Cloning and partial characterization of Tetrahymena thermophila mitogen activated protein kinase 3 (TtMPK3) gene

In this study, a full-length cDNA fragment encoding putative mitogen activated protein kinase (MAPK) from Tetrahymena thermophila was cloned by using RT-PCR with degenerate oligonucleotide primers. Based upon the presence of 11 subdorriains and TEY dual phosphorylation motif in protein kinase subdomain VIII, it is named as Tetrahymena thermophila mitogen activated protein kinase 3 (TtMPK3). TtMPK3 cDNA fragment has 1176 bp long single open reading frame (ÖRF) encoding a polypeptide of 391 amino acids with predicted size of 45.5 kDa. TtMPK3 is highly homologous to SLT2 of budding yeast and Erkl of Giardia intestinalis. Southern blot analysis indicated that TtMPK3 is a single copy gene in Tetrahymena macronuclear genome. Western blot analyses with anti-carboxyl TtMPK3p peptide serum showed that TtMPK3 polyclonal antibody specifically recognizes only about 44 kDa expected size protein. Immunoprecipitates of anti-carboxyl polypeptide of TtMPK3p antibody tested in in vitro kinase assay showed kinase activity two to four times more than the control preimmune serum. Until today, four possible TtMPK genes including TMPK3 were identified and the Tetrahymena Genome Project identified additional 11 members from p38 Erk and Erk7 subgroup of MAPK family in Tetrahymena genome.

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[1]. Corliss JO. 1984. Organism and populations. The kingdom protista and its 54 phyla. BioSystems, 17: 87- 126.
[2]. Furgason WH. 1940. The significant cytostomal pattern of the Glaucoma-Colpidium group, and a proposed new genus and species, Tetrahymena geleii. Archiv fur Protistenkunde, 94: 224-266.
[3]. Hill DL. 1972.The biochemistry and physiology of Tetrahymena. Cell Biology. Academic Press. XII. New York,USA.
[4]. Frankel J. 2000. Cell Biology of Tetrahymena thermophila. Methods in Cell Biology, 62: 27-125.
[5]. Gall J. 1986. The Molecular Biology of Ciliated Protozoa. Academic Press. Orlando, USA.
[6]. Hutton JC. 1997. Tetrahymena: the key to the genetic analysis of the regulated pathway of polypeptide secretion, Proc Natl Acad Sci USA, 94(20): 10490-2.
[7]. Saad Y, Doerder FP. 1995. Immobilization antigen variation in natural isolates of Tetrahymena thermophila. European Journal of Protistology, 31: 45-53.
[8]. Doerder FP, Gates MA, Eberhardt FP, Arslanyolu M. 1995. High frequency of sex and equal frequencies of mating types in natural populations of Tetrahymena thermophila. Proc. Nat. Acad. Sci. USA., 92: 8715-8718.
[9]. Doerder FP, Arslanyolu M, Saad Y, Kaczmarek M, Mendoza M, Mita B. 1996. Ecological genetics of Tetrahymena thermophila: mating types, i-antigens, multiple alleles and epistasis. Journal of Eukaryotic Microbiology, 43: 95-100.
[10]. Arslanyolu M, Doerder, FP. 2000. Genetic and environmental factors affecting mating type frequency in natural isolates of Tetrahymena thermophila. Journal of Eukaryotic Microbiology. 47(4): 412-418.
[11]. Madhani HD, Fink GR. 1998. The riddle of MAP kinase signaling specifity. Trends in Genetics, 14(4): 151-155.
[12]. Seger R, Krebs EG. 1995. The MAPK signaling cascade. FASEB Journal, 9: 726-735.
[13]. Kultz D. 1998. Phylogenetic and functional classification of mitogen- and stress-activated protein kinases. Journal of Molecular Evolution, 46: 571-588.
[14]. Hanks SK, Hunter T. 1995. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J., 9: p. 576-596.
[15]. Schaeffer HJ, Weber M. 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Molecular Cell Biology, 18(12): 7371- 7382.
[16]. Sambrook J, Fritsch EF, ManiatisT. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, USA.
[17]. Wilks AF. 1991. Cloning members of protein kinase family using polymerase chain reaction. Methods in Enzymology. 200: 533-547.
[18]. Loh EY, Elliott JF, Cwirls S, Lanier LL, Davis MM. 1989. Polymerase Chain Reaction with single-sided specifity: Analysis of T cell receptor gama chain. Science. 243(13 January): 217-243.
[19]. Doolittle R, 1986. Of URFS and ORFS: a primer on how to analyze derived amino acid sequences. University Science Books. 1-103.
[20]. McKimm-Breschkin JL. 1990. Tne use of tetrametylbenzidine for solif phase immunoassay. Journal of Immunological Methods. 135: 277-280.
[21]. Altschul SF, Gish W, Miller W, Myers EW, Lipman D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology. 215: 403-410.
[22]. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research. 22: 4673-4680.
[23]. Felsenstein J. 1988. Phylogenies from molecular sequences: inference and reliability. Annual Review of Genetics. .22: 521-565.
[24], Peitsch MC. 1995. Protein modeling by E-mail. Bio/Technology. 13: 658-660.
[25]. Schwede T, Kopp J, Guex N, Peitsch MC. 2003. SWISS-MODEL: an automated protein homologymodeling server. Nucleic Acids Research. 31: 3381- 3385.
[26]. Hanks SK, Linberg RA. 1991. Use of degenerate oligonucleotide probes to identify clones that encode protein kinases. Methods in Enzymology. 200: 525- 532.
[27]. Wuitschick JD, Karrer KM. 1999. Analysis of genomic G + C content, codon usage, initiator codon context and translation termination sites in Tetrahymena thermophila. J Eukaryotic Microbiology. 46(3): 239-47.
[28]. Wuitschick JD, Karrer KM. 2000. Codon usage in Tetrahymena thermophila. Methods in Cell Biology. 62: 565-8.
[29]. Lozupone CA, Knight RD, Landweber LF. 2000. The molecular basis of nuclear genetic code change in ciliates. Current Biology, 11: 65-74.
[30]. Wang S, Nakashima S, Numata O, Fujiu K, Nozawa Y. 1999. Molecular cloning and cell-cycle-dependent expression of the acetyl-CoA synthetase gene in Tetrahymena cells. Biochemistry Journal, 343(Pt 2): 479-485.
[31]. Zarzov P, Mazzoni C, Mann C. 1996. The SLT2 (MPK1) MAP kinase is activated during periods of polarized cell growth in yeast. The EMBO Journal. 15(1): 83-91.
[32]. Canagarajah BJ, Khokhlatchev A, Cobb MH, Goldsmith EJ. 1997. Activation mechanism of the MAP Kinase ERK2 by dual phosphorylation. Cell. 90: 859-869.
[33]. Clinton GM, Brown NA. 1991. Generation and use of anti-peptide antibodies directed againts catalytic domain of protein kinases. Methods in Enzymology, 200: 463-474.
[34]. Duerr B, Gawienowski M, Ropp T, Jacobs T. 1993. MsErkl: a mitogen-activated protein kinase from a flowering plants. The Plant Cell, 5: 87-96.
[35]. Sangwan V, Dhindsa RS. 2002. In vivo and in vitro activation of temperature responsive plant map kinases. FEBS Letters. 531: 561-564.
[36]. Erickson AK, Payne DM, Martino PA, Rossomando AJ, Shabanowitz J, Weber MJ, Hunt DF, Sturgill TW. 1990. Identification by mass spectrometry of threonine 97 in bovine myelinbasic proteinas aspecific phosphorylation site for mitogen activated protein kinase.- Journal of Biological Chemistry. 265: 19728- 19735.
[37]. Nakashima S, Wang S, Hisamoto N, Sakai H, Andoh M, Matsumoto K, Nozawa Y. 1999. Molecular cloning and expression of a stress-responsive mitogenactivated protein kinase-related kinase from tetrahymena cells. Journal of Biological Chemistry.274(15): 9976-9983.
[38]. Allen SL. 2000. Genetic nomenclature rules for Tetrahymena thermophila. Methods in Cell Biology, 62:561-3.
[39]. Shiozaki K, Shiozaki M, Russell P. 1998. Heat Stress Activates Fission Yeast Spcl/Styl MAPK by a MEKK-Independent Mechanism. Molecular Biology of the Cell. 9: 339-1349.
[40]. Banuett F. 1998. Signalling in the yeast: an informational cascade with links to the filamentous fungi. Microbiology and Molecular Biology Reviews, (June): 249-274.
[41]. Shiozaki K, Russell P. 1996. Conjugation, meiosis, and the osmotic stress response are regulated by Spcl kinase through Atfl transcription factor in fission yeast. Genes and Development. 10; 2276-2288.
[42]. Eisen JA et al. 2006. Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote. PLoS Biology September, 4:9, 1620-1642.