Biomedical subjects

Leena Ukil

Publications and source records attributed to Leena Ukil.

3 recordsLinked to original sources

PubMed2005

A versatile and efficient gene-targeting system for Aspergillus nidulans.

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

Antigens, Nuclear↗

PubMed2004

Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans.

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.

Aspergillus nidulans↗

PubMed2003

The PHOA and PHOB cyclin-dependent kinases perform an essential function in Aspergillus nidulans.

Unlike Pho85 of Saccharomyces cerevisiae, the highly related PHOA cyclin-dependent kinase (CDK) of Aspergillus nidulans plays no role in regulation of enzymes involved in phosphorous acquisition but instead modulates differentiation in response to environmental conditions, including limited phosphorous. Like PHO85, Aspergillus phoA is a nonessential gene. However, we find that expression of dominant-negative PHOA inhibits growth, suggesting it may have an essential but redundant function. Supporting this we have identified another cyclin-dependent kinase, PHOB, which is 77% identical to PHOA. Deletion of phoB causes no phenotype, even under phosphorous-limited growth conditions. To investigate the function of phoA/phoB, double mutants were selected from a cross of strains containing null alleles and by generating a temperature-sensitive allele of phoA in a deltaphoB background. Double-deleted ascospores were able to germinate but had a limited capacity for nuclear division, suggesting a cell cycle defect. Longer germination revealed morphological defects. The temperature-sensitive phoA allele caused both nuclear division and polarity defects at restrictive temperature, which could be complemented by expression of mammalian CDK5. Therefore, an essential function exists in A. nidulans for the Pho85-like kinase pair PHOA and PHOB, which may involve cell cycle control and morphogenesis.

Amino Acid Sequence↗