Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1997 Mar 1;322(Pt 2):403–410. doi: 10.1042/bj3220403

Cloning and characterization of murine carnitine acetyltransferase: evidence for a requirement during cell cycle progression.

S Brunner 1, K Kramar 1, D T Denhardt 1, R Hofbauer 1
PMCID: PMC1218205  PMID: 9065756

Abstract

We have employed a newly developed differential screening technique (reverse strand priming) to identify murine carnitine acetyltransferase (CARAT) as a growth- and cell cycle-regulated gene in S3T3 mouse fibroblasts. Sequence analysis of the full-length cDNA clone and homology comparisons have revealed 87% homology to the human CARAT gene. On Northern blots we were able to measure a 2-3-fold induction 18 h after a mitogenic stimulus following serum deprivation as well as after release from a sodium butyrate block. The cell cycle induction pattern of the CARAT gene was analysed in mouse fibroblasts at different stages of the unperturbed cell cycle. Fractions obtained by elutriation of an exponentially growing culture showed a biphasic maximum of transcript abundance in the G1 and G2 phases of the cell cycle. CARAT expression was investigated in several organs of the adult mouse. Among those measured, CARAT expression was highest, relative to liver, in heart muscle (56-fold) and testis (21-fold). Using both conventional antisense oligodeoxynucleotides and novel single-stranded antisense phagemid DNA, we obtained evidence that the CARAT enzyme function is necessary for progression through G1 and into the S-phase of the cell cycle.

Full Text

The Full Text of this article is available as a PDF (669.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agius L., Wright P. D., Alberti K. G. Carnitine acyltransferases and acyl-CoA hydrolases in human and rat liver. Clin Sci (Lond) 1987 Jul;73(1):3–10. doi: 10.1042/cs0730003. [DOI] [PubMed] [Google Scholar]
  2. Almendral J. M., Sommer D., Macdonald-Bravo H., Burckhardt J., Perera J., Bravo R. Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol Cell Biol. 1988 May;8(5):2140–2148. doi: 10.1128/mcb.8.5.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aureli T., Miccheli A., Ricciolini R., Di Cocco M. E., Ramacci M. T., Angelucci L., Ghirardi O., Conti F. Aging brain: effect of acetyl-L-carnitine treatment on rat brain energy and phospholipid metabolism. A study by 31P and 1H NMR spectroscopy. Brain Res. 1990 Aug 27;526(1):108–112. doi: 10.1016/0006-8993(90)90255-a. [DOI] [PubMed] [Google Scholar]
  4. Bakker A., Biermans W., Van Belle H., De Bie M., Bernaert I., Jacob W. Ultrastructural localisation of carnitine acetyltransferase activity in mitochondria of rat myocardium. Biochim Biophys Acta. 1994 Mar 29;1185(1):97–102. doi: 10.1016/0005-2728(94)90199-6. [DOI] [PubMed] [Google Scholar]
  5. Bieber L. L. Carnitine. Annu Rev Biochem. 1988;57:261–283. doi: 10.1146/annurev.bi.57.070188.001401. [DOI] [PubMed] [Google Scholar]
  6. Bonnefont J. P., Haas R., Wolff J., Thuy L. P., Buchta R., Carroll J. E., Saudubray J. M., Demaugre F., Nyhan W. L. Deficiency of carnitine palmitoyltransferase I. J Child Neurol. 1989 Jul;4(3):198–203. doi: 10.1177/088307388900400310. [DOI] [PubMed] [Google Scholar]
  7. Bougnères P. F., Saudubray J. M., Marsac C., Bernard O., Odièvre M., Girard J. Fasting hypoglycemia resulting from hepatic carnitine palmitoyl transferase deficiency. J Pediatr. 1981 May;98(5):742–746. doi: 10.1016/s0022-3476(81)80834-7. [DOI] [PubMed] [Google Scholar]
  8. Brice A., Berrard S., Raynaud B., Ansieau S., Coppola T., Weber M. J., Mallet J. Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression. J Neurosci Res. 1989 Jul;23(3):266–273. doi: 10.1002/jnr.490230304. [DOI] [PubMed] [Google Scholar]
  9. Carta A., Calvani M., Bravi D., Bhuachalla S. N. Acetyl-L-carnitine and Alzheimer's disease: pharmacological considerations beyond the cholinergic sphere. Ann N Y Acad Sci. 1993 Sep 24;695:324–326. doi: 10.1111/j.1749-6632.1993.tb23077.x. [DOI] [PubMed] [Google Scholar]
  10. Chatterjee B., Song C. S., Kim J. M., Roy A. K. Cloning, sequencing, and regulation of rat liver carnitine octanoyltransferase: transcriptional stimulation of the enzyme during peroxisome proliferation. Biochemistry. 1988 Dec 13;27(25):9000–9006. doi: 10.1021/bi00425a018. [DOI] [PubMed] [Google Scholar]
  11. Corti O., Finocchiaro G., Rossi E., Zuffardi O., DiDonato S. Molecular cloning of cDNAs encoding human carnitine acetyltransferase and mapping of the corresponding gene to chromosome 9q34.1. Genomics. 1994 Sep 1;23(1):94–99. doi: 10.1006/geno.1994.1463. [DOI] [PubMed] [Google Scholar]
  12. D'Anna J. A., Tobey R. A., Gurley L. R. Concentration-dependent effects of sodium butyrate in Chinese hamster cells: cell-cycle progression, inner-histone acetylation, histone H1 dephosphorylation, and induction of an H1-like protein. Biochemistry. 1980 Jun 10;19(12):2656–2671. doi: 10.1021/bi00553a019. [DOI] [PubMed] [Google Scholar]
  13. Demaugre F., Bonnefont J. P., Colonna M., Cepanec C., Leroux J. P., Saudubray J. M. Infantile form of carnitine palmitoyltransferase II deficiency with hepatomuscular symptoms and sudden death. Physiopathological approach to carnitine palmitoyltransferase II deficiencies. J Clin Invest. 1991 Mar;87(3):859–864. doi: 10.1172/JCI115090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DiDonato S., Rimoldi M., Moise A., Bertagnoglio B., Uziel G. Fatal ataxic encephalopathy and carnitine acetyltransferase deficiency: a functional defect of pyruvate oxidation? Neurology. 1979 Dec;29(12):1578–1583. doi: 10.1212/wnl.29.12.1578. [DOI] [PubMed] [Google Scholar]
  15. Elgersma Y., van Roermund C. W., Wanders R. J., Tabak H. F. Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene. EMBO J. 1995 Jul 17;14(14):3472–3479. doi: 10.1002/j.1460-2075.1995.tb07353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Esser V., Britton C. H., Weis B. C., Foster D. W., McGarry J. D. Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function. J Biol Chem. 1993 Mar 15;268(8):5817–5822. [PubMed] [Google Scholar]
  17. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  18. Finocchiaro G., Taroni F., Rocchi M., Martin A. L., Colombo I., Tarelli G. T., DiDonato S. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):661–665. doi: 10.1073/pnas.88.2.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Frangioni J. V., Neel B. G. Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem. 1993 Apr;210(1):179–187. doi: 10.1006/abio.1993.1170. [DOI] [PubMed] [Google Scholar]
  20. Gadaleta M. N., Petruzzella V., Renis M., Fracasso F., Cantatore P. Reduced transcription of mitochondrial DNA in the senescent rat. Tissue dependence and effect of L-carnitine. Eur J Biochem. 1990 Feb 14;187(3):501–506. doi: 10.1111/j.1432-1033.1990.tb15331.x. [DOI] [PubMed] [Google Scholar]
  21. Gadaleta M. N., Petruzzella V., Renis M., Fracasso F., Cantatore P. Reduced transcription of mitochondrial DNA in the senescent rat. Tissue dependence and effect of L-carnitine. Eur J Biochem. 1990 Feb 14;187(3):501–506. doi: 10.1111/j.1432-1033.1990.tb15331.x. [DOI] [PubMed] [Google Scholar]
  22. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  23. Hofbauer R., Denhardt D. T. Cell cycle-regulated and proliferation stimulus-responsive genes. Crit Rev Eukaryot Gene Expr. 1991;1(4):247–300. [PubMed] [Google Scholar]
  24. Johnson T. M., Kocher H. P., Anderson R. C., Nemecek G. M. Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase. Biochem J. 1995 Jan 15;305(Pt 2):439–444. doi: 10.1042/bj3050439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kalaria R. N., Harik S. I. Carnitine acetyltransferase activity in the human brain and its microvessels is decreased in Alzheimer's disease. Ann Neurol. 1992 Oct;32(4):583–586. doi: 10.1002/ana.410320417. [DOI] [PubMed] [Google Scholar]
  26. Karlseder J., Rotheneder H., Wintersberger E. Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. Mol Cell Biol. 1996 Apr;16(4):1659–1667. doi: 10.1128/mcb.16.4.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kispal G., Sumegi B., Dietmeier K., Bock I., Gajdos G., Tomcsanyi T., Sandor A. Cloning and sequencing of a cDNA encoding Saccharomyces cerevisiae carnitine acetyltransferase. Use of the cDNA in gene disruption studies. J Biol Chem. 1993 Jan 25;268(3):1824–1829. [PubMed] [Google Scholar]
  28. Lau L. F., Nathans D. Identification of a set of genes expressed during the G0/G1 transition of cultured mouse cells. EMBO J. 1985 Dec 1;4(12):3145–3151. doi: 10.1002/j.1460-2075.1985.tb04057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  30. Makar T. K., Cooper A. J., Tofel-Grehl B., Thaler H. T., Blass J. P. Carnitine, carnitine acetyltransferase, and glutathione in Alzheimer brain. Neurochem Res. 1995 Jun;20(6):705–711. doi: 10.1007/BF01705539. [DOI] [PubMed] [Google Scholar]
  31. Makar T. K., Hungund B. L., Cook G. A., Kashfi K., Cooper A. J. Lipid metabolism and membrane composition are altered in the brains of type II diabetic mice. J Neurochem. 1995 May;64(5):2159–2168. doi: 10.1046/j.1471-4159.1995.64052159.x. [DOI] [PubMed] [Google Scholar]
  32. Parfett C. L., Hofbauer R., Brudzynski K., Edwards D. R., Denhardt D. T. Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs. Gene. 1989 Oct 30;82(2):291–303. doi: 10.1016/0378-1119(89)90054-1. [DOI] [PubMed] [Google Scholar]
  33. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Przyrembel H. Therapy of mitochondrial disorders. J Inherit Metab Dis. 1987;10 (Suppl 1):129–146. doi: 10.1007/BF01812853. [DOI] [PubMed] [Google Scholar]
  35. Ratheiser K., Schneeweiss B., Waldhäusl W., Fasching P., Korn A., Nowotny P., Rohac M., Wolf H. P. Inhibition by etomoxir of carnitine palmitoyltransferase I reduces hepatic glucose production and plasma lipids in non-insulin-dependent diabetes mellitus. Metabolism. 1991 Nov;40(11):1185–1190. doi: 10.1016/0026-0495(91)90214-h. [DOI] [PubMed] [Google Scholar]
  36. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  37. Sargent T. D. Isolation of differentially expressed genes. Methods Enzymol. 1987;152:423–432. doi: 10.1016/0076-6879(87)52049-3. [DOI] [PubMed] [Google Scholar]
  38. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  39. Taroni F., Verderio E., Fiorucci S., Cavadini P., Finocchiaro G., Uziel G., Lamantea E., Gellera C., DiDonato S. Molecular characterization of inherited carnitine palmitoyltransferase II deficiency. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8429–8433. doi: 10.1073/pnas.89.18.8429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Uziel G., Garavaglia B., Di Donato S. Carnitine stimulation of pyruvate dehydrogenase complex (PDHC) in isolated human skeletal muscle mitochondria. Muscle Nerve. 1988 Jul;11(7):720–724. doi: 10.1002/mus.880110708. [DOI] [PubMed] [Google Scholar]
  41. Wintersberger E., Mudrak I., Wintersberger U. Butyrate inhibits mouse fibroblasts at a control point in the G1 phase. J Cell Biochem. 1983;21(3):239–247. doi: 10.1002/jcb.240210306. [DOI] [PubMed] [Google Scholar]
  42. Woeltje K. F., Esser V., Weis B. C., Sen A., Cox W. F., McPhaul M. J., Slaughter C. A., Foster D. W., McGarry J. D. Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II. J Biol Chem. 1990 Jun 25;265(18):10720–10725. [PubMed] [Google Scholar]
  43. Wolf H. P. Possible new therapeutic approach in diabetes mellitus by inhibition of carnitine palmitoyltransferase 1 (CPT1). Horm Metab Res Suppl. 1992;26:62–67. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES