tRNA Genes Affect Multiple Aspects of Local Chromosome Behavior.

In eukaryotic organisms the tRNA genes, which exist in multiple copies, are spread throughout the genome. Although it had been assumed that these genes are spread throughout the nucleus, it has recently shown that tRNA genes are actually clustered at the nucleolus. Since the tRNA genes seem to play a role in organizing the entire genome, their transcription may have dramatic influences. The three studies presented here were undergone to provide insight into these influences and their maintenance. The first study provides evidence that tRNA gene transcription influences recombination in the genome. The study examined two different circumstances. The first situation was used to tested if the transcription of repetitive tRNA genes influenced their tendency to undergo homologous recombination and the second situation was used to test how a tRNA gene influences the homologous recombination of two nearby repetitive sequences. It was determined that two transcriptionally active tRNA genes had a five times greater rate of recombination than if one or both tRNA genes were inactivated, however tRNA gene transcription had no discernible influence over the recombination between nearby repetitive elements. This could have implications in the human genome, which contains over 1 million RNA polymerase III transcribed SINE elements. The second study explores how tRNA gene transcription influences their clustering and localization at the nucleolus. This study demonstrated that the condensin complex associates with tRNA genes and physically interacts with RNA polymerase III transcription factors. Condensin’s interactions with these two elements may lead to the clustering and localization of the tRNA genes, which could play an important role in the organization of the entire genome in eukaryotes. The third study found a gene deletion, mod5Δ, that results in the alleviation of silencing near tRNA genes. The results of this study indicate that Mod5p’s catalytic activity is necessary for tgm silencing. The protein was also shown to be present at tRNA genes and seems to maintain the silencing in cis. These results indicate that Mod5p performs an activity necessary for the repression of pol II transcription and this may have implications for the regulation of many genes in higher eukaryotes.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60716/1/mhyatt_1.pd

Increased Recombination Between Active tRNA Genes

Transfer RNA genes are distributed throughout eukaryotic genomes, and are frequently found as multicopy families. In Saccharomyces cerevisiae, tRNA gene transcription by RNA polymerase III suppresses nearby transcription by RNA polymerase II, partially because the tRNA genes are clustered near the nucleolus. We have tested whether active transcription of tRNA genes might also suppress recombination, since recombination between identical copies of the repetitive tRNA genes could delete intervening genes and be detrimental to survival. The opposite proved to be the case. Recombination between active tRNA genes was elevated, but only when both genes are transcribed. We also tested the effects of tRNA genes on recombination between the direct terminal repeats of a neighboring retrotransposon, since most Ty retrotransposons reside next to tRNA genes, and the selective advantage of this arrangement is not known.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63113/1/dna.2006.25.359.pd

Clustering of yeast tRNA genes is mediated by specific association of condensin with tRNA gene transcription complexes.

http://deepblue.lib.umich.edu/bitstream/2027.42/85738/1/Haeusler-Pratt-Hystt-Engelke-2008.pd

Mechanism-Based Inactivation of Human CYP2E1 by Diethyldithocarbamate

Although the ability of disulfiram to inactivate CYP2E1 has been known for more than 20 years, the mechanism has not yet been elucidated. A metabolite of disulfiram, diethyldithocarbamate (DDC), is converted by CYP2E1 to a reactive intermediate that subsequently inactivates the protein, leading to mechanism-based inactivation. Mass spectral analysis of the inactivated human 2E1 protein demonstrates that the inactivation is due to the formation of an adduct of the reactive metabolite of DDC with the apoprotein. These data, along with mass spectral analysis of a reactive intermediate trapped with GSH, indicate the involvement of a reactive intermediate with a molecular mass of 116 Da. Our results suggest that this binding involves formation of a disulfide bond with one of the eight cysteines in CYP2E1. The inactivation of wild-type CYP2E1 as well as two of its polymorphic mutants, CYP2E1*2 and CYP2E1*4, was also investigated. For wild-type CYP2E1, the KI was 12.2 μM and the kinact was 0.02 min−1. The KI values for the two polymorphic mutants were 227.6 and 12.4 μM for CYP2E1.2 and CYP2E1.4, and the kinact values were 0.0061 and 0.0187 min−1, respectively. These data indicate that DDC is a much less efficient inactivator of CYP2E1.2 than it is of either the wild-type or the CYP2E1.4 variant