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Experiments were used the wild type zip1, SK1 and BR2495 yeast strains. Most experiments were carried out using the altered diploid yeast strain BR2495 . This strain carried the cdc28-63 mutation and cells were induced for meiosis. A fusion gene ysw1::lacZ was introduced into the BR2495 strain, by a plasmid called pTP36. YSW1 is a late meiotic gene not expressed in pachytene arrested cells. Expression of the fusion gene was observed to find proteins which stop the sporulation defect seen in zip1 cells.
A multicopy plasmid pTP121 was created using a number of processes for NDT80 overexpression. pTP121 contains sequences which encode the hemagglutinin (HA) epitope, (which can be screened for using antibodies). This plasmid was then used to replace BR2495 chromosomal NDT80 with Ndt80-HA. Western blotting was then used to observe how Ndt80 was regulated in wild type and Zip1 mutants. Immunoprecipitation of Ndt80-HA using calf intestinal phosphatase was carried out, after which the samples were used for immunoblotting.
Results - The sporulation defect in zip1 mutants is suppressed by the overproduction of Ndt80.
The zip1 mutant BR2495 strain fails to sporulate at all because it undergoes pachytene induced checkpoint arrest. A strain of zip1 BR2495 containing a multicopy plasmid which overproduced Ndt80 in fact sporulated but with less effectiveness than the wild type (shown in figure 12A and 12B). In SK1 strains, zip1 sporulated with reduced effectiveness and took longer to sporulate than in wild type cells. SK1 strains which had overproduction of Ndt80 progressed through meiosis and formed spores faster than the SK1 zip1 mutant strain (Figure 12C and 12D). Cells of this strain took longer to sporulate and in reduced numbers compared with the wild type. It was also seen that Ndt80 overproduction in dmc1 mutants increased spore production, but this did not occur in the hop2 mutant. Wild type cells sporulated quicker and in greater amounts than wild type cells with an over production of Ndt80 (figures 12A,B,C,D). This is due to a reduction in the amount of meiotic cells entering Meiosis I. Tung et al hypothesize this may be due to large amounts of Ndt80 hindering progression through meiosis and thus sporulation.
Ndt80 overproduction thus increases the production of spores both in time and amount either by bypassing the pachytene checkpoint or by preventing zip1 faults in the chromosomes.
In order to check which of these mechanisms increase sporulation, Tung et al compared the BR2495 zip1 mutant strain to the zip1 mutant strain which overproduced Ndt80 using electron microscopy. In both strains the axial elements were underdeveloped and chromosomes failed to synapse correctly. Compared with SK1 wild type strains, zip1 mutant SK1 has reduced production of chiasmata which is not fixed by overproduction of Ndt80. From these observations, it can be concluded that Ndt80 overproduction doesn't correct chromosomal defects but instead overrides the pachytene checkpoint.
Western blotting techniques were used to find out how Ndt80 was regulated in wild type and zip1 cells during the pachytene checkpoint. It was found that there is an inhibition of Ndt80 phosphorylation. Wild type cells had far more Ndt80 than zip1 cells as can be seen in figure 13A-D. It was also seen during SDS-PAGE that wild type Ndt80 proteins moved slower across the gel as meiosis progressed compared with zip1 which doesn't appear to have any change in movement. The reason for this change was discovered using immunoprecipitation by phopshorylating wild type, dmc1, hop2 and zip1 mutant extracts with phosphatase. It was found that wild type Ndt80 are regulated by being phopshorylated, wheras dmc1, hop2 and zip1 cells almost not phosphorylated at all (seen in figure 13 C and D). This difference in phosphorylation is why mutants arrest at the pachytene checkpoint. In order for zip1 mutants to produce spores, they must be allowed to phopshorylate Ndt80 and thus bypass the pachytene checkpoint. Mutations within pch2 and mek1 allow the phosphorylation of Ndt80 in zip1 mutants (San-Segundo & Roeder 1999 and Xu et al 1997). As can be seen in figure 13D the phosphorylation and build up of Ndt80 in zip1 due to pch2 and mek1 mutants is the same amount of wild type Ndt80 phosphorylation and accumulation. The paper shows that Cdc28 must be inactivated by phosphorylation to induce pachytene arrest, and it may function without interacting with Ndt80. In Cdc28-63 mutants arrested at pachtene, Ndt80 is still found and undergoes phosphorlylation (Figure 13D).
Conclusion- Ndt80 is regulated at the pachytene checkpoint via phosphorylation.
Phosphorylation of Ndt80 is critical in its regulation, activating it to work as a transcription factor. The activation of Ndt80 casuses expression in its target genes such as CLB1, CLB2 and causes the dissociation of Zip1 from chromosomes thus exiting the pachytene stage. Cells wich incur a pachytene-induced arrest, are not phosphorylated and thus Ndt80 is inactivated. In pachytene arrested cells, Ndt80 is unable to transcribe its target genes as it is less phosphorylated and present in much lower numbers. This paper fails to mention the localisation of Ndt80 during pachytene, and thus its role (if any) in regulation.