RESUMO
BACKGROUND: Ara h 2 is a major peanut allergen recognized by IgE in more than 90% of patients. After electrophoretic separation the purified protein exists as a doublet, and sequences of one incomplete cDNA and one genomic clone for this allergen have been reported. METHODS: Ara h 2 isoforms were purified and analyzed by mass spectroscopy, and PCR amplification products of Ara h 2 were cloned and sequenced. RESULTS: Mass spectroscopy of purified Ara h 2 clearly identified a molecular doublet of 16,670 and 18,050 Daltons. Amplification of a peanut cDNA library using PCR primer pairs located at the amino- and carboxy-terminus revealed 2 bands separated by 50 base pairs, which we cloned and sequenced. Two types of complete cDNA clones were obtained, Ara h 2.01 and Ara h 2.02. Compared to Ara h 2.01 and the previously reported cDNA sequences, Ara h 2.02 is characterized by a 12 amino acid insertion starting at position 75 that contains a third repeat of the major IgE binding epitope DPYSPS. CONCLUSION: We demonstrated the molecular and genetic characteristics of two Ara h 2 isoforms, revealing that one, Ara h 2.02, might be the more potent allergen.
Assuntos
DNA Complementar/classificação , DNA Complementar/isolamento & purificação , Glicoproteínas/classificação , Glicoproteínas/isolamento & purificação , Albuminas 2S de Plantas , Sequência de Aminoácidos , Antígenos de Plantas , Clonagem Molecular , Códon de Iniciação/classificação , Códon de Iniciação/genética , Códon de Iniciação/isolamento & purificação , Códon de Terminação/classificação , Códon de Terminação/genética , Códon de Terminação/isolamento & purificação , Primers do DNA/classificação , Primers do DNA/genética , Primers do DNA/isolamento & purificação , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/imunologia , DNA Complementar/genética , Glicoproteínas/genética , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Proteínas de Plantas , Reação em Cadeia da Polimerase , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Sequências Repetidas Terminais/genética , Sequências Repetidas Terminais/imunologiaRESUMO
Fusobacterium nucleatum is a gram-negative non-spore-forming, non-motile, obligate anaerobic rod that is normally isolated from the oral cavity. Several studies have reported a significant heterogeneity within the F. nucleatum species. The aim of the present study was to analyze the clonal diversity of F. nucleatum strains isolated from intracanal infections and to evaluate the presence of Enterobacterial Repetitive Intergenic Consensus (ERIC)-like sequences in the genome of F. nucleatum. Samples were collected from 13 single-root teeth from adult patients, all having carious lesions, necrotic pulps and radiographic evidence of periradicular bone loss. F. nucleatum was isolated from two different patients (subjects 5 and 7) by culture. Amplification of 19 colonies from subject 5 and 15 colonies from subject 7 using ERIC primers resulted in four clonal types, two per subject. An intense amplicon of approximately 700 bp was generated by ERIC-PCR for all F. nucleatum isolates and F. nucleatum ssp. polymorphum ATCC 10953. The amplification reaction using primer 1254 confirmed the results obtained with the ERIC primer. Our findings indicate that DNA fingerprints provided by ERIC- and Arbitrarily Primed (AP)-PCR may constitute a powerful tool for investigating F. nucleatum clonal diversity.
Assuntos
Cavidade Pulpar/microbiologia , Necrose da Polpa Dentária/microbiologia , Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/genética , Adulto , Perda do Osso Alveolar/microbiologia , Pareamento de Bases/genética , Células Clonais , Sequência Consenso/genética , Impressões Digitais de DNA , Primers do DNA/classificação , Cárie Dentária/microbiologia , Amplificação de Genes , Genoma Bacteriano , Genótipo , Humanos , Sequências Repetitivas Dispersas/genética , Doenças Periapicais/microbiologia , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
MOTIVATION: DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. RESULTS: MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.
Assuntos
Metilação de DNA , Primers do DNA , Primers do DNA/química , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Software , Sequência de Bases , Desenho Assistido por Computador , Ilhas de CpG/genética , Primers do DNA/classificação , Primers do DNA/genética , Desenho de Equipamento/métodos , Receptor beta de Estrogênio , Humanos , Internet , Dados de Sequência Molecular , Multimídia , Projetos Piloto , Reação em Cadeia da Polimerase/instrumentação , Controle de Qualidade , Receptores de Estrogênio/genética , Sensibilidade e Especificidade , Alinhamento de Sequência/métodos , Interface Usuário-ComputadorRESUMO
To get a better understanding of the effect of interelement selection on the variation of long terminal repeat retrotransposon families, we have investigated the evolutionary history of blood in the Drosophila melanogaster species complex. We carried out a PCR approach to amplify the 5' untranslated region from blood in the four species of the complex. This procedure revealed two main classes of size variants. Phylogenetic analyses of nucleotide sequences from these variants and blood elements from the Drosophila Genome Projects database show that elements are grouped according to their size, so that they probably correspond to two subfamilies. These two subfamilies arose prior to the split of the complex, and several facts indicate that the expansion of one of them is leading to the competitive exclusion of the other, at least from the euchromatic regions of the genome.
Assuntos
Drosophila melanogaster/genética , Filogenia , Retroelementos , Sequências Repetidas Terminais , Regiões 5' não Traduzidas/classificação , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Primers do DNA/classificação , Primers do DNA/genética , Evolução Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Expression of the coxsackie-adenovirus receptor (CAR) is a critical determinant in cellular susceptibility to infection with adenovirus-based gene transfer vectors. This study is focused on the hypothesis that manipulation of the cytoplasmic tail and transmembrane regions of CAR can be used to change cell surface levels of CAR and, consequently, to alter the efficiency of Ad-mediated gene transfer. To accomplish this, Flag-tagged ([F]) human CAR ([F]CAR), [F]tailless-CAR (lacking the cytoplasmic tail), and [F]GPI-CAR (containing a GPI lipid anchor instead of the transmembrane and cytoplasmic regions) were exogenously expressed in CHO cells. Analysis of (125)I-labeled anti-Flag antibody binding to transfected cells revealed that [F]tailless-CAR and [F]GPI-CAR were expressed on the cell surface in 1.8- to 2.5-fold higher amounts than [F]CAR, while the total expression levels were similar. Infection with replication-deficient adenovirus encoding beta-galactosidase (Ad-betagal) demonstrated 1.5- to 2-fold higher levels of transgene expression in CHO cells expressing [F]tailless-CAR or [F]GPI-CAR, respectively, compared with cells containing [F]CAR. The form of CAR expressed did not affect the transport of fluorescent Cy3-Ad particles from the cell surface to the nuclear region. These observations indicate that transduction of target cells by Ad vectors can be optimized by increasing cell surface levels of CAR through functional deletion of the tail and membrane protein domains.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , Enterovirus/fisiologia , Receptores Virais/metabolismo , Animais , Células CHO/metabolismo , Células COS , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Cricetinae , Primers do DNA/química , Primers do DNA/classificação , Corantes Fluorescentes , Técnicas de Transferência de Genes , Vetores Genéticos , Glicosilfosfatidilinositóis/metabolismo , Humanos , Oligopeptídeos , Peptídeos/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , RNA Mensageiro/análise , Fosfolipases Tipo C/farmacologia , beta-Galactosidase/genéticaRESUMO
Barley (Hordeum vulgare L.) is potentially a new source of genes for wheat (Triticum aestivum L.) improvement. Wheat-barley chromosome recombinant lines provide a means for introgressing barley genes to wheat genome by chromosome engineering, and since these are expected to occur only rarely in special cytogenetic stocks, an efficient selection skill is necessary to identify them. To convert RFLP markers to barley allele-specific PCR markers useful for effective production of wheat-barley recombinant lines, 91 primer sets derived from RFLP clones which were previously mapped to the barley chromosomes were examined for PCR amplification using 'Chinese Spring' wheat, 'Betzes' barley and the wheat-barley chromosome addition lines. The polymorphisms were detected by an agarose gel electrophoresis of the PCR products without digestion with restriction enzymes. Out of 81 primer sets producing polymorphisms between the wheat and barley genomes, 26 amplified barley chromosome-specific DNAs which were confirmed to be located on the same chromosome as the RFLP markers by using the wheat-barley chromosome addition lines. These amplified DNAs represent barley allele-specific amplicons, which distinguish barley alleles from their wheat homoeologous counterparts. The present investigation revealed a higher probability for obtaining allele-specific amplicons from genomic DNA-derived RFLP markers than from cDNA-derived ones. The barley allele-specific amplicons developed in this study, namely, four for chromosome 2H, two for 3H, seven for 4H, eight for 5H, one for 6H and four for 7H, are suitable for identifying 'Chinese Spring' wheat- 'Betzes' barley recombinant chromosomes. However, one out of eight barley allele-specific amplicons on chromosome 5H did not detect a unique barley band in a 'New Golden' barley chromosome 5H addition line of 'Shinchunaga' wheat, indicating there may be a need to reconstruct allele-specific amplicons with different barley cultivars.
