Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2.

Abstract

Fertilization ofXenopus laevis eggs triggers a period of rapid cell division comprising 12 nearly synchronous mitoses. Protein synthesis is required for these divisions, and new proteins appear after fertilization. Others proteins, however, which are synthesized in the unfertilized egg, are no longer made in the early embryo. To identify such proteins, a differential screen of an egg cDNA library gave nine clones corresponding to mRNAs that are deadenylylated soon after fertilization. The sequence ofone of these clones (Egi) revealed a high homology to p34Wdc2 the kinase subunit of maturationpromoting factor. Only 12 amino acids in the deduced amino acid sequence were unique to Egl when its sequence was compared to all other known examples of cdc2. Despite this strong similarity, however, Egl was unable to complement a yeast cdc2-mutant in Schizosaccharomyces pombe or a cdc28 mutant of Saccharomyces cerevisiae. Four Egl transcripts, two major and two minor, were found in Xenopus oocytes and early embryos. These RNAs appeared very early (stage I) in oogenesis and their level remained constant until the midlastfula transition, at which time they dedined. Egi RNA is found in the poly(A)+ fraction of oocytes only between the time of meiotic maturation and fertilization-that is to say, in the unfertilized egg. At fertilization the RNA loses its poly(A) tail and at the same time leaves the polyribosomes.

Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes.

“…In addition to cyclin B, cyclin A has been shown to complex with p34 DC2 in higher eukaryotic cells and may also perform a function during mitosis in both embryonic and somatic cells Lehner and O'Farrell, 1990;Minshull et al, 1990;Devault et al, 1992;Pagano et al, 1992b). However, human cyclin A complexes predominantly with p33CDK2, a CDC2-related kinase catalytic subunit that functions during S-phase (Girard et al, 1991;Paris et al, 1991;Tsai et al, 1991;Pagano et al, 1992b).…”

“…In vertebrates, whereas p34CDC2 plays an essential role in mitosis (Riabowol et al, 1989; reviewed by Draetta, 1990), the function of p34CDC2 in Gl and S phases appears to be mediated not by a single kinase but by a family of CDC2-related proteins. Several of these putative kinases have been isolated from human and other vertebrates using methods that identify very closely related proteins (Paris et al, 1991;Meyerson et al, 1992;Xiong et al, 1992b). Although the functions of most of these kinases in the cell cycle remain unknown, substantial evidence suggests that some of the CDC2-related kinases are involved in the regulation of the Gl and S phases in the cell cycle (Fang and Newport, 1991;Tsai et al, 1991 Elledge et al, 1992;Pagano et al, 1992a,b;Rosenblatt et al, 1992;Xiong et al, 1992b).…”

Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation.

“…Through genetic complementation testing and identification of associated cyclin regulatory subunits, at least five cyclin-dependent kinases have been identified (Fang and Newport 1991;Paris et al 1991;Tsai et al 1991;Elledge et al 1992;Matsushime et al 1992;Meyerson et al 1992;Pagano et al 1992a, b;Rosenblatt et al 1992;Xiong et al 1992b). By use of PCR and low-stringency hybridization techniques, further potential members of the cdc2 family have been identified on the basis of their protein sequence similarities to cdc2 (Meyerson et al 1992;Okuda et al 1992).…”

Activation of the p34 CDC2 protein kinase at the start of S phase in the human cell cycle.

“…The p34 CDC2 protein recently has been shown to be one member of a family of related proteins (Elledge and Spottswood, 1991;Koff et al, 1991;Paris et al, 1991), and one of these proteins, CDK2, has been implicated in Gl/S phase regulation (Fang and Newport, 1991;Koff et al, 1991;Tsai et al, 1991). Also, three new human cyclins have been cloned, and all can substitute for the S. cerevisiae CLN proteins in functional assays (Koff et al, 1991;Lew et al, 1991;Motokura et al, 1991;Xiong et al, 1991).…”

“…Activation of the Cyclin A/p34 CDC2 Complex at the Start of S Phase The above experiment did not directly demonstrate activation of the p34 CDC2 kinase at the start of S phase because it was possible that kinases related to CDC2, such as CDK2 (Elledge and Spottswood, 1991;Koff et al, 1991;Paris et al, 1991;Tsai et al, 1991), would also phosphorylate the T-antigen peptide kinase substrate. Also, it was not apparent whether the cyclin A-associated form of p34 CDC2 was active.…”