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Cell cycle by emphasizing DNA replication - Research Paper Example

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Isolation and characterization of dominant negative mutant alleles of CDC6 done to comprehend extensively the importance and regulation of CDC6 in DNA replication, CDC6 gene containing plasmid mutagenized randomly under GALI 10 promoter by passage through E. Coli strain, The…
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Cell cycle by emphasizing DNA replication
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Download file to see previous pages Increase in the number of cells with 2C-DNA content attests that over expression of wild type CDC6 impacts on the cell cycle kinetics. However, this over expression has no effect on cell proliferation. After S phase is complete, CDC6-d2 on the other hand blocks cell cycle progression and inhibits proliferation as cells with 2C DNA pile up; CDC6-d2 does not inhibit DNA replication but it does passage via mitosis.
When SCF CDC4 is inactive, dephosphorylation of B subunit can be driven by over expression of the wild type CDC6 in nocodazole arrested cells. This however is not the case as with when the SCF CDC4 is active as over expression of the cells does not lead to dephosphorylation of the B subunit because over expression of CDC6-d2 blocks cells in G2/M by inactivating CDC28-Clb kinase and in a CDC4+ background produces phenotype very much like that produced by the over expression of wild type CDC6 in a CDC4 mutant background.
Unlike CDC6-d2 which is stable in nocodazole arrested cells, the wild type protein is not and quickly disappears into the cells thus showing CDC6-d2 as being resistant to mode 3 proteolysis. It is noted however that CDC6-d2 is not affected for mode 1 proteolysis or mode 2 proteolysis but is affected as a substrate for mode 3 proteolysis; a single point mutation yielding transformation from C to T at bp 1103 causing substitution of threonine at amino acid 368 with methionine was pointed out after DNA analysis of the CDC6-d2. There are three glycine and six proline residues in a 50 amino acid residue surrounding this region, triggering belief that this region could be unstructured (Perkins, Lusy & John, 4837).
Two other regions were equally examined to check whether their muattions also had some effects on the stability of CDC6. Two CDC6 alleles were constructed using site directed mutagenesis where serine 372 was transformed to alanine (S372A) and in the other, serine 354 was changed alanine ...Download file to see next pagesRead More
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