Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72–80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cryl-AI::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage; using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere- proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cryl-A2::TRP1 cry2-AI::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRYI: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a Crys phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a Crys phenotype. (2) Haploids containing the cryl-A2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-AI::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of 13-galactosidase are expressed from a CRYI-lacZ gene fusion than from a CRY2-1acZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cryl), the CryR phenotype of cry2 mutants is only expressed in strains containing a cryl-A null allele.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development. Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of the Cdkrelated kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments. Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced. Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity.

The Huntington's disease (HD) gene encodes a protein, huntingtin, with no known function and no detectable sequence similarity to other proteins in current databases. To gain insight into the normal biological role of huntingtin,we isolated and sequenced a cDNAencoding a protein that is a likely homolog of theHD gene product in Drosophila melanogaster. We also determined the complete sequence of 43 125 contiguous base pairs of genomic DNA that encompass the Drosophila HD gene, allowing the intron-exon structure and 5'- and 3'-flanking regions to be delineated. The predicted Drosophila huntingtin protein has 3583 amino acids, which is several hundred amino acids larger than any other previously characterized member of the HDfamily. Analysis of the genomic and cDNA sequences indicates that Drosophila HD has 29 exons, compared with the 67 exons present in vertebrate HD genes, and that Drosophila huntingtin lacks the polyglutamine and polyproline stretches present in its mammalian counterparts. TheDrosophila HD mRNA is expressed in a broad range of developmental stages and in the adult, a temporal pattern of expression similar to that observed for mammalian HD transcripts. We can discern five regions of high similarity from multiple sequence alignments between Drosophila and vertebrate huntingtins. These regions may define functionally important domains within the protein.

Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase that displays kinase activity predominantly in neurons, is activated by two non-cyclin activators, p35 or p39. Here, we report a physical and functional interaction between the Cdk5/p35 complex and mouse Sds3 (mSds3), an essential component of mSin3-histone deacetylase (HDAC) co-repressor complex. mSds3 binds to p35 both in vitro and in vivo, enabling active Cdk5 to phosphorylate mSds3 at serine 228. A mSds3 S228A mutant retained mSin3 binding activity, but its dimerization was not greatly enhanced by p35 when compared with wild type. Notably, p35 overexpression augmented mSds3-mediated transcriptional repression in vitro. Interestingly, mutational studies revealed that the ability of exogenous mSds3 to rescue cell growth and viability in mSds3 null cells correlates with its ability to be phosphorylated by Cdk5. The identification of mSds3 as a substrate of the Cdk5/p35 complex reveals a new regulatory mechanism in controlling the mSin3-HDAC transcriptional repressor activity and provides a new potential therapeutic means to inhibit specific HDAC activities in disease.