The goals of this project are to determine the genome organization and genetic regulatory sequences controlling expression of nuclear genes for cytochromoe c oxidase subunits in humans. Mammalian cytochrome oxidase consist of at least twelve subunits, which are the products of two separate genomes. The three largest polypeptides (I-III) are mitochondrial gene products, whereas the smaller polypeptides (IV-VIII) are nuclear gene products. Futhermore, many of the nuclear encoded oxidase subunits have tissue-specific forms, implying the existence of more than one expressed gene for these subunits.
The specific aims are to determine the pattern of tissue-specific expression and the genome organization of subunit IV genes. These goals will be accomplished by isolating and sequencing cDNAs for skeletal muscle and fetal heart libraries and comparing these sequences with that of human liver cDNA. The pattern of expression of subunit IV genes in liver, heart, muscle and kidney will be determined by analyzing transcripts by Northern blot analysis, S1-nuclease mapping and primer extension experiments. The genome organization will be determined by characterizing and sequencing genomic clones. Regulatory sequences will be analyzed by means of gene fusions with the CAT gene. These fusions will be introduced into several different human and rodent gene(s) will be mapped to human chromosomes by means of somatic celll hybrids and human DNA will be analyzed for restriction fragment length polymerphisms. Finally, cDNA probes for other oxidase subunits will be isolated by constructing and screening a beef heart expression library with antibody to beef heart oxidase and with synthetic oligonucleotide probes. These studies are crucial to our understanding of regulatory signals involved in controlling expression of this essential enzyme complex and in understanding the genetic basis of inherited cytochrome oxidase deficiencies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037086-03
Application #
3292063
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1986-09-01
Project End
1990-08-31
Budget Start
1988-09-01
Budget End
1990-08-31
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Smith, E O; Lomax, M I (1993) Structural organization of the bovine gene for the heart/muscle isoform of cytochrome c oxidase subunit VIa. Biochim Biophys Acta 1174:63-71
Lomax, M I; Hewett-Emmett, D; Yang, T L et al. (1992) Rapid evolution of the human gene for cytochrome c oxidase subunit IV. Proc Natl Acad Sci U S A 89:5266-70
Smith, E O; BeMent, D M; Grossman, L I et al. (1991) The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence. Biochim Biophys Acta 1089:266-8
Lomax, M I; Welch, M D; Darras, B T et al. (1990) Novel use of a chimpanzee pseudogene for chromosomal mapping of human cytochrome c oxidase subunit IV. Gene 86:209-16
Lomax, M I; Coucouvanis, E; Schon, E A et al. (1990) Differential expression of nuclear genes for cytochrome c oxidase during myogenesis. Muscle Nerve 13:330-7
Koga, Y; Fabrizi, G M; Mita, S et al. (1990) Sequence of a cDNA specifying subunit VIIc of human cytochrome c oxidase. Nucleic Acids Res 18:684
Aqua, M S; Lomax, M I; Schon, E A et al. (1989) Nucleotide sequence of a cDNA for bovine cytochrome c oxidase subunit VIIc. Nucleic Acids Res 17:8376
Seelan, R S; Scheuner, D; Lomax, M I et al. (1989) Nucleotide sequence of a cDNA for bovine cytochrome c oxidase subunit VIIa. Nucleic Acids Res 17:6410