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Analysis of the human genome draft sequences has revealed a more complete portrait of the olfactory receptor gene repertoire in humans than was available previously. The new information provides a basis for deeper analysis of the functions of the receptors, and promises new insights into the evolutionary history of the family.
Several lines of evidence suggest that the X chromosome of various animal species has an unusual complement of genes with sex-biased or sex-specific expression. However, the study of the X chromosome gene content in different organisms provided conflicting results. The most striking contrast concerns the male-biased genes, which were reported to be almost depleted from the X chromosome in Drosophila but overrepresented on the X chromosome in mammals. To elucidate the reason for these discrepancies, we analysed the gene content of the Z chromosome in chicken. Our analysis of the publicly available expressed sequence tags (EST) data and genome draft sequence revealed a significant underrepresentation of ovary-specific genes on the chicken Z chromosome. For the brain-expressed genes, we found a significant enrichment of male-biased genes but an indication of underrepresentation of female-biased genes on the Z chromosome. This is the first report on the nonrandom gene content in a homogametic sex chromosome of a species with heterogametic female individuals. Further comparison of gene contents of the independently evolved X and Z sex chromosomes may offer new insight into the evolutionary processes leading to the nonrandom genomic distribution of sex-biased and sex-specific genes.
HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.
PURPOSE: Identification of causative mutations for retinal blinding disorders is often limited by restricted understanding of gene expression and underlying molecular mechanisms that trigger degenerative processes. This study was conducted to develop a catalog of canine retina-expressed genes that would provide a unique tool to investigate normal and altered function in the adult retina. Because of the conserved syntenies between the dog and human, this approach would identify new potential disease candidate genes for both species. METHODS: A canine normalized retinal cDNA library was produced and analyzed by using a modified PhredPhrap algorithm. Computerized annotation provided gene homology and chromosomal location for individual clones and contigs in a Web-accessible database. RESULTS: From 6316 cDNA clones, 3980 retinal expressed sequence tags (ESTs) were derived. Homology to the canine genome draft sequence was found for more than 99% of all ESTs, but only for 32% when compared with annotated canine cDNAs. Functional analysis suggests an enrichment of this library for genes involved with eye function and development, chaperone, or ribosomal functions when compared with mouse and human National Center for Biotechnology Information (NCBI) RefSeq entries. CONCLUSIONS: A combination of annotation approaches with ongoing mapping and expression studies provide functional data covering at least 27% to 30% of the currently proposed canine catalog of genes expressed in the retina. This is an essential first step toward establishing an integrated network for gene identification and expression patterns suitable for functional genetics, comparative genomics and evolutionary analysis of genes and gene families with respect to the developmental and degenerative processes of the retina.
The genomes of three strains of Listeria monocytogenes that have been associated with food-borne illness in the USA were subjected to whole genome comparative analysis. A total of 51, 97 and 69 strain-specific genes were identified in L.monocytogenes strains F2365 (serotype 4b, cheese isolate), F6854 (serotype 1/2a, frankfurter isolate) and H7858 (serotype 4b, meat isolate), respectively. Eighty-three genes were restricted to serotype 1/2a and 51 to serotype 4b strains. These strain- and serotype-specific genes probably contribute to observed differences in pathogenicity, and the ability of the organisms to survive and grow in their respective environmental niches. The serotype 1/2a-specific genes include an operon that encodes the rhamnose biosynthetic pathway that is associated with teichoic acid biosynthesis, as well as operons for five glycosyl transferases and an adenine-specific DNA methyltransferase. A total of 8603 and 105 050 high quality single nucleotide polymorphisms (SNPs) were found on the draft genome sequences of strain H7858 and strain F6854, respectively, when compared with strain F2365. Whole genome comparative analyses revealed that the L.monocytogenes genomes are essentially syntenic, with the majority of genomic differences consisting of phage insertions, transposable elements and SNPs.
Many Gram-negative plant and animal pathogens share a common virulence strategy that relies on the specialized type III secretion system. This apparatus is used to secrete virulence factors, called effectors, into the extracellular host environment and directly into the cytoplasm of host cells. Effectors interfere with host signaling and host metabolism to create an optimal environment for pathogen replication. The identification of effectors in plant pathogens was limited for many years to those effectors that elicit strong plant defenses on some hosts. The members of this subset, called avirulence proteins, can be readily identified because they dominantly confer strong defense-inducing properties to a heterologous virulent strain. This chapter describes two methods to identify type III-secreted effectors in plant pathogens independently of their phenotype. The first method consists of an in vivo molecular genetic screen that uses the activity of an avirulence protein to identify effectors without avirulence activity. It should be possible to apply this method to most Gram-negative plant pathogens. The second method consists of a bioinformatic approach applicable to those pathogens for which at least a draft genome sequence is available.
Olfactory receptors likely constitute the largest gene superfamily in the vertebrate genome. Here we present the nearly complete human olfactory subgenome elucidated by mining the genome draft with gene discovery algorithms. Over 900 olfactory receptor genes and pseudogenes (ORs) were identified, two-thirds of which were not annotated previously. The number of extrapolated ORs is in good agreement with previous theoretical predictions. The sequence of at least 63% of the ORs is disrupted by what appears to be a random process of pseudogene formation. ORs constitute 17 gene families, 4 of which contain more than 100 members each. "Fish-like" Class I ORs, previously considered a relic in higher tetrapods, constitute as much as 10% of the human repertoire, all in one large cluster on chromosome 11. Their lower pseudogene fraction suggests a functional significance. ORs are disposed on all human chromosomes except 20 and Y, and nearly 80% are found in clusters of 6-138 genes. A novel comparative cluster analysis was used to trace the evolutionary path that may have led to OR proliferation and diversification throughout the genome. The results of this analysis suggest the following genome expansion history: first, the generation of a "tetrapod-specific" Class II OR cluster on chromosome 11 by local duplication, then a single-step duplication of this cluster to chromosome 1, and finally an avalanche of duplication events out of chromosome 1 to most other chromosomes. The results of the data mining and characterization of ORs can be accessed at the Human Olfactory Receptor Data Exploratorium Web site (http://bioinfo.weizmann.ac.il/HORDE).
Microarray analyses is applied for prognosis of gastric cancer, including risk assessments of lymph-node metastasis and peritoneal dissemination. EST AA769445 derived from an unknown gene is reported to be frequently down-regulated in intestinal-type gastric cancer based on microarray analyses. Here, we identified and characterized the gene corresponding to EST AA769445 by using bioinformatics. EST AA769445 overlapped with MGC29506 cDNA (BC021275) and PACAP cDNA (AF338109). MGC29506 protein (189 aa) and PACAP protein (123 aa) were identical in codon 1-59, but were divergent in the C-terminal region due to a frame shift caused by sixteen-base insertion in PACAP cDNA. MGC29506 and PACAP were derived from the same gene, consisting of four exons, due to alternative splicing of alternative splice-acceptor-site type. Fourteen human ESTs were the MGC29506 type, while one human EST was the PACAP type. MGC29506 was the major isoform of the MGC29506/PACAP gene on human chromosome 5q31.2. MGC29506 orthologs in other species were next searched for. AK088094 and AK008016 cDNAs with nucleotide substitutions resulting in four-amino-acid substitutions were derived from mouse Mgc29506 gene. Rat Mgc29506 gene was identified in the rat genome draft sequence AC135285. ESTs AJ396310 and AJ441435 were derived from chicken Mgc29506 gene, while ESTs BW315632 and BW251767 from ciona mgc29506 gene. N-terminal signal peptide, six cysteine residues and other amino acids were conserved among human, mouse, rat, chicken, and ciona MGC29506 proteins. MGC29506 gene, frequently down-regulated in intestinal-type gastric cancer, was found to encode secreted-type protein with six conserved cysteine residues.
A Gram-stain-negative, aerobic, non-motile, catalase- and oxidase-positive, white rod-shaped strain, RF13T, was isolated from water samples of the Qingliang River in Fucheng County, Hebei Province, China, and was grown at 15-42 °C (optimum 35 °C), pH 6.0-8.0 (optimum pH 7), and 0-0.5% (w/v) NaCl (optimum concentration 0%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RF13T belonged to the genus Albidovulum, with closest sequence similarity to Albidovulum salinarum MCCC 1K0602T (97.2%), Frigidibacter oleivorans CGMCC 1.3778T (97.2%), Allgaiera indica MCCC 1A01802T (96.8%), and Pseudothioclava arenosa KCTC 52190T (96.4%). The genome size of strain RF13T was 3.7 Mb, and the DNA G+C content was 64.6%. The DNA-DNA hybridisation value (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI) between strain RF13T and the reference strain were less than 20.0%, 78.8%, and 72.8%, respectively. Chemotaxonomic analysis revealed Summed feature 8 (48.4%) (C18:1 ω6c and/or C18:1 ω7c), C18:1 ω7c 11-methyl (22.1%), C18:0 3OH (7.9%), and C10:0 3OH (5.0%) as predominant fatty acids. The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, two unidentified aminolipids, two unidentified phospholipids, and three unidentified lipids. The predominant isoprenoid quinone was ubiquinone-10 (Q-10), and a small amount of Q-9 was also detected. In addition, strain RF13T exhibited a minimum inhibitory concentration (MIC) of 20 mM for molybdate in R2A broth medium and was capable of reducing molybdate to molybdenum blue. Based on the results of biochemical, physiological, phylogenetic, and chemotaxonomic analyses, combined with 16S rRNA gene sequence analyses and draft genome sequence comparisons, strain RF13T was considered to represent a novel species of the genus Albidovulum, and was therefore named Albidovulum molybdatiresistens sp. nov. The type strain was RF13T (= GDMCC 1.3414T= JCM 35643T).
The PPP1R1B-STARD3-TCAP-PNMT-MGC9753-ERBB2-MGC14832-GRB7 locus on human chromosome 17q12 is frequently amplified in human gastric and breast cancer. We have recently identified and characterized human MGC9753 (also known as wild-type CAB2) and mouse Mgc9753. Here, we identified and characterized mouse Erbb2 gene by using bioinformatics. BLAST programs revealed that mouse AK031099 cDNA was derived from mouse Erbb2 gene. Because AK031099 cDNA showed 806 C-->A nucleotide substitution compared with mouse genome draft sequences and mouse Erbb2 ESTs, the nucleotide sequence of mouse Erbb2 cDNA was determined in silico by correcting 806 A of AK031099 cDNA to C. Nucleotide position 48-3818 of mouse Erbb2 cDNA was the coding region. Mouse Erbb2 gene, consisting of 27 exons, was located within the Ppp1r1b-Grb7 locus on the mouse chromosome 11. Mouse Erbb2 protein (1256 aa) showed 87.5% total-amino-acid identity with human ERBB2 protein, and 95.2% total-amino-acid identity with rat Erbb2 protein. Mouse Ppp1r1b-Grb7 locus and human Ppp1r1b-Grb7 locus were evolutionarily conserved in the order and the orientation of genes therein. Nucleotide and amino-acid substitution rates of Neurod2 located centromeric to the Ppp1r1b-Grb7 locus were significantly lower than others within the Ppp1r1b-Grb7 locus. This is the first report on the complete coding sequence of mouse Erbb2 gene as well as on the comprehensive comparison of Ppp1r1b-Grb7 locus within the human and mouse genomes.
Human Frizzled-7 (FZD7) and human FzE3, showing 98.8% nucleotide identity, encode almost identical WNT receptors with nine amino-acid substitutions. FzE3 is claimed to be expressed specifically in esophageal cancer. We determined the structure of the FZD7 gene and the FZD7 cDNA expressed in esophageal cancer. The FZD7 gene without intron and the FZD7 cDNAs isolated from esophageal cancer cell lines TE4 and TE5 were found to encode WNT receptor identical to FZD7, but not to FzE3. Nucleotide sequence of FzE3 was not identified on the human genome draft sequence. Thus, we could not obtain any data suggesting the existence of FzE3. Expression profile of FZD7 was also investigated. FZD7 was expressed throughout normal gastrointestinal tract, from esophagus to rectum. Among human esophageal and gastric cancer cell lines, expression level of FZD7 was relatively lower in esophageal cancer cell lines, and was highest in the gastric cancer cell line MKN7. FZD7 was up-regulated in one out of six cases of human primary gastric cancer. As over-expression of Frizzled-7 leads to activation of the WNT-beta-catenin-TCF pathway, up-regulation of FZD7 in human gastric cancer might play key roles in carcinogenesis through activation of the WNT-beta-catenin-TCF pathway.
WNT signals are transduced to the JNK pathway, the Ca2+-releasing pathway, or the beta-catenin - TCF pathway through seven-transmembrane-type WNT receptors encoded by Frizzled genes (FZD1-FZD10). WRCH1/ARHV and CDC42 are potentially implicated in the WNT-JNK pathway. Here, WRCH2/ARHV cDNAs were isolated by using bioinformatics and cDNA-PCR. WRCH2 gene, consisting of at least 3 exons, encoded a 236-amino-acid protein with proline-rich domain and GTPase domain. WRCH2 was homologous to WRCH1 (55.4% total-amino-acid identity) and CDC42 (43.5% total-amino-acid identity). WRCH2 gene was located on human chromosome 15q15, which is one of fragile sites in the human genome. A single nucleotide substitution (632 Gright curved arrow A) was identified between WRCH2 cDNA and human genome draft sequences, which resulted in Arg177Lys amino-acid substitution. WRCH2 mRNA was relatively highly expressed in pancreas, placenta, and fetal brain. WRCH2 mRNA was over-expressed in TMK1 (gastric cancer), Hs700T (pancreatic cancer), HeLa S3 (cervical cancer), and A549 (lung cancer). WRCH2 mRNA was moderately expressed in MKN74, MKN45, MKN28, KATO-III (gastric cancer), HL-60 (pro-myelocytic leukemia), Raji (Burkitt's lymphoma), and SW480 (colorectal cancer). WRCH2 mRNA was up-regulated in 3 out of 8 cases of primary gastric cancer. Because Wrch1 can activate PAK1 and JNK1, and induce filopodium formation and stress fiber dissolution, over-expression of WRCH2 mRNA in human cancer cells might also lead to more malignant phenotype.
Although approximately one-quarter of the roughly 4,000 genetically inherited diseases currently recorded in respective databases (LocusLink, OMIM) are already linked to a region of the human genome, about 450 have no known associated gene. Finding disease-related genes requires laborious examination of hundreds of possible candidate genes (sometimes, these are not even annotated; see, for example, refs 3,4). The public availability of the human genome draft sequence has fostered new strategies to map molecular functional features of gene products to complex phenotypic descriptions, such as those of genetically inherited diseases. Owing to recent progress in the systematic annotation of genes using controlled vocabularies, we have developed a scoring system for the possible functional relationships of human genes to 455 genetically inherited diseases that have been mapped to chromosomal regions without assignment of a particular gene. In a benchmark of the system with 100 known disease-associated genes, the disease-associated gene was among the 8 best-scoring genes with a 25% chance, and among the best 30 genes with a 50% chance, showing that there is a relationship between the score of a gene and its likelihood of being associated with a particular disease. The scoring also indicates that for some diseases, the chance of identifying the underlying gene is higher.
Processed pseudogenes were created by reverse-transcription of mRNAs; they provide snapshots of ancient genes existing millions of years ago in the genome. To find them in the present-day human, we developed a pipeline using features such as intron-absence, frame-disruption, polyadenylation, and truncation. This has enabled us to identify in recent genome drafts approximately 8000 processed pseudogenes (distributed from http://pseudogene.org). Overall, processed pseudogenes are very similar to their closest corresponding human gene, being 94% complete in coding regions, with sequence similarity of 75% for amino acids and 86% for nucleotides. Their chromosomal distribution appears random and dispersed, with the numbers on chromosomes proportional to length, suggesting sustained "bombardment" over evolution. However, it does vary with GC-content: Processed pseudogenes occur mostly in intermediate GC-content regions. This is similar to Alus but contrasts with functional genes and L1-repeats. Pseudogenes, moreover, have age profiles similar to Alus. The number of pseudogenes associated with a given gene follows a power-law relationship, with a few genes giving rise to many pseudogenes and most giving rise to few. The prevalence of processed pseudogenes agrees well with germ-line gene expression. Highly expressed ribosomal proteins account for approximately 20% of the total. Other notables include cyclophilin-A, keratin, GAPDH, and cytochrome c.
Neuroblast undergoes asymmetrical cell division to produce the neuroblast itself and ganglion mother cell along the apical-basal axis. Inscuteable (Insc) and Partner of Inscuteable (Pins) are translocated to the apical cell cortex during asymmetrical cell division of Drosophila neuroblast. Insc is implicated in the apical-basal orientation of mitotic spindle and the basal localization of Prospero (Pros) and Numb. Here, we identified and characterized human Inscuteable (INSC) gene using bioinformatics. Human INSC gene, consisting of at least 13 exons, was located within human genome draft sequence AC090744.5 (around nucleotide position 150581-16936 in reverse orientation). Human INSC gene, closely linked to CALCB gene with an interval of about 30 kb, was assigned to human chromosome 11p15.2-p15.1. Amino-acid sequence of human INSC polypeptide (579 aa) was determined based on exon sequences of human INSC gene. C. elegans hypothetical protein F43E2.3 (NP_495539), homologous to human INSC, was designated C. elegans Insc. Central INSC homologous (ISH) domain and C-terminal PDZ-binding motif were evolutionary conserved among INSC proteins. The former part of ISH domain is implicated in Pros localization, while function of the latter part of ISH domain and C-terminal PDZ-binding motif remain to be elucidated. Human INSC mRNA was expressed in eye, kidney, fetal cochlea, parathyroid tumor, chondrosarcoma, epidermoid carcinoma, and skin tumor. Because LGN/Pins, PARD3/Par-3Bazooka, PARD6A/Par-6 and PRKCZ/aPKC genes implicated in asymmetrical cell division are evolutionarily and functionally conserved, human INSC protein might be implicated in asymmetrical cell division of human neural stem cells and other stem cells.
WNT signaling pathway is implicated in carcinogenesis and embryogenesis. WNT signal is transduced to the beta-catenin - TCF pathway, the JNK pathway, or the Ca2+-releasing pathway through seven-transmembrane-type WNT receptors encoded by Frizzled (FZD) genes. Xenopus Strabismus (Stbm) is a tetra-spanning transmembrane protein interacting with Dishevelled, and is a negative regulator of the WNT - beta-catenin - TCF signaling pathway. STB1/KIAA1215/VANGL2 is a human orthologue of Xenopus Stbm (90.6% total-amino-acid identity). Here, STB2/VANGL1 gene fragments were identified in human genome draft sequences by using bioinformatics, and STB2 cDNAs were isolated by using cDNA-PCR. STB2 gene consisted of at lest 7 exons, and encoded a 524-amino-acid protein with 4 transmembrane domains and the C-terminal Ser/Thr-X-Val motif. Human STB2 was homologous to human STB1 (73.1% total-amino-acid identity) and Xenopus Stbm (72.7% total-amino-acid identity). STB2 gene was clustered with Calsequestrin 2 (CASQ2) gene in tail-to-tail manner (interval less than 5.0 kb), and CASQ2 gene is mapped to human chromosome 1p11-p13.3 or linked to human chromosome 1p13-p21. STB2 mRNAs of 4.8- and 6.8-kb in size were expressed almost ubiquitously in various normal tissues. STB2 mRNA was significantly up-regulated in gastric cancer cell lines MKN28, MKN74, pancreatic cancer cell lines BxPC-3, PSN-1 and Hs766T. On the other hand, STB2 mRNA was significantly down-regulated in a pancreatic cancer cell line AsPC-1. This is the first report on molecular cloning and characterization of STB2.
Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.
WNT2 gene on human chromosome 7q31 is a paralog of the WNT2B gene on human chromosome 1p13. Rat Wnt2 gene was identified within rat genome draft sequence AC095247.4. Human WNT2 showed 96.4% total-amino-acid identity with rat Wnt2, 96.1% with mouse Wnt2, 68.6% with zebrafish wnt2, and 67.8% with fugu wnt2. WNT2 is an evolutionarily conserved secreted-type glycoprotein belonging to the WNT family. WNT2 mRNA is expressed in human fetal lung and placenta, but almost undetectable in normal gastrointestinal tract. WNT2 mRNA is frequently up-regulated in human gastric cancer due to tumor-stromal interaction, and WNT2 gene is rarely amplified in human gastric cancer. WNT2 mRNA is also frequently up-regulated in colorectal polyps, primary colorectal cancer of stage A-C, and also in liver metastasis from colorectal cancer. Putative biding sites for estrogen receptor, GATA-1, AP-2, TCF-1, BHLH, MBF-I, p53, and HNF-5 are located within the 5'-flanking region of human WNT2 gene. WNT2 is up-regulated by beta-estradiol in human MCF-7 cells; however, the mechanism of WNT2 up-regulation in most cases of gastrointestinal cancer remains to be elucidated. WNT2 is a tumor marker of gastric and colorectal cancer. Detection of theWNT2 protein in feces by immunohistochemistry or ELISA and WNT2 mRNA in feces by cDNA-PCR or custom-made microarray could be applied for screening of colorectal cancer. Because WNT2 up-regulation leads to carcinogenesis through activation of the WNT-beta-catenin pathway, WNT2 specific antagonist might be applied for chemoprevention or treatment of gastrointestinal cancer. WNT2 gene is one of the targets for pharmacogenomics in the field of oncology.