Doctoral Dissertations

Date of Award

8-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Eugene M. Rinchik

Abstract

Complementation analyses of radiation-induced deletion mutations involving the albino (c) locus in chromosome 7 of the mouse have identified several loci, both proximal and distal to c, that have important roles in development. Embryos homozygous for a deletion of the most proximal of these loci, "mesoderm-deficient" (msd), fail to develop mesoderm and die at Day 11.5 of gestation. Animals homozygous for a deletion of the "hepatocyte-specific developmental regulation-1" (hsdr-1) locus, located between the msd and c loci, develop morphological, ultrastructural, and biochemical abnormalities of the liver and kidney, and die shortly after birth. It has been postulated that the hsdr-1 locus encodes a trans-acting regulatory factor(s) required for normal liver- and kidney-specific gene expression of various enzymes and proteins. Cloning the breakpoints of various albino deletions that define the extents of the msd and hsdr-1 loci is one way of generating molecular probes for studying the particular gene(s) involved in these developmental functions. Mapping of the anonymous locus D7Was12 with respect to the panel of albino deletions suggested that it, like the previously cloned Emv-23 locus, might be a useful access point for cloning some of these deletion breakpoints. Using pulsed-field-gel (CHEF) analysis, a long-range map of wild-type DNA spanning about 1.7 megabases surrounding the c, D7Was12, and Emv-23 loci was constructed. The molecular identification (with pulsed-field-gel electrophoresis) of structural rearrangements caused by the breakpoints of five albino deletions involved in the hsdr-1 and msd phenotypes, as well as of two viable albino deletions that remove only part of the tyrosinase gene and extend distally, enabled the localization of the deletion breakpoints on the long-range map. This analysis revealed that cloning the breakpoints of the viable deletions can generate additional DNA probes that will, in turn, facilitate the cloning of the breakpoints of some of the deletions defining the hsdr-1 and msd functions. A deletion-breakpoint-fusion fragment cloned from c24R75M/c24R75M DNA with the MTY811 probe proved to be instrumental in cloning the breakpoint of a neonatally lethal deletion, c14CoS (complementation designation Al), one of the original mutations used to define the hsdr-1 locus. DNA probes, isolated from a portion of the A1-breakpoint- fusion fragment located proximal to the breakpoint (i.e., within the hsdr-1 region), detect transcripts that are most abundant in the liver and kidney. Liver cDNA clones, isolated with one of these probes, were found to encode fumarylacetoacetate hydrolase (FAH). All of the deletions eliciting the hsdr-1 phenotype prevent expression of this Fah gene, and most disrupt the coding segment of the gene. Therefore, the Fah gene maps within the hsdr-1 locus, as defined by deletion breakpoints, and disruption of this gene may be responsible for the hsdr-1 phenotype. Mutations in the human FAH gene are associated with type 1 hereditary tyrosinemia (HT), a disorder which, like that observed in mice homozygous for deletions of hsdr-1, causes morphological abnormalities and biochemical dysfunctions of the liver and kidney. However, there may be other gene(s) at the hsdr-1 locus that are associated with the specific biochemical and ultrastructural abnormalities, observed in deletion homozygotes, that have not yet been reported in HT patients. Further studies, particularly correction-of-phenotype experiments with transgenic mice, will be required to determine whether or not loss of the Fah gene is responsible for the abnormalities present in mice homozygous for several different albino deletions.

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