA RDA should be a useful adjunct strategy to expedite the cloning of human genes when a YAC contig is available across a candidate interval.Microarray analysis: The goals of this study were to assess whether microarrays could be useful for disease gene identification, genomic structural analysis and determination of other genes potentially contributing to a disease phenotype. A model system was developed based on Niemann-Pick type C (NP-C) and the gene NPC1 that is mutated in this disease. First, NPC1 exons were identified using an array of genomic fragments from a bacterial artificial chromosome (BAC), 108N2, which encodes NPC1. The NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and were used to determine part of the NPC1 genomic structure. Next, to demonstrate that NPC1 could be identified based upon differential gene expression, sub-arrays of 108N2 fragments were probed with cDNA generated from hamster cell lines differentially expressing NPC1. The NP-C cell line CT60 probe did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by the probe from the non-NP-C cell line 911D5A13, derived from CT60 and expressing NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Finally, to identify genes differentially expressed in NP-C, fibroblast cDNAs from NP-C and unaffected individuals were hybridized to arrays containing human expressed sequence tags. As expected, activation of endogenous cholesterol biosynthesis was indicated by increased expression of enzymes in this pathway. Among the other differentially expressed genes was an unexpected candidate, CD9, a tetra-membrane spanning protein of unknown function that is present in the myelin sheath and may play role in Schwann cell migration. These data suggest the potential usefulness of array analysis to investigate the genomic structure of a disease gene and to identify candidate genes with potential roles in disease pathogenesis, using NP-C as a model system.Ongoing work involves gene expression analysis in tissues from NP-C mice compared to aged matched wild type controls and various NP-C human cell lines compared to age/sex matched controls. - cholesterol research genetics human genome research
