RESUMEN
Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.
Asunto(s)
Adenosina Trifosfatasas , Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Evolución Molecular , Alelos , Proteínas Bacterianas/genética , Escherichia coli/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Genotipo , Proteínas MutL , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Reacción en Cadena de la Polimerasa , Pirofosfatasas , Recombinación Genética , Salmonella typhimurium/genéticaRESUMEN
An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Meiosis , Profase , Recombinación Genética , Zea mays/genética , Secuencia de Aminoácidos , Clonación Molecular , Intercambio Genético , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Evolución Molecular , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Modelos Genéticos , Modelos Estructurales , Datos de Secuencia Molecular , Recombinasa Rad51 , Homología de Secuencia de Aminoácido , Complejo SinaptonémicoRESUMEN
We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 13 , Cósmidos/genética , Lugares Marcados de Secuencia , Animales , Cromosomas Artificiales de Levadura , Redes de Comunicación de Computadores , Cricetinae , Humanos , Células Híbridas , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.
Asunto(s)
Reparación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Recombinación Genética , Saccharomyces cerevisiae/genética , Cinética , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52 , Proteínas de Saccharomyces cerevisiaeRESUMEN
Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.
Asunto(s)
Neoplasias de la Mama/genética , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 13/genética , Proteínas de Neoplasias/genética , Programas Informáticos , Factores de Transcripción/genética , Proteína BRCA2 , Secuencia de Bases , Cromosomas Artificiales de Levadura/genética , Cromosomas Humanos Par 13/ultraestructura , Cósmidos/genética , ADN Complementario/genética , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Selección GenéticaRESUMEN
Three different ways of detecting insertion/deletion (Ins/Del) genotypes of the apolipoprotein B gene encoding signal peptide were compared. Unequal quantities of the two alleles were observed in 50 Serbian subjects. The low abundance allele was clearly visible only when a modified method of silver staining for polyacrylamide gels was used. This method is more reliable for Ins/Del polymorphism genotyping, avoiding misclassifications by conventional ethidium bromide staining of both agarose and polyacrylamide gels.
Asunto(s)
Apolipoproteínas B/genética , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Mutagénesis Insercional , Señales de Clasificación de Proteína/genética , Eliminación de Secuencia , Alelos , Secuencia de Bases , Cartilla de ADN , Genotipo , Humanos , Datos de Secuencia Molecular , Tinción con Nitrato de PlataAsunto(s)
Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Ratones/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia de Consenso , Genes , Genes Fúngicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteína Recombinante y Reparadora de ADN Rad52 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Vertebrados/genética , Levaduras/genéticaRESUMEN
We have developed an efficient method for assembling ordered cosmid contigs aligned to mega-YACs and midi-YACs (average insert sizes of 1.0 and 0.35 Mb, respectively) and used this general method to initiate high-resolution physical mapping of human chromosome 13 (Chr 13). Chr 13-enriched midi-YAC (mYAC) and mega-YAC (MYAC) sublibraries were obtained from corresponding CEPH total human YAC libraries by selecting colonies with inter-Alu PCR probes derived from Chr 13 monochromosomal cell hybrid DNA. These sublibraries were arrayed on filters at high density. In our approach, the MYAC 13 sublibrary is screened by hybridization with cytogenetically assigned Chr 13 DNA probes to select one or a small subset of MYACs. Inter-Alu PCR products from each MYAC are then hybridized to the MYAC and mYAC sublibraries to identify overlapping YACs and to an arrayed Chr 13-specific cosmid library to select corresponding cosmids. The set of selected cosmids, gridded on filters at high density, is hybridized with inter-Alu PCR products from each of the overlapping YACs to identify subsets of cosmids and also with riboprobes from each cosmid of the arrayed set ("cosmid matrix cross-hybridization"). From these data, cosmid contigs are assembled by a specifically designed computer program. Application of this method generates cosmid contigs spanning the length of a MYAC with few gaps. To provide a high-resolution map, ends of cosmids are sequenced at preselected sites to position densely spaced sequence-tagged sites.
Asunto(s)
Cromosomas Humanos Par 13 , Cósmidos , ADN/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Cartilla de ADN , Sondas de ADN , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Lugares Marcados de SecuenciaRESUMEN
The promoter of human K14 keratin gene, specific for the basal layer of stratified epithelia, is regulated by nuclear receptors for retinoic acid and thyroid hormone. However, the DNA sequences responsible for this regulation have not yet been identified. To identify the retinoic acid-responsive site, we have devised a simple site-specific mutagenesis method and introduced mutations into the K14 keratin gene promoter. These mutations identify the retinoic acid-responsive site. The site consists of a cluster of consensus palindrome half-sites in various orientations. As shown previously, retinoic acid and thyroid hormone receptors can recognize and bind common sequences in regulated genes. Here, we describe mutations that abolish regulation by both receptors. Interestingly, the hormone-dependent and -independent regulatory sites of the thyroid hormone nuclear receptor can be separated. Clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5 and K10 keratin gene promoters. Similar clusters may be responsible for retinoic acid-mediated transcription regulation in epidermis.
Asunto(s)
Proteínas Portadoras/genética , Queratinas/genética , Receptores de Hormona Tiroidea/genética , Secuencia de Bases , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Regiones Promotoras Genéticas , Receptores de Ácido RetinoicoRESUMEN
Effects of nitrate (doses of 600 and 1200 mg/kg/day during 14 days) and sodium nitrite (60 and 120 mg/kg/day during 14 days) on germ cells of male mice were investigated. The mode of application was stomach intubation. The germ cell stages analysed were spermatids (for the heritable effects) and differentiating and stem-cell spermatogonia (for direct effects). A lack of heritable translocations, sperm abnormalities, as well as morphological changes, such as changes in eyes, coat colour, testes and body weight, was demonstrated in F1 males originating from treated P males. Significant effects in treated males were found with respect to: (1) sex-chromosomal univalency in the diakinesis-methaphase I stage after the treatment of stem spermatogonia (both doses of sodium nitrate and the higher dose of sodium nitrite), (2) sperm-head abnormalities after treatment of differentiating spermatogonia (the higher dose of sodium nitrate and both doses of sodium nitrite), and (3) fertility after treatment of spermatids (the higher dose of sodium nitrite). Nonmutagenic effects and possible carcinogenic potential of the tested doses are discussed.
Asunto(s)
Nitratos/toxicidad , Nitritos/toxicidad , Espermatogénesis/efectos de los fármacos , Espermatogonias/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Animales , Fertilidad/efectos de los fármacos , Técnicas In Vitro , Tamaño de la Camada , Masculino , Ratones , Mutación/efectos de los fármacos , Razón de Masculinidad , Cabeza del Espermatozoide/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Translocación GenéticaRESUMEN
Effects of both sodium nitrate (doses of 600 and 1200 mg/kg/day for 3 days) and sodium nitrite (doses of 60 and 120 mg/kg/day for 3 days) on spermatids of mice were investigated by measuring unscheduled DNA synthesis (UDS) 17 days after the end of treatment, and sperm-head abnormality 11 and 17 days after the end of treatment. Neither chemical induced the UDS response in early to mid spermatids (17 days). The only positive result in the sperm-head abnormality test was obtained for the dose of 120 mg/kg/day of sodium nitrite both at 11 and 17 days after treatment. The results presented are in accordance with those of our earlier experiments with the same chemicals, suggesting their nonmutagenic action on the tested germ-cell stages of male mice.
Asunto(s)
Daño del ADN , Reparación del ADN/efectos de los fármacos , Nitratos/toxicidad , Nitritos/toxicidad , Espermátides/efectos de los fármacos , Animales , ADN/biosíntesis , Técnicas In Vitro , Masculino , Ratones , Cabeza del Espermatozoide/efectos de los fármacos , Cabeza del Espermatozoide/ultraestructuraRESUMEN
The yield of radiation-induced micronuclei in human lymphocytes was assessed by two methods, i.e., by incorporating bromodeoxyuridine or by inhibiting cytokinesis by cytochalasin for identification of cells which have undergone one cell division. The cytochalasin block method was found to be more efficient with a capacity to detect between 60 and 90% of the induced fragments. Dose-response characteristics and the results of fractionation experiments indicate that the yield of micronuclei reflects both classes of acentric fragments, i.e., those associated and independent of exchange type of aberrations.