Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1986 Aug; 6(8): 2855–2864.
PMCID: PMC367853

Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts.


The muscle-specific form of creatine kinase (MCK) is induced in differentiating myoblast cultures, and a dramatic increase in mRNA levels precedes and parallels the increase in MCK protein. To study this induction, the complete MCK gene was cloned and characterized. The transcription unit was shown to span 11 kilobases and to contain seven introns. The splice junctions were identified and shown to conform to the appropriate consensus sequences. Close homology with branchpoint consensuses was found upstream of the 3' splice sites in six of seven cases. Transcriptional regulation of the gene in differentiating myoblast cultures was demonstrated by nuclear run-on experiments; increases in transcription accounted for a major part of the increased mRNA levels. Regulated expression of a transfected MCK gene containing the entire transcription unit with 3.3 kilobases of 5'-flanking sequence was also demonstrated during differentiation of the MM14 mouse myoblast cell line. The MCK 5'-flanking region was sufficient to confer transcriptional regulation to a heterologous structural gene, since chloramphenicol acetyl transferase activity was induced during differentiation of cultures transfected with an MCK-chloramphenicol acetyl transferase fusion construct. Examination of the DNA sequence immediately upstream of the transcription start site revealed a 17-nucleotide element which occurred three times. Comparisons with other muscle-specific genes which are also transcriptionally regulated during myogenesis revealed upstream homologies in the alpha-actin and myosin heavy chain genes, but not in the myosin light-chain genes, with the regions containing these repeats. We suggest that coordinate control of a subset of muscle genes may occur via recognition of these common sequences.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Benfield PA, Zivin RA, Miller LS, Sowder R, Smythers GW, Henderson L, Oroszlan S, Pearson ML. Isolation and sequence analysis of cDNA clones coding for rat skeletal muscle creatine kinase. J Biol Chem. 1984 Dec 10;259(23):14979–14984. [PubMed]
  • Benton WD, Davis RW. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. [PubMed]
  • Bessman SP, Geiger PJ. Transport of energy in muscle: the phosphorylcreatine shuttle. Science. 1981 Jan 30;211(4481):448–452. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Buskin JN, Jaynes JB, Chamberlain JS, Hauschka SD. The mouse muscle creatine kinase cDNA and deduced amino acid sequences: comparison to evolutionarily related enzymes. J Mol Evol. 1985;22(4):334–341. [PubMed]
  • Caravatti M, Minty A, Robert B, Montarras D, Weydert A, Cohen A, Daubas P, Buckingham M. Regulation of muscle gene expression. The accumulation of messenger RNAs coding for muscle-specific proteins during myogenesis in a mouse cell line. J Mol Biol. 1982 Sep;160(1):59–76. [PubMed]
  • Chamberlain JS, Jaynes JB, Hauschka SD. Regulation of creatine kinase induction in differentiating mouse myoblasts. Mol Cell Biol. 1985 Mar;5(3):484–492. [PMC free article] [PubMed]
  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. [PubMed]
  • Chu G, Sharp PA. SV40 DNA transfection of cells in suspension: analysis of efficiency of transcription and translation of T-antigen. Gene. 1981 Mar;13(2):197–202. [PubMed]
  • Devlin RB, Emerson CP., Jr Coordinate accumulation of contractile protein mRNAs during myoblast differentiation. Dev Biol. 1979 Mar;69(1):202–216. [PubMed]
  • Freytag SO, Beaudet AL, Bock HG, O'Brien WE. Molecular structure of the human argininosuccinate synthetase gene: occurrence of alternative mRNA splicing. Mol Cell Biol. 1984 Oct;4(10):1978–1984. [PMC free article] [PubMed]
  • Gorman CM, Merlino GT, Willingham MC, Pastan I, Howard BH. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. [PMC free article] [PubMed]
  • Grichnik JM, Bergsma DJ, Schwartz RJ. Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5' end of the chicken alpha-skeletal actin gene. Nucleic Acids Res. 1986 Feb 25;14(4):1683–1701. [PMC free article] [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Hastings KE, Emerson CP., Jr cDNA clone analysis of six co-regulated mRNAs encoding skeletal muscle contractile proteins. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1553–1557. [PMC free article] [PubMed]
  • Keller EB, Noon WA. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417–7420. [PMC free article] [PubMed]
  • Kimura A, Israël A, Le Bail O, Kourilsky P. Detailed analysis of the mouse H-2Kb promoter: enhancer-like sequences and their role in the regulation of class I gene expression. Cell. 1986 Jan 31;44(2):261–272. [PubMed]
  • Konieczny SF, Emerson CP., Jr Differentiation, not determination, regulates muscle gene activation: transfection of troponin I genes into multipotential and muscle lineages of 10T1/2 cells. Mol Cell Biol. 1985 Sep;5(9):2423–2432. [PMC free article] [PubMed]
  • Land H, Grez M, Hauser H, Lindenmaier W, Schütz G. 5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucleic Acids Res. 1981 May 25;9(10):2251–2266. [PMC free article] [PubMed]
  • Lebherz HG. Ontogeny and regulation of fructose diphosphate aldolase isoenzymes in "red" and "white" skeletal muscles of the chick. J Biol Chem. 1975 Aug 10;250(15):5976–5981. [PubMed]
  • Linkhart TA, Clegg CH, Hauschka SD. Control of mouse myoblast commitment to terminal differentiation by mitogens. J Supramol Struct. 1980;14(4):483–498. [PubMed]
  • Linkhart TA, Clegg CH, Hauschika SD. Myogenic differentiation in permanent clonal mouse myoblast cell lines: regulation by macromolecular growth factors in the culture medium. Dev Biol. 1981 Aug;86(1):19–30. [PubMed]
  • Mahdavi V, Chambers AP, Nadal-Ginard B. Cardiac alpha- and beta-myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci U S A. 1984 May;81(9):2626–2630. [PMC free article] [PubMed]
  • McKnight GL, McConaughy BL. Selection of functional cDNAs by complementation in yeast. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4412–4416. [PMC free article] [PubMed]
  • McKnight GS, Palmiter RD. Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J Biol Chem. 1979 Sep 25;254(18):9050–9058. [PubMed]
  • Medford RM, Nguyen HT, Nadal-Ginard B. Transcriptional and cell cycle-mediated regulation of myosin heavy chain gene expression during muscle cell differentiation. J Biol Chem. 1983 Sep 25;258(18):11063–11073. [PubMed]
  • Melloul D, Aloni B, Calvo J, Yaffe D, Nudel U. Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells. EMBO J. 1984 May;3(5):983–990. [PMC free article] [PubMed]
  • Merrill GF, Hauschka SD, McKnight SL. tk Enzyme expression in differentiating muscle cells is regulated through an internal segment of the cellular tk gene. Mol Cell Biol. 1984 Sep;4(9):1777–1784. [PMC free article] [PubMed]
  • Messing J, Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. [PubMed]
  • Miller J, Bothwell A, Storb U. Physical linkage of the constant region genes for immunoglobulins lambda I and lambda III. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3829–3833. [PMC free article] [PubMed]
  • Mount SM. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. [PMC free article] [PubMed]
  • Mulvihill ER, Palmiter RD. Relationship of nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. J Biol Chem. 1977 Mar 25;252(6):2060–2068. [PubMed]
  • Nguyen HT, Medford RM, Nadal-Ginard B. Reversibility of muscle differentiation in the absence of commitment: analysis of a myogenic cell line temperature-sensitive for commitment. Cell. 1983 Aug;34(1):281–293. [PubMed]
  • Nudel U, Calvo JM, Shani M, Levy Z. The nucleotide sequence of a rat myosin light chain 2 gene. Nucleic Acids Res. 1984 Sep 25;12(18):7175–7186. [PMC free article] [PubMed]
  • Nudel U, Greenberg D, Ordahl CP, Saxel O, Neuman S, Yaffe D. Developmentally regulated expression of a chicken muscle-specific gene in stably transfected rat myogenic cells. Proc Natl Acad Sci U S A. 1985 May;82(10):3106–3109. [PMC free article] [PubMed]
  • Okayama H, Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. [PMC free article] [PubMed]
  • Perriard JC, Caravatti M, Perriard ER, Eppenberger HM. Quantitation of creatine kinase isoenzyme transition in differentiating chicken embryonic breast muscle and myogenic cell cultures by immunoadsorption. Arch Biochem Biophys. 1978 Nov;191(1):90–100. [PubMed]
  • Reiss NA, Kaye AM. Identification of the major component of the estrogen-induced protein of rat uterus as the BB isozyme of creatine kinase. J Biol Chem. 1981 Jun 10;256(11):5741–5749. [PubMed]
  • Robert B, Daubas P, Akimenko MA, Cohen A, Garner I, Guenet JL, Buckingham M. A single locus in the mouse encodes both myosin light chains 1 and 3, a second locus corresponds to a related pseudogene. Cell. 1984 Nov;39(1):129–140. [PubMed]
  • Ruskin B, Krainer AR, Maniatis T, Green MR. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Shani M, Zevin-Sonkin D, Saxel O, Carmon Y, Katcoff D, Nudel U, Yaffe D. The correlation between the synthesis of skeletal muscle actin, myosin heavy chain, and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis. Dev Biol. 1981 Sep;86(2):483–492. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Strehler EE, Periasamy M, Strehler-Page MA, Nadal-Ginard B. Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates. Mol Cell Biol. 1985 Nov;5(11):3168–3182. [PMC free article] [PubMed]
  • Stuart GW, Searle PF, Chen HY, Brinster RL, Palmiter RD. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. [PMC free article] [PubMed]
  • Tombes RM, Shapiro BM. Metabolite channeling: a phosphorylcreatine shuttle to mediate high energy phosphate transport between sperm mitochondrion and tail. Cell. 1985 May;41(1):325–334. [PubMed]
  • Wallimann T, Pelloni G, Turner DC, Eppenberger HM. Monovalent antibodies against MM-creatine kinase remove the M line from myofibrils. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4296–4300. [PMC free article] [PubMed]
  • Whalen RG, Sell SM, Butler-Browne GS, Schwartz K, Bouveret P, Pinset-Härstöm I. Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature. 1981 Aug 27;292(5826):805–809. [PubMed]
  • Zakut R, Shani M, Givol D, Neuman S, Yaffe D, Nudel U. Nucleotide sequence of the rat skeletal muscle actin gene. Nature. 1982 Aug 26;298(5877):857–859. [PubMed]
  • Zimmern D, Kaesberg P. 3'-terminal nucleotide sequence of encephalomyocarditis virus RNA determined by reverse transcriptase and chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4257–4261. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...