tRNA Pseudogenes Research Articles

Mitochondrial genomes and 28S rDNA contradict the proposed obsoletion of the order Tetraonchidea (Platyhelminthes: Monogenea)

Due to the incongruence of morphology-based hypotheses and scarcity of molecular data, validity of the order Tetraonchidea remains contentious. The only complete mitogenome currently available for the entire order is that of Paratetraonchoides inermis (Tetraonchoididae). To study the phylogeny of Tetraonchidea from mitogenomic perspective, we sequenced the first mitogenome for the family Tetraonchidae: Tetraonchus monenteron (Tetraonchidea). To get a nuclear-data perspective, we also sequenced nuclear 28S rDNA gene of both species. The mitogenome of T. monenteron does not have high A + T content, nor tRNA pseudo-genes, both of which were unique features reported in P. inermis. However, T. monenteron exhibits a unique gene order, with a large number of tRNA rearrangements in comparison to P. inermis and other monogeneans. Phylogenetic analyses conducted using Bayesian inference and maximum likelihood methods, complemented with partitioning, consistently support the sister-group relationship of T. monenteron (Tetraonchidae) and P. inermis (Tetraonchoididae). This is also partially supported by the 28S rDNA data and two morphologic apomorphies. This close relationship of Tetraonchidae and Tetraonchoididae challenges the latest major morphology-based classification, which proposed obsoletion of the Tetraonchidea order, and grouped Tetraonchoididae into the Gyrodactylidea clade. The validity of this order shall have to be further confirmed with more data.

The mitochondrial genome of the black-tailed dasyure (Murexia melanurus)

In this study, we report the mitochondrial genome of the black-tailed dasyure (Murexia melanurus) of New Guinea. The circular genome is 17,736 bp in length and has an AT content of 60.5%. Its gene content – 13 protein-coding genes (PCGs), 2 ribosomal (rRNA) genes, 21 transfer RNA (tRNA) genes, a tRNA pseudogene (tRNALys), and a non-coding control region (CR) – and gene arrangement are consistent with previous marsupial mitogenome assemblies.

Mitochondrial genomes of two diplectanids (Platyhelminthes: Monogenea) expose paraphyly of the order Dactylogyridea and extensive tRNA gene rearrangements

BackgroundRecent mitochondrial phylogenomics studies have reported a sister-group relationship of the orders Capsalidea and Dactylogyridea, which is inconsistent with previous morphology- and molecular-based phylogenies. As Dactylogyridea mitochondrial genomes (mitogenomes) are currently represented by only one family, to improve the phylogenetic resolution, we sequenced and characterized two dactylogyridean parasites, Lamellodiscus spari and Lepidotrema longipenis, belonging to a non-represented family Diplectanidae.ResultsThe L. longipenis mitogenome (15,433 bp) contains the standard 36 flatworm mitochondrial genes (atp8 is absent), whereas we failed to detect trnS1, trnC and trnG in L. spari (14,614 bp). Both mitogenomes exhibit unique gene orders (among the Monogenea), with a number of tRNA rearrangements. Both long non-coding regions contain a number of different (partially overlapping) repeat sequences. Intriguingly, these include putative tRNA pseudogenes in a tandem array (17 trnV pseudogenes in L. longipenis, 13 trnY pseudogenes in L. spari). Combined nucleotide diversity, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently showed that nad2, nad5 and nad4 were the most variable PCGs, whereas cox1, cox2 and cytb were the most conserved. Phylogenomic analysis showed that the newly sequenced species of the family Diplectanidae formed a sister-group with the Dactylogyridae + Capsalidae clade. Thus Dactylogyridea (represented by the Diplectanidae and Dactylogyridae) was rendered paraphyletic (with high statistical support) by the nested Capsalidea (represented by the Capsalidae) clade.ConclusionsOur results show that nad2, nad5 and nad4 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in the Diplectanidae. The unique gene order pattern further suggests discontinuous evolution of mitogenomic gene order arrangement in the Class Monogenea. This first report of paraphyly of the Dactylogyridea highlights the need to generate more molecular data for monogenean parasites, in order to be able to clarify their relationships using large datasets, as single-gene markers appear to provide a phylogenetic resolution which is too low for the task.

Complete sequences of the mitochondrial DNA of the Petalonia binghamiae

Here, the complete mitogenome of Petalonia binghamiae (J. Agardh) K. L. Vinogradova was determined and analyzed. The length of the complete P. binghamiae mitogenome was determined to be 37,460 bp. The mitogenome contains a set of 68 genes, including 35 protein-coding genes, 3 rRNA genes, 24 tRNA genes, 1 tRNA pseudogene, and 5 unidentified open reading frames (ORFs). The noncoding sequence constitutes 7.03% of the P. binghamiae mtDNA. Twenty-seven of the 35 (77.14%) protein-coding genes ended with a TAA stop codon, 6 (17.14%) with TAG, and 2 (5.71%) with TGA. All protein-coding genes in P. binghamiae were inferred to use the start codon ATG, except for nad11. The mitogenome phylogenetic analysis, based on 35 protein-coding genes, reveals that P. binghamia showed that P. binghamiae was clustered together with P. fascia. The complete mitochondrial genome sequence provided here would be useful for further understanding the evolution of Petalonia.

Sequence Assembly of Yarrowia lipolytica Strain W29/CLIB89 Shows Transposable Element Diversity.

Yarrowia lipolytica, an oleaginous yeast, is capable of accumulating significant cellular mass in lipid making it an important source of biosustainable hydrocarbon-based chemicals. In spite of a similar number of protein-coding genes to that in other Hemiascomycetes, the Y. lipolytica genome is almost double that of model yeasts. Despite its economic importance and several distinct strains in common use, an independent genome assembly exists for only one strain. We report here a de novo annotated assembly of the chromosomal genome of an industrially-relevant strain, W29/CLIB89, determined by hybrid next-generation sequencing. For the first time, each Y. lipolytica chromosome is represented by a single contig. The telomeric rDNA repeats were localized by Irys long-range genome mapping and one complete copy of the rDNA sequence is reported. Two large structural variants and retroelement differences with reference strain CLIB122 including a full-length, novel Ty3/Gypsy long terminal repeat (LTR) retrotransposon and multiple LTR-like sequences are described. Strikingly, several of these are adjacent to RNA polymerase III-transcribed genes, which are almost double in number in Y. lipolytica compared to other Hemiascomycetes. In addition to previously-reported dimeric RNA polymerase III-transcribed genes, tRNA pseudogenes were identified. Multiple full-length and truncated LINE elements are also present. Therefore, although identified transposons do not constitute a significant fraction of the Y. lipolytica genome, they could have played an active role in its evolution. Differences between the sequence of this strain and of the existing reference strain underscore the utility of an additional independent genome assembly for this economically important organism.

A Ralstonia solanacearum phage ϕRP15 is closely related to Viunalikeviruses and encodes 19 tRNA-related sequences

A T4-related phage infecting Ralstonia solanacearum was isolated in Thailand (ϕRP15). ϕRP15 virions showed unique morphology with star-like protrusions attached to the base plate via a stalk. The 168kb genome sequence of ϕRP15 was determined and found to encode 17 tRNAs (plus two tRNA pseudogenes). Ten of these phage-encoded tRNAs corresponded to codons that are relatively abundant in the phage but rare in the host, while others matched to codons frequent in both phage and host. Phylogenetic and proteomic analyses demonstrate that ϕRP15 forms a clade with Delftia phage ϕW-14, which in turn is placed as a sister group of viunalikeviruses. The hosts of ϕRP15 and ϕW-14 (R. solanacearum and Delftia acidovorans, respectively) belong to Betaproteobacteria, while most of the hosts of viunalikeviruses are of the Enterobacteriaceae belonging to Gammaproteobacteria. These phages may have evolved closely associated with their hosts that have very different life-styles in the natural habitats.

Molecular analysis of the 16S-23S rDNA internal spacer region (ISR) and truncated tRNAAla gene segments in Campylobacter lari

Following PCR amplification and sequencing, nucleotide sequence alignment analyses demonstrated the presence of two kinds of 16S-23S rDNA internal spacer regions (ISRs), namely, long length ISRs of 837-844 base pair (bp) [n = six for urease-negative (UN) Campylobacter lari isolates, UN C. lari JCM2530(T), RM2100, 176, 293, 299 and 448] and short length ISRs of 679-725 bp [n = six for UN C. lari: n = 14 for urease-positive thermophilic Campylobacter (UPTC) isolates]. The analyses also indicated that the short length ISRs mainly lacked the 156 bp sequence from the nucleotide positions 122-277 bp in long length ISRs for UN C. lari JCM2530(T). The 156 bp sequences shared 94.9-96.8 % sequence similarity among six isolates. Surprisingly, atypical tRNA(Ala) gene segment (5' end 35 bp), which was extremely truncated, occurred within the 156 bp sequences in the long length ISRs, as an unexpected tRNA(Ala) pseudogene. An order of the intercistronic tRNA genes within the short nucleotide spacer of 5'-16S rDNA-tRNA(Ala)-tRNA(Ile)-23S rDNA-3' occurred in all the C. lari isolates examined.

A global picture of tRNA genes in plant genomes

Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for tRNA(Tyr) and tRNA(Mete) . By contrast, in green algae, more than half of the tRNA genes contain an intron. This suggests divergent means of intron acquisition and the splicing process between green algae and land plants. Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in flowering plants. The only exceptions are tRNA(Gly) -snoRNA and tRNA(Mete) -snoRNA cotranscripts in dicots and monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and plastidial tRNA gene populations is also provided.

Genomic organization of eukaryotic tRNAs.

BackgroundSurprisingly little is known about the organization and distribution of tRNA genes and tRNA-related sequences on a genome-wide scale. While tRNA gene complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNA gene in Entamoeba histolytica have been described in some detail, systematic comparative studies are rare and mostly restricted to bacteria. We therefore set out to survey the genomic arrangement of tRNA genes and pseudogenes in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities.ResultsIn line with previous reports, we find that tRNA complements evolve rapidly and tRNA gene and pseudogene locations are subject to rapid turnover. At phylum level, the distributions of the number of tRNA genes and pseudogenes numbers are very broad, with standard deviations on the order of the mean. Even among closely related species we observe dramatic changes in local organization. For instance, 65% and 87% of the tRNA genes and pseudogenes are located in genomic clusters in zebrafish and stickleback, resp., while such arrangements are relatively rare in the other three sequenced teleost fish genomes. Among basal metazoa, Trichoplax adhaerens has hardly any duplicated tRNA gene, while the sea anemone Nematostella vectensis boasts more than 17000 tRNA genes and pseudogenes. Dramatic variations are observed even within the eutherian mammals. Higher primates, for instance, have 616 ± 120 tRNA genes and pseudogenes of which 17% to 36% are arranged in clusters, while the genome of the bushbaby Otolemur garnetti has 45225 tRNA genes and pseudogenes of which only 5.6% appear in clusters. In contrast, the distribution is surprisingly uniform across plant genomes. Consistent with this variability, syntenic conservation of tRNA genes and pseudogenes is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. Despite this large variation in abundance in Eukarya we observe a significant correlation between the number of tRNA genes, tRNA pseudogenes, and genome size.ConclusionsThe genomic organization of tRNA genes and pseudogenes shows complex lineage-specific patterns characterized by an extensive variability that is in striking contrast to the extreme levels of sequence-conservation of the tRNAs themselves. The comprehensive analysis of the genomic organization of tRNA genes and pseudogenes in Eukarya provides a basis for further studies into the interplay of tRNA gene arrangements and genome organization in general.

The complete mitochondrial genome of the large-headed frog, Limnonectes bannaensis (Amphibia: Anura), and a novel gene organization in the vertebrate mtDNA

We determined the complete nucleotide sequence of the mitochondrial (mt) genome of the large-headed frog, Limnonectes bannaensis (Amphibia, Anura) by using polymerase chain reaction (PCR). The entire mtDNA sequence is 16,867 bp in length with a novel case of tRNAs in vertebrates. This mt genome is characterized by three distinctive features: (1) a tandem duplication of tRNA Met gene is observed, (2) the tRNA Ala, tRNA Asn, tRNA Cys and tRNA Glu genes coded on the L-strand are absent from the L. bannaensis mtDNA, the tRNA Cys and tRNA Glu genes change into tRNA pseudogenes by reason of degenerative anticodon, and a noncoding sequence of 206 nt long (NC1) has replaced the original position of other two tRNAs, (3) besides NC1, another three noncoding spacers (NC2–4) longer than 50 bp are found in the broken WANCY region and the region NC3-ND5-NC4-ND6-ΨE-Cytb-CR of the new sequence. These features could be explained by a model of gene duplication and deletion. The new sequence data was used to assess the phylogenetic relationships among 25 species of Anura using neighbor-joining, Bayesian, and maximum likelihood methods, and the phylogenetic tree shows the rice frog Fejervarya limnocharis is closest to L. bannaensis in the study.

Molecular analyses of mitochondrial pseudogenes within the nuclear genome of arvicoline rodents

Nuclear sequences of mitochondrial origin (numts) are common among animals and plants. The mechanism(s) by which numts transfer from the mitochondrion to the nucleus is uncertain, but their insertions may be mediated in part by chromosomal repair mechanisms. If so, then lineages where chromosomal rearrangements are common should be good models for the study of numt evolution. Arvicoline rodents are known for their karyotypic plasticity and numt pseudogenes have been discovered in this group. Here, we characterize a 4 kb numt pseudogene in the arvicoline vole Microtus rossiaemeridionalis. This sequence is among the largest numts described for a mammal lacking a completely sequenced genome. It encompasses three protein-coding and six tRNA pseudogenes that span approximately 25% of the entire mammalian mitochondrial genome. It is bordered by a dinucleotide microsatellite repeat and contains four transposable elements within its sequence and flanking regions. To determine the phylogenetic distribution of this numt among the arvicolines, we characterized one of the mitochondrial pseudogenes (cytochrome b) in 21 additional arvicoline species. Average rates of nucleotide substitution in this arvicoline pseudogene are estimated as 2.3 x 10(-8) substitutions/per site/per year. Furthermore, we performed comparative analyses among all species to estimate the age of this mitochondrial transfer at nearly 4 MYA, predating the origin of most arvicolines.

Links Between Repeated Sequences

L1 and Alu elements are long and short interspersed retrotransposable elements (LINEs and SINEs) in humans, respectively. Proteins encoded in the autonomous L1 mediate retrotransposition of the nonautonomous Alu and cellular mRNAs. Alu is the only active SINE in the human genome and is derived from 7SL RNA of signal recognition particle. In the other eukaryotic genomes, various tRNA- and 5S rRNA-derived SINEs are found. Some of the tRNA- and 5S rRNA-derived SINEs have partner LINEs of which 3′ sequences are similar to those of the SINEs. One of the tRNA-derived SINEs is shown to be mobilized by its partner LINE. Many copies of tRNA and 5S rRNA pseudogenes are present in the human genome. These pseudogenes may have been generated via the retrotransposition process using L1 proteins. Although there are no sequence similarities between L1 and Alu, L1 functionally links with Alu and even cellular genes, impacting on our genome shaping.

Gene Rearrangements and Evolution of tRNA Pseudogenes in the Mitochondrial Genome of the Parrotfish (Teleostei: Perciformes: Scaridae)

Genomic size of animal mitochondrial DNA is usually minimized over time. Thus, when regional duplications occur, they are followed by a rapid elimination of redundant material. In contrast to this general view, we report here long-sustained tRNA pseudogenes in the mitochondrial genome (mitogenome) of teleost fishes of the family Scaridae (parrotfishes). During the course of a molecular phylogenetic study of the suborder Labroidei, we determined the complete nucleotide sequence of the mitogenome for a parrotfish, Chlorurus sordidus, and found a gene rearrangement accompanied by a tRNA pseudogene. In the typical gene order of vertebrates, a tRNA-gene cluster between ND1 and ND2 genes includes tRNA(Ile) (I), tRNA(Gln) (Q), and tRNA(Met) (M) genes in this order (IQM). However, in the mitogenome of the parrotfish, the tRNA(Met) gene was inserted between the tRNA(Ile) and the tRNA(Gln) genes, and the tRNA(Gln) gene was followed by a putative tRNA(Met) pseudogene (psiM). Such a tRNA gene rearrangement including a pseudogene (IMQpsiM) was found in all of the 10 examined species, representing 7 of the 10 currently recognized scarid genera. All sister groups examined (20 species of Labridae and a single species of Odacidae) had the typical gene order of vertebrate mitogenomes. Phylogenetic analysis of the tRNA(Met) genes and the resulting pseudogenes demonstrated that the ancestral tRNA(Met) gene was duplicated in a common ancestor of the parrotfish. Based on the fossil record, these results indicate that the pseudogenes have survived at least 14 million years. Most of the vertebrate mitochondrial gene rearrangements involving the IQM region have held the tRNA(Met) gene just upstream of the ND2 gene, and even in a few exceptional cases, including the present ones, the tRNA pseudogenes have been found in that position. In addition, most of these tRNA(Met) pseudogenes maintained clover-leaf secondary structures, with the remainder sustaining the clover-leaf structure in the "top half (TpsiC and acceptor arms). Considering their potential secondary structures (holding "top halves" of the clover-leaf structures), locations within mitogenomes (flanking the 5' ends of the ND2 genes) and stabilities over time (survived at least 14 Myr), it is likely that the tRNA pseudogenes retain function as punctuation marks for mitochondrial ND2 mRNA processing.

Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of > 40 kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is approximately 19 kb and the right junction > 20 kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re-formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (PsitRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.

A tRNA pseudogene in the archaeon Methanococcus jannaschii.

While searching the first completely sequenced genome of the archaeon Methanococcus jannaschii for a small RNA gene, we discovered a 5' truncated gene of a transfer RNA (tRNA(Ser-UCR)) at position 334,431-334,486; including the CCA-end that exactly matched the 3' terminal domain of the annotated M. jannaschii tRNA(Ser-UCR) gene located at position 303,992-304,081. This truncated tRNA gene covering 56 nucleotides (about 2/3) of the genuine tRNA represents, to the best of our knowledge, the first described tRNA pseudogene in the archaeal domain.

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions.

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.

Nucleotide sequence of nuclear tRNA(Gly) genes and tRNA(Gly) pseudogenes from yellow lupin (Lupinus luteus): expression of the tRNA(Gly) genes in vitro and in vivo.

A nuclear DNA fragment (7.8 kb) from yellow lupin (L. luteus) was sequenced and shown to contain tRNA(Gly) (GGC) genes and tRNAGly (GGC) pseudogenes organized in three tandemly repeated units: of 2565 bp and 2564 bp, and one, truncated from its 3' end, of 1212 bp. Each unit contains an identical pair of a tRNA(Gly) gene and a pseudogene, both having the same polarity. The nucleotide sequence of the gene appears colinear to L. luteus cytoplasmic tRNA(Gly) (GGC) primary structure. All three genes are efficiently transcribed in HeLa-cell nuclear extract giving two primary transcripts. The main, longer primary transcripts have each an extremely long 3' trailer of about 100 nucleotides, the structure of which is specific only for tRNAGly genes and pseudogenes (80% homology) of the studied tandem (but not for other tRNA(Gly) genes of the yellow lupin genome) as it has been shown by Southern hybridization. This distinctive feature allowed to isolate putative tRNAGly precursor(s) encoded by at least one of the three tRNA(Gly) (GGC) genes from L. luteus seedlings.

TRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence.

We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.

Pre-tRNA 3'-processing in Saccharomyces cerevisiae. Purification and characterization of exo- and endoribonucleases.

We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre-tRNA 3'-processing in vitro. Two pre-tRNA 3'-exonucleases with molecular masses of 33 and 60 kDa, two pre-tRNA 3'-endonucleases with molecular masses of 45 kDa/60 kDa and 55 kDa and 70-kDa 3'-pre-tRNase were purified from yeast whole cell extracts by several successive chromatographic purification steps. The purified exonucleases are non-processive 3'-exonucleases that catalyze the exonucleolytic processing of 3'-trailer sequences of pre-tRNAs to produce mature tRNAs. The 45-kDa/60-kDa 3'-endonuclease is tRNA-specific and catalyzes the processing of pre-tRNAs in a single endonucleolytic step. Two isoenzymes of this activity (p45 and p60) were identified by chromatography. The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3'-end. All of the purified 3'-pre-tRNases accept homologous as well as heterologous pre-tRNA substrates. Pre-tRNAs carrying a 5'-leader are processed with almost the same efficiency as those lacking this 5'-leader. Mature tRNAs carrying the CCA 3'-sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3'-exonucleases, not by the 3'-endonucleases. The specific endonuclease p45/p60 discriminates between UUUOH as a 3'-flank, which is cleaved, and the CCA 3'-end of mature tRNAs, which is not cleaved. This study suggests that several 3'-pre-tRNases are active on tRNA precursors in vitro and might therefore in pre-tRNA 3'-processing in yeast, partly in a cooperative manner.

The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria

The complete 27,694-bp mitochondrial (mt) DNA sequence of Hansenula wingei, which is a typical budding yeast and contains circular mitochondrial DNA, has been determined. The mt sequence contains genes encoding large and small ribosomal RNAs, 25 tRNAs, three subunits of cytochrome c oxidase (subunits 1, 2 and 3), three subunits of ATPase (subunits 6, 8 and 9), apocytochrome b, seven subunits of NADH dehydrogenase (subunits 1, 2, 3, 4, 4L, 5 and 6), and a ribosomal protein, VAR1. The VAR1 gene is considered to be a typical yeast type. This is consistent with data on DNA and the deduced amino-acid sequence homology comparisons of genes ubiquitous in yeast and fungi. However, we have identified seven genes encoding NADH dehydrogenase subunits, which are not found in other yeast mitochondrial genomes, thus placing the H. wingei mitochondrial genome in a unique position. In addition the H. wingei mitochondrial genome also encodes one tRNA pseudogene and one short unidentified ORF. The genome is compact with only two introns both of which contain an ORF. One intron lies in the large rRNA gene while the other is situated in the cytochrome c oxidase subunit-1 gene. The conserved nonanucleotide motif (A/T)TATAAG (T/A)(A/T), which is a transcription start signal in Saccharomyces cerevisiae mitochondria, has also been found in the H. wingei mitochondrial genome. The codon assignments for ATA and CTN in H. wingei mitochondria are different from those in S. cerevisiae mitochondria. These results indicate a unique and novel structure for the H. wingei mitochondrial genome in terms of characteristics which are typical for both yeast and for filamentous fungi. This is the first complete mt DNA sequence report in yeast.