Characterization of a Mouse Gene (Fbxw6) That Encodes a Homologue of Caenorhabditis elegans SEL-10

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box–WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.     

Biodiversity, genomes, and DNA sequence databases

There are ∼1.4 million organisms on this planet that have been described morphologically but there is no comparable coverage of biodiversity at the molecular level. Little more than 1% of the known species have been subject to any molecular scrutiny and eukaryotic genome projects have focused on a group of closely related model organisms. The past year, however, has seen an ∼80% increase in the number of species represented in sequence databases and the completion of the sequencing of three prokaryotic genomes. Large-scale sequencing projects seem set to begin coverage of a wider range of the eukaryotic diversity, including green plants, microsporidians and diplomonads.     

Unravelling the Leishmania genome

The past few years have seen significant advances in our understanding of eukaryotic genomes. In the field of parasitology, this is best exemplified by the application of genome mapping techniques to the study of genome structure and function in the protozoan parasite, Leishmania. Although much is known about the organism and the diseases it causes, molecular genetics has only recently begun to play a major part in elucidating some of the unusual characteristics of this interesting parasite. Mapping of the small (35 Mb) genome and determination of the functional role of genes by the application of in vitro homologous gene targeting techniques are revealing novel avenues for the development of prophylactic measures.     

Will genetics really revolutionize the drug discovery process?

Genetics has played only a modest role in drug discovery, but new technologies will radically change this. Whole genome sequencing will identify new drug discovery targets, and emerging methods for the determination of gene function will increase the ability to select robust targets. Detection of single nucleotide polymorphisms and common polymorphisms will enhance the investigation of polygenic diseases and the use of genetics in drug development. Oligonucleotide arraying technologies will allow analysis of gene expression patterns in novel ways.     

RGSZ1 and Ret RGS: Two of Several Splice Variants from the Gene RGS20

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

Genome research and evolution in trypanosomes

Trypanosoma brucei and Trypanosoma cruzi cause different human diseases. As strategies for immune evasion. T. brucei undergoes antigenic variation whereas T. cruzi becomes an intracellular organism. This fundamental difference is reflected by major differences in their genome organizations. Recent comparisons of their gene sequences indicate that these two trypanosome species are highly divergent evolutionarily.     

A High-Resolution Genetic, Physical, and Comparative Gene Map of the Doublefoot (Dbf) Region of Mouse Chromosome 1 and the Region of Conserved Synteny on Human Chromosome 2q35

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0. 4-cM (±0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.     

Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster

There is a rich diversity of paucimannose N-glycans in worms and flies, and these may play a role in the survival of these organisms. Although paucimannose N-glycans are not expressed in vertebrates, complex N-glycans may take over some of the functions of paucimannose N-glycans. Identification of the target proteins of β-1,2-N-acetylglucosaminyltransferase I (GnTI) in worms and flies and elucidation of their functions may thus lead to a better understanding of the role of GnTI-dependent glycoproteins in the survival/longevity of both invertebrates and vertebrates.     

单核细胞增生李斯特菌对秀丽隐杆线虫致病性的研究

单核细胞增生李斯特菌(Listeria monocytogenes,Lm)是李斯特菌病的病原细菌。用Lm野生株EGDe、弱毒株ΔprfA、毒力回复株+prfA和高毒株+prfA^*喂饲模式生物秀丽隐杆线虫N2,并以线虫的良好食源大肠埃希菌OP50以及非致病的无害李斯特菌（Listeria innocua）作为对照,检测Lm对线虫发育周期、寿命和产卵数的影响。结果显示：当以无害李斯特菌、Lm野生株以及PrfA突变株为食时,线虫的产卵数虽有所下降,但线虫不仅能够正常产卵,而且其发育周期和寿命均较以OP50为食时显著延长（P≤0.05）;线虫体表和消化道中均可检测到大量李斯特菌,但粪便中的活菌数极少。以上结果说明Lm不能杀死秀丽隐杆线虫,对线虫也没有显著致病性,不适合作为研究Lm致病机制的模型;Lm可在线虫体表和消化道存在,暗示Lm可借助线虫在土壤环境中生存和传播。     

Insights into the mechanisms of homologous recombination from the structure of RuvA

The recent structure determination of RuvA has provided the first insights into the structural basis for its interaction with Holliday junction DNA. Multiple copies of a helix-hairpin-helix motif which line the four grooves between the monomers in the tetrameric structure are thought to be involved in the interaction of the protein with its DNA target. This suggests that the four arms of the junction are held by RuvA in a fourfold symmetric arrangement and has fuelled ideas on the way in which components of the Ruv complex combine to catalyse the process of homologous recombination     

Identification of New Regulatory Sequences Far Upstream of the Mouse Monocyte Chemoattractant Protein-1 Gene

We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.     

Homology between a 173-kb Region from Mouse Chromosome 10, Telomeric to the Ifng Locus, and Human Chromosome 12q15

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).     

Single-Nucleotide Polymorphism Alleles in the Insulin Receptor Gene Are Associated with Typical Migraine

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.     

The dynamics of cyclin dependent kinase structure

In the past year, several new crystal structures have provided exciting insights into the conformational changes underlying the regulation of cyclin-dependent kinases. We now understand the structural basis of many of the mechanisms by which cyclin-dependent kinases are regulated, including activation by cycling binding and phosphorylation, inhibition by the inhibitor p27, and binding by the CKS proteins.     

秀丽隐杆线虫模型在抗衰老研究中的应用

随着人口老龄化问题的凸显,衰老相关的研究越来越被重视。秀丽隐杆线虫（Caenorhabditis elegans）是抗衰老研究领域中非常重要的生物模型,具有生命周期短、易于培养和观察等优点,但与其他哺乳动物模型相比仍有一些局限性,如DNA甲基化的缺乏等。本文主要综述了秀丽隐杆线虫模型在抗衰老研究和药物筛选中的应用,包括抗衰老药物对线虫寿命和抗性的测定与评估、药物筛选以及健康衰老研究中的应用,并概括了该模型的优势和局限性,为秀丽隐杆线虫模型在抗衰老研究中的应用提供理论依据。     

The integration of microarray information in the drug development process

In the past year, microarray technologies have moved beyond the proof-of-principle stage. Microarrays are now being used for genome-wide expression monitoring, large-scale polymorphism screening and mapping, and for the evaluation of drug candidates.