We propose to investigate two related aspects of mammalian mitochondrial molecular biology. First, we will establish the extent, origin, and potential function of intracellular mitochondrial DNA (mtDNA) sequence heterogeneity, with particular emphasis on a sequence involved in controlling gene expression which we have shown is heterogeneous in tissue. We will compare the magnitude and distribution of mtDNA sequence polymorphs as a function of adult and fetal tissue in the dairy cow. Potential nuclear effects will be assessed by a similar analysis as a function of bovine lineage, breed, and artiodactyl species. The clonal origin of mtDNA heterogeneity will be tested by studying tissue culture cells cloned from bovine, human, and mouse lines. Finally, the relative template activity of individual polymorphs will be studied in vitro using mtRNA polymerase. Second, to better understand mtDNA sequences controlling initiation of transcription and replication, we will analyze the in vitro functon of artiodactyl mtRNA polymerases on homologous and non-homologous mtDNA templates. We will finish determining the D-loop region sequence of ten artiodactyls, accurately locate; the termini of D-loop region RNA and DNA, partially purify several artiodactyl mtRNA polymerases and size in vitro transcription products to correlate with in vivo RNA initiation sites. This study will define what sequence features regulate mtRNA polymerase initiation. It will also afford a unique analysis of the co-evolution of a DNA sequence and its interactive protein over a relatively short evolutionary span, but one in which significant DNA sequence change has occurred.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034825-03
Application #
3286487
Study Section
Molecular Biology Study Section (MBY)
Project Start
1985-09-01
Project End
1988-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Florida
Department
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Ghivizzani, S C; Madsen, C S; Nelen, M R et al. (1994) In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication. Mol Cell Biol 14:7717-30
Ghivizzani, S C; Madsen, C S; Hauswirth, W W (1993) In organello footprinting. Analysis of protein binding at regulatory regions in bovine mitochondrial DNA. J Biol Chem 268:8675-82
Madsen, C S; Ghivizzani, S C; Hauswirth, W W (1993) Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region. Mol Cell Biol 13:2162-71
Madsen, C S; Ghivizzani, S C; Hauswirth, W W (1993) In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria. Proc Natl Acad Sci U S A 90:7671-5
Ghivizzani, S C; Mackay, S L; Madsen, C S et al. (1993) Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region. J Mol Evol 37:36-7
Hehman, G L; Hauswirth, W W (1992) DNA helicase from mammalian mitochondria. Proc Natl Acad Sci U S A 89:8562-6
Muise, R C; Hauswirth, W W (1992) Transcription in maize mitochondria: effects of tissue and mitochondrial genotype. Curr Genet 22:235-42
Chan, L; Zuker, M; Jacobson, A B (1991) A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences. Nucleic Acids Res 19:353-8
Ashley, M V; Laipis, P J; Hauswirth, W W (1989) Rapid segregation of heteroplasmic bovine mitochondria. Nucleic Acids Res 17:7325-31
Laipis, P J; Van de Walle, M J; Hauswirth, W W (1988) Unequal partitioning of bovine mitochondrial genotypes among siblings. Proc Natl Acad Sci U S A 85:8107-10

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