Cell cycle dependent expression of Plk1 in synchronized porcine fetal fibroblasts.
Enzymes of the Polo-like kinase (Plk) family are active in the pathways controlling mitosis in several species. We have cloned cDNA fragments of the porcine homologues of Plk1, Plk2, and Plk3 employing fetal fibroblasts as source. All three partial cDNAs showed high sequence homology with their mouse and human counterparts and contained the Polo box, a domain characteristic for all Polo kinases. The expression levels of Plk1 mRNA at various points of the cell cycle in synchronized porcine fetal fibroblasts were analyzed by both RT-PCR and the ribonuclease protection assay. Plk1 mRNA was barely detectable in G0 and G1, increased during S phase and peaked after the G2/M transition. A monoclonal antibody was generated against an in vitro expressed porcine Plk1-protein fragment and used to detect changes in Plk1 expression at the protein level. Plk1 protein was first detected by immunoblotting at the beginning of S phase and was highest after the G2/M transition. In summary, the Plk1 expression pattern in the pig is similar to that reported for other species. The absence of Plk1 mRNA and protein appears to be a good marker for G0/G1 and thus for the selection of donor cells for nuclear transfer based somatic cloning.
Anger
Martin
M
Institute of Animal Physiology and Genetics, Libechov, Czech Republic.
Kues
Wilfried A
WA
Klima
Jiri
J
Mielenz
Manfred
M
Kubelka
Michal
M
Motlik
Jan
J
Esner
Milan
M
Dvorak
Petr
P
Carnwath
Joseph W
JW
Niemann
Heiner
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D005314:Embryonic and Fetal Development / Q000502:physiology*; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D025521:Tumor Suppressor Proteins; D014981:Xenopus; D029867:Xenopus Proteins / Q000235:genetics; D000097869:Polo-Like Kinase 1; D000097868:Polo-like Kinases
D018797:Cell Cycle Proteins; D017931:DNA Primers; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1; C420219:PLK3 protein, Xenopus; C486256:PLK3 protein, human; D000097868:Polo-like Kinases; C438873:PlK2 protein, Xenopus; C093328:Plk3 protein, mouse