Generation of new snxAHrb1 alleles by a non-complementation assay

Emily Kohlbrenner 

Advisor:  Dr. Steven JamesEmily Kohlbrenner

nimXCdc2 in Aspergillus nidulans encodes the catalytic subunit of CDK1, the canonical cdc2-cyclinB kinase whose activation in late G2 phase triggers entry into mitosis. Within the snxA gene (suppressor-of-nimXCdc2), snxA1 was isolated as an extragenic suppressor of nimX2F223L and suppresses the heat-sensitive G2 arrest of nimX2 and permits mitosis to occur at the restrictive temperature of 42oC. snxA1 alone also confers a cold-sensitive arrest in G1 at 22oC or below. snxA encodes the A. nidulans ortholog of Saccharomyces cerevisiae Hrb1/Gbp2, nonessential shuttling mRNA binding proteins belonging to the SR (Serine-Arginine Rich) and RRM (RNA Recognition Motif) protein family. These proteins were not previously known as regulators of cell division. Deletion of snxA phenocopies snxA1, conferring cold-sensitivity and suppressing nimX2, albeit with a more severe, growth- impaired phenotype than snxA1. It was determined that the snxA1 mutation lies outside the coding region and most likely is in a proximal element such as enhancer that makes it difficult to identify. These findings suggest that (1) snxA1 is likely to be a hypomorphic mutation resulting from diminished levels of the wild-type protein, and (2) at normal levels, snxA acts to restrain the rate and timing of cell division. A non-complementation strategy was used to isolate 10 new candidate snxA mutations as well as linkage analysis, complementation testing, and DNA sequencing to verify bona fide snxA alleles and to identify the mutations in each. It is of interest to continue investigating the snxA novel mechanism in cell proliferation because it is possible that snxA may play an essential role cancerous unrestrained cell division.