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Vol. 288, No. 1, July, pp. 97-106, 1991

Cloning and Characterization c Oxidase Subunit IV Gene”* Robert

S. Carter3 and Narayan

of the Mouse Cytochrome

G. Avadhani4

Laboratories of Biochemistry, Department University of Pennsylvania, Philadelphia,

of Animal Biology, School of Veterinary Pennsylvania 191046046

Medicine,

December 13, 1990, and in revised form March 1, 1991

cDNA for mouse cytocbrome c oxidase subunit IV (COXIV) was isolated by screening mouse liver and kidney cDNA libraries with a bovine COXIV cDNA probe. The 679-nucleotide nearly full length cDNA codes for a 22-amino acid presequence and a 147-amino acid mature protein which show 77 to 95% positional identity with the predicted sequences of human, bovine, and rat subunits. Screening of mouse genomic X EMBLS library using the mouse cDNA probe yielded two overlapping clones. Restriction mapping and sequencing of the clones show that the mouse COXIV mRNA sequences are contained in five exons ranging from 58 to 236 base pairs, and four introns in a 7-kilobase region of the mouse genome. Southern blot analysis of restriction-digested genomic DNA indicates the presence of a single gene for COXIV in the mouse genome. Primer extension analysis using a synthetic 22-mer oligonucleotide, together with the 0.68-kilobase size of the mRNA shown by the Nortbern blot analysis, indicates that the major transcription start site of the COXIV gene is located 59 nucleotides upstream of the translation start site. The COXIV gene is highly GC rich and lacks TATA and CAAT elements in the immediate upstream region of the transcription start site. The putative promoter region, however, contains a number of GC boxes similar to those involved in the binding of Spl transcription factor. The unique features of the gene, as well as its characteristics common to other nuclear genes coding for different mitocbondrial proteins, have been discussed. D 1991AcademicPWSS, IIIC.

1 This research was supported in part by NIH grant GM-29037. ‘The nucleic acid sequences in this paper have been submitted to GenBank under Accession Numbers M37829, M37830, and M37831. ’ The results presented here will be part of a thesis to be submitted by R.S.C. in partial fulfillment of the Ph.D. degree in Biochemistry at the University of Pennsylvania. ’ To whom correspondence should be addressed.

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Cytochrome oxidase, the terminal enzyme of the respiratory chain is a mitochondrial inner membrane protein functioning in the transfer of electrons from reduced cytochrome c to water. The catalytic functions of cytochrome c oxidation, electron transport, and proton pumping by cytochrome oxidase have been assigned to the three largest subunits of the enzyme, all of which are encoded by the mitochondrial genome in eukaryotic cells (6,8). The enzyme contains an additional number of nuclear encoded subunits ranging from four in slime molds, six in yeast, to as many as ten in animal cells (5, 7). Although the role of these nuclear-encoded subunits remains unclear, yeast genetic studies have revealed that certain subunits are required for function and/or assembly of the complex (24). Biochemical characterization and molecular cloning of the nuclear-encoded polypeptides (16) have demonstrated the existence of tissue-specific isoforms of certain cytochrome oxidase subunits, suggesting a possible basis for the different kinetic properties of the enzyme observed in various cell types (4, 23). The genes for all six nuclear-encoded subunits of cytochrome oxidase in yeast have been cloned and in some cases the genomic DNA sequences and trans-acting factors involved in transcriptional regulation have been described (29, 30, 43). Although cDNAs for a number of mammalian subunits have been described (2, 10-12, 19, 32, 33, 39, 45, 46), the detailed characterization of functional genes from higher eukaryotes has been complicated due to the presence of multiple genes and pseudogenes for most subunits. The functional gene for rat subunit VIc was recently reported (38), and while this work was in progress, the genes for rat subunit IV (44), and mouse Vb (A. Basu and N. G. Avadhani, submitted for publication) have been characterized. Molecular analysis of these genes may reveal the means by which the synthesis of these nuclear-encoded cytochrome oxidase subunits is regulated and perhaps suggest functions for these polypeptides. Furthermore, the genetic study of nuclear-encoded mitochondrial proteins may help reveal the mech-

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anism by which the nuclear and organelle genomes are coordinately regulated. In the present paper we report the cloning of identical cDNA species for mouse kidney and liver cytochrome oxidase subunit IV and the characterization of the complete gene for this subunit. MATERIALS

AND

METHODS

Probe preparation and hybridization method. Double-stranded DNA sequences to be used as probes were isolated by restriction digestion and fractionation on agarose gels; DNA bands were visualized by ethidium bromide staining, excised, and purified by adsorption to “Geneclean,” a cationic glass resin (Bio 101, La Jolla, CA). The DNA was denatured by boiling and labeled by Klenow fragment extension of random hexameric oligonucleotides (Pharmacia-LKB Biotechnologies Inc., Piscataway, NJ) with added [3ZP]dCTP (6000 Ci/mmol, Amersham Corp., Arlington Heights, IL) to a specific act,ivity of >109 cpm/pg. All filter hybridizations, including plaque screenings and Northern and Southern analysis, were performed as described (22) in 50% formamide, 6X SSC’ (1X SSC = 0.15 M NaCl, 0.015 M Na3CSH507, pH 7.0), 5X Denhardt’s (1X Denhardt’s = 0.02% Ficoll, 0.02% polyvinyl-pyrrolidone, and 0.02% bovine serum albumin), 0.1% SDS, and 200 pg/ml denatured salmon sperm DNA. Following prehybridization of filters for 6-12 h, probe was added to a concentration of 1 to 2 X 10’ cpm/ml in fresh hybridization buffer. Unless noted hybridizations were performed overnight at 42°C followed by washes in 2X SSC, 0.2% SDS at 65°C. Reduced stringency screening of the mouse liver cDNA library with COXIV bovine probe was performed in the above solutions with a hybridization temperature of 37°C and washes at 55°C. Isolation of cDNA and genomic recombinant phages. Mouse DBA/ 25 adult liver genomic library in XEMBLB and mouse liver cDNA Xgtll library were obtained from Clontech Laboratories. Mouse kidney Xgtll library with size fractionated cDNA (

Cloning and characterization of the mouse cytochrome c oxidase subunit IV gene.

cDNA for mouse cytochrome c oxidase subunit IV (COXIV) was isolated by screening mouse liver and kidney cDNA libraries with a bovine COXIV cDNA probe...
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