Poster abstracts

Poster number 18 submitted by Heather Edgerton

Identification and initial phenotypic analysis of mitotic regulatory proteins in Aspergillus nidulans

Heather D. Edgerton (Molecular Genetics, Ohio State University), Tania Nayak (Molecular Genetics, Ohio State University), Edyta Szewczyk (Molecular Genetics, Ohio State University), Berl R. Oakley (Molecular Genetics, Ohio State University)

Abstract:
γ-tubulin mutants of the filamentous fungus Aspergillus nidulans alter mitotic regulation and we are, consequently, examining the functions of mitotic regulatory proteins and their localizations in wild-type and γ-tubulin mutant strains of this organism. Here, we report the identification, localization and deletion of the A. nidulans homologs of three important mitotic regulatory proteins, Mps1, Cdh1, and Cdc20. Mps1 is a member of the spindle assembly checkpoint and is also involved in centrosome/spindle pole body (SPB) duplication. Cdh1 and Cdc20 are regulators of the anaphase promoting complex (APC). We have identified A. nidulans genes that encode homologs of each protein, and we designate the A. nidulans proteins AnMPS1, AnCDH1 and AnCDC20. Using fusion PCR and a subsequent transformation procedure, we have created C-terminal GFP fusions of each of these while keeping them under the control of their endogenous promoters. AnMPS1-GFP and AnCDH1-GFP localize to the SPB. Time lapse microscopy reveals that the AnMPS1-GFP signal at the SPB begins to become visible in late G1 or S, increases in intensity through G2, and its localization to the SPB is lost 3-5 minutes prior to anaphase. AnCDH1-GFP fluorescence at the SPB is most intense in G1. Neither protein’s localization is altered in a strain carrying the γ-tubulin mutation mipAD159 that alters mitotic regulation. We have deleted the genes that encode each of these proteins and have found that AnMPS1 and AnCDC20 are essential for viability while AnCDH1 is not essential. Supported by the NIGMS.

References:
Jung, M.K., Prigozhina, N., Oakley, C.E., Nogales, E. & Oakley, B.R. (2001) Mol. Biol. Cell 12, 2119-36.
Nayak, T., Szewczyk, E., Oakley, C.E., Osmani, A., Ukil, L., Murray, S.L., Hynes, M.J., Osmani, S.A., & Oakley, B.R. (2006) Genetics 172, 1557-66.
Oakley, B.R., Oakley, C.E., Yoon, Y., & Jung, M.K (1990) Cell 61, 1289-301.
Oakley, C.E. & Oakley, B.R. (1989) Nature 338, 662-4.
Prigozhina, N.L., Oakley, C.E., Lewis, A.M., Nayak, T., Osmani, S.A., & Oakley, B.R. (2004) Mol. Biol. Cell 15, 1374-86.
Szewczyk, E., Nayak, T., Oakley, C.E., Edgerton, H., Xiong, Y., Taheri-Talesh, N., Osmani, S.A., & Oakley, B.R. (2006) Nat. Protoc. 1, 3111-20.

Keywords: cell cycle, mitosis, filamentous fungi