Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.

Eurycoma longifolia Jack. is a treelet that grows in the forests of Southeast Asia and is widely used throughout the region because of its reported medicinal properties. Widespread harvesting of wild-grown trees has led to rapid thinning of natural populations, causing a potential decrease in genetic diversity amongE. longifolia. Suitable genetic markers would be very useful for propagation and breeding programs to support conservation of this species, although no such markers currently exist. To meet this need, we have applied a genome complexity reduction strategy to identify a series of single nucleotide polymorphisms (SNPs) within the genomes of several E.longifolia accessions. We have found that the occurrence of these SNPs reflects the geographic origins of individual plants and can distinguish different natural populations. This work demonstrates the rapid development of molecular genetic markers in species for which little or no genomic sequence information is available. The SNP markers that we have developed in this study will also be useful for identifying genetic fingerprints that correlate with other properties of E. longifolia, such as high regenerability or the appearance of bioactive metabolites.

Efficient single nucleotide polymorphism (SNP) genotyping methods are necessary to accomplish many current gene discovery goals. A crucial element in large-scale SNP genotyping is the number of individual biochemical reactions that must be performed. An efficient method that can be used to simultaneously amplify a set of genetic loci across a genome with high reliability can provide a valuable tool for large-scale SNP genotyping studies. In this paper we describe and characterize a method that addresses this goal. We have developed a strategy for reducing genome complexity by using degenerate oligonucleotide primer (DOP)-PCR and applied this strategy to SNP genotyping in three complex eukaryotic genomes; human, mouse, and Arabidopsis thaliana. Using a single DOP-PCR primer, SNP loci spread throughout a genome can be amplified and accurately genotyped directly from a DOP-PCR product mixture. DOP-PCRs are extremely reproducible. The DOP-PCR method is transferable to many species of interest. Finally, we describe an in silico approach that can effectively predict the SNP loci amplified in a given DOP-PCR, permitting the design of an efficient set of reactions for large-scale, genome-wide SNP studies. Single nucleotide polymorphisms (SNPs) are the most abundant resource of genetic variation among individuals of a species. They are the focus of large-scale genotyping projects in both humans and model organisms and the projected demands for human genome-wide association studies range into the tens and hundreds of millions of genotypes (1, 2). Large-scale mutagenesis projects and genetic modifier screens in model organisms such as Mus musculus and Arabidopsis thaliana are also driving the need for low-cost, high-efficiency genotyping. SNPs have the inherent potential for highly automated genotyping necessary for these studies and a broad range of SNP-based genotyping methodologies have been developed to date (for a recent review, see ref.3).However, SNP genotyping protocols developed to date require individual amplification of SNP-containing loci by PCR or some other biochemical reaction. The cost and labor required to carry out individual enzymatic reactions have led to a desire to combine, or multiplex, biochemical reactions. Multiplexing can improve efficiency, but predicting compatibility of reactions is increasingly challenging as the number of loci increases (4). An alternative strategy to address this problem is to develop a biochemical reaction that is preoptimized to achieve a similar goal to that of multiplexing.One approach that has been applied to this problem has been to use PCR primers complementary to interspersed repetitive sequences, such as the human alu sequence, to amplify subsets of unique genomic sequences with a single PCR primer. Although this approach has great utility in many contexts, it is limited in the subset of genomic sequences that can be amplified by the position of interspersed repetitive sequences in the genome. To achieve a broader representation of possible sequences that can be am...