Morehead State University

Poster Title

Creating the First Genetically Engineered Eukaryote with Circular Chromosomes

Major

n/a

Minor

n/a

Institution

Morehead State University

KY House District #

98

KY Senate District #

18

Department

Department of Biology and Chemistry

Abstract

Telomeres are repetitive sections of DNA at the termini of linear chromosomes that protect the ends from damage and unlimited cell division. However, misregulation of telomeres underlies two of our greatest medical challenges: cancer and aging. >85% of cancers upregulate telomerase, the enzyme that maintains telomeres, while telomeres progressively shorten during aging. Not all organisms have linear chromosomes and the issues that come with telomeres; prokaryotes like bacteria generally contain a singular circular chromosome. To experimentally investigate the evolution and function of telomeres, we are circularizing chromosome VII in a strain of the single-celled eukaryote Saccharomyces cerevisiae, also known as brewer’s or baker’s yeast. S. cerevisiae has proven to be a powerful model organism due to its affordability and genetic manipulability. Yeast share similar chromosome and telomere structure to humans, allowing extrapolation of our findings to humans. To circularize the chromosomes, we created two pieces of DNA called cassettes: one for each arm of chromosome VII. Both cassettes contain a region of sequence similarity with the chromosome, a selectable marker gene, and opposite halves of a URA3 gene. The cassettes were inserted into the chromosome distal to any functional genes. The two halves of the URA3 gene also contain a region of sequence similarity, allowing recombination between the cassettes that will result in a functional URA3 gene and a circularized chromosome. We have obtained several candidate yeast colonies for both cassette integrations and are currently testing for insertion using a technique called PCR. The long-term goal of this project is to create strains of S. cerevisiae with all sixteen of its chromosomes circularized. This research will help answer fundamental questions about how telomeres evolved and their roles in cancer and aging in humans.

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Creating the First Genetically Engineered Eukaryote with Circular Chromosomes

Telomeres are repetitive sections of DNA at the termini of linear chromosomes that protect the ends from damage and unlimited cell division. However, misregulation of telomeres underlies two of our greatest medical challenges: cancer and aging. >85% of cancers upregulate telomerase, the enzyme that maintains telomeres, while telomeres progressively shorten during aging. Not all organisms have linear chromosomes and the issues that come with telomeres; prokaryotes like bacteria generally contain a singular circular chromosome. To experimentally investigate the evolution and function of telomeres, we are circularizing chromosome VII in a strain of the single-celled eukaryote Saccharomyces cerevisiae, also known as brewer’s or baker’s yeast. S. cerevisiae has proven to be a powerful model organism due to its affordability and genetic manipulability. Yeast share similar chromosome and telomere structure to humans, allowing extrapolation of our findings to humans. To circularize the chromosomes, we created two pieces of DNA called cassettes: one for each arm of chromosome VII. Both cassettes contain a region of sequence similarity with the chromosome, a selectable marker gene, and opposite halves of a URA3 gene. The cassettes were inserted into the chromosome distal to any functional genes. The two halves of the URA3 gene also contain a region of sequence similarity, allowing recombination between the cassettes that will result in a functional URA3 gene and a circularized chromosome. We have obtained several candidate yeast colonies for both cassette integrations and are currently testing for insertion using a technique called PCR. The long-term goal of this project is to create strains of S. cerevisiae with all sixteen of its chromosomes circularized. This research will help answer fundamental questions about how telomeres evolved and their roles in cancer and aging in humans.