RESUMO
The study of regulatory small RNAs, such as siRNAs and microRNAs in plants, has necessitated methods tailored to their unique features. Their analysis demands the use of sensitive and quantitative methods for their detection. The use of Northern blot hybridization offers an attractive alternative to address qualitative as well as quantitative features. We highlight the advantages and shortcomings of this method and offer a detailed description of the techniques that best work in our hands, considering their use for the study of several small RNAs in multiple samples. We enumerate relevant details as well as cautionary comments in cases where we have detected potential difficulties.
Assuntos
MicroRNAs/genética , Plantas/genética , RNA de Plantas/genética , Northern Blotting/métodos , Regulação da Expressão Gênica de Plantas/genética , Hibridização de Ácido Nucleico/genética , RNA Interferente Pequeno/genéticaRESUMO
There are two major centres of genetic diversification of common bean (Phaseolus vilgaris L.), the Mesoamerican and the Andean, and the legume is capable of establishing nitrogen-fixing symbioses with several rhizobia; Rhizobium etli seems to be the dominant species in both centres. Another genetic pool of common bean, in Peru and Ecuador, is receiving increasing attention, and studies of microsymbionts from the region can help to increase our knowledge about coevolution of this symbiosis. We have previously reported several putative new lineages from this region and here present data indicating that strains belonging to one of them, PEL4, represent a novel species. Based on 16S rRNA gene sequence phylogeny, PEL4 strains are positioned in the Rhizobium phaseoli/R. etli/Rhizobium leguminosarum clade, but show unique properties in several morphological, physiological and biochemical analyses, as well as in BOX-PCR profiles ( < 75% similarity with related species). PEL4 strains also differed from related species based on multilocus sequence analysis of three housekeeping genes (glnII, gyrB and recA). Nucleotide identities of the three concatenated genes between PEL4 strains and related species ranged from 91.8 to 94.2%, being highest with Rhizobium fabae. DNA-DNA hybridization ( < 47% DNA relatedness) and average nucleotide identity values of the whole genomes ( < 90.2%) also supported the novel species status. The PEL4 strains were effective in nodulating and fixing N2 with common beans. The data supported the view that PEL4 strains represent a novel species, Rhizobium ecuadorense sp. nov. The type strain is CNPSo 671(T) ( = UMR 1450(T) = PIMAMPIRS I 5(T) = LMG 27578(T)).
Assuntos
Rhizobium , DNA Bacteriano/genética , Equador , Ácidos Graxos/química , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico/genética , Peru , Phaseolus , Filogenia , RNA Ribossômico 16S/genética , Rhizobium/classificação , Análise de Sequência de DNA , Simbiose/genéticaRESUMO
Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-ß-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.
Assuntos
Regulação Fúngica da Expressão Gênica , Óleos de Plantas/metabolismo , Trichophyton/genética , Trichophyton/metabolismo , Carbono/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Hibridização de Ácido Nucleico/genética , Análise de Sequência com Séries de Oligonucleotídeos , Azeite de Oliva , Trichophyton/crescimento & desenvolvimentoRESUMO
Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.
Assuntos
Aberrações Cromossômicas , DNA/análise , Endometriose/genética , Doenças Ovarianas/genética , Adulto , Endometriose/patologia , Feminino , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico/genética , Doenças Ovarianas/patologia , Reação em Cadeia da PolimeraseRESUMO
Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.
Assuntos
Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Aberrações Cromossômicas , DNA , Endometriose/genética , Doenças Ovarianas/genética , Endometriose/patologia , Perda de Heterozigosidade , Hibridização de Ácido Nucleico/genética , Doenças Ovarianas/patologia , Reação em Cadeia da PolimeraseRESUMO
INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA(R) v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.
Assuntos
Genótipo , Hepacivirus/genética , Hibridização de Ácido Nucleico/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões 5' não Traduzidas/genética , Genoma Viral/genética , Hepacivirus/classificação , Humanos , Proteínas não Estruturais Virais/genéticaRESUMO
INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.
INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA® v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.
Assuntos
Humanos , Genótipo , Hepacivirus/genética , Hibridização de Ácido Nucleico/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , /genética , Genoma Viral/genética , Hepacivirus/classificação , Proteínas não Estruturais Virais/genéticaRESUMO
Transferability of microsatellite loci between closely related species has been reported in several species. This helps reduce costs involved with the development of primers for newly investigated species. Fifteen microsatellite primers developed for Rangifer tarandus, Cervus elaphus, C. axis, and Moschus berezovskii were tested on five species of Brazilian brocket deer of the genus Mazama (M. americana, M. bororo, M. gouazoubira, M. nana, and M. nemorivaga). These primers were tested with DNA extracted from blood samples of two individuals of each species obtained from the Núcleo de Pesquisa e Conservação de Cervídeos (NUPECCE) of Universidade Estadual Paulista (UNESP). Fourteen of the 15 primers tested amplified microsatellite regions of all five species of Mazama, confirmed by sequencing of the amplified fragments. We conclude that these primers could be used for population studies of brocket deer.
Assuntos
Cervos/genética , Loci Gênicos/genética , Repetições de Microssatélites/genética , Animais , Brasil , Primers do DNA/economia , Primers do DNA/genética , Cervos/classificação , Hibridização de Ácido Nucleico/genéticaRESUMO
OBJECTIVES: To describe the molecular analysis through comparative genomic hybridization (CGH) of fetuses with gastroschisis, and to observe if this technique could improve the resolution of the conventional cytogenetic techniques. METHODS: Amniotic analysis of fetuses with gastroschisis, using both conventional (G-banding) and molecular (CGH) cytogenetics assays. RESULTS: All of the seven fetuses studied displayed a normal G-band karyotype. Six fetuses displayed a normal disomic profile through CGH and one sample has displayed ish cgh enh 3q26-->qter result (ICSN). The fetus with this imbalance of chromosome 3 was re-classified as a ruptured omphalocele, instead of gastroschisis, after birth. CONCLUSIONS: The molecular investigation through CGH technique can improve the resolution of the conventional karyotye analysis in cases of abdominal wall defects.
Assuntos
Parede Abdominal/anormalidades , Perfilação da Expressão Gênica/métodos , Hibridização de Ácido Nucleico/métodos , Diagnóstico Pré-Natal/métodos , Parede Abdominal/fisiologia , Amniocentese/métodos , Feminino , Gastrosquise/diagnóstico , Gastrosquise/genética , Humanos , Hibridização de Ácido Nucleico/genética , GravidezRESUMO
FG syndrome is an X-linked multiple congenital anomalies (MCA) syndrome. It has been mapped to four distinct loci FGS1-4, through linkage analysis (Xq13, Xp22.3, and Xp11.4-p11.3) and based on the breakpoints of an X chromosome inversion (Xq11:Xq28), but so far no gene has been identified. We describe a boy with FG syndrome who has an inherited duplication at band Xq22.3 detected by comparative genomic hybridization microarray (Array-CGH). These duplication maps outside all four loci described so far for FG syndrome, representing therefore a new locus, which we propose to be called FGS5. MID2, a gene closely related to MID1, which is known to be mutated in Opitz G/BBB syndrome, maps within the duplicated segment of our patient. Since FG and Opitz G/BBB syndromes share many manifestations we considered MID2 a candidate gene for FG syndrome. We also discuss the involvement of other potential genes within the duplicated segment and its relationship with clinical symptoms of our patient, as well as the laboratory abnormalities found in his mother, a carrier of the duplication.
Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos X/genética , Face/anormalidades , Deficiência Intelectual/genética , Proteínas Associadas aos Microtúbulos/genética , Aberrações dos Cromossomos Sexuais , Transtornos dos Cromossomos Sexuais/patologia , Fatores de Transcrição/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Repetições de Microssatélites/genética , Hibridização de Ácido Nucleico/genética , Linhagem , Transtornos dos Cromossomos Sexuais/genéticaRESUMO
We applied a combination of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH), to characterize the genetic aberrations in three osteosarcomas (OS) and one Ewing's sarcoma. CGH identified recurrent chromosomal losses at 10p14-pter and gains at 8q22.3-24.1 in OS. Interphase FISH allowed to confirm 8q gain in two cases. A high amplification level of 11q12-qter was detected in one OS. The Ewing's sarcoma showed gain at 1p32-36.1 as the sole chromosome alteration. These studies demonstrate the value of molecular cytogenetic methods in the characterization of recurrent genomic alterations in bone tumor tissue
Assuntos
Humanos , Masculino , Feminino , Neoplasias Ósseas , Hibridização de Ácido Nucleico/genética , Hibridização in Situ Fluorescente , Osteossarcoma , Sarcoma de Ewing , Aberrações Cromossômicas , CitogenéticaRESUMO
In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.
Assuntos
Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Nicotiana/genética , Proteínas de Plantas/genética , Proteínas de Ligação a RNA/genética , Perfilação da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/genética , Reprodução/genética , Reprodução/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de Proteína , Análise de Sequência de RNA , Nicotiana/fisiologiaRESUMO
En este artículo se exponen los principales descubrimientos de la nueva ciencia denominada genómicas y sus principales repercusiones para el diagnóstico microbiológico, tales como la nueva tecnología de los ADN chips
Assuntos
Humanos , Doenças Transmissíveis/genética , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Bactérias/genética , Doenças Transmissíveis/diagnóstico , Hibridização de Ácido Nucleico/genéticaRESUMO
El advenimiento de las técnicas de DNA recombinante abrió una nueva era en los estudios genéticos. Por primera vez se hizo posible aislar, amplificar e incluso manipular segmentos de DNA de genes individuales. Estos métodos, junto con herramientas poderosas como los métodos de secuenciación, reacción en cadena de la polimerasa y el chips de DNA, han permitido el conocimiento de la estructura, función y regulación de los genes. Actualmente, las técnicas del DNA recombinante tienen muchas aplicaciones importantes, entre las que destacan la localización y clonación de genes responsables de enfermedades, lo que permite su diagnóstico molecular y la identificación precisa de individuos a través de sus huellas genómicas. Además, esta metodología ha permitido la producción comercial de genes y proteínas mediante ingeniería genética para su empleo en medicina y la creación de animales transgénicos
Assuntos
DNA Recombinante/genética , DNA Recombinante , Análise de Sequência/métodos , Análise de Sequência , Clonagem Molecular/métodos , Hibridização de Ácido Nucleico/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Biologia Molecular , Biologia Molecular/tendências , Biblioteca GenômicaRESUMO
A survey for citrus viroids was conducted in the major citrus commercial growing areas in Costa Rica. Screening of 36 sweet orange and 12 lemon trees resulted in the detection of members of four of the five citrus viroid groups as determined by nucleic acid hybridization using specific RNA probes and polymerase chain reaction (PCR) using specific oligonucleotide primers. CEVd, CVd-IIa, CVD-IIb and CVd-III viroids were found to be widespread in the three main regions of commercial citrus production. CVd-Ib was only found in lemon in Nicoya
Assuntos
Citrus/virologia , Hibridização de Ácido Nucleico/genética , Viroides/isolamento & purificação , Costa Rica , Reação em Cadeia da Polimerase , Sondas RNA , Viroides/genéticaRESUMO
A survey for citrus viroids was conducted in the major citrus commercial growing areas in Costa Rica. Screening of 36 sweet orange and 12 lemon trees resulted in the detection of members of four of the five citrus viroid groups as determined by nucleic acid hybridization using specific RNA probes and polymerase chain reaction (PCR) using specific oligonucleotide primers. CEVd, CVd-IIa, CVD-IIb and CVd-III viroids were found to be widespread in the three main regions of commercial citrus production. CVd-Ib was only found in lemon in Nicoya.
Assuntos
Citrus/virologia , Hibridização de Ácido Nucleico/genética , Viroides/isolamento & purificação , Costa Rica , Reação em Cadeia da Polimerase , Sondas RNA , Viroides/genéticaRESUMO
Usando seis diferentes probes de cromossomo X, estimou-se a freqüência de polimorfismo de comprimento de fragmento de restriçäo (RFLP) com endonucleases de restriçäo em pessoas näo parentes, em uma populaçäo espanhola de Valência. As freqüências de alelos foram semelhantes às de outras populaçöes européias, em particular francesas e turcas. Um alto grau de polimorfismo foi encontrado para todos os marcadores, sendo que a freqüência do alelo raro variou de 0,484 a 0,357 e um alto nível de heterozigose dos marcadores L1.28, 754, OTC e p58.1 foi encontrado nesta populaçäo, confirmando sua utilidade para a diagnose.
Assuntos
Humanos , Masculino , Feminino , Aberrações dos Cromossomos Sexuais/genética , Cromossomo X/genética , Alelos , Sondas de DNA , Enzimas de Restrição do DNA , Marcadores Genéticos , Hibridização de Ácido Nucleico/genética , Polimorfismo de Fragmento de RestriçãoRESUMO
No presente trabalho estudaram-se 4 protocolos diferentes de detecçäo näo radioativa de ácidos nucleicos utilizando sonda genética biotinilada. A razäo deste trabalho säo as inúmeras dificuldades encontradas na padronizaçäo dos ensaios de hibridizaçäo utilizando o método quimioluminiscência principalmente devido ao grande background presente nos filtros. Devido às grandes vantagens que a utilizaçäo de sondas näo radioativas nos oferece, utilizando DNA plasmídico pBR-322, descreveram-se uma metodologia simples, reprodutível e de baixo custo de hibridizaçäo molecular utilizando a técnica de quimioluminiscência
Assuntos
Biotina , Sondas de DNA , Hibridização de Ácido Nucleico/genéticaRESUMO
Del 6 al 15 de los casos son síndrome de Turner presentan un cariotipo en mosaico con una línea 45,X, y otra con un pequeño cromosoma sexual marcador de origen no determinado, que puede ser un anillo o un fragmento céntrico. La identificación de su origen, que frecuentemente se dificulta por la falta de resolución de las técnicas citogenéticas y la variabilidad en el fenotipo clínico , es de vital importancia ya que, si provienen de un cromosoma Y, el paciente tiene un riesgo elevado de desarrollar gonadoblastoma. El objetivo del presente trabajo fue determinar el origen de pequeños cromosomas sexuales marcadores mediante la técnica de hibridación in situ con fluorescencia (FISH). Se estudiaron ocho pacientes: siete mostraban fenotipo Turner con genitales externos femeninos, y la octava tenía talla baja con ambigüedad de genitales. En todos los casos se identificó, en el cariotipo de sangre periférica, un mosaico con una línea celular que presentaba un cromosoma sexual marcador; en tres se corroboró el mosaico en fibroblastos. Para la técnica de FISH se utilizaron sondas de DNA marcadas con biotina, con secuencias complementarias a la región centroamérica alfa satélite de los cromosomas X y Y. Con la sonda alfa satélite del X, se estableció que el cromosoma marcador provenía del X en siete pacientes. En la paciente con genitales ambiguos, se determinó que el marcador derivaba del cromosoma Y. Consecuentemenete la técnica FISH fue capaz de identificar el origen de los cromosomas sexuales marcadores en las ocho pacientes