Codons AGA and AGG are read as glycine in ascidian mitochondria

Summary A 1.2-kb DNA fragment of the cytochrome oxidase subunit I (CO I) gene of mitochondria isolated from an ascidian,Halocynthia roretzi, was amplified by polymerase chain reaction (PCR) and sequenced. Codons AGA and AGG appeared in its reading frame, indicating that these are sense codons in this organelle. Sequence comparisons with the corresponding regions of other animal mitochondrial CO I genes suggest that codons AGA and AGG correspond to glycine in the ascidian mitochondrial genome, but not to serine as in most invertebrate genomes, nor to stops as in vertebrate genomes. The other codons are identical to those of vertebrate mitochondria.     

Acceptor stem and anticodon RNA hairpin helix interactions with glutamine tRNA synthetase

The class I glutamine (Gln) tRNA synthetase interacts with the anticodon and acceptor stem of glutamine tRNA. RNA hairpin helices were designed to probe acceptor stem and anticodon stem-loop contacts. A seven-base pair RNA microhelix derived from the acceptor stem of tRNA^Gln was aminoacylated by Gln tRNA synthetase. Variants of the glutamine acceptor stem microhelix implicated the discriminator base as a major identity element for glutaminylation of the RNA helix. A second RNA microhelix representing the anticodon stem-loop competitively inhibited tRNA^Gln charging. However, the anticodon stem-loop microhelix did not enhance aminoacylation of the acceptor stem microhelix. Thus, transduction of the anticodon identity signal may require covalent continuity of the tRNA chain to trigger efficient aminoacylation.     

核酸去甲基酶AlkB在毕赤酵母中的表达纯化及在tRNA相关研究中的应用

[目的]核酸的甲基化修饰是一种常见的化学修饰形式,具有重要的生物学功能,却也在一定程度上给一些核酸研究过程带来了技术难度。tRNA上具有的大量甲基化修饰会阻碍逆转录进程,从而降低荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)和高通量测序对其的检测效率。来自大肠杆菌(Escherichia coli)的AlkB蛋白是一种多功能的脱烷基化酶,可以去除DNA和RNA上多种甲基化为代表的修饰,有望解决以上问题。[方法]针对大肠杆菌来源的AlkB,分别尝试在大肠杆菌和毕赤酵母(Pichia pastoris)表达系统中进行诱导表达和纯化,对纯化获得的AlkB进行酶学性质测定。最后以tRNA_UAU^Ile等两种tRNA为代表,研究AlkB的处理对于荧光定量PCR法检测tRNA表达水平的影响。[结果]AlkB在大肠杆菌中表达时多以包涵体形式存在,但是在毕赤酵母中可以成功分泌表达。使用镍柱分离纯化后获得了纯度高于95%的AlkB蛋白,其酶学性质参数如...     

Isolation and preliminary characterization of gas1-1, a mutation causing partial suppression of the phenotype conferred by the gibberellin-insensitive (gai) mutation in Arabidopsis thaliana (L.) Heyhn

The semi-dominant gai mutation of arabidopsis confers a dark-green dwarf phenotype resembling that of gibberellin (GA)-deficient mutants. In contrast to GA-deficient mutants, gai mutants do not respond to GA treatments and accumulate higher levels of bioactive GAs than are found in wild-type controls. The gai mutation thus alters the responses of plant cells to GA, indicating that the GAI (wild-type) gene product is involved in GA reception and/or signal transduction. Here we describe the isolation and preliminary characterization of a mutation, gas1-1, which is not linked to gai and which partially suppresses the effect of the gai mutation. Double mutant, gai gas1-1, homozygotes are less severely dwarfed and lighter green than gai GAS1 controls. However, comparisons of the effects of treatments with exogenous GA demonstrate that gas1-1 does not increase the GA responsiveness of the gai mutant. Thus the gas1-1 mutation appears to reduce the GA-dependency of plant growth, and identifies a gene (GAS1) whose product is a candidate GA signal-transduction component.Abbreviations GA gibberellin - GA₃ gibberellic acid We thank Maarten Koornneef (Wageningen Agricultural University, The Netherlands) for providing mutant seed stocks; Mark Aarts and Bernard Mulligan (University of Nottingham, UK) for performing the -irradiation. This work was made possible by AFRC/BBSRC PMB Grants PG208/520 and PG208/0600, and by a grant from the Gatsby Charitable Foundation. P.C. was supported by a Human Capital and Mobility Fellowship from the EC.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

Structure,function and evolution of seryl-tRNA synthetases: Implications for the evolution of aminoacyl-tRNA synthetases and the genetic code

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA^ser in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 Amnoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3 end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence. Correspondence to: S. CusackBased on a presentation made at a workshop-Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.