p22 dynactin light chain (DCTN3): Positional cloning and investigation for involvement in cartilage-hair hypoplasia

The goal of this project was to implement a positional cloning strategy to identify novel genes from human chromosome region 9p13 that may be involved in human disease. Human 9p13 has been previously implicated in several cancers and the inherited disease cartilage-hair hypopasia. We constructed an integrated large-insert clone-based physical map of human 9p13 comprised of 131 clones and 135 markers. The markers included 42 novel expressed sequence tags and 23 known genes. During the course of this project we found an interstitial subtelomeric-related sequence in 9p13 that also mapped to chromosomes 4, 5, 6, 13, and X.;The gene for the p22 dynactin light chain (DCTN3) was identified using positional cloning methods. DCTN3 was localized to human 9p13 by radiation hybrid mapping and fluorescence in situ hybridization. Rat comparative mapping localized Dctn3, and two flanking genes, Galt and Cntfr, to rat chromosome 5. The DCTN3 gene consists of 7 exons and 6 introns and spans approximately 6.9 kb. Exon sequencing failed to identify functional mutations in cartilage-hair hypopasia-affected patients. A neutral (C/T) single-nucleotide polymorphism (SNP) was identified at nucleotide position 34 of exon 1. Single strand conformation polymorphism (SSCP) screening of 100 individuals demonstrated a low 2% frequency for this SNP. The role of this gene is now being examined in cancer.;We have developed a YAC-based complementation strategy to identify target candidate genes by gene transfer. Eight YAC clones were selected from our physical map that spans human 9p13. These clones were retrofitted with the neo resistance plasmid pDC47. Southern blotting and PCR data demonstrate that pDC47 properly integrated into the left arm of each YAC clone. A spheroplast fusion protocol was optimized that successfully transferred each YAC into the hamster CHTG cell line providing functional evidence that the integrated plasmid confers G418 resistance to resulting fusion colonies. The functional complementation assay is being used with traditional positional cloning methods to identify additional genes that may be involved in proliferative disorders.