The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CANI and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.Retrotransposons such as Ty are common in eucaryotes. While they are not infectious, they otherwise resemble retroviruses in their structural organization and replication in viruslike particles via an RNA intermediate (for a review, see reference 1). The finding that two-to fourfold increases in the transcription of Ty message caused disproportionately large increases in Ty transposition led to speculation that the cell may exert controls on transposition at a posttranscriptional level (7). Possibly the extra Ty mRNA leads to a complete saturation of this hypothetical posttranscriptional regulatory system. Other evidence that the rate of Ty transposition is not simply proportional to the Ty message level comes from the observation that growth at a low temperature (17 versus 30°C) causes a 50-to 100-fold increase in the rate of Ty transposition without changing the level of Ty mRNA (24,25). One explanation of this observation is that the reverse transcriptase activity is optimal at low temperatures (10).Mutations in the Saccharomyces cerevisiae RAD6 gene may result in several phenotypes, including extreme sensitivity to DNA-damaging agents, lack of induced mutagenesis, defective sporulation, increased mitotic recombination, and an increased rate of spontaneous mutation (for a review, see reference 13). RAD6 encodes an E22ok enzyme which polyubiquitinates histones H2A and H2B in vitro (16, 35) and which may influence chromatin structure (16,29,35). Here we show that among the spontaneous mutations stimulated by rad6 are mutations due to the transposition of Ty elements. Since the yeast RAD6 protein may be analogous to a mammalian 20-kilodalton E2 ubiquitin-conjugating enzyme (16,26), these studies may be relevant to studies of the cellular mechanisms used to control the movements of retrotransposons and retroviruses in other eucaryotes. LP2752-4B and LP2752-4Brad6A and plasmid pR67 were kindly provided by Louise Prakash. N-152, also from Louise Prakash, is a rad6-3 mutant (28) and was derived from B-635, a cycl-115 mutant derived from D311-3A (a Iys2 hisi trp2), both kindly provided by Fred Sherman.Mutant isolation. A large number of independent colonies from each strain were used to inoculate individual slants of complete medium, YPD (32), which were grown at 30°C. Cells suspended from these slants were plated either to synthetic complete medium lacking arginine and containing 60 ,ug of canavanine per ml (-Arg +Can) (32) or to medium containin...
The accurate and efficient translation of proteins is of fundamental importance to both bacteria and higher organisms. Most of our knowledge about the control of translational fidelity comes from studies of Escherichia coli. In particular, ram (ribosomal ambiguity) mutations in structural genes of E. coli ribosomal proteins S4 and S5 have been shown to increase translational error frequencies. We describe the first sequence Analyses of informational suppressors in Escherichia coli have elucidated the components involved in determining the fidelity of translation. In most instances, the factors identified have been either tRNAs or ribosomal proteins (31,69,84). Alterations in five different ribosomal proteins have been shown to influence translational accuracy. Mutations in the structural genes for S12 (30), S17 (8), and L6 (53) can cause an increase in accuracy, while mutations in the structural genes for L7/L12 (47), S4 (77), and S5 (13, 71) can decrease fidelity. Such mutations that affect S4 or S5 are known as ram (ribosomal ambiguity) mutations because they cause a general ambiguity of translation, suppressing nonsense as well as missense and frameshift mutations (3, 73). More recently, nonsense suppressor phenotypes have also been shown to be associated with mutations in elongation factor EF-Tu (85, 89).We and others have used a similar genetic approach to identify components of the eucaryotic protein synthesis machinery that control translational fidelity (2,21,25,54,65,88,91,95 sup45, SUP44, and SUP46 strains increase in vitro misreading of polyuridylate templates (25,62).Despite the suggestive biochemical evidence, the first two yeast omnipotent suppressor genes to be cloned and sequenced (SUP35+ and SUP45+) do not encode ribosomal proteins (10,11,17,18,38,54,57,95). The SUP45+ gene is predicted to encode a protein of molecular mass 49 kDa (10). Regions of weak similarity to several tRNA synthetases were found, but the function of the gene product is still not known. The SUP35+ gene is predicted to encode a protein of 76.5 kDa that shows a high degree of similarity to EF-la (45, 54, 95) but is not identical to any of the three biochemically characterized elongation factors from yeast cells. Neither SUP35+ nor SUP45+ appears likely to encode a ribosomal protein, since both predicted gene products are much larger than ribosomal proteins, and their codon usage patterns indicate that, unlike ribosomal protein genes, they are not highly expressed (10,54,95).Here, we describe the sequence of a third yeast omnipotent suppressor gene, sup44+. This suppressor does encode a ribosomal protein. Furthermore, the SUP44 protein shows substantial sequence similarity to the E. coli S5 ram protein. MATERIALS AND METHODSStrains and genetic methods. The following Saccharomyces cerevisiae strains were used: SL815-26B [et met8-J leu2-1 trpl-J (his5-2 and/or his3-J) Iys2-J ura3-52 SUP44] and SL-982 [ala ura3-521ura3-52 met8-J/met8-J leu2-111eu2-1 ade3-261+ his5-21+ his3-All+ tyr7-J1+ trpl-l1+]. Standard yeast genetic procedu...
Nitration of tyrosine and other aromatic amino acid residues in proteins occurs in the setting of inflammatory, neurodegenerative, and cardiovascular diseases—importantly, this modification has been implicated in the pathogenesis of diverse diseases and the physiological process of aging. To understand the biological consequences of aromatic nitration in both health and disease, it is critical to molecularly identify the proteins that undergo nitration, specify their cognate modification sites and quantify their extent of nitration. To date, unbiased identification of nitrated proteins has often involved painstaking 2D-gel electrophoresis followed by Western Blotting with an anti-nitrotyrosine antibody for detection. Apart from being relatively slow and laborious, this method suffers from limited coverage, the potential for false-positive identifications, and failure to reveal specific amino acid modification sites. To overcome these shortcomings, we have developed a solid-phase, chemical-capture approach for unbiased and high-throughput discovery of nitrotyrosine and nitrotryptophan sites in proteins. Utilizing this method, we have successfully identified several endogenously nitrated proteins in rat brain and a total of 244 nitrated peptides from 145 proteins following in vitro exposure of rat brain homogenates to the nitrating agent peroxynitrite (1 mM). As expected, Tyr residues constituted the great majority of peroxynitrite-mediated protein nitration sites; however, we were surprised to discover several brain proteins that contain nitrated Trp residues. By incorporating a stable-isotope labeling step, this new Aromatic Nitration Site IDentification (ANSID) method was also adapted for relative quantification of nitration site abundances in proteins. Application of the ANSID method offers great potential to advance our understanding of the role of protein nitration in disease pathogenesis and normal physiology.
We describe a patient at 20-22 weeks gestation, with a known difficult airway, who underwent eight sessions of electroconvulsive therapy using the ProSeal laryngeal mask airway and controlled ventilation. The airway management options for brief periods of general anaesthesia in patients with increased gastric volume are discussed.
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