No abstract

Chromosome ends contain repetitive, protein‐nucleotide sequences called telomeres that protect against DNA loss during replication. These noncoding sequences can vary in nucleotide identity as well as repeat sequence length. Our model system, Aspergillus nidulans, has a telomere sequence of 5’‐TTAGGG‐3’, which is the same as the human repeat sequence. Unlike in humans, who have a somewhat variable telomere length, those of A. nidulans are tightly regulated at roughly 110 bp. Using A. nidulans as a model, we previously designed an inexpensive and effective method termed telomere‐anchored PCR as an alternative for current telomere length methods that can be labor intensive and relatively expensive. However, the adaptability of this method and its efficiency at determining telomere length for organisms that might exhibit less stringent telomere length regulation remains uncharacterized. We initially examined the validity of this method in a similar species, Aspergillus oryzae. A. oryzae has a telomere repeat identity of 5’‐TTAGGGTCAACA‐3’ and has been shown to contain similar telomere lengths to A. nidulans of about 120 bp. Preliminary results suggest that telomere anchored PCR can function as an assay to determine telomere length in A. oryzae. This provides incentive to pursue optimization of the protocol for other Aspergillus species as well as additional organisms like Tetrahymena thermophila to further extend the applications of this quick and easy method.

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.

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of telomerase in the fungus Aspergillus nidulans. Previous research on TER indicated secondary structural similarities to both Saccharomyces cerevisiae and mammalian cells. However, it was previously unknown whether TER migrates into the cytoplasm during the assembly of telomerase, as in S. cerevisiae, or remains in the nucleus, as in mammalian cells. We now have employed a molecular tool, the unique multi‐nucleate state of A. nidulans termed heterokaryon, to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, determined whether assembly occurs in the nucleus or cytoplasm, and verified those results with DAPI staining. Our results suggest assembly in the nucleus, a scenario that is similar to humans, but not to yeast. These results further suggest the strength of A. nidulans as a model organism in studying the localization of telomerase components.

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of TER in Aspergillus nidulans, a molecule that is in part structurally similar to that of Saccharomyces cerevisiae and partly to that of mammalian cells. Research in S. cerevisiae has shown that the telomerase RNA appears to leave the nucleus and is potentially assembled in the cytoplasm. Whether TER migrates to the cytoplasm is not known in A. nidulans, but its ability to form a multi‐nucleate state known as a heterokaryon can be used to determine the localization of TER. We assay for the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, and determined if assembly of the active enzyme occurs in the nucleus or the cytoplasm. Our results suggest assembly in the nucleus, a scenario that is similar to humans but not to yeast. These results further suggest the potential of A. nidulans as a useful model organism to study the localization of telomerase components.