Lynn Doucette-Stamm

A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes

The Huntingtons disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

Towards a proteome-scale map of the human protein-protein interaction network.

Systematic mapping of protein–protein interactions, or ‘interactome’ mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein–protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of ∼8,100 currently available Gateway-cloned open reading frames and detected ∼2,800 interactions. This data set, called CCSB-HI1, has a verification rate of ∼78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by ∼70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum

The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.

A protein–protein interaction map of the Caenorhabditis elegans 26S proteasome

The ubiquitin‐proteasome proteolytic pathway is pivotal in most biological processes. Despite a great level of information available for the eukaryotic 26S proteasome—the protease responsible for the degradation of ubiquitylated proteins—several structural and functional questions remain unanswered. To gain more insight into the assembly and function of the metazoan 26S proteasome, a two‐hybrid‐based protein interaction map was generated using 30 Caenorhabditis elegans proteasome subunits. The results recapitulate interactions reported for other organisms and reveal new potential interactions both within the 19S regulatory complex and between the 19S and 20S subcomplexes. Moreover, novel potential proteasome interactors were identified, including an E3 ubiquitin ligase, transcription factors, chaperone proteins and other proteins not yet functionally annotated. By providing a wealth of novel biological hypotheses, this interaction map constitutes a framework for further analysis of the ubiquitin‐proteasome pathway in a multicellular organism amenable to both classical genetics and functional genomics.

Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or “ORFeomes,” need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.

Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping

Yeast one-hybrid (Y1H) assays provide a gene-centered method for the identification of interactions between gene promoters and regulatory transcription factors (TFs). To date, Y1H assays have involved library screens that are relatively expensive and laborious. We present two Y1H strategies that allow immediate prey identification: matrix assays that use an array of 755 individual Caenorhabditis elegans TFs, and smart-pool assays that use TF multiplexing. Both strategies simplify the Y1H pipeline and reduce the cost of protein-DNA interaction identification. We used a Steiner triple system (STS) to create smart pools of 4–25 TFs. Notably, we uniplexed a small number of highly connected TFs to allow efficient assay deconvolution. Both strategies outperform library screens in terms of coverage, confidence and throughput. These versatile strategies can be adapted both to TFs in other systems and, likely, to other biomolecules and assays as well.

Functional modularity of nuclear hormone receptors in a Caenorhabditis elegans metabolic gene regulatory network

Gene regulatory networks (GRNs) provide insights into the mechanisms of differential gene expression at a systems level. GRNs that relate to metazoan development have been studied extensively. However, little is still known about the design principles, organization and functionality of GRNs that control physiological processes such as metabolism, homeostasis and responses to environmental cues. In this study, we report the first experimentally mapped metazoan GRN of Caenorhabditis elegans metabolic genes. This network is enriched for nuclear hormone receptors (NHRs). The NHR family has greatly expanded in nematodes: humans have 48 NHRs, but C. elegans has 284, most of which are uncharacterized. We find that the C. elegans metabolic GRN is highly modular and that two GRN modules predominantly consist of NHRs. Network modularity has been proposed to facilitate a rapid response to different cues. As NHRs are metabolic sensors that are poised to respond to ligands, this suggests that C. elegans GRNs evolved to enable rapid and adaptive responses to different cues by a concurrence of NHR family expansion and modular GRN wiring.

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.

Generation and characterization of irradiation hybrids of human chromosome 4

In recent years investigators have attempted to develop more rapid and precise methods to isolate specific chromosomal DNA regions. In this paper we demonstrate a modification of the method first developed by Goss and Harris for generation of irradiation hybrids. The gene encoding the dominant selectable marker for resistance to neomycin was introduced into human chromosome 4 using retroviral insertion into human fibroblasts. Transfer of these chromosomes via microcells into the mouse cell line NIH3T6 produced a somatic cell line containing chromosome 4 as the only human chromosome. Irradiation of this cell line followed by fusion with the hamster cell line CHTG49 generated hybrids containing only small portions of chromosome 4p on a hamster background. The use of selection produced stable hybrids that retained chromosome 4 fragments over long periods of tissue culture passage. To obtain new polymorphic markers for Huntingtons disease, one of these hybrids was used to isolate new genomic fragments. We identified 41 single-copy fragments, of which 27 have been mapped to specific regions of chromosome 4; 52% of these fragments map to the region of chromosome 4 containing theHD gene.

Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region.

The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome. We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene. A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb. Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168. The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA. These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region.