Sunday, April 29, 2007
1B-66

Examination of adjacent gene orientation and multiple promoter use on transcript expression and enzymatic activities

Ju Yun Bae, Molecular and Environmental Toxicology Program, University of Wisconsin, US Forest Service, Forest Products Laboratory, Madison, WI 53726, José Laplaza, US Forest Service, Forest Products Laboratory, US Forest Service, Forest Products Laboratory, Madison, WI 53726, and Thomas W. Jeffries, Molecular and Environmental Toxicology Program, University of Wisconsin-Madison and US Forest Service, Forest Products Laboratory, US Forest Service, Forest Products Laboratory, Madison, WI 53726.

Orientation of adjacent genes has been reported to affect their expression, so we examined the possible effect of gene orientation and repeated use of a single promoter on gene expression and enzymatic activity. D-xylose reductase (XYL1) and D-xylitol dehydrogenase (XYL2) were assembled in four ways. Each pair of genes was assembled into two different tandem (1→2 or 2→1), convergent (1→←2), and divergent (←12→) orientations. TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed in Saccharomyces cerevisiae. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then co-expressed XYL2 from either a single or multicopy plasmid that was also driven by same promoter. XYL2 transcript and enzyme expression increased with promoter strength while the expression of XYL1 was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids, however, other factors could affect expression of adjacent genes in chromosomes.

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