Identification and cellular localization of human PFTAIRE1
Introduction
Cdc2-related protein kinases play important roles in growth and differentiation from yeast to human. The Cdks (cyclin-dependent kinases) family controls the transitions between successive phases of the cell cycles in all eukaryotic cells (Morgan, 1997, Reed, 1992, Nasmyth, 1993). All Cdks are structurally related to each other and all require associated cyclin proteins for activity (Nigg, 1995). The first vertebrate Cdk, Cdc2 (also referred to as Cdk1), is shown to be the catalytic subunit of M-phase promoting factor (MPF), a universal inducer of mitosis (King et al., 1994). Additional vertebrate Cdks have been identified subsequently. Cdc2 (Cdk1) and Cdk2 are functionally homologous to yeast cdc2/Cdc28 and are clearly involved in central cell cycle functions (Norbury et al., 1991). The fission yeast cdc2 protein kinase is essential for the control of the mitotic cell cycle, being required for G1 to S phase transition (Nurse and Bissett, 1981) as well as entrance into mitosis (Nurse et al., 1976). Cdc28 is the main Cdk implicated not only in cell cycle control (Nasmyth, 1996) but also in the filamentous growth in S. cerevisiae (Edgington et al., 1999). Many other Cdks play the other cellular processes rather than cell division. For example, Cdk7-CyclinH-Mat1 complex possesses Cdk-activation kinase (CAK) activity responsible for phosphorylating Cdks. (Nigg, 1996); Cdk7-CyclinH-Mat1 and Cdk8-CyclinC are associated with RNA Polymerase II and possess CTD kinase activity (Nigg, 1996, Svejstrup et al., 1996, Tassan et al., 1995); Cdk5, whose activation requires an association with p35, is expressed in post-mitotic cells of the central nervous system and is required for neural differentiation (Ohshima et al., 1996). There are also a group of Cdc2-related protein kinases such as PCTAIRE, PFTAIRE, KKIALRE, MAK (Male-germ cell Associated Kinase) and MRK (MAK Related Kinase) etc. The cyclin partners for these kinases have not been identified yet (Meyerson et al., 1992, Abe et al., 1995, Matsushime et al., 1990). PCTAIRE kinases have been cloned from humans (Meyerson et al., 1992), mice (Okuda et al., 1992) and other organisms (Hirose et al., 1997) based on their similarity to Cdc2. Two PFTAIRE genes from mice have been reported (Lazzaro et al., 1997, Besset et al., 1998). mPFTAIRE1 is ubiquitously expressed in murine tissues, and high expression level was detected in brain, testis and embryo (Besset et al., 1999). mPFTAIRE is implicated in the process of meiosis as well as neuron differentiation (Besset et al., 1998). MRK, isolated from a rat heart cDNA library (Abe et al., 1995) is ubiquitously expressed in adult rat tissues. However, the expression of MRK in embryos was restricted to embryonic myocardium during early organogenesis, suggesting that the MRK may be involved in cardiac development. All these show that Cdc2-related protein kinases family has broad and diverse functions in various cellular processes.
To identify novel members of Cdc2-related protein kinase, we used degenerate oligonucleotide primers based on the conserved sequences of known Cdc2-related protein kinases (Hanks et al., 1988). 100 ESTs were subcloned and sequenced. The ESTs were used as probes to screen novel Cdc2-related protein kinase genes. In this study, we report the cloning of human PFTAIRE1. The expression patterns, cellular and chromosomal localization of this novel gene were also demonstrated.
Section snippets
Cloning of a human PFTAIRE1
mRNA was prepared from human laryngeal cancer tissue, which was from a male adult at the age of 63 in Shanghai, China, using a PolyATtract System 1000 kit (Promega). The first-strand cDNA was synthesized with AMV reverse transcriptase (Promega) and oligo-dT (12–18) as the primer. The cDNA was then used as the template for subsequent rounds of PCR amplification. Two degenerate oligonucleotide primers for the PCR reaction were designed according to the conserved subdomains of Cdc2-related protein
Cloning and chromosomal mapping of PFTAIRE1
Using the degenerated RT-PCR DNA fragment highly homologous with mouse mPFTAIRE1 as the probe to screen a Hela cDNA library, we obtained three positive clones. The longest clone contains a single open reading frame with a predicted protein of 469 amino acids (Fig. 1A). The predicted initiation codon beginning at base 1 is preceded by in-frame stop codons. The predicted amino acid sequence shares the highest similarity with the mouse PFTAIRE1 with 95% identities through the entire coding region (
Conclusions
- 1.
A 2043 bp cDNA encoding a human PFTAIRE1 was cloned from a Hela cDNA library. The predicted protein shares the highest similarities with the mouse PFTAIRE1 with 95% identities. The hPFTAIRE1 gene is located at chromosome 7q21.13 determined by using a Stanford G3 Radiation Hybrid panel kit.
- 2.
Northern blot analysis revealed that a 6 kb hPFTAIRE1 transcript is expressed predominantly in brain, pancreas, kidney, heart, testis and ovary. The 6 kb hPFTAIRE1 transcript is also detected at a lower level
Acknowledgements
We thank Dr Haoping Liu for critical reading of the manuscript. We also thank Dr Ke Tang and Dr Gaoxiang Ge for the transfection of Hela cells. This work was supported by grants from Chinese National Natural Science Foundation (Grant 39625009) and Chinese Academy of Sciences Applied Research Foundation (Grant 951A13010205).
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