Construction and characterization of a human-specific Xpter-Xq27.3 yeast artificial chromosome library; and chromosome walking to the Fragile X site

Jeannie Tsun-Huei Lee, University of Pennsylvania


The Fragile X Syndrome is the most common cause of heritable mental retardation, affecting one in 2000 male and one in 5000 female newborns. In affected families, mental retardation segregates as an X-linked dominant disorder with incomplete penetrance. As its name implies, the Fragile X Syndrome is associated with a rare fragile site at Xq27.3 that is inducible in cultured lymphoblasts under thymidine starvation. Whether fragility is causally associated with the clinical disorder or is, rather, merely a secondary consequence of the disease process is currently not known, but at the initiation of this thesis work, fragility was the only tangible marker available for the study of the Fragile X Syndrome. Thus, an understanding of this mental retardation syndrome required the cloning and characterization of the Xq27.3 fragile site and the subsequent identification of associated genes whose changes are causally related to the development of the disease. In this thesis, we undertook the task of cloning the fragile site. The hybrid cell line Micro-21D contains a human Fragile X chromosome which has been induced to break at Xq27.3 (presumably the fragile site) and then translocated onto a hamster chromosome. We have exploited the species chimerism at the breakpoint junction as a landmark readily identifiable by biochemical means. As part of our approach to cloning the breakpoint, we constructed a yeast artificial chromosome (YAC) library from Micro-21D. Characterization of this library revealed a coverage of 4.5 X-chromosome equivalents, an average YAC size of 280 kb, an a co-cloning rate of 11%. We then employed two complementary strategies to clone the Micro-21D breakpoint: first, the identification of a hamster-human double-positive clone derived from the Micro-21D breakpoint; and second, YAC-walking from tightly linked proximal markers to the fragile site. Walking from the proximal marker, pRS46-1.1, allowed us to generate a proximal YAC contig of 630 kb and eventually led us to identify XTY26, a YAC extending across the breakpoint of Micro-21D. From this YAC, others have isolated an unstable repeat sequence (GGC)n mapping just distal to the Micro-21D breakpoint. Our study of its inheritance pattern within Fragile X families suggests that expansion of this trinucleotide repeat occurs in affected individuals. The results of our findings and that of others are discussed in the context of the clinical phenotype and the existing models of Fragile X disease mechanism.

Subject Area

Molecular biology

Recommended Citation

Lee, Jeannie Tsun-Huei, "Construction and characterization of a human-specific Xpter-Xq27.3 yeast artificial chromosome library; and chromosome walking to the Fragile X site" (1993). Dissertations available from ProQuest. AAI9321430.