RESUMEN
We report here the molecular cloning of an approximately 1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 20 , Amplificación de Genes , Proteínas de Neoplasias/genética , Transactivadores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cromosomas Artificiales de Levadura , Clonación Molecular , Cartilla de ADN , Exones , Humanos , Hibridación Fluorescente in Situ , Intrones , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Transactivadores/química , Transcripción Genética , Células Tumorales Cultivadas , Dedos de Zinc/genéticaRESUMEN
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.
Asunto(s)
Cromosomas Humanos Par 1 , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factor de Crecimiento Nervioso/genética , Bacteriófago P1 , Secuencia de Bases , Mapeo Cromosómico , Biblioteca de Genes , Vectores Genéticos , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Microscopía , Datos de Secuencia Molecular , Receptor trkARESUMEN
Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.
Asunto(s)
Mapeo Cromosómico , ADN/genética , Muridae/genética , Cromosoma X , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Sondas de ADN , Fibroblastos , Citometría de Flujo/métodos , Biblioteca de Genes , Hibridación Fluorescente in Situ , Datos de Secuencia MolecularRESUMEN
Propionibacterium acnes (Corynebacterium parvum) is being implicated more frequently as a cause of intraocular inflammation following cataract surgery. In addition to its role as an infectious agent, P. acnes also may possess adjuvant-like or adjuvant-enhancing properties. The presence of this organism in an eye with residual lens material after extracapsular cataract surgery could augment inflammation resulting from a phacoantigenic (phacoanaphylactic) response. We have modified an established rat model of lens-induced granulomatous uveitis (LIGU) to examine the adjuvant properties of P. acnes. Our results suggest that P. acnes effectively potentiates LIGU.