RESUMO
The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 20/genética , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Sequência de Bases , Ligação Genética , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sitios de Sequências RotuladasRESUMO
Physical mapping of small genomic DNA fragments or expressed sequences by in situ hybridization is typically limited by the size of the target DNA sequence. Isolation of large insert DNA clones from libraries containing the target DNA sequence facilitates physical mapping by fluorescence in situ hybridization and allows rapid assignment of genes to cytogenetic bands. Here, we demonstrate the scheme by mapping the human protooncogene trk (NTRK1), a tyrosine kinase receptor type I gene that has earlier been assigned to two different cytogenetic loci. Large DNA insert library screening was carried out by in vitro DNA amplification using oligonucleotide primers flanking exon 4 of trk. The scheme presented here can easily be generalized to map physically very small nonrepetitive genomic DNA fragments or incomplete cDNAs.
Assuntos
Cromossomos Humanos Par 1 , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator de Crescimento Neural/genética , Bacteriófago P1 , Sequência de Bases , Mapeamento Cromossômico , Biblioteca Gênica , Vetores Genéticos , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Microscopia , Dados de Sequência Molecular , Receptor trkARESUMO
Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Aberrações Cromossômicas/genética , Cromossomos Humanos Par 20 , Mapeamento Cromossômico , Feminino , Humanos , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Células Tumorais CultivadasRESUMO
A fluorescence in situ hybridization (FISH) map of human chromosome 21 was compiled using yeast artificial chromosome (YAC) DNA probes that encode 28 markers physically and/or genetically mapped on the chromosome. Probes that recognize the centromere and rDNA repeat sequences in the p arm were also placed as reference markers on the FISH map. For each probe, the location of the fluorescence hybridization signal was measured on metaphase chromosomes with respect to fractional chromosome length (FL) from p-ter. The location of the markers was established with a standard error of +/- 1.9 Mb using from 9 to 63 FL measurements for each probe. The relative order and separation of the markers as determined by FISH are shown to correspond well to those of other maps of the chromosome. Fifty-one additional YAC and 86 cosmid clones were also localized by FISH with respect to the 30 markers on the chromosome. The cosmids, chosen at random from a flow-sorter chromosome 21 cosmid library, show some biases in chromosome distribution.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 21 , Hibridização in Situ Fluorescente , Cromossomos Fúngicos , Cosmídeos , Sondas de DNA , Biblioteca Gênica , Marcadores Genéticos , Genoma Humano , Humanos , Células Híbridas , Sensibilidade e EspecificidadeRESUMO
We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.