Despite evidence that the tendency to develop obesity in humans is inherited and like to be polygenic in nature, the individual genes contributing to this neuroendocrine predisposition are largely unknown. Several mutations in exhibiting obesity phenotypes represent targets for the so-called """"""""reverse genetic"""""""" approach which requires no knowledge or assumptions concerning the biological nature of the defect and seeks ultimately to identify genes based solely on their chromosomal location.
The specific aims of this project are(1) to isolate additional anonymous DNA markers closely linked to the db locus, (2) provide more detailed resolution to the genetic linkage map of the db gene locus on mouse chromosome 4, (3) to derive the physical map of the db locus, (4) to examine the db locus and two other autosomal mutant gene loci that lead to obesity in the mouse for evidence of structural genomic DNA abnormalities, and (5) to begin studies ultimately to isolate these obesity genes. The explicit approaches will include the isolation of anonymous DNA markers from a phage lambda genomic library created from a hybrid cell line containing mouse chromosome 4 as its only mouse-derived genetic material, the deletion and mapping analysis of a panel of irradiation-reduced hybrid cell lines, RFLP analysis of interspecific and recombinant inbred mouse crosses, and separating and analyzing very large DNA molecules by pulsed field gel electrophoresis (PFGE) and blotting. Genomic DNA preparation (including samples in agarose plugs suitable for PFGE analysis) from mouse tissues and cell lines will be isolated. Coventional DNA Southern blots and genetic linkage experiments will be performed to provide more probes and mapping resources intended to narrow the focus on mouse chromosome 4 to identify the db gene. Blot transfers of PGFE experiments will be hybridized with various mouse and human DNA probes which are known to map closely to the db mutation and the other obesity mutations in mice. The examination of these large scale Southern blots will focus on (1) providing a physical genetic map for a complex of mutations near the db locus and (2) identifying potential structural abnormalities in DNA from db/db mice, mutants mapping near the db locus on mouse chromosome 4, and other single gene obesity mutants mapping to different but accessible mouse chromosomal loci. The long term objective are ultimately to identify the genes responsible for these genetic autosomal obesity syndromes. This grant proposal is of medical significance because any insights gained into these mouse mutations may help identify genes in mice and potential homologues in man contributing to the inherited tendency to develop obesity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044074-04
Application #
3245560
Study Section
Special Emphasis Panel (SRC (05))
Project Start
1991-07-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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McAllister, K A; Ramachandran, S; Haugen-Strano, A et al. (1997) Genetic mapping of the Brca2 breast cancer susceptibility gene on mouse chromosome 5. Mamm Genome 8:540-1
Boney, C M; Fiedorek Jr, F T; Paul, S R et al. (1996) Regulation of preadipocyte factor-1 gene expression during 3T3-L1 cell differentiation. Endocrinology 137:2923-8
Vinik, B S; Kay, E S; Fiedorek Jr, F T (1995) Mapping of the MEK kinase gene (Mekk) to mouse chromosome 13 and human chromosome 5. Mamm Genome 6:782-3
Bennett, L M; Haugen-Strano, A; Cochran, C et al. (1995) Isolation of the mouse homologue of BRCA1 and genetic mapping to mouse chromosome 11. Genomics 29:576-81
Fiedorek Jr, F T; Kay, E S (1995) Mapping of the focal adhesion kinase (Fadk) gene to mouse chromosome 15 and human chromosome 8. Mamm Genome 6:123-6
Semenkovich, C F; Coleman, T; Fiedorek Jr, F T (1995) Human fatty acid synthase mRNA: tissue distribution, genetic mapping, and kinetics of decay after glucose deprivation. J Lipid Res 36:1507-21
Fiedorek Jr, F T; Kay, E S (1995) Mapping of the insulin promoter factor 1 gene (Ipf1) to distal mouse chromosome 5. Genomics 28:581-4
Fiedorek Jr, F T; Kay, E S (1994) Mapping of PCR-based markers for mouse chromosome 4 on a backcross penetrant for the misty (m) mutation. Mamm Genome 5:479-85