RESUMO
Type 1 diabetes is a complex disorder with interaction of both genetic and environmental factors. One of the loci, IDDM4, has been mapped to chromosome 11q13, with evidence of association to two markers, D11S1917 and H0570polyA. To identify putative candidate genes for IDDM4, we have constructed a 400-kb clone contig in this region and sequenced the clones. We have also sequenced the orthologous DNA from mouse. Previously, we identified a cDNA for the low-density lipoprotein receptor-related protein 5 gene (LRP5) 3 kb distal to H0570polyA. We have now determined the exon-intron structure of this gene. Detailed sequence analysis has identified a further three genes in this region: the CGI-85 gene (previously identified by W.-C. Lin) and two novel genes, C11orf24 and C11orf23. The C11orf24 gene has no known similarity to other genes, and its function is unknown. C11orf23 has similarity to the SIT4 (sporulation-induced transcript 4)-associated protein (SAP) family of yeast proteins, which are involved in regulation of the cell cycle. The full-length C11orf23 cDNA is the first mammalian orthologue of the yeast SAP family to be identified. Identification of these four genes in a 400-kb region of the IDDM4 region underpins our strategy to identify the IDDM4 locus.
Assuntos
Cromossomos Humanos Par 11/genética , DNA/genética , Diabetes Mellitus Tipo 1/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , DNA/química , Éxons , Feminino , Expressão Gênica , Genes/genética , Humanos , Íntrons , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Masculino , Proteínas de Membrana/genética , Repetições de Microssatélites , Dados de Sequência Molecular , Fosfoproteínas Fosfatases , Mapeamento Físico do Cromossomo , Isoformas de Proteínas/genética , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.
Assuntos
Proteínas do Olho/genética , Genes Dominantes/genética , Degeneração Macular/genética , Proteínas de Membrana/genética , Deleção de Sequência/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 6/genética , Clonagem Molecular , Análise Mutacional de DNA , Éxons/genética , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Feminino , Humanos , Hibridização In Situ , Íntrons/genética , Escore Lod , Macaca mulatta/genética , Degeneração Macular/patologia , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Linhagem , RNA Mensageiro/análise , RNA Mensageiro/genética , Retina/metabolismo , Retina/patologia , Alinhamento de SequênciaRESUMO
A mixed 'clone-by-clone' and 'whole-genome shotgun' strategy will be used to determine the genomic sequence of the mouse. This method will allow a phase of rapid annotation of the contemporaneous human sequence draft, through whole-genome 'sample sequence comparisons'.
Assuntos
Genoma , Homologia de Sequência do Ácido Nucleico , Animais , Clonagem Molecular , Biologia Computacional/métodos , Feminino , Biblioteca Genômica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Análise de Sequência de DNA/métodosRESUMO
Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.
Assuntos
Adenoviridae/fisiologia , Vírus Defeituosos/fisiologia , Vetores Genéticos/fisiologia , Vírus Auxiliares/fisiologia , Adenoviridae/genética , Animais , Linhagem Celular , Sequência Consenso , Citomegalovirus/genética , DNA Recombinante/química , DNA Recombinante/genética , Vírus Defeituosos/genética , Eritropoetina/genética , Eritropoetina/metabolismo , Escherichia coli , Genes Reporter , Genes Sintéticos , Vetores Genéticos/genética , Células HeLa , Humanos , Imunocompetência , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Recombinação Genética , Segurança , Transfecção , Montagem de Vírus , Replicação ViralRESUMO
Fas-mediated apoptosis is an important regulator of cell survival, and abnormalities in this system have been shown to result in a number of human pathological conditions. A secreted member of the tumor necrosis factor receptor superfamily, DcR3, was recently reported to be amplified in human lung and colon cancers as a negative regulator of Fas-mediated apoptosis. We identified this gene, which we call M68. M68 genomic DNA, mRNA, and protein levels were examined in a series of human gastrointestinal tract tumors. Using M68 immunohistochemistry and a scoring system similar to that used for HER-2/neu, we found that M68 protein was overexpressed in 30 of 68 (44%) human adenocarcinomas of the esophagus, stomach, colon, and rectum. Tumors examined by Northern blot revealed M68 mRNA highly elevated in a similar fraction of primary tumors from the same gastrointestinal tract regions, as well as in the colon adenocarcinoma cell lines SW480 and SW1116. Further, we found M68 protein to be overexpressed in a substantial number of tumors in which gene amplification could not be detected by fluorescence in situ hybridization or quantitative genomic PCR, suggesting that overexpression of M68 may precede amplification in tumors. Finally, we find that M68 lies within a four-gene cluster that includes a novel helicase-like gene (NHL) related to RAD3/ERCC2, a plasma membrane Ras-related GTPase and a member of the stathmin family, amplification or overexpression of which may also contribute to cell growth and tumor progression.
Assuntos
Fatores de Ribosilação do ADP , Cromossomos Humanos Par 20/genética , Neoplasias Esofágicas/genética , Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Receptores de Superfície Celular/biossíntese , Sequência de Aminoácidos , Apoptose , Mapeamento Cromossômico , DNA Helicases/genética , DNA Complementar/genética , Etiquetas de Sequências Expressas , GTP Fosfo-Hidrolases/genética , Amplificação de Genes , Genes , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Fatores de Crescimento Neural/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Neoplásico/genética , Receptores de Superfície Celular/genética , Receptores do Fator de Necrose Tumoral , Membro 6b de Receptores do Fator de Necrose Tumoral , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estatmina , Receptor fas/biossíntese , Receptor fas/fisiologiaRESUMO
Vitelliform macular dystrophy (VMD2, Best disease, MIM153700) is an early onset, autosomal, dominant macular degeneration characterized by the deposition of lipofuscin-like material within and below the retinal pigment epithelium (RPE); it is associated with degeneration of the RPE and overlying photoreceptors. Recently, we cloned the gene bestrophin, which is responsible for the disease, and identified a number of causative mutations in families with VMD2. Here, we report that the analysis of bestrophin in a collection of 259 age-related macular degeneration (AMD) patients provides evidence that mutations in the Best disease gene do not play a significant role in the predisposition of individuals to AMD. However, our results suggest that, in addition to Best disease, mutations within the bestrophin gene could be responsible for other forms of maculopathy with phenotypic characteristics similar to Best disease and for other diseases not included in the VMD category.
Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Adulto , Fatores Etários , Idade de Início , Idoso , Alelos , Animais , Bestrofinas , Canais de Cloreto , Variação Genética , Humanos , Canais Iônicos , Degeneração Macular/patologia , Camundongos , Fenótipo , Mutação PuntualAssuntos
Compostos de Boro , Mapeamento Cromossômico/métodos , Primers do DNA , Corantes Fluorescentes , Análise de Sequência de DNA , Compostos de Boro/síntese química , Compostos de Boro/química , Primers do DNA/síntese química , Primers do DNA/química , Armazenamento de Medicamentos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , RobóticaRESUMO
Here, we describe a novel strategy called enzymatic "Mop-Up" that efficiently removes contaminating dNTPs from reverse-phase, high-performance liquid chromatography (RP-HPLC) purified 3'-O-modified dNTP syntheses. Enzymatic mop-up takes advantage of the high selectivity of DNA polymerases for the former nucleoside triphosphates over the latter nucleotide analogs. We demonstrate the selective removal of contaminating dATP and dTTP from RP-HPLC purified 3'-O-methyl-dATP and 3'-O-(2-nitrobenzyl)-dTTP syntheses, respectively. These data highlight the importance of natural nucleotide contamination when interpreting enzymatic incorporation data and provide an alternative hypothesis for the observed property of catalytic editing of DNA polymerases. Moreover, the effective removal of natural nucleotides from 3'-O-modified analogs addresses the important issue of nucleotide read-through for stop-start DNA sequencing strategies, such as the base addition sequencing scheme (BASS).
Assuntos
DNA Polimerase Dirigida por DNA , DNA/biossíntese , Desoxirribonucleotídeos/isolamento & purificação , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Desoxirribonucleotídeos/síntese químicaRESUMO
Direct DNA sequencing of human immunodeficiency virus type 1 (HIV-1) pol and env gene regions was characterized for accuracy and precision. Restricted maximum likelihood (REML) analysis of molecular clone reconstruction experiments using a primer-walking strategy showed that the BODIPY and BODIPY energy-transfer (BET) dye primers sets were significantly more accurate in quantitating heterogenous base mixtures than the fluorescein/rhodamine dye primers. Of the three sets examined, the BET dye primers were the most accurate. The improved accuracy correlated with the reduced emission band-widths of BODIPY and BET dye primers and the more uniform signal intensities of BET dye primers. However, comparing % coefficients of variation (CV) for the three dye primer sets, revealed that BODIPY dye primers gave better precision than both BET and fluorescein/rhodamine dye primer sets. Several sequence-dependent motifs were identified that showed specific nucleotide-biased incorporation and were determined to be the major variable component of the total %CV. Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations.
Assuntos
Compostos de Boro/química , Primers do DNA/genética , HIV-1/genética , Análise de Sequência de DNA/métodos , Primers do DNA/química , DNA Viral/química , DNA Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genes env/genética , Genes pol/genética , Heterogeneidade Genética , Variação Genética , Genoma Viral , Proteína gp120 do Envelope de HIV/genética , HIV-1/química , Humanos , Reprodutibilidade dos TestesRESUMO
A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.
Assuntos
Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Cromossomos Humanos Par 11/genética , Clonagem Molecular , Cosmídeos/genética , DNA/química , DNA/genética , DNA Bacteriano/genética , DNA Complementar/química , DNA Complementar/genética , Diabetes Mellitus Tipo 1/genética , Expressão Gênica/genética , Predisposição Genética para Doença , Biblioteca Genômica , Humanos , Proteínas Relacionadas a Receptor de LDL , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/genética , Camundongos , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
A novel member of the low density lipoprotein receptor (LDLR) gene family has been identified and characterized. This gene, termed LDL receptor-related protein 6 (LRP6), encodes a transmembrane protein which has 71% identity and is structurally similar to the protein encoded by LRP5, a proposed candidate gene for type 1 diabetes located on human chromosome 11q13. LRP6 maps to human chromosome 12p11-p13. Mouse Lrp6 encodes a protein that has 98% identity to human LRP6 and maps to chromosome 6. Unlike other members of the LDLR family, LRP6 and LRP5 display a unique pattern of four epidermal growth factor (EGF) and three LDLR repeats in the extracellular domain. The cytoplasmic domain of LRP6 is not similar to other members of the LDLR family, while comparison with LRP5 reveals proline-rich motifs that may mediate protein-protein interactions. Thus, it is likely that LRP6 and LRP5 comprise a new class of the LDLR family.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 12 , Diabetes Mellitus Tipo 1/genética , Receptores Imunológicos/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Fator de Crescimento Epidérmico/química , Biblioteca Gênica , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Receptores Imunológicos/biossíntese , Receptores Imunológicos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Best macular dystrophy (BMD), also known as vitelliform macular dystrophy (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration characterized by an abnormal accumulation of lipofuscin within and beneath the retinal pigment epithelium cells. In pursuit of the disease gene, we limited the minimum genetic region by recombination breakpoint analysis and mapped to this region a novel retina-specific gene (VMD2). Genetic mapping data, identification of five independent disease-specific mutations and expression studies provide evidence that mutations within the candidate gene are a cause of BMD. The 3' UTR of the candidate gene contains a region of antisense complementarity to the 3' UTR of the ferritin heavy-chain gene (FTH1), indicating the possibility of antisense interaction between VMD2 and FTH1 transcripts.
Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bestrofinas , Caenorhabditis elegans/genética , Canais de Cloreto , Mapeamento Cromossômico , Clonagem Molecular , Análise Mutacional de DNA , DNA Complementar , Feminino , Ferritinas/genética , Homologia de Genes , Humanos , Canais Iônicos , Masculino , Camundongos , Dados de Sequência Molecular , LinhagemRESUMO
A class of dyes, BODIPY fluorophores, has been identified for automated DNA sequencing that has improved spectral characteristics compared with conventional fluorescein and rhodamine dyes. Single and double BODIPY dye primers were characterized in commercially available DNA sequencers and showed uniform electrophoretic mobilities and high fluorescence intensities. The improved physical properties of BODIPY dye primers were demonstrated by direct base-calling from the unprocessed fluorescent signals and improved heterozygote analyses of mixed-base populations. The high sensitivity of BODIPY dye primers requires at least 33 percent less reagent consumed per reaction than conventional dye primers, which should affect the costs of large genome-sequencing efforts.
Assuntos
Compostos de Boro , Corantes Fluorescentes , Análise de Sequência de DNA/métodos , Autoanálise , Sequência de Bases , Compostos de Boro/química , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Dados de Sequência Molecular , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
GelScann(1.0) is a user-friendly program that accurately quantitates DNA from CCD imaged agarose gels. The algorithm automatically locates lanes, locates bands within a given lane, and quantitates the intensity of each band. GelScann (1.0) uses a statistical method to discriminate DNA from local background fluorescence. The sensitivity of the LaneFinder and BandFinder can be adjusted by the user interacting with GelScann (1.0)'s option box. We demonstrate that GelScann(1.0) can accurately determine DNA that GelScann(1.0) can accurately determine DNA concentrations for routine molecular biology applications and determine the relative intensities of PCR amplified fragments for genetic testing.
Assuntos
Algoritmos , DNA Viral/análise , Eletroforese em Gel de Ágar/métodos , Processamento de Imagem Assistida por Computador , Bacteriófago M13/genética , Feminino , Triagem de Portadores Genéticos , Infecções por HIV/virologia , HIV-1/genética , Humanos , Microcomputadores , Distrofias Musculares/genética , Reação em Cadeia da PolimeraseRESUMO
Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.
Assuntos
DNA/biossíntese , Desoxirribonucleotídeos/farmacologia , Sequência de Bases , Cromatografia Líquida de Alta Pressão , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Moldes GenéticosRESUMO
A severe shock is produced in 18 mongrel dogs by standardized bone trauma and haemorrhagic shock. Impairment of microcirculation is demonstrated by acidosis, reduced oxygen uptake and disseminated intravascular coagulation (DIC). Further, a specific increase in pulmonary vascular resistance--independent of the reduction in blood flow--is evoked, concomitantly with the occurrence of microthrombi, oedema and haemorrhage in lung tissue. Pretreatment with heparin inhibits disseminated intravascular coagulation and reduces impairment of microcirculation and the consecutive damage of intestinal organs. However increase of pulmonary vascular resistance is unchanged and pulmonary haemorrhage is pronounced. Therefore heparin pretreatment enhances pulmonary histologic impairment after trauma and haemorrhagic shock.
Assuntos
Heparina/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Ferimentos e Lesões/tratamento farmacológico , Acidose/fisiopatologia , Animais , Coagulação Intravascular Disseminada/prevenção & controle , Cães , Hemodinâmica/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacosRESUMO
Eleven mongrel dogs were observed for 72 h following a standardized traumatic hemorrhagic shock. The decrease of plasma albumin concentration and blood 125-I-albumin activity with a concomitant increase in lung I-albumin content is possibly caused by capillary leakage. A rise in the extravascular lung water content was measured by a gravimetric postmortem method. An in vivo double indicator technique, however, failed to document these findings.
Assuntos
Permeabilidade Capilar , Síndrome do Desconforto Respiratório/metabolismo , Choque Hemorrágico/metabolismo , Choque Traumático/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Cães , Espaço Extracelular/metabolismoRESUMO
In six beagles femoral head blood flow is measured with radioactive microspheres. When intra-articular pressure of the hip joint is increased by 50 mm Hg, blood flow is diminished to 48%. Reducing intra-articular pressure to the initial level increases femoral head blood flow to 78%. The results support the demand for immediate pressure relief of hemarthrosis.
Assuntos
Cabeça do Fêmur/irrigação sanguínea , Animais , Cães , Emergências , Hemartrose/terapia , Pressão , Fluxo Sanguíneo RegionalRESUMO
Regional myocardial blood flow (MBF) and left ventricular (LV) function were measured in 18 dogs after 60 min hypothermic (14-16 degrees C) arrest with (group I) and without cardioplegia (group II). Regardless whether postischemic LV function was severely (group II) or only moderately (group I) impaired, the amount of MBF and its transmural distribution was not significantly altered after 30 min reperfusion. In contrast to topical hypothermia, additional cardioplegia maintained metabolic regulation of coronary flow.
