Differential Expression of the Mouse Mitochondrial Genes and the Mitochondrial RNA-Processing Endoribonuclease RNA by Androgens

Gail A. Cornwall,





Center for Reproductive Biology Research (G.A.C., M.-C.O.-C.) Department of Cell Bioloav (G.A.C., M.-C.O.-C., S.R.H.) Vanderbilt University Sch.O), 50 rni sodium phosphate buffer, pH 6.6, 0.1% SDS, 0.1% sodium pyrophosphate, 5x Denhardt’s solution, 50 mg/ml salmon sperm DNA, and 5% dextran sulfate. The filters were then washed in 2x SSC, 0.1% SDS at 42 C, followed by washes in 0.1 x SSC, 0.1% SDS at 42 C and 65 C. Clones representing putative androgen-regulated cDNAs were then selected and subjected to two additional rounds of subtraction with a subtracted cDNA probe to remove any false-positive clones. Using the standard Stratagene protocol, the cloned inserts were in viva excised, and the resulting phagemid was plated out with E. co/i to prepare double-stranded DNA for subsequent sequencing and expression analysis. Northern

Blot Analysis

RNA was separated on a 1% agarose gel containing borate buffer, pH 8.2, and 0.66 M formaldehyde. The RNA samples were heated at 95 C for 2 min and then loaded onto the gel and electrophoresed. To verify equal loading of RNA in each lane of the gel, the gels were stained with ethidium bromide. The gels were washed extensively in water to remove formaldehyde before transferring to a nylon membrane (Nytran, Schleicher and Schuell, Keene, NH) overnight in the presence of 1 Ox SSC. The blots were then washed for 5 min in 5x SSC before UV cross-linking the RNA to the blot (Stratalinker, Stratagene). The blots were prehybridized for 2 h at 42 C in hybridization buffer containing 50% formamide, 5x SSC, 0.2 mg/ml salmoti sperm DNA, 0.4 mg/ml yeast RNA, 50 pg/ml


BSA, 0.1% SDS, and 12.5 mM sodium phosphate buffer, pH 6.6, followed by hybridization overnight at 42 C in the presence of 3 x lo5 cpm probe/ml hybridization buffer. cDNA probes were prepared using a random primer labeling method (PrimeIt, Stratagene) modified from that described by Feinberg and Vogelstein (39). After hybridization, the blots were washed in 2x SSC at room temperature for 10 min followed by washing in 2x SSC, 1% SDS at 42 C for 30-60 min and then at 65 C before exposure to film. To again verify equal amounts and transfer of RNA in each lane of the blot, the cDNA probe was removed from the blot by two washes at 55 C in 0.1 x SSC, 1% SDS that had been brought to boiling before being added to the blots and then rehybridized under the same conditions described above with a cDNA probe to 18s rRNA. A synthetic oligonucleotide complementary to the RNAase MRP RNA was commercially prepared by Bio-Synthesis Inc. (Denton, TX). The 44-mer, 5’-GACGGATGACGCCCCGCTGCACGCCGCTCAGCTCGCCCCGGAGG-3’ was complementary to nucleotides 159-202 of the mouse RNAase MRP RNA (11). The oligomer was end-labeled following a protocol from Pharmacia (Piscataway, NJ) which uses [y-3ZP]ATP and T4 kinase. Hybridization of Northern blots was carried out at 42 C in the same hybridization buffer described above but containing 40% formamide. The blots were then washed in 1 X SSC at 50 C for 30 min.

DNA Slot Blot Analysis One microgram of DNA was added to 400 ~1 Tris/EDTA buffer containing 3 M NaOH. The DNA solution was then heated at 65 C for 1 h, after which the solution was neutralized by the addition of 2 M ammonium acetate. The DNA was then loaded onto nylon membrane (Nytran) using the Schleicher and Schuell slot blot apparatus. The DNA was then UV crosslinked to the membrane and prehybridized for 2 h in the hybridization buffer described above for Northern blot analysis. Hybridization with a random-primed cDNA probe to the mitochondrial genes D-loop-cytochrome b-NADH dehydrogenase 5 was then carried out as described for Northern blot analysis. To confirm equal loading of the DNA, the blot was reprobed with an epididymal-specific cDNA (cs 82) isolated during other screenings of the library. Sequence


Double-stranded sequence analysis was performed using [35S] ATP and the Sequenase II sequencing kit (US. Biochemical Co., Cleveland, OH), which uses the Sanger (40) method of dideoxy chain termination.

Acknowledgments The authors wish technical assistance

to gratefully of Suzanne

acknowledge the excellent Justis and Elizabeth Wolf.

Received February 14, 1992. Revision received April 15, 1992. Accepted April 28, 1992. Address requests for reprints to: Dr. Stephen R. Hann, C2310 MCN, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. This work was supported by NIH Grant HD-03820 (to M.CO.-C), the Life and Health Insurance Research Fund (to S.R.H.), a grant from the Andrew W. Mellon Foundation (to M.-CO.-C.), and National Research Service Award HD-07376 (to G.A.C.).

REFERENCES 1. Brunk CF 1981 Mitochondrial proliferation esis. Exp Cell Res 136:305-309



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2. Webster KA, Gunning P, Hardeman E, Wallace DC, Kedes L 1990 Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts. J Cell Physiol 142:566-573 3. Kagawa Y, Ohta S 1990 Regulation of mitochondrial ATP synthesis in mammalian cells by transcriptional control. Int J Biochem 22:219-229 4. Nelson BD 1987 Biogenesis of mammalian mitochondria. Curr Top Bioenergetics 35:221-272 5. Mutvei A, Kuzela S, Nelson BD 1989 Control of mitochondrial transcription by thyroid hormone. Eur J Biochem 180:235-240 6. Clayton DA 1984 Transcription of the mammalian mitochondrial genome. Annu Rev Biochem 53:573-594 7. Williams RS 1986 Mitochondrial gene expression in mammalian striated muscle. J Biol Chem 261 :12390-12394 8. Cantatore P, Loguercio PP, Fracasso F, Flagella Z, Gadaleta MN 1986 Quantitation of mitochondrial RNA species during rat liver development: the concentration of cytochrome oxidase subunit I (col) mRNA increases at birth. Ceil Diff 19:125-132 9. Gelfand R, Attardi G 1981 Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable. Mol Cell Biol 1:497-511 10. Cantatore P, Flagella Z, Fracasso F, Lezza AM, Gadaleta MN, de Montalvo A 1987 Synthesis and turnover rates of four rat liver mitochondrial RNA species. FEBS Lett 213:144-148 11, Attardi G, Chomyn A, King MP, Kruse B, Polosa PL, Murdter NN 1989 Biogenesis and assembly of the mitochondrial respiratory chain: structural, genetic, and pathological aspects. Biochem Sot Trans 18:509-513 12. Chang DD, Clayton DA 1987 A novel endoribonuclease cleaves at a priming site of mouse mitochondrial DNA replication. EMBO J 6:409-417 13. Chang DD, Clayton DA 1987 A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science 235:1178-l 184 14. Chang DD, Clayton DA 1989 Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate. Cell 56:131-l 39 15. Fisher RP, Clayton DA 1988 Purification and characterization of human mitochondrial transcription factor 1. Mol Cell Biol 8:3496-3509 16. Nagley P 1991 Coordination of gene expression in the formation of mammalian mitochondria. Trends Genet 7:14 17. Attardi G, Schatz G 1988 Biogenesis of mitochondria. Annu Rev Cell Biol 4:289-333 18. Kessler MA, Lamm L, Jarnagin K, DeLuca HF 1986 1,25Dihydroxyvitamin Da-stimulated mRNAs in rat small intestine. Arch Biochem Biophys 251:403-412 19. Van ltallie CM, Dannies PS 1988 Estrogen induces accumulation of the mitochondrial ribonucleic acid for subunit II of cytochrome oxidase in pituitary tumor cells. Mol Endocrinol 2:332-337 20. Ku CY, Lu Q, Ussuf KK, Weinstock GM, Sanborn BM 1991 Hormonal regulation of cytochrome oxidase subunit messenger RNAs in rat Sertoli cells. Mol Endocrinol 5:1669-l 676 21. Shan B, Vazquez E, Lewis JA 1990 Interferon selectively inhibits the expression of mitochondrial genes: a novel pathway for interferon-mediated responses. EMBO J 9:4307-4314 22. Mazo AM, Minchenko AG, Avdonina TA, Gause GG, Pusyriov AT 1983 Discrete poly (A)- RNA species from

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rat liver mitochondria are fragments of 16s mitochondrial rRNA carrying its 5’-termini. Mol Biol Rep 9:155-l 61 Meziane AE, Callen J-C, Mounolou J-C 1989 Mitochondrial gene expression during Xenopus laevis development: a molecular studv. EMBO J 8:1649-1655 Montoya J, Gaines GL, Attardi G 1983 The pattern of transcription of the human mitochondrial rRNA genes reveals two overlapping transcription units. Cell 34:151159 Bartsch G, Oberholzer M, Holliger 0, Weber J, Weber A, Rohr HP 1978 Stereology: a new quantitative morphological method to study epididymal function. Andrologia lo:31 -42 Brooks DE 1978 Activity and androgenic control of enzymes associated with the tricarboxvlic cvcle, lipid oxid&ion and mitochondrial shuttles in the epididymis and eoididvmal soermatozoa of the rat. Biochem J 174:741752 ‘ Nelson BD, Mutvei A, Joste V 1984 Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone. Arch Biochem Biophys 228:41-48 Tata JR, Ernster L, Lindberg 0, Arrhenius E, Pedersen S, Hedman R 1963 The action of thyroid hormone at the cell level. Biochem J 86:408-428 Schleicher G, Stumpf WE, Gurley JM, Drews U 1989 Differential nuclear binding of [3H]testosterone and its metabolites to androgen and estrogen receptors in brain, pituitary, heart, kidney, and accessory sex glands of the mouse: an autoradiographic study. J Steroid Biochem 33:581-587 Gustafsson J-A, Pousette A 1975 Demonstration and partial characterization of cytosol receptors for testosterone. Biochemistry 14:3094-3101 Janne OA, Kontula KK, lsomaa VV, Torkeli TK, Bardin CW 1983 Androgen receptor-dependent regulation of ornithine decarboxvlase gene expression in mouse kidney. In: Eriksson H, Gustafsson J:A (eds) Steroid Hormone Receptors: Structure and Function. Elsevier Science Publishers, New York, p 461 Koenig H, Goldstone A, Blume G, Lu CY 1980 Testosterone-mediated sexual dimorphism of mitochondria and lysosomes in mouse kidney proximal tubules. Science 209:1023-l 026 Goldstone AD, Koenig H, Lu CY 1981 Androgens regulate cell growth, lysosomal hydrolases and mitochondrial cytochrome c oxidase in mouse aorta. Biochim Biophys Acta 673:170-l 76 Sterling K, Milch PO, Brenner MA, Lazarus JH 1977 Thyroid hormone action: the mitochondrial pathway. Science 197:996-999 Lansman RA, Clayton DA 1975 Mitochondrial protein synthesis in mouse L-cells: effect of selective nicking of mitochondrial DNA. J Mol Biol 99:777-793 Dyson ALMB, Orgebin-Crist M-C 1973 Effect of hypophysectomy, castration, and androgen replacement upon the fertilizing ability of rat epididymal spermatozoa. Endocrinology 93:391-402 Robaire B, Hermo L 1988 Efferent ducts, epididymis, and vas deferens: structure, functions and their requlation. In: Knobil E, Neil1 J (eds) The Physiology of Reproduction. Raven Press, Ltd.. New York, vol 1:999-l 080 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-l 59 Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-l 3 Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 7415463-5467 Attardi G 1986 The elucidation of the human mitochondrial genome: A historical perspective. BioEssays 5:34-39

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Differential expression of the mouse mitochondrial genes and the mitochondrial RNA-processing endoribonuclease RNA by androgens.

Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represen...
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