Complete genomic sequence is available for 36 eukaryotic species, including the major model research organisms from yeast to mouse. Determination of the sequence of an additional 700 new eukaryotic genomes is currently in progress. Despite knowing most of the protein domains encoded in the genome of a given organism, the majority of genes have not been directly linked to a specific biological process. The classical approach of forward genetic screens persists as an effective approach to link genes to specific cellular processes. Knowledge of both the structural and biochemical activities of gene products predicted from sequence, and functional requirements deduced from genetics, provides valuable clues to direct mechanistic studies to determine which gene products participate in specific biological activities or contribute to disease states.
The work described in this dissertation was motivated by the results of a forward genetic screen in the nematode Caenorhabditis elegans , to identify gene products that are involved in the maintenance and organization of the unique cytoskeletal architecture at the apical side of intestinal cells. The genetic screen uncovered four mutant animals that share the same tissue anomaly, namely, numerous invaginations of the apical cell membrane that faces the intestinal lumen. Prior analysis revealed that two of the four mutant animals are due to single gene defects that disrupt the ifd-2 gene, known to encode a subunit of the polymeric intermediate filament cytoskeleton of intestinal cells. A third mutant disrupts the dal-1 gene, that encodes a protein weakly similar to a family of proteins that bind polymeric intermediate filaments. The goal of this dissertation is to complete the initial molecular analysis of the four intestinal mutants by pursuing two specific aims: (1) genetic mapping and molecular identification of the gene defective in the remaining uncharacterized mutant, dal(dt2400) and (2) generation of specific antibodies to the ifd-2 and dal-1 gene products in order to compare their subcellular localization and integrity in normal and mutant animals. A series of genetic mapping studies revealed that the gene defective in dal(dt2400) mutant animals is located near map position -17.7 on the X chromosome. The genetic mapping reduces the number of genes that could correspond to the mutant from approximately 22,000 to 60, and thus paves the way for ongoing, targeted studies to identify the specific molecular defect that gives rise to the intestinal defects in this mutant strain. With regard to aim 2, antibodies specific for the IFD-2 and DAL-1 gene products were generated in guinea pig and rabbit hosts, respectively. The antibodies should permit detailed molecular analysis of the level, location, and relative organization of the IFD-2 and DAL-1 proteins in animals in both normal and gene-specific disease states. Given the cellular and molecular similarities between the nematode and human intestines, the work presented here provides a solid foundation to advance a systematic molecular analysis of how animals regulate the topology and integrity of the thin, mechanical barrier that separates the body from food, drugs, toxins and other material ingested from the environment.
|Advisor:||Waddle, James A.|
|Commitee:||Ruben, Larry, Vik, Steven B.|
|School:||Southern Methodist University|
|School Location:||United States -- Texas|
|Source:||MAI 51/01M(E), Masters Abstracts International|
|Subjects:||Biology, Molecular biology, Genetics|
|Keywords:||Barrier function, Caenorhabditis elegans, Intermediate filaments, Intestinal morphology, Terminal web|
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