RESUMO
Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Cromossomos de Plantas/genética , DNA de Plantas/genética , Gossypium/genética , Análise de Sequência de DNA/métodos , Mapeamento de Sequências Contíguas , DNA de Plantas/química , Genoma de Planta/genética , Biblioteca Genômica , Poliploidia , Reprodutibilidade dos Testes , Retroelementos/genéticaRESUMO
We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.
Assuntos
Resistência à Doença , Evolução Molecular , Glycine max/genética , Família Multigênica , Phaseolus/genética , Sequência de Aminoácidos , Teorema de Bayes , Diploide , Genes de Plantas , Phaseolus/química , Phaseolus/imunologia , Phaseolus/microbiologia , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Domínios e Motivos de Interação entre Proteínas , Pseudomonas/imunologia , Pseudomonas/patogenicidade , Recombinação Genética , Seleção Genética , Alinhamento de Sequência , Glycine max/química , Glycine max/imunologia , Glycine max/microbiologia , TetraploidiaRESUMO
Native virus-plant interactions require more understanding and their study will provide a basis from which to identify potential sources of emerging destructive viruses in crops. A novel tymovirus sequence was detected in Asclepias viridis (green milkweed), a perennial growing in a natural setting in the Tallgrass Prairie Preserve (TGPP) of Oklahoma. It was abundant within and frequent among A. viridis plants and, to varying extents, within other dicotyledonous and one grass (Panicum virgatum) species obtained from the TGPP. Extracts from A. viridis containing the sequence were infectious to a limited number of species. The virus genome was cloned and determined to be closely related to Kennedya yellow mosaic virus. The persistence of the virus within the Oklahoma A. viridis population was monitored for five successive years. Virus was present in a high percentage of plants within representative areas of the TGPP in all years and was spreading to additional plants. Virus was present in regions adjacent to the TGPP but not in plants sampled from central and south-central Oklahoma. Virus was present in the underground caudex of the plant during the winter, suggesting overwintering in this tissue. The RNA sequence encoding the virus coat protein varied considerably between individual plants (≈3%), likely due to drift rather than selection. An infectious clone was constructed and the virus was named Asclepias asymptomatic virus (AsAV) due to the absence of obvious symptoms on A. viridis.
Assuntos
Asclepias/virologia , Genoma Viral/genética , Doenças das Plantas/virologia , Tymovirus/isolamento & purificação , Sequência de Bases , Proteínas do Capsídeo/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Deriva Genética , Variação Genética , Geografia , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Oklahoma , Filogenia , Folhas de Planta/virologia , RNA Viral/genética , Análise de Sequência de DNA , Nicotiana/virologia , Tymovirus/classificação , Tymovirus/genéticaRESUMO
BACKGROUND: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.
Assuntos
Metilação de DNA , Genoma Humano , Estudo de Associação Genômica Ampla , Linfoma Folicular/genética , Análise de Sequência de DNA/métodos , Linfócitos B/metabolismo , Humanos , Linfoma Folicular/metabolismo , Regiões Promotoras Genéticas , Sulfitos/químicaRESUMO
Enterococcus faecalis has emerged as a prominent healthcare-associated pathogen frequently encountered in bacteremia, endocarditis, urinary tract infection, and as a leading cause of antibiotic-resistant infections. We recently demonstrated a capacity for high-level biofilm formation by a clinical E. faecalis isolate, E99. This high biofilm-forming phenotype was attributable to a novel locus, designated bee, specifying a pilus at the bacterial cell surface and localized to a large approximately 80 kb conjugative plasmid. To better understand the origin of the bee locus, as well as to potentially identify additional factors important to the biology and pathogenesis of strain E99, we sequenced the entire plasmid. The nucleotide sequence of the plasmid, designated pBEE99, revealed large regions of identity to the previously characterized conjugative plasmid pCF10. In addition to the bee locus, pBEE99 possesses an open reading frame potentially encoding aggregation substance, as well as open reading frames putatively encoding polypeptides with 60% to 99% identity at the amino acid level to proteins involved in regulation of the pheromone response and conjugal transfer of pCF10. However, strain E99 did not respond to the cCF10 pheromone in clumping assays. While pBEE99 was found to be devoid of any readily recognizable antibiotic resistance determinants, it carries two non-identical impB/mucB/samB-type genes, as well as genes potentially encoding a two-component bacteriocin similar to that encoded on pYI14. Although no bacteriocin activity was detected from an OG1RF transconjugant carrying pBEE99 against strain FA2-2, it was approximately an order of magnitude more resistant to ultraviolet radiation. Moreover, curing strain E99 of this plasmid significantly reduced its ability to survive UV exposure. Therefore, pBEE99 represents a novel conjugative plasmid that confers biofilm-forming and enhanced UV resistance traits that might potentially impact the virulence and/or fitness of E. faecalis.
Assuntos
Conjugação Genética/efeitos da radiação , Enterococcus faecalis/genética , Enterococcus faecalis/efeitos da radiação , Plasmídeos/genética , Tolerância a Radiação/genética , Raios Ultravioleta , Bacteriocinas/farmacologia , Sequência de Bases , Conjugação Genética/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Oligopeptídeos/genética , Fases de Leitura Aberta/genética , Feromônios/genética , Mapeamento Físico do Cromossomo , Tolerância a Radiação/efeitos da radiaçãoRESUMO
The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.
Assuntos
Evolução Molecular , Duplicação Gênica , Genes de Plantas , Glycine max/genética , Poliploidia , Retroelementos , Centrômero/genética , Cromossomos Artificiais Bacterianos , DNA de Plantas/química , Deleção de Genes , Genoma de Planta , Imunidade Inata/genética , Família Multigênica , Mutagênese Insercional , Phaseolus/genética , Filogenia , Doenças das Plantas/genética , Análise de Sequência de DNARESUMO
The Dmbt1 gene encodes alternatively spliced glycoproteins that are either membrane-associated or secreted epithelial products. Functions proposed for Dmbt1 include it being a tumor suppressor, having roles in innate immune defense and inflammation, and being a Golgi-sorting receptor in the exocrine pancreas. The heavily sulfated membrane glycoprotein mucin-like glycoprotein (Muclin) is a Dmbt1 product that is strongly expressed in organs of the gastrointestinal (GI) system. To explore Muclin's functions in the GI system, the Dmbt1 gene was targeted to produce Muclin-deficient mice. Muclin-deficient mice have normal body weight gain and are fertile. The Muclin-deficient mice did not develop GI tumors, even when crossed with mice lacking the known tumor suppressor p53. When colitis was induced by dextran sulfate sodium, there was no significant difference in disease severity in Muclin-deficient mice. Also, when acute pancreatitis was induced with supraphysiological caerulein, there was no difference in disease severity in the Muclin-deficient mice. Exocrine pancreatic function was impaired, as measured by attenuated neurohormonal-stimulated amylase release from Muclin-deficient acinar cells. Also, by [(35)S]Met/Cys pulse-chase analysis, traffic of newly synthesized protein to the stimulus-releasable pool was significantly retarded in Muclin-deficient cells compared with wild type. Thus Muclin deficiency impairs trafficking of regulated proteins to a stimulus-releasable pool in the exocrine pancreas.
Assuntos
Trato Gastrointestinal/fisiologia , Mucinas/deficiência , Amilases/metabolismo , Animais , Western Blotting , Proteínas de Ligação ao Cálcio , Ceruletídeo , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Proteínas de Ligação a DNA , Sulfato de Dextrana , Eletroforese em Gel de Poliacrilamida , Neoplasias Gastrointestinais/epidemiologia , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/genética , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Soybean, Glycine max (L.) Merr., is a well documented paleopolyploid. What remains relatively under characterized is the level of sequence identity in retained homeologous regions of the genome. Recently, the Department of Energy Joint Genome Institute and United States Department of Agriculture jointly announced the sequencing of the soybean genome. One of the initial concerns is to what extent sequence identity in homeologous regions would have on whole genome shotgun sequence assembly. RESULTS: Seventeen BACs representing approximately 2.03 Mb were sequenced as representative potential homeologous regions from the soybean genome. Genetic mapping of each BAC shows that 11 of the 20 chromosomes are represented. Sequence comparisons between homeologous BACs shows that the soybean genome is a mosaic of retained paleopolyploid regions. Some regions appear to be highly conserved while other regions have diverged significantly. Large-scale "batch" reassembly of all 17 BACs combined showed that even the most homeologous BACs with upwards of 95% sequence identity resolve into their respective homeologous sequences. Potential assembly errors were generated by tandemly duplicated pentatricopeptide repeat containing genes and long simple sequence repeats. Analysis of a whole-genome shotgun assembly of 80,000 randomly chosen JGI-DOE sequence traces reveals some new soybean-specific repeat sequences. CONCLUSION: This analysis investigated both the structure of the paleopolyploid soybean genome and the potential effects retained homeology will have on assembling the whole genome shotgun sequence. Based upon these results, homeologous regions similar to those characterized here will not cause major assembly issues.
Assuntos
Genes Duplicados/genética , Genoma de Planta/genética , Glycine max/genética , Mapeamento Físico do Cromossomo/métodos , Poliploidia , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA/métodos , Sequência de Bases/genética , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Plantas/genética , Evolução Molecular , Marcadores Genéticos , Repetições de Microssatélites , Filogenia , Polimorfismo Genético/genética , Software , Especificidade da Espécie , Sintenia/genéticaRESUMO
BACKGROUND: The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. RESULTS: A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. CONCLUSION: A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.
Assuntos
Acondroplasia/genética , Osso e Ossos/anormalidades , Peptídeo Natriurético Tipo C/genética , Mutação Puntual , Precursores de Proteínas/genética , Animais , Fator Natriurético Atrial , Tamanho Corporal , Primers do DNA , Feminino , Marcadores Genéticos , Genótipo , Masculino , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.
Assuntos
Regulação Enzimológica da Expressão Gênica , Lisossomos/química , N-Acetilglucosaminiltransferases/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Ácido Aspártico/química , Northern Blotting , Domínio Catalítico , Bovinos , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Biblioteca Gênica , Humanos , Lisina/química , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos/química , Peptídeos/química , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , TransfecçãoRESUMO
The waddles (wdl) mouse is a unique animal model that exhibits ataxia and appendicular dystonia without pathological abnormalities of either the central or the peripheral nervous systems. A 19-bp deletion in exon 8 of the carbonic anhydrase-related protein VIII gene (Car8) was detected by high-throughput temperature-gradient capillary electrophoresis heteroduplex analysis of PCR amplicons of genes and ESTs within the wdl locus on mouse chromosome 4. Although regarded as a member of the carbonic anhydrase gene family, the encoded protein (CAR8) has no reported enzymatic activity. In normal mice, CAR8 is abundantly expressed in cerebellar Purkinje cells as well as in several other cell groups. Compatible with nonsense-mediated decay of mutant transcripts, CAR8 is virtually absent in mice homozygous for the wdl mutation. These data indicate that the wdl mouse is a Car8 null mutant and that CAR8 plays a central role in motor control.
Assuntos
Anidrases Carbônicas/deficiência , Coxeadura Animal/genética , Proteínas do Tecido Nervoso/deficiência , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais , Anidrases Carbônicas/genética , Mapeamento Cromossômico , Genes Recessivos , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genéticaRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder in humans that causes the formation of fluid-filled renal cysts, often leading to renal failure. PKD1 mutations cause 85% of ADPKD. Feline PKD is autosomal dominant and has clinical presentations similar to humans. PKD affects approximately 38% of Persian cats worldwide, which is approximately 6% of cats, making it the most prominent inherited feline disease. Previous analyses have shown significant linkage between the PKD phenotype and microsatellite markers linked to the feline homolog for PKD1. In this report, the feline PKD1 gene was scanned for causative mutations and a C>A transversion was identified at c.10063 (human ref NM_000296) in exon 29, resulting in a stop mutation at position 3284, which suggests a loss of approximately 25% of the C-terminus of the protein. The same mutation has not been identified in humans, although similar regions of the protein are truncated. The C>A transversion has been identified in the heterozygous state in 48 affected cats examined, including 41 Persians, a Siamese, and several other breeds that have been known to outcross with Persians. In addition, the mutation is segregating concordantly in all available PKD families. No unaffected cats have been identified with the mutation. No homozygous cats have been identified, supporting the suggestion that the mutation is embryonic lethal. These data suggest that the stop mutation causes feline PKD, providing a test to identify cats that will develop PKD and demonstrating that the domestic cat is an ideal model for human PKD.
Assuntos
Predisposição Genética para Doença , Mutação , Rim Policístico Autossômico Dominante/genética , Proteínas/genética , Animais , Sequência de Bases , Gatos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Genótipo , Masculino , Proteínas de Membrana/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Valores de Referência , Sensibilidade e Especificidade , Canais de Cátion TRPPRESUMO
Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.
Assuntos
Fungos/genética , Íntrons , Splicing de RNA/genética , Aspergillus nidulans/genética , Sequência de Bases , Sequência Consenso , Cryptococcus neoformans/genética , DNA Fúngico/química , DNA Fúngico/genética , Éxons , Etiquetas de Sequências Expressas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Genoma Fúngico , Neurospora crassa/genética , Filogenia , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Especificidade da Espécie , Spliceossomos/metabolismoRESUMO
FELINES (Finding and Examining Lots of Intron 'N' Exon Sequences) is a utility written to automate construction and analysis of high quality intron and exon sequence databases produced from EST (expressed sequence tag) to genomic sequence alignments. We demonstrated the various programs of the FELINES utility by creating intron and exon sequence databases for the fungal organism Schizosaccharomyces pombe from alignments of EST to genomic sequences. In addition, we analyzed our constructed S.pombe sequence databases and the well-established Saccharomyces cerevisiae intron database from Manuel Ares' Laboratory for conserved sequence motifs. FELINES was shown to be useful for characterizing branchsites, polypyrimidine tracts and 5' and 3' splice sites in the intron databases and exonic splicing enhancers (ESEs) in S.pombe exons. FELINES is available at http://www.genome.ou.edu/informatics.html.
Assuntos
Éxons/genética , Etiquetas de Sequências Expressas , Íntrons/genética , Software , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Elementos Facilitadores Genéticos/genética , Splicing de RNA , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Alinhamento de SequênciaRESUMO
We analyzed 137 kb covering human neurofibromatosis 2 ( NF2) tumor suppressor locus and orthologous loci from baboon, mouse, rat, and pufferfish Takifugu rubripes. A predominant feature of human-rodent conservation is a very similar distribution of conserved islands, regarding length, position, and degree of identity. By use of a threshold of 75% identity over > or =100 bp of gap-free alignment, comparisons of human-mouse sequences resulted in 3.58% for extra-exonic conservation, which can be compared to 4.5% of exonic sequence content within the human locus. We identified a duplication of neurofibromin 2 in pufferfish, which resulted in two putative proteins with 74% and 76% identity to the human protein. One distinct island (called inter 1), conserved between all analyzed species, was located between promoters of the NIPSNAP1 and NF2 genes. Inter 1 might represent a novel regulatory element, important for the function of this locus. The high level of intronic conservation in the NF2 locus suggests that a number of unknown regulatory elements might exist within this gene. These elements could be affected by disease-causing mutations in NF2 patients and NF2-associated tumors. Alternatively, this conservation might be explained by presence of not yet characterized transcriptional unit(s) within this locus.
Assuntos
Sequência Conservada/genética , Genes da Neurofibromatose 2 , Mamíferos/genética , Takifugu/genética , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Duplicação Gênica , Biblioteca Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Proteínas/genética , Alinhamento de Sequência , Homologia de SequênciaRESUMO
Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Drosophila , Síndrome de Hermanski-Pudlak/genética , Proteínas de Insetos/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Mutação/genética , Proteínas/genética , Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Adulto , Sequência de Aminoácidos , Animais , Células COS , Pré-Escolar , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais de Bacteriófago P1/genética , Modelos Animais de Doenças , Feminino , Síndrome de Hermanski-Pudlak/metabolismo , Síndrome de Hermanski-Pudlak/patologia , Humanos , Masculino , Melanossomas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Oligopeptídeos , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas c-myc/imunologia , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11/genética , DNA de Neoplasias/análise , Genes Supressores de Tumor , Proteínas Proto-Oncogênicas , Neoplasias do Colo do Útero/genética , Centrômero/genética , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais Bacterianos/metabolismo , Primers do DNA/química , Endométrio/patologia , Feminino , Células HeLa , Humanos , Células Híbridas , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Cariotipagem , Perda de Heterozigosidade , Metáfase , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Polimorfismo Conformacional de Fita Simples , Neoplasias do Colo do Útero/patologiaRESUMO
OBJECTIVE: The objective of this study was to delineate a precise molecular map of the commonly deleted region (CDR) on mouse chr2 in radiation-induced mouse acute myeloid leukemic (AML) cells. MATERIALS AND METHODS: We used a PCR-based loss of heterozygosity (LOH) assay to map the chr2-CDR in AML cells isolated from F1 hybrid mice (BALB/cJ x CBA/CaJ) which developed AML following exposure to a single dose of 3 Gy of 137Cs gamma rays. A total of 30 polymorphic microsatellite markers, mapping within or close to chr2(D-E), were used under optimized PCR conditions that generate a single major band for each marker on a nondenaturing polyacrylamide gel. RESULTS: Detailed LOH mapping identified two distinct AML-CDRs: one localized to a 4.6 centiMorgan (cM) interval between markers D2Mit272 and D2Mit394; the other mapped to a 0.8 cM interval between markers D2Mit276 and D2Mit444. Both CDRs span the mouse chr2E region. CONCLUSION: The data present, for the first time, evidence for two distinctly noncontiguous CDRs on mouse chr2 harboring gene(s) involved in AML development. These CDRs are orthologous to human chromosomes 11p11-13 and 15q11-15 that have been implicated in subsets of AML. This finding indicates the region of mouse chr2 that must be searched for candidate genes involved in radiation-induced AML.
Assuntos
Mapeamento Cromossômico , Raios gama , Deleção de Genes , Leucemia Mieloide/genética , Leucemia Induzida por Radiação/genética , Repetições de Microssatélites/efeitos da radiação , Doença Aguda , Animais , Marcadores Genéticos , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
The muted (mu) mouse is a model for Hermansky-Pudlak Syndrome (HPS), an inherited disorder of humans causing hypopigmentation, hemorrhaging and early death due to lung abnormalities. The mu gene regulates the synthesis of specialized mammalian organelles such as melanosomes, platelet dense granules and lysosomes. Further, balance defects indicate that it controls the synthesis of otoliths of the inner ear. The mu gene has been identified by a positional/candidate approach involving large mouse interspecific backcrosses. It encodes a novel ubiquitously expressed transcript, specifying a predicted 185 amino acid protein, whose expression is abrogated in the mu allele which contains an insertion of an early transposon (ETn) retrotransposon. Expression is likewise expected to be lost in the mu( J) allele which contains a deletion of a single base pair within the coding region. The presence of structurally aberrant melanosomes within the eyes of mutant mice together with localization of the muted protein within vesicles in both the cell body and dendrites of transfected melan-a melanocytes emphasizes the role of the mu gene in vesicle trafficking. The mu gene is present only in mice and humans among analyzed genomes. As is true for several other recently identified mouse HPS genes, the mu gene is absent in lower eukaryotes. Therefore, the mu gene is a member of the novel gene set that has evolved in higher eukaryotes to regulate the synthesis/function of highly specialized subcellular organelles such as melanosomes and platelet dense granules.