In silico analysis of nonsynonymous single-nucleotide polymorphisms (nsSNPs) of the SMPX gene

Mutations in the SMPX gene can disrupt the regular activity of the SMPX protein, which is involved in the hearing process. Recent reports showing a link between nonsynonymous single-nucleotide polymorphisms (nsSNPs) in SMPX and hearing loss, thus classifying deleterious SNPs in SMPX will be an uphill task before designing a more extensive population study. In this study, damaging nsSNPs of SMPX from the dbSNP database were identified by using 13 bioinformatics tools. Initially, the impact of nsSNPs in the SMPX gene were evaluated through different in silico predictors; and the deleterious convergent changes were analyzed by energy-minimization-guided residual network analysis. In addition, the pathogenic effects of mutations in SMPX-mediated protein–protein interactions were also characterized by structural modeling and binding energy calculations. A total of four mutations (N19D, A29T, K54N, and S71L) were found to be highly deleterious by all the tools, which are located at highly conserved regions. Furthermore, all four mutants showed structural alterations, and the communities of amino acids for mutant proteins were readily changed, compared to the wild-type. Among them, A29T (rs772775896) was revealed as the most damaging nsSNP, which caused significant structural deviation of the SMPX protein, as a result reducing the binding affinity to other functional partners. These findings reflect the computational insights into the deleterious role of nsSNPs in SMPX, which might be helpful for subjecting wet-lab confirmatory analysis.     

In silico characterization and comparative genomic analysis of the Culex quinquefasciatus glutathione S-transferase (GST) supergene family

The glutathione S-transferases (GSTs) are phase II class of detoxification enzymes that are involved both directly and indirectly in insecticide resistance mechanisms. The Culex quinquefasciatus GST superfamily was analyzed by utilizing the public domain Culex genome sequence. In total, 35 cytosolic (seven classes) and 5 microsomal putatively active GSTs were retrieved, classified, and annotated. The study revealed the presence of three unclassified GSTs. Of 35 cytosolic GSTs, 65% contributed by insect specific Delta–Epsilon classes. Gene cluster analysis revealed that most of the genes of Delta, Epsilon, and Theta classes were organized into gene clusters. The gene organization analysis revealed the dominance of phase “0” introns in the Culex GST family. The studies on intron loss and gain events revealed that the Delta GSTs have experienced a higher number of loss and gains during their evolution. A positive correlation was observed between the phylogenetic relationship of members of the GST superfamily and their corresponding exon–intron organization. In addition, the genes within the gene clusters revealed the monophyletic phylogenetic relationship implying the importance of gene duplication events in the gene families' evolution. Finally, the comparative genomic analysis has shown a complex evolutionary scenario associated with the GST supergene family evolution in insects.     

Unique features of different members of the human guanylate-binding protein family.

Guanylate-binding proteins (GBPs) are the most abundant cellular proteins expressed in response to interferon-gamma (IFN-gamma), with seven highly homologous members in humans, termed HuGBP-1 to HuGBP-7. To date, differential features that may indicate differential functions of these proteins have not been described. Here, we investigated the expression and subcellular localization of the different HuGBPs in endothelial cells (EC). IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) induced the expression of HuGBP-1, HuGBP-2, and HuGBP-3 at similar high levels. In contrast, expression of HuGBP-4 and HuGBP-5 was robustly induced only by IFN-gamma and not by TNF-alpha and IL-1beta. Expression of HuGBP-6 and HuGBP-7 was not detected in EC under the various conditions examined. Investigating subcellular localization of the EC-expressed HuGBPs, HuGBP-1, HuGBP-3, and HuGBP-5 were exclusively detected in the cytoplasm, whereas HuGBP-2 and HuGBP-4 displayed a nucleocytoplasmic distribution. Treatment of the cells with IFN-gamma and aluminum fluoride caused rapid enrichment of HuGBP-1 and HuGBP-2 in the Golgi apparatus, as demonstrated by time-lapse microscopy and fluorescence analyses of GFP-tagged HuGBPs. HuGBP-3 and HuGBP-4 were never detected in the Golgi apparatus, whereas HuGBP-5 was constitutively enriched in this cytosolic compartment, irrespective of stimulation. These results assign a characteristic pattern of expression and subcellular localization to each of the HuGBPs, indicating for the first time that these proteins may have different cellular functions.     

Chromosomal localization and complete genomic sequence of the murine autoimmune regulator gene (Aire)

We have recently cloned the murine autoimmune regulator (Aire) gene, the homologue of human AIRE responsible for the autoimmune polyglandular syndrome type 1 (APS1) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). Here, we report the genomic sequence (18,413 bp) for the entire Aire gene and its 5' flanking region, which contains putative regulatory sequences. Comparison of the genomic and cDNA sequences indicates that the Aire gene is composed of 14 exons and the coding sequence shares high similarities between mouse and human. The sizes of the homologous introns in the two species are conserved; however, the introns do not share significant sequence homologies except the sequences near the splice donor and acceptor sites. Sequence analyses of the 5' regulatory region and the complete coding region in three mouse strains (B6, NOD and SJL) did not reveal any sequence variation, suggesting sequence conservation between different inbred mouse strains. Using one of the six microsatellite markers identified by genomic sequencing and a B6 x Cast backcross mapping panel, we mapped the mouse Aire gene to chromosome 10, a syntenic region containing the Cdl18 and Pfkl genes on human chromosome 21q22.     

Porcine Fas-ligand gene: genomic sequence analysis and comparison with human gene.

Thymic Fas-ligand (FasL) cDNA and hepatic FasL genomic sequences were obtained from a 2-month-old LW pig. From these nucleotide sequences, amino acid sequence was deduced and compared with FasL sequences obtained from various animals. This comparison reveals that porcine FasL is closer to that of human, macaca and cat, and differs more from mouse and rat. The extracelluar domains of porcine and human FasL proteins appear to be functionally compatible. The complete genomic DNA sequence of porcine FasL was also compared with its human counterpart. Exons showed 80-89% nucleotide homology between pig and human, while introns showed 64-69% nucleotide homology. Sequence comparison by Harr plot analysis revealed many stretches within introns having identical sequences, suggesting that the sites may have unidentified common functions. One potential extra exon between exons 2 and 3 was located within porcine intron 2. This potential exon has no counterpart in human FasL intron 2. Whether or not this extra exon can be expressed and could cause additional immunological responses remains to be investigated. For future xenotransplantation, it is important to compare porcine and human genomic sequences, and to investigate their system compatibilities.     

Complete genomic sequence of the goat prion protein gene (PRNP)

Prion protein genetics plays a central role in transmissible spongiform encephalopathies, a disease occurring in human and animals. Here we report a 27-kb genomic sequence containing the goat PRNP gene. It shows, both in structure and content, a remarkable similarity with its sheep ortholog and can serve as a basis for future (comparative) studies with reference to the regulation of PRNP gene expression and the search for genetic tools to prevent/control/eradicate (goat) TSE. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper has been submitted to the GenBank nucleotide sequence database and has been assigned the accession number EU870890.     

Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters.

The CC chemokines are a closely related subfamily of the chemokine superfamily. Most of the CC chemokine genes form a cluster on chromosome 11 in mice and chromosome 17 in humans. To date, 11 and 16 functional genes have been localized within the mouse and human clusters, respectively. Notably, some of the genes within these clusters appear to have no counterparts between the two species, and the orthologous relationships of some of the genes are difficult to establish solely on the basis of amino acid similarity. In this study, we have taken a comparative genomic approach to reveal some of the features that may be involved in the dynamic evolution of these gene clusters. We sequenced a 122-kb region containing five chemokine genes of the mouse CC cluster. This mouse sequence was combined with those determined by the Mouse Genome Sequencing Project, and the entire sequence of the mouse CC cluster was compared with that of the corresponding cluster in the human genome by percent identity plot and dot-plot analyses. Although no additional chemokine genes have been found in these clusters, our analysis has revealed that numerous gene rearrangements have occurred even after the diversification of rodents and primates, resulting in several species-specific chemokine genes and pseudogenes. In addition, phylogenetic analysis and comparison of the genomic sequences unambiguously identified the orthologous relationships of some of the chemokine genes in the mouse and human CC gene clusters.     

Comparative DNA sequence analysis of mouse and human protocadherin gene clusters

The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.     

Characterization and genomic sequence of the murine 60 kD Ro gene

Autoantibodies binding 60 kD Ro (or SS-A) are commonly found in patients with systemic lupus erythematosus and Sj?gren's syndrome. While many studies have examined the autoimmune response directed against this RNA-protein, its function is still uncertain. As part of a broad effort to better understand animal models of anti-Ro autoimmunity we have characterized the murine 60 kD Ro gene. Southern blot analysis of mouse genomic DNA suggests that the 60 kD Ro gene is a single copy gene. The complete sequence of the gene was determined from three overlapping genomic lambda phage clones (GenBank accession number AF065398). The murine 60 kD Ro gene spans approximately 23 kb and consists of 8 or 9 exons. DNA sequence analysis revealed the presence of multiple B1 repetitive units. It maps in synteny with the human 60 kD Ro gene. Therefore, the isolation and characterization of the 60 kD Ro gene will be instrumental for future studies on protein function and the role this protein plays in the development of autoimmune responses.     

Identification and characterization of human ZPBP-like gene in silico

PP1R1B-ERBB2-GRB7 locus on human chromo-some 17q12 is frequently amplified in gastric and breast cancer. Because recombination hot spot or fragile site is located around the terminus of amplified region (amplicon), we searched for a novel gene closely linked to the teromeric end of the ERBB2 amplicon. Here, we identified and characterized the ZPBP-like (ZPBPL) gene by using bioinformatics. ZPBPL gene, corresponding to BC043152 cDNA, was found to consist of seven exons. ZPBPL (316 aa) and ZPBP (351 aa) proteins, showing 34.8% total amino-acid identity, shared the zona pellucida binding protein homologous (ZPBH) domain with conserved 15 cysteine residues. ZPBPL was a secreted-type glycoprotein with the ZPBH domain, while ZPBP was a type 2 transmembrane protein with the extracellular ZPBH domain. ZPBPL mRNA was co-expressed with ZPBP mRNA in testis, germ cell tumor, and brain medulla. ZPBPL might be implicated in the gamete interaction during fertilization just like ZPBP. The MGC9753-ERBB2-MGC14832-GRB7-ZNFN1A3-ZPBPL-PRO2521-ORMDL3-GSDM locus on human chromosome 17q12-q21 and the ZPBP-ZNFN1A1-FIGNL1-DDC-GRB10-COBL-SEC61G-EGFR-LANCL2 locus on human chromosome 7p12-p11 were next compared. Comparative genomics revealed that ZPBPL-ZNFN1A3-GRB7-ERBB2 and ZPBP-ZNFN1A1-GRB10-EGFR loci were paralogous regions within the human genome. This is the first report on identification and characterization of the ZPBPL gene.     

Identification and characterization of human Inscuteable gene in silico

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.     

Identification and characterization of the human FMN1 gene in silico

Mouse Formin (Fmn1) protein plays a key role in limb morphogenesis. Fmn1 is one of the actin regulators with scaffold function, interacting with Profilin, SRC, EMS1, FNBP1, FNBP2, FNBP3, FNBP4, WBP4 and alpha-catenin. Fmn1, Fmn2, FHOD1, FHOD3, GRID2IP and FHDC1 are non-FDD-type Formin homology proteins, while FMNL1, FMNL2, FMNL3, DIAPH1, DIAPH2, DIAPH3, DAAM1 and DAAM2 are FDD-type Formin homology proteins. Here, we identified the human FMN1 gene by using bioinformatics. The complete coding sequence of human FMN1 cDNA was determined by assembling AC055874.8 genome sequence (nucleotide position 178207-180073), AI040235 EST (complementary sequence for nucleotide position 331-156) and FLJ45135 cDNA (nucleotide position 319-3310). FMN1 isoform 1 (exons 1-18) and FMN isoform 2 (exons 1b and 3-18) were transcribed due to alternative splicing of the alternative promoter type. The FMN1 gene at human chromosome 15q13.3 was located between CKTSF1B1 (Gremlin) and RYR3 genes. The Xenopus fmn1 gene was identified within the Xenopus genome sequence CH216-24N20 (AC147835.1). The FMH1 domain (codon 1-120 of FMN1) and FMH2 domain (codon 683-835 of FMN1) were identified as novel regions conserved among human FMN1, mouse Fmn1, and Xenopus fmn1. The FMH2 domain was almost identical to the alpha-catenin binding domain of mouse Fmn1. Human FMN1 (1419 aa), showing 77.1% total amino-acid identity with mouse Fmn1, was found consisting of FMH1, FMH2, FH1 and FH2 domains. This is the first report on the identification and characterization of the human FMN1 gene as well as the FMH1 and FMH2 domains.     

Murine GBP-5, a new member of the murine guanylate-binding protein family, is coordinately regulated with other GBPs in vivo and in vitro.

A new murine member of the interferon (IFN)-inducible guanylate-binding protein (GBP) family was cloned in a search for glucocorticoid-attenuated response genes induced in the lung during endotoxemia. The full-length MuGBP-5 cDNA encodes a 590 amino acid residue protein with GTP binding motifs identical to those in human GBP-1 (HuGBP-1) and a similar isoprenylation sequence at the C-terminus. An alternatively spliced form of MuGBP-5 that lacks the second GTP binding motif and differs at the C-terminus was also identified. The MuGBP-5 gene is located on chromosome 3, near MuGBP-3 and MuGBP-2, and has a genomic organization similar to other GBP genes. To facilitate the evaluation of GBP family message expression, we constructed RNase protection assay probes for MuGBP-1, MuGBP-2, MuGBP-3, MuGBP-4/Mag-2 (macrophage activation gene-2), and MuGBP-5 and validated their use in Swiss Webster, BALB/c, and C57BL/6 mice. In BALB/c mice, all five MuGBPs were induced in multiple organs during endotoxemia, and all had a similar pattern of expression in different tissues. With minor quantitative differences, the MuGBPs also had similar patterns of response to IFN-gamma, lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) in RAW 264.7 and Swiss 3T3 cells. The coordinate expression of the MuGBPs suggests that they share common mechanisms of regulation.     

The genomic structure of the human skeletal muscle sodium channel gene.

Electrical excitability of neurons and muscle cells reflects the actions of a family of structurally related sodium channels. Mutations in the adult skeletal muscle sodium channel have been associated with the inherited neuromuscular disorders paramyotonia congenita (PMC) and hyperkalemic periodic paralysis (HPP). We have deciphered the entire genomic structure of the human skeletal muscle sodium channel gene and developed a restriction map of the locus. SCN4A consists of 24 exons spanning 35 kb of distance on chromosome 17q. We describe the sequence of all intron/exon boundaries, the presence of several polymorphisms in the coding sequence, and the locations within introns of two dinucleotide repeat polymorphisms. This is the first sodium channel for which the entire genomic structure has been resolved. The organization of the SCN4A exons relative to the proposed protein structure is presented and represents a foundation for functional and evolutionary comparisons of sodium channels. Knowledge of the exon structure and flanking intron sequences for SCN4A will permit a systematic search for mutations in PMC and HPP.