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

Abstract

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 T\MPK3 were identified and the Tetrahymena Genome Project identified additional 11 members from p38 Erk and Erk7 subgroup of MAPK family in Tetrahymena genome.

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 T\MPK3 were identified and the Tetrahymena Genome Project identified additional 11 members from p38 Erk and Erk7 subgroup of MAPK family in Tetrahymena genome.