Proc. Natl. Acad. Sci. USA Vol. 81, pp. 7313-7317, December 1984 Biochemistry Complete nucleotide sequence of Torpedo marmorata mRNA coding for the 43,000-dalton v2 protein: Muscle-specific creatine kinase (cDNA cloning/hybrid-selected translation/oligonucleotide-specific primer extension) JRR6ME GIRAUDAT*, ANNE DEVILLERS-THIERY*, JEAN-CLAUDE PERRIARDt, AND JEAN-PIERRE CHANGEUX* *Unitd de Neurobiologie Moldculaire et Laboratoire Associd 270, Centre National de la Recherche Scientifique Interactions Moldculaires et Cellulaires, Institut Pasteur, 25 rue du Docteur Roux, 75 015 Paris, France; and lInstitute for Cell Biology, Swiss Federal Institute for Technology, Honggerberg, CH 8093 Zurich, Switzerland Contributed by Jean-Pierre Changeux, August 1, 1984 ABSTRACT DNA sequences coding for the muscle-specif- charge. We previously reported the construction of cDNA ic subunit of creatine kinase have been isolated from cDNA libraries from Torpedo marmorata electric organ, which en- libraries constructed from Torpedo marmorata electric organ. abled us to isolate cDNA clones coding for the a-subunit of Clones were screened by differential in situ hybridization and the acetylcholine receptor (12, 13). We describe here the iso- hybrid-selected translation. The in vitro translation product of lation of cDNA clones coding for the T. marmorata muscle- the selected mRNA was immunoprecipitated by anti-chicken specific CK and report the complete nucleotide sequence of creatine kinase antibodies and comigrated with Torpedo mus- the corresponding mRNA. cle creatine kinase on two-dimensional gels at the same posi- tion as the cytosolic 43,000-dalton protein referred to as v2. MATERIALS AND METHODS The cDNA inserts hybridized to a mRNA species present in Materials. T. marmorata were obtained live from the Ma- adult Torpedo muscle but not in brain. The complete sequence rine Biological Station of Arcachon, France. Unless other- of the mRNA was determined on one of the clones except for wise noted, the enzymes were from PL Biochemicals or the 78 nucleotides of the mRNA 5' terminal sequence, which Boehringer Mannheim. Reverse transcriptase was from Life were identified by the primer extension method. The amino Sciences (St. Petersburg, FL) and Stehelin (Basel, Switzer- acid sequence of muscle-specific creatine kinase from T. mar- land). Restriction endonucleases were products of New En- morata was deduced and analyzed. It includes the known se- gland Biolabs, Boehringer Mannheim, or Bethesda Research quence of a peptide from the active site of rabbit muscle-specif- Laboratories. Radioactive molecules were from the Radio- ic creatine kinase. chemical Centre. 3-([3-Cholamidopropyl]dimethylam- monio)-1-propanesulfonate (CHAPS) was from Sigma. Ni- Creatine kinase (CK), by maintaining a constant level of trocellulose paper used for protein transfer was from Milli- ATP, plays a critical role in the energy metabolism ofthe cell pore, and paper for DNA or RNA transfers was from (1). It is abundant in muscle and in brain but is present at Schleicher and Schuell. Diaminobenzidine was from Sigma, lower levels in other tissues. Three active CK isozymes have horseradish peroxidase-conjugated IgG was from Institut been distinguished on the basis of their electrophoretic mo- Pasteur, and protein A-Sepharose was from Pharmindustrie bilities. They correspond to the various dimeric combina- (Paris, France). tions of two different subunits M and B: MM, BB, and MB. Protein Preparations. Organs were homogenized as de- Adult brain contains exclusively BB-CK, as do myoblasts. scribed (14). The supernatant of a high-speed centrifugation During the course of muscle differentiation, the progressive of electric organ homogenate was used as a cytosolic prepa- appearance of the muscle-specific M-CK leads to the tran- ration. Acetylcholine receptor-rich membrane fragments sient presence of MB-CK, and, finally, in the adult skeletal were prepared as described (14). muscle, to the exclusive occurrence of MM-CK (2). Purification of Poly(A)+ RNA. Polyadenylylated RNA was The M- and B-CK subunits are both close to 40,000 dal- purified from frozen organs as described (12). tons but can be distinguished by immunological techniques In Vitro Protein Synthesis and Immunoprecipitation. In vi- (3) or by two-dimensional gel electrophoresis (4). In chicken, tro translation of mRNAs was performed using the reticulo- distinct mRNA species encode the M- and B-CK subunits cyte lysate translation system (15) in the presence of and the presence of a single CK subunit has been correlated, [35S]methionine. Immunoprecipitation of the translation at least in some tissues, with the selective expression of the products was carried out as described (12). Typically, 5 Al of corresponding mRNA species (5, 6). Apart from the amino antibodies were added to 25 A.l of translation mixture. The acid sequence of a few peptides isolated from rabbit M-CK anti-chicken muscle or brain CK antibodies used here were or B-CK (1), the primary structure of the CK subunits and affinity-purified antibodies, and displayed no cross-reactiv- their degree of homology is not known. Isolation of cDNA ity between chicken CK isoenzymes (3). clones containing partial sequences of chicken and rat M-CK Positive mRNA Hybridization Selection. The method de- has been reported (6, 7). scribed by Ploegh et al. (16) with the hybridization and wash- The electric organ of Torpedo derives embryologically ing conditions described in ref. 12 was used. Typically, 4 Ag from skeletal muscle. One of its major cytosolic compo- of purified DNA insert from clone pCK-1 was immobilized nents, the 43,000-dalton vi protein (8, 9), often found in on a 10-mm2 nitrocellulose filter and hybridized with 40 ,g crude preparations of subsynaptic membrane fragments [but of electric organ mRNA. The eluted mRNA was assayed in unambiguously distinct from the membrane-bound PI protein 50 1.l of in vitro translation mixture. (9, 10, 33)] has been identified as CK (10, 11), and this en- Polyacrylamide Gel Electrophoresis and Immunoblots. zyme might play a role in the energetics of the electrical dis- Isoelectrofocusing was carried out according to O'Farrell The publication costs of this article were defrayed in part by page charge Abbreviations: CK, creatine kinase; M-CK, subunit of muscle-spe- payment. This article must therefore be hereby marked "advertisement" cific MM-CK isozyme; B-CK, subunit of brain-specific BB-CK iso- in accordance with 18 U.S.C. §1734 solely to indicate this fact. zyme. 7313 Downloaded by guest on September 25, 2021 7314 Biochemistry: Giraudat et al. Proc. NatL Acad Sd USA 81 (1984) (17), using a mixture of Ampholine (pH 3-10) and Servalyte fragment of the coding sequence for CK. Construction of (pH 4-9) (1:4, vol/vol). CHAPS detergent was used in place plasmids with longer cDNA inserts was then undertaken by of Nonidet P-40. NaDodSO4 gel electrophoresis was per- a cloning procedure derived from that of Okayama and Berg formed according to Laemmli (18) on 10% acrylamide/0.27% (22). bisacrylamide slab gels. Proteins were transferred to nitro- A poly(A)+ RNA fraction enriched in CK mRNA was ob- cellulose paper and immunostained as described (19). tained by sucrose gradient fractionation of total poly(A)+ RNA Blot Analysis. Glyoxal-denatured RNA was electro- RNA isolated from adult T. marmorata electric organ (12). phoresed and blotted as described (20). After prehybridiza- Starting from 56 ng of this enriched fraction, 4400 recombi- tion, the blot was hybridized with a 32P-labeled DNA probe nant clones were isolated. Of these, 49 gave a strong positive (2 x 108 cpm/Ag) (21). Washing conditions were as follows: signal on hybridization with the 32P-labeled insert of pCK-1. 4 times for 5 min in 0.3 M NaClI0.03 M Na citrate/0.1% Na- Positive colonies were further examined by restriction en- DodSO4 at room temperature and 2 times for 15 min in 15 zyme analysis. The largest recombinant clone, designated mM NaCl/1.5 mM Na citrate/0.1% NaDodSO4 at 500C. pCK-2, contained a DNA insert of 1450 base pairs. The in- Construction of Clone pCK-2. A cDNA library was con- serts of all other positive pCK clones (including pCK-1) were structed from adult T. marmorata electric organ mRNA. The contained in the pCK-2 cDNA. protocol used relied on an idea from D. Caput and contained Characterization of the pCK Clones by Positive mRNA Hy- all the essential steps of the Okayama and Berg procedure bridization-Selection and in Vitro Translation. As shown in (22). The EcoRI/Pst I fragment from pBR322, tailed with Fig. 1 (lanes 1-3), the cDNA insert from clone pCK-1 selec- (dT)50_60 at the Pst I end, was used as a primer. A 2-fold mo- tively hybridized to a mRNA that yielded after in vitro trans- lar excess of poly(A)+ RNA was added, and single-stranded lation a protein band of 43,000 daltons on NaDodSO4/poly- cDNA was synthesized with reverse transcriptase as de- acrylamide gel electrophoresis. This band comigrated with scribed (23). The reaction product was then elongated with the it protein prepared from electric organ. As this region of 10 dC residues. Removal of the tailed end of the vector DNA the gel contains several protein species (8, 9), further analy- was achieved by cleavage with BamHI restriction enzyme. sis was carried out on two-dimensional gels. Samples of elec- The Pst I/BamHI fragment from pBR322, elongated with tric organ cytoplasm and purified acetylcholine receptor-rich (dG)10 20 at the Pst I site, was used as a linker. The vector membranes provided standards and were mixed with the cDNA-mRNA hybrids carrying a cohesive BamHI end and translation product prior to electrophoresis. The gel was an oligo(dC) tail on the other side were hybridized to the stained with Coomassie blue and then autoradiographed.