One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientationdependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.The eukaryotic mRNAs produced by RNA polymerase II (Pol II) are capped with an inverted 7-methyl-guanosine (m 7 G) linked to the first residue of the mRNA (36). This event occurs by a series of three enzymatic reactions; The 5Ј triphosphate end of the nascent RNA Pol II transcript is cleaved by 5Ј RNA triphosphatase to produce the diphosphate end. RNA guanylyltransferase forms a covalent enzyme-GMP complex and subsequently caps the RNA substrate by adding a guanosine residue in a 5Ј-5Ј triphosphate linkage. The cap is then methylated by RNA (guanine-7) methyltransferase (23, 39). In higher eukaryotes, a bifunctional monomeric polypeptide carries both the RNA triphosphatase and guanylyltransferase activities, while the capping enzyme from yeast is a complex of RNA triphosphatase and guanylyltransferase subunits (40). The polypeptides are encoded by the CET1 and CEG1 genes, respectively in Saccharomyces cerevisiae, and both are essential for the cell viability.Capping, the first mRNA modification, occurs by the time the transcript is only 25 to 30 nucleotides long in an early transcription phase. Such cotranscriptional capping is mediated by recruitment of capping enzyme machinery to the phosphorylated carboxy-terminal domain (CTD) of the largest subunit of Pol II (7,15,22,51). The CTD of Pol II has an unusual structure with many heptapeptide repeats (YSPTSPS). The capping enzyme binds directly and specifically to the phosphorylated CTD of Pol II via the Ceg1 subunit (yeast) or the guanylyltransferase domain (metazoan). Furthermore, the guanylyltransferase activity of Ceg1 associated with phosphorylated CTD is allosterically regulated by both the Cet1 and phosphorylated CTD to ensure it has a coordin...

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PurposeTo evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium.MethodsThis study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy.ResultsCorneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal.ConclusionsThe main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.

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The TASS animal model seems to be a useful means to evaluate corneal endothelial cell damage caused by toxic substances to find ways to protect or reduce endothelial cell damage. Dispersive viscoelastics were shown to have partial protective effects against corneal endothelial cell damage caused by a toxic disinfectant.