Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Forestry, Wildlife, and Fisheries

First Advisor

Terrence R. Tiersch

Abstract

This research presents studies on cytogenetics and molecular genetics of channel catfish (Ictalurus punctatus), the most important fish species cultured in the United States. The goals of this project were to analyze the genome of the channel catfish and to develop a direct method of mapping single-locus genes. A series of techniques were developed to facilitate these studies, including culture of fibroblast cells, preparation of chromosomes from catfish of different ages, staining for nucleolus organizer regions (NOR) and heterochromatin (C-banding), restriction enzyme banding, replication R-banding, simultaneous detection of sister-chromatid exchange (SCE) and C-banding, fluorescent in-situ hybridization (FISH) and indirect and direct in-situ polymerase chain reaction (ISPCR). A primary cell line was established from caudal fin tissue. The cell line was fibroblastic, and strongly positive for fibronectin and collagens type I and III in the cytoplasm. These cells maintained a normal karyotype after 42 generations and were positive for the channel catfish gene Ig H that codes for immunoglobulin. Individual chromosomes were identified by location of the NOR and C-banding, and by restriction enzyme and replication R-banding. The 29 chromosome pairs were divided into 8 distinct groups based on morphology and size. Standard C-banding and replication R-banding karyotypes were established, and ideograms were prepared for the first time for this species. A procedure for simultaneous detection of the SCE and C-banding was developed, which may allow measurement of the distance between exchange sites and centromeres. The baseline occurrence of SCE in the absence of mutagenic materials, were measured to be 3.6 $\pm$ 1.6% of chromosomes in each cell. In addition, FISH and ISPCR procedures were developed for analysis of the single-locus Ig H gene on whole-cell, nuclear, and chromosomal preparations. Two copies of the gene were revealed in each positive interphase nucleus. The chromosomal location of the Ig H gene was detected. However, the identity of the chromosome remains unknown because the banding pattern was not analyzable after hybridization. Application of the ISPCR in chromosomal mapping is new for fish species and is only in initial stages for higher vertebrates.

ISBN

9780591133943

Pages

153

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