Determining the End Nucleotide of Telomeres in Aspergillus nidulans

Abstract

Telomeres are noncoding, repetitive DNA sequences at the ends of chromosomes that prevent DNA loss during replication. Telomerase is the ribonucleoprotein that extends telomeres by adding on telomere repeats. To do so, telomerase must recognize and bind to a specific sequence in the telomere repeats, which is often uncharacterized. In humans and our model system, Aspergillus nidulans, the telomere repeat is 5’‐TTAGGG‐3′. However, it is difficult to predict the order these nucleotides appear in the final telomere repeat because of end‐processing mechanisms that can occur at the chromosome ends. Previous work in Euplotes crassus and Tetrahymena thermophila has shown that their telomeres preferentially end 5’‐GGTTTTGG‐3′ and 5’‐TGGGGT‐’3 respectively. In humans however, the end nucleotide is much more varied on the G strand. It is currently unknown what the end nucleotide is in Aspergillus nidulans. To investigate this question, we employ a combination of telomere anchored PCR and quantitative PCR (qPCR) to preferentially amplify telomeres with specific endings. Initial results suggested all endings were present in genomic DNA, but to verify these results a control system was necessary. We have developed an oligonucleotide system consisting of synthetic telomere templates to test the validity of our method. With this control, we have observed that our system preferentially amplifies matching template and primer combinations, although there is some non‐specific binding between noncomplementary pairs. Thus, we have found suggestive preliminary evidence that we can use this system to assist in characterizing the end nucleotide of Aspergillus nidulans.