The genes coding for tRNATyr of Drosophila melanogaster: Localization and determination of the gene numbers

Transfer RNA^Tyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNA^Tyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA^Tyr genes. The regions 22F and 85A each contain four to five tRNA^Tyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA^Tyr genes.     

Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.     

tRNA genes protect a reporter gene from epigenetic silencing in mouse cells

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.     

Structure and Evolution of the Atypical Mitochondrial Genome of <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Isopoda,Crustacea)

The crustacean isopod Armadillidium vulgare is characterized by an unusual ∼42-kb-long mitochondrial genome consisting of two molecules co-occurring in mitochondria: a circular ∼28-kb dimer formed by two ∼14-kb monomers fused in opposite polarities and a linear ∼14-kb monomer. Here we determined the nucleotide sequence of the fundamental monomeric unit of A. vulgare mitochondrial genome, to gain new insight into its structure and evolution. Our results suggest that the junction zone between monomers of the dimer structure is located in or near the control region. Direct sequencing indicated that the nucleotide sequences of the different monomer units are virtually identical. This suggests that gene conversion and/or replication processes play an important role in shaping nucleotide sequence variation in this mitochondrial genome. The only heteroplasmic site we identified predicts an alloacceptor tRNA change from tRNA^Ala to tRNA^Val. Therefore, in A. vulgare, tRNA^Ala and tRNA^Val are found at the same locus in different monomers, ensuring that both tRNAs are present in mitochondria. The presence of this heteroplasmic site in all sequenced individuals suggests that the polymorphism is selectively maintained, probably because of the necessity of both tRNAs for maintaining proper mitochondrial functions. Thus, our results provide empirical evidence for the tRNA gene recruitment model of tRNA evolution. Moreover, interspecific comparisons showed that the A. vulgare mitochondrial gene order is highly derived compared to the putative ancestral arthropod type. By contrast, an overall high conservation of mitochondrial gene order is observed within crustacean isopods.     

The sequence,organization, and evolution of the Locusta migratoria mitochondrial genome

The sequencing of the cloned Locusta migratoria mitochondrial genome has been completed. The sequence is 15,722 by in length and contains 75.3% A+T, the lowest value in any of the five insect mitochondrial sequences so far determined. The protein coding genes have a similar A+T content (74.1%) but are distinguished by a high cytosine content at the third codon position. The gene content and organization are the same as in Drosophila yakuba except for a rearrangement of the two tRNA genes tRNA^lys and tRNA^asp. The A+T-rich region has a lower A+T nucleotide content than in other insects, and this is largely due to the presence of two G+C-rich 155-bp repetitive sequences at the 5 end of this section and the beginning of the adjacent small rRNA gene. The sizes of the large and small rRNA genes are 1,314 and 827 bp, respectively, and both sequences can be folded to form secondary structures similar to those previously predicted for Drosophila. The tRNA genes have also been modeled and these show a strong resemblance to the dipteran tRNAs, all anticodons apparently being conserved between the two species. A comparison of the protein coding nucleotide sequences of the locust DNA with the homologous sequences of five other arthropods (Drosophila yakuba, Anopheles quadrimaculatus, Anopheles gambiae, Apis mellifera, and Artemia franciscana) was performed. The amino acid composition of the encoded proteins in Locusta is similar to that of Drosophila, with a Dayhoff distance twice that of the distance between the fruit fly and the mosquitoes. A phylogenetic analysis revealed the locust genes to be more similar to those of the Dipterans than to those of the honeybee at both the nucleotide and amino acid levels. A comparative analysis of tRNA orders, using crustacean mtDNAs as outgroups, supported this. This high level of divergence in the Apis genome has been noted elsewhere and is possibly an effect of directional mutation pressure having resulted in an accelerated pattern of sequence evolution. If the general assumption that the Holometabola are monophyletic holds, then these results emphasize the difficulties of reconstructing phylogenies that include lineages with variable substitution rates and base composition biases. The need to exercise caution in using information about tRNA gene orders in phylogenetic analysis is also illustrated. However, if the honeybee sequence is excluded, the correspondence between the other five arthropod sequences supports the findings of previous studies which have endorsed the use of mtDNA sequences for studies of phylogeny at deep levels of taxonomy when mutation rates are equivalent. Correspondence to: P.K. Flook     

Nuclear tRNATyr genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana

Summary We have examined the organization of tRNA^Tyr genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 × 107 bp. Three tRNA^Tyr gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRl fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA^Tyr genes flanked by a tRNA^Ser gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA^Tyr genes encode the known cytoplasmic tRNA_GA ^Tyr. Both genes contain a 12 by long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRl-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggests that the tRNA^Ser/tRNA^Tyr cluster evolved 1–5 million years ago, after the Brassicaceae diverged from the other flowering plants about 5–10 million years ago.     

综述: 高等植物转座元件功能研究进展

转座元件是指在基因组中能够移动、复制并重新整合到基因组新位点的DNA片段．转座元件一度被视为基因组内的“垃圾”或“自私DNA”,长期以来,转座元件的研究主要集中于阐释转座元件在宿主中的复制或表观沉默机制,而转座元件的调控功能并未得到全面探讨．已有研究表明,转座元件的比例与物种基因组大小存在正相关性,从而为C值悖论的解释提供了依据．近年来,越来越多的证据表明转座元件可以作为宿主基因组的“控制元件”发挥重要的调控作用．在作物中研究发现,转座元件既可以通过顺式或反式作用方式调控基因表达,也可以诱导表观等位基因的产生,从而促使固着生长的植物更好地适应外界环境的变化．本文拟就高等植物转座元件的作用及其对未来作物育种的意义进行总结．     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNAf ^Met) gene was identified on a 347 bpEco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAsf ^Met or tRNAf ^Met genes studied so far. Whereas the tRNAf ^Met gene is present as a single copy in the potato mt genome, a tRNA pseudogene corresponding to 60% of a complete tRNA (from the 5 end to the variable region) and located at 105 nucleotides upstream of the tRNAf ^Met gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNAf ^Met gene in the mt genome is very different among plants, which suggests that the tRNAf ^Met gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.     

The Chloroplast trnT–trnF Region in the Seed Plant Lineage Gnetales

The trnT–trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT–trnL and trnL–trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25–35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL–trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL–trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA^Thr(UGU), tRNA^Leu(UAA), and tRNA^Phe (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints. [Reviewing Editor: Debashish Bhattacharya]     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.     

tRNA genes protect a reporter gene from epigenetic silencing in mouse cells

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.Key words: barrier elements, tRNA genes, Pol III-transcribed genes     

Structures of tobacco chloroplast genes for tRNAIle (CAU), tRNALeu (CAA), tRNACys (GCA), tRNASer (UGA) and tRNAThr (GGU): a compilation of tRNA genes from tobacco chloroplasts

Summary The location and nucleotide sequences of tobacco chloroplast genes for tRNA^Ile (CAU), tRNA^Leu (CAA), tRNA^Cys (GCA), tRNA^Ser (UGA) and tRNA^Thr (GGU) (trnI-CAU, trnL-CAA, trnC-GCA, trnS-UGA and trnT-GGU, respectively) have been determined. The trnI and trnL are located in the inverted repeat region. The trnC, trnS and trnT are present in the large single copy region. These five tRNA genes together with the 25 different tRNA genes previously published have been compiled and compared. These 30 tRNA genes corresponding to 20 amino acids are most likely to be all of the tRNA genes encoded in tobacco chloroplast genome.This paper is dedicated to Professor Morio Ikehara on the occasion of his retirement from Osaka University in March 1986.     

Sequencing chromosome 5D of Aegilops tauschii and comparison with its allopolyploid descendant bread wheat (Triticum aestivum)

Flow cytometric sorting of individual chromosomes and chromosome‐based sequencing reduces the complexity of large, repetitive Triticeae genomes. We flow‐sorted chromosome 5D of Aegilops tauschii, the D genome donor of bread wheat and sequenced it by Roche 454 GS FLX platform to approximately 2.2x coverage. Repetitive sequences represent 81.09% of the survey sequences of this chromosome, and Class I retroelements are the prominent type, with a particular abundance of LTR/Gypsy superfamily. Nonrepetitive sequences were assembled to cover 17.76% of the total chromosome regions. Up to 6188 nonrepetitive gene loci were predicted to be encoded by the 5D chromosome. The numbers and chromosomal distribution patterns of tRNA genes suggest abundance in tRNA^Lys and tRNA^Met species, while the nonrepetitive assembly reveals tRNA^Ala species as the most abundant type. A comparative analysis of the genomic sequences of bread wheat and Aegilops chromosome 5D indicates conservation of gene content. Orthologous unique genes, matching Aegilops 5D sequences, numbered 3730 in barley, 5063 in Brachypodium, 4872 in sorghum and 4209 in rice. In this study, we provide a chromosome‐specific view into the structure and organization of the 5D chromosome of Ae. tauschii, the D genome ancestor of bread wheat. This study contributes to our understanding of the chromosome‐level evolution of the wheat genome and presents a valuable resource in wheat genomics due to the recent hybridization of Ae. tauschii genome with its tetraploid ancestor.     

Discovery and assembly of repeat family pseudomolecules from sparse genomic sequence data using the Assisted Automated Assembler of Repeat Families (AAARF) algorithm

Background  

Higher eukaryotic genomes are typically large, complex and filled with both genes and multiple classes of repetitive DNA. The repetitive DNAs, primarily transposable elements, are a rapidly evolving genome component that can provide the raw material for novel selected functions and also indicate the mechanisms and history of genome evolution in any ancestral lineage. Despite their abundance, universality and significance, studies of genomic repeat content have been largely limited to analyses of the repeats in fully sequenced genomes.     

Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This transposition-deletion system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Organization of the Mitochondrial Genome of a Deep-Sea Fish, Gonostoma gracile (Teleostei: Stomiiformes): First Example of Transfer RNA Gene Rearrangements in Bony Fishes

We determined the complete nucleotide sequence of the mitochondrial genome (except for a portion of the putative control region) for a deep-sea fish, Gonostoma gracile. The entire mitochondrial genome was purified by gene amplification using long polymerase chain reaction (long PCR), and the products were subsequently used as templates for PCR with 30 sets of newly designed, fish-universal primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products showed that the genome contained the same 37 mitochondrial structural genes as found in other vertebrates (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes), with the order of all rRNA and protein-coding genes, and 19 tRNA genes being identical to that in typical vertebrates. The gene order of the three tRNAs (tRNA^Glu, tRNA^Thr, and tRNA^Pro) relative to cytochrome b, however, differed from that determined in other vertebrates. Two steps of tandem duplication of gene regions, each followed by deletions of genes, can be invoked as mechanisms generating such rearrangements of tRNAs. This is the first example of tRNA gene rearrangements in a bony fish mitochondrial genome. Received August 5, 1998; accepted February 19, 1999.     

Molecular domestication of mobile elements

Transposable elements are ubiquitous in all organisms and represent a dynamic component of their genomes, causing mutations and thereby genetic variation. Because of their independent and expansive replication strategy, these elements are called selfish and were thought to have no impact on the adaptive evolution of their host organisms. Although most TE-induced mutations seem to exert only negative effects on the fitness of their carrier, recent evidence indicates that in the course of evolution at least some TE-mediated changes have become established features of the host genome. For example, the insertion of TEs may provide novel cis-regulatory regions to preexisting host genes or TE-derived trans-acting factors may undergo a molecular transition into novel host genes through a process described as molecular domestication. The stationary P element related gene clusters of D. guanche, D. madeirensis and D. subobscura provide an excellent model system to study the evolutionary impact of TEs on genome evolution. Each cluster unit consists of a cis-regulating section composed of different insertion sequences followed by the first three exons of a P element that are coding for a 66 kDa ‘repressor-like’ protein. This revised version was published online in August 2006 with corrections to the Cover Date.     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.