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Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQmediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of Caenorhabditis elegans. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 ؎ 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform in vivo suppression of polyQ-mediated neuronal dysfunction.

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, we performed a series of preliminary experiments aimed at developing a suitable protocol. We found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.To physically map genomes of complex organisms a number of techniques have been developed. These techniques include pulsed-field gel electrophoresis (1, 2), chromosome jumping (3), and cloning of large DNA fragments in yeast artificial chromosomes (YACs) (4,5). Physical mapping, isolation of large genes plus all regulatory sites, and description of long-range genomic organization will depend on isolating and analyzing fragments of several hundred kilobase pairs of contiguous DNA. Intuitively appealing, the YAC system must be rigorously examined to understand cloning efficiency as a function of size and the extent to which the clones faithfully represent their genomic origin. Until recently (6), all YAC experiments had been carried out with DNA purified and manipulated in solution. In these early experiments one major problem has been small mean size of the YACs. This is probably due to extended handling ofDNA in solution. As can1-100 ura3-1 leul gall 4+ SUQS), W839-1C (a ade2-1 can1-100 or canl-J00,x his3-11,JS leu2-3,112 trpl-1 ura3-1 radl ::LEU2+ radS2-8::TRPI +), Y90 (a ura3-52 lys2-801 ade2-101 trpl-901), Y93 (a ura3-52 lys2-801 ade2-101 trpl-901 his3-A200), and YNN281 (a trpl-A his3-A200 ura3-52 lys2-801 ade2-1 gallb). Media. All yeast cultures were grown on either complete medium (YPD) or selective medium lacking uracil or uracil and tryptophan. These media have been described by Sherman et al. (10 4256The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.