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T. Footz, S. Dubois, J. Johnson, V. Raymond, M. Walter; A Yeast Model System for Assessing Functional Defects From POAG-Associated Alterations in WDR36/UTP21. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1652.
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To establish that the small subunit (SSU) rRNA processome protein Utp21p is the yeast orthologue of human WDR36, the product of the GLC1G candidate gene for adult-onset primary open angle glaucoma (POAG). To investigate the feasibility of using baker’s yeast for genetic manipulations of the essential gene UTP21 to mimic DNA alterations in POAG patients. To determine the effects of expressing mutant Utp21p protein in a utp21Δ background. To correlate potential functional protein defects with POAG patient genotype.
A haploid yeast test strain was created with its chromosomal copy of UTP21 deleted but carrying the corresponding ORF on a plasmid. Plasmids expressing recombinant WDR36 or in vitro-mutagenized UTP21 constructs were "shuffled" into the test strain and scored for growth in a plate assay, and steady-state rRNA levels were examined by Northern analysis.
WDR36 was unable to complement the utp21Δ strain; this may be due to functional divergence of the proteins, or to inability of yeast to correctly express the human ORF. Eleven POAG-associated base substitutions in WDR36 were introduced into homologous positions in UTP21 (eight are identical residues), in addition to a utp21 point mutation that was identified in a screen for genetic interactions with STI1 (Flom et. al, 2005, Curr. Genet., 47:368). The STI1-interacting mutation displayed a slow-growth phenotype that was most prevalent at 37°C. The eleven POAG-associated changes did not produce any growth defects. None of the twelve mutant strains exhibited elevated levels of precursor or intermediate species of rRNA relative to 18S, the SSU processome product.
To date, our yeast model system has failed to pinpoint any functional defects in UTP21 constructs derived by introducing POAG-associated substitutions in the homologous gene WDR36. Since many of these DNA alterations have also been found at low frequencies in normal controls, it is of great importance to validate their role in the onset or progression of POAG. We are continuing to explore other assays in which to test the function of UTP21 mutants in our knockout strain.
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