Doctoral Dissertations

Date of Award

8-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

E. M. Rinchik

Abstract

Molecular and phenotypic analyses of allelic mutations that disrupt normal development are extremely useful tools for dissecting the roles particular genes play in development. Saturation mutagenesis of a large region surrounding the albino (c) locus of mouse chromosome 7, employing the point-mutagen N-ethyl-N-nitrosourea (ENU), has resulted in the identification of five independent mutations of a gene, designated fitness 1 (fit1), which is necessary for normal pre- and postnatal growth and viability. By design of the mutagenesis strategy, fit1, as well as other ENU-induced mutations recovered in this screen, map within the extensive set of deletion mutations that surround the c locus. Therefore, given the inherent advantages of deletion mutations for rapidly mapping DNA probes and for gaining access to distant loci by cloning deletion breakpoints, fit1 is a candidate for a positional-cloning strategy, which allows the molecular identification of a gene based on its map position rather than on functional information. Molecular characterization of the fit1 gene, in conjunction with biological analysis of the fit1 phenotype, should provide key insights into the processes involved in normal growth and development. Additionally, analysis of the five independent fit1 mutations may provide information relevant to associating biological functions to specific DNA sequences. The fit1 gene has been mapped to a specific subregion of the c deletion complex, defined by the distal breakpoints of several deletion mutations. Molectdar access to the fit1 region was gained by cloning one of the deletion breakpoints associated with the proximeil border of the fit1 interval. Physical mapping of the fit1 region, with this proximal probe and with an existing DNA probe that maps distal to fit1, has allowed determination of the maximum size of the fit1 region, and has further distinguished the proximal border of the fit1 locus and the distal border of at least three loci mapping proximal to fit1. Two yeast artificial chromosomes (YACs) were obtained, one of which spans an interval known to contain at least part of the fit1 gene. Characterization of these YAC clones has provided additional DNA probes at the fit1 locus, has narrowed the region containing at least part of the fit1 gene, and has suggested several putative CpG island sites at the fit1 locus. Characterization of the fit1 phenotype revealed that fit1 mutants are growth retarded at least as early as 14.5 days of gestation and remain dwarfed throughout their shortened lifespan. Also, these growth analyses indicate that there is a range of severity among the five fit1 mutations. Additionally, fit1 mutants were found to be anemic, displayed numerous peripheral blood defects, and were deficient in early hematopoietic progenitor cell populations. Not only were erythroid and myeloid cells reduced, but the number of lymphoid lineage cells were also lower. These results implicate fit1 involvement in normal hematopoietic differentiation and suggest that, in conjunction with molecular analyses, further characterization of the fit1 gene, and these five allelic presumed point mutations of the gene, will lead to an improved understanding of normal pre- and postnatal development and hematopoietic differentiation.

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