The goal of this project is to map DNA sequences to chromosomes in zebrafish. The diploid chromosome number is fifty (2n=50). The zebrafish offers the following advantages for research in vertebrate developmental genetics: Its small size, its rapid life cycle, its clear embryos and its haploid development. Recently over 2000 developmental mutants have been isolated. A genetic linkage map has been constructed. The proposed work will attempt to identify specific chromosomes and to perform a molecular cytogenetic characterization of them. The PI proposes to obtain the following different sequences for localization onto zebrafish chromosomes: 1) highly repetitive sequences already isolated, from zebrafish chromosomes; 2) additional repetitive sequences that she plans to isolate herself by digestion with frequent cutting restriction enzymes, electrophoretic separation, cloning of and sequencing of the repeated sequences; 3) moderately repetitive sequences such as rDNA, histones and actin genes which she will produce by PCR amplification using primers whose sequences are based on information from zebrafish or other fish; 4) single copy sequences from known linkage groups. These genes will be isolated from a cosmid library by screening for clones to selected genes. She proposes to identify specific chromosomes using SINE banding or replication banding. Fluorescence in situ hybridization (FISH) using SINE sequences will be used to aid chromosome identification. The above sequences will be labeled with biotinylated nucleotides and used for localization on metaphase chromosomes by FISH. The metaphase chromosomes will be obtained from cultured fin and kidney cells. The PI proposes to use mapping methodology which she had previously used for mapping similar sequences to the chromosomes of several species of salmonid fishes. They include FISH, primed in situ hybridization (PRINS) and microdissection of specific chromosomes. A month before submitting the proposal she began working with zebra fish to apply the FISH technologies to zebrafish. Apart from the need to construct a physical map of the zebrafish genome the rationale was that these studies would complement the toxicology projects with zebrafish ongoing at the same Marine Sciences Freshwater Center. Identification of the chromosomes will be done by replication banding methods using synchronized cultures and SINE banding using Alu 1 and Tc1 fragments as probes for FISH, to band chromosomes .
| Phillips, R B; Reed, K M (2000) Localization of repetitive DNAs to zebrafish (Danio rerio) chromosomes by fluorescence in situ hybridization (FISH). Chromosome Res 8:27-35 |
