Positional cloning of l7Rn3 and phenotypic analysis of l7Rn3¹⁷⁷⁷SB and l7Rn3²⁵²¹SB

Author

Sharon E. Thomas

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Engineering

Major Professor

Monica J. Justice

Committee Members

Bruce D. Mckee

Abstract

An-Nethyl-Nnitrosourea (ENU) .mutagenesis screen .using the .deletions at theAlbino locus on mouse Chromosome 7 revealed a number of embryonic lethal mutations.The focus of this work is on one of these Ioci, l7Rn3 and two of its six ENU-induced alleles(Rinchiket al., 1990). Homozygotes for the presumed amorphic allele {l7Rn3^1777SB) fail to gastrulate and lack a patterned visceral endoderm and epiblast. Hemizygotes for the viable allele(l7Rn3^2521SB)are small, exhibit subtle skeletal abnormalities of incomplete penetrance and variable expressivity and a slight anemia. Heterozygotes and homozygotes for the viable allele also exhibit a deletion-specific maternal effect that results in embryonic lethality at mid-gestation (Rinchik and Carpenter, 1999). Blastocyst transfers and histology suggest that the probable cause of death is an improper development of extraembryonic stmctures.In order to identify the gene mutated in the l7Rn3 alleles, positional cloning was undertaken. A 129/SvJ BAC contig was constructed and sample sequencing identified Odd Oz homolog 4 (Odz4). To confirm candidacy Northern blot analysis, DHPLC analysis and sequencing of RT-PCR products were performed. One mutation in five of the available alleles has been detected to date.DHPLC and sequencing revealed that l7Rn3^4323SB contains a GC to CO transversion resulting in an alanine to proline amino acid change in the globular C terminus portion of the protein. Northern blot analysis, however, failed to detect any alterations in transcript size or levels in the mutants. Odz4 is a large and complex gene spanning several hundred kilobases of genomic DNA. A, BAC contig that spans this entire region was sequenced, and the genomic information obtained will be useful for additional isoform identification and future mutation analysis.The allelic series of l7Rn3 reveals several roles of Odz4 in embryonic development including the induction and/or maintenance of the primitive streak and possibly anterior posterior patterning of the embryo, somite patterning and perhaps in the establishment and/or maintenance of extraembryonic structures. The allelic series for this locus, therefore,is an invaluable tool for elucidating the many functions of Odz4 in embryonic development.

Recommended Citation

Thomas, Sharon E., "Positional cloning of l7Rn3 and phenotypic analysis of l7Rn3¹⁷⁷⁷SB and l7Rn3²⁵²¹SB. " PhD diss., University of Tennessee, 2000.
https://trace.tennessee.edu/utk_graddiss/8430