Conservatism of sites of tRNA loci among the linkage groups of severalDrosophila species

Summary The sites of seven tRNA genes (Arg-2, Lys-2, Ser-2b, Ser-7, Thr-3, Thr-4, Val-3b) were studied by in situ hybridization.¹²⁵I-labeled tRNA probes fromDrosophila melanogaster were hybridized to spreads of polytene chromosomes prepared from fourDrosophila species representing different evolutionary lineages (D. melanogaster, Drosophila hydei, Drosophila pseudoobscura, andDrosophila virilis). Most tRNA loci occurred on homologous chromosomal elements of all four species. In some cases the number of hybridization sites within an element varied and sites on nonhomologous elements were found. It was observed that both tRNA ₂ ^Arg and tRNA ₂ ^Lys hybridized to the same site on homologous elements in several species. These data suggest a limited amount of exchange among different linkage groups during the evolution ofDrosophila species.     

Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

Summary The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.     

Genetic evidence for functional specificity of the yeast GCN2 kinase

In yeast the GCN2 kinase mediates translational control ofGCN4 by phosphorylating the subunit of eIF-2 in response to extracellular amino acid limitation. Although phosphorylation of eIF-2 has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect onGCN4 mRNA translation. Under the same conditions, translation of other mRNAs appears only slightly affected. The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear. Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitatesGCN4-specific translational regulation.     

Cloning and characterization of the platypus mitochondrial genome

The vertebrate mitochondrial genome is highly conserved in size and gene content. Among the chordates there appears to be one basic gene arrangement, but rearrangements in the mitochondrial gene order of the avian lineages have indicated that the mitochondrial genome may be more variable than once thought. Different gene orders in marsupials and eutherian mammals leave the ancestral mammalian order in some doubt. We have investigated the mitochondrial gene order in the platypus (Ornithorhynchus anatinus), a representative of the third major group of mammals, to determine which mitochondrial gene arrangement is ancestral in mammals. We have found that the platypus mtDNA conforms to the basic chordate gene arrangement, common to fish, amphibians, and eutherian mammals, indicating that this arrangement was the original mammalian arrangement, and that the unusual rearrangements observed in the avians and marsupials are probably lineage-specific. Correspondence to: N.J. Gemmell     

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...