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.     

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.     

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.     

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.     

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.     

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.     

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     

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).     

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.     

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.     

Comparative Genomic Analysis of Genes Encoding Translation Elongation Factor 1Bα in Human and Mouse Shows EEF1B1 to Be a Recent Retrotransposition Event

We have characterized genomic loci encoding translation elongation factor 1Bα (eEF1Bα) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.     

Pax genes and their roles in cell differentiation and development

Members of the Pax gene family are expressed in various tissues during ontogenesis. Evidence for their crucial role in morphogenesis, organogenesis, cell differentiation and oncogenesis is provided by rodent mutants and human diseases. Additionally, recent experimental in vivo and in vitro approaches have led to the identification of molecules that interact with Pax proteins.     

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Résumé

À l'inverse de la situation des vertébrés et de la majorité des insectes, chez qui un gène unique code pour l'acétylcholinestérase (AChE), quatre gènes d'AChE ont été clones et caractérisés chez Caenorhabditis elegans. Le gène ace-1 (localisé sur le chromosome X) et le gène ace-2 (chromosome I) assurent respectivement l'expression de l'AChE dans les tissus musculaire (ace-1) et nerveux (ace-2). Les gènes ace-x et ace-y ne sont séparés que de quelques centaines de paires de bases sur le chromosome II et leur rôle est pour l'instant inconnu.     

Identification of an estrogenic hormone receptor in Caenorhabditis elegans

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.