RESUMEN
While current sequencing efforts consider the detection of alpha satellite repeats as logical end points for map construction, detailed maps of most pericentromeric regions are lacking to confirm this hypothesis. Here we identify the different alpha satellite families present at the pericentromeric region of chromosome 12. The order, size and location of these repeats is established using radiation hybrid analysis, pulsed field gel analysis and FISH and the maps are integrated with current sequence information. For the different classes of alpha satellites present at the chromosome 12 centromere the paralogs in the human genome were mapped by FISH. Unique sequences flanking the alpha satellite repeats were identified, some of which are not represented in the current draft sequence. This mapping effort localises the different alpha satellite repeats within the pericentromeric region and anchors them in the current maps. The novel sequences identified may serve as the end point for the ongoing sequencing efforts.
Asunto(s)
Centrómero , Cromosomas Humanos Par 12 , Southern Blotting , Aberraciones Cromosómicas , Cromosomas Humanos Par 12/ultraestructura , Cósmidos/análisis , Cósmidos/química , Cósmidos/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Mapeo de Híbrido por Radiación , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.
Asunto(s)
Avena/genética , ADN de Plantas/genética , Genoma de Planta , Secuencias Repetitivas Esparcidas , Secuencias Repetitivas de Ácidos Nucleicos , Cromosomas , Clonación Molecular , Cósmidos/análisis , Sondas de ADN , ADN de Plantas/aislamiento & purificación , Biblioteca Genómica , Hibridación Fluorescente in Situ , Especificidad de la EspecieRESUMEN
Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743-745, 2000)
Asunto(s)
ADN/análisis , Hibridación Fluorescente in Situ/métodos , Mapeo Cromosómico/métodos , Cósmidos/análisis , Desnaturalización de Ácido Nucleico , Coloración y Etiquetado/métodosRESUMEN
Expression sequence tags (EST) obtained by sequencing a randomly primed cDNA library and gene signatures (GS) obtained by sequencing a 3'-directed cDNA library can identify genes that are active in the source cells. Eight ESTs and ten GSs which represent novel human genes, except for one GS, and which have been assigned to human chromosome 11 were used to select cosmids from a chromosome 11-specific cosmid library. These cosmids were regionally mapped using the fluorescence in situ hybridization technique.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 11/química , Cromosomas Humanos Par 11/genética , ADN Complementario/genética , Animales , Línea Celular , Cromosomas Humanos Par 12 , Cromosomas Humanos Par 15 , Cósmidos/análisis , Cósmidos/genética , Cricetinae , Cricetulus , ADN Complementario/química , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The nucleotide sequence of a 26.7 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome IV is presented. An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene. These ORFs include the genes encoding the large subunit of RNA polymerase II, the biotin apo-protein ligase, an ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor, and the sequence encoding the protein phosphatase 2A. Further sequence analysis revealed a short ORF encoding the ribosomal protein YL41B, an intron in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome. The potential biological relevance of these findings is discussed.