Novel telomere-anchored PCR approach for studying sexual stage telomeres in Aspergillus nidulans.

Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed "telomere-anchored PCR," measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.

Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans.

Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.

Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid.

A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum,Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5-2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.

The early impact of genetics on our understanding of cell cycle regulation in Aspergillus nidulans.

The application of genetic analysis was crucial to the rapid progress that has been made in cell cycle research. Ron Morris, one of the first to apply genetics to cell cycle research, developed Aspergillus nidulans into an important model system for the analysis of many aspects of cell biology. Within the area of cell cycle research, Ron's laboratory is noted for development of novel cell biological and molecular genetic approaches as well as seminal insights regarding the regulation of mitosis, checkpoint regulation of the cell cycle, and the role of microtubule-based motors in chromosome segregation. In this special edition of FGB dedicated to Ron Morris, and in light of the recent progress in fungal genomics, we review the outstanding contributions his work made to our understanding of mitotic regulation. Indeed, his efforts have provided many mutants and experimental tools along with the conceptual framework for current and future studies of mitosis in A. nidulans.