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Kokkanti – Defining a Minimal Yeast Centromere

 

Defining a Minimal Yeast Centromere (2024)

Undergraduate: Aryan Kokkanti


Faculty Advisor: Kerry Bloom
Department: Biology

A minimal centromere is defined as the smallest sequence that allows for proper segregation of chromosomes in anaphase. To determine the minimal centromere of S. cerevisiae, we engineered multiple centromere VIII (CEN8) sequences that have their 118 base pair consensus sequence along with additional DNA flanking the 118 bp upstream, downstream, or both. We inserted the CEN8 sequences into chromosome III under the control of the GAL promoter to conditionally activate a dicentric chromosome. Analysis of colony growth on dextrose (centromere active) and galactose (centromere inactive) shows that the 118 bp consensus does not function as a centromere. Flanking sequences downstream of the 118 bp consensus confers centromere function. Adding DNA upstream of the 118 bp consensus has no significant effect on function. The distribution of repairs events arising from broken dicentric chromosomes shows an increased frequency of NHEJ and HR with the latter dominating as more DNA is added to either flank. When the engineered CEN8 sequences are introduced into yeast plasmids with selection markers, there is an increase in centromere function with increased DNA downstream of the 118 bp consensus. We conclude that CEN8 requires another 19 bases pairs downstream of the 118 bp consensus to function as a centromere.