Altered dosage of the Saccharomyces cerevisiae spindleploe body duplication gene, NDC1, leads to aneuploidy and p

olyploidy

881622 apia Wang

11/8

Background

• Aneuploidy--multipolar mitosis, monopolar mitosis, and nondisjunction.

• Centrosomes--aberrant structure, aneuploidy aurora2/STK15/BTAK.

• SPB--centrosome-equivalent organelle, early G1.

• NDC1--SPB duplication. SPBs and NPCs, ndc1–1, chromosomes segregate with the single, functional SPB

• Yeast cells--sensitive to both increased and decreased NDC1

• Gene dosage, SPB duplication, and genetic stability.

Material and Method

• 5-fluoroorotic acid (5-FOA), alpha-factor

• ndc1 null allele, plasmid-borne copy of NDC1

• LEU2 vector containing NDC1, URA3 vectors containing either NDC1

• flow cytometer, Indirect immunofluorescence microscopy

Result• The NDC1 Locus Is Haploinsufficient. Be able to lose a plasmid-borne copy

of NDC1--ndc1(delta) LEU2 vector containing NDC1

• Characterization of NDC1 Haploinsufficiency Survival Mechanisms.

1.Aneuploidy 2.mitotic recombination 3.extragenic suppressor mutation 4.mutation of the URA3 marker on the plasmid.

Did not appear to affect viability.

• Moderate Constitutive Overexpression of NDC1 Causes Haploid Cells to Increase in Ploidy.

1. Transformed with the vector alone 2. Transformed with the vecter contai

ning NDC1 Did not appear to affect viability

• Induced NDC1 Overexpression in Synchronized Cells Leads to Monopolar Spindle Phenotypes.

GAL1-inducible promoter(GAL-NDC1–3xpk)

alpha-factor--G1:effect of excess Ndc1p on SPB duplication.

G2:large-budded

• examined microtubule staining and the localization of an SPB component.

(Spc42p-GFP) overexpressing NDC1–3xpk-- contained two spots of Spc42p-GFP sign

al. similar to ndc1–1--monopolar spindles a

nd defective SPBs

• Ndc1p Is Mislocalized When Overexpressed.

Corresponding to its localization to both NPCs and SPBs

Large spots in the cytoplasm

• Oncogenes typically encode positive regulators of cell cycle progression in which either gain-of-function mutations or overexpression of the wild-type gene leads to cellular transformation.

• Decreased p53 gene dosage can lead to tumorigenesis

The End

Bonus