Cockerill, M.J.; (1996) D-type cyclins in Xenopus laevis. Doctoral thesis , University of London.

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Abstract

I have isolated two D-type cyclins and a putative kinase partner related to Cdk4 from the frog Xenopus laevis. Three D-type cyclins have previously been isolated from mammalian species, where they are thought to be involved in the control of cell proliferation via the regulation the GO-+G 1 and G 1--IS transitions. The RNA and protein levels of D-type cyclins and Cdk4 were followed during Xenopus early embryonic development. The two D-type cyclins and Cdk4 are absent during the early rapid cleavage phase of Xenopus development. Cyclin D1 mRNA and protein can first be detected after the mid-blastula transition (MBT), and while Cdk4 mRNA is present throughout development the protein is only detectable after MBT. I have been unable to detect cyclin D2 mRNA or protein in embryos or adult tissues, although the D2 clone was originally isolated from an oocyte cDNA library. When translated in vitro, Xenopus cyclins D1 and D2 preferentially associate with recombinant Cdk4 protein, and bind less strongly to Cdc2 and Cdk2. Cdk4 in combination with either cyclin Dl or cyclin D2 is also able to form a ternary complex with retinoblastoma protein (pRb), and cyclin D2-Cdk4 was able to phosphorylate the pRb to which it was bound. Whole-mount in situ hybridization revealed that cyclin Dl, Cdk4 and pRb mRNAs are localised to distinct regions of the developing embryo, in contrast to other cyclins which tend to be distributed rather homogeneously, if they are present at all. In particular, cyclin D1 mRNA is strongly localised to the developing eye and other neural regions of the developing embryonic head. Dominant-negative mutants of Cdk4 were constructed by mutating the essential lysine 33 amino acid residue to arginine. The mRNA encoding this kinase-dead Cdk4 was injected into fertilized eggs to look for effects resulting from lack of Cdk4 function after MBT, when Cdk4 protein is first expressed. No discernible abnormal phenotype was seen, however, in agreement with previously reported results obtained in human cultured cells.

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