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PubMed1988

Functional dissection of 5' and 3' extragenic control regions of human tRNA(Val) genes reveals two different regulatory effects.

Two natural human tRNA(Val) genes, pHtV1 and pHtV3, differ in their transcription efficiency by an order of magnitude. The extragenic control regions (ECRs) responsible for this effect were compared with respect to the kinetics and thermodynamics of transcription complex formation. The 5' ECR of pHtV1 acts by increasing both the rate of stable complex formation and the equilibrium constant of association between tDNA and at least one transcription factor present in the stable complex. The stability of the preinitiation complexes is not affected by ECRs. For the formation of a stable preinitiation complex, we suggest a two-step mechanism, comprising (i) the ECR-controlled association of at least one transcription factor (TFIIIC) with the tDNA, and (ii) an ECR-independent conformational change of this tDNA-protein complex. The function of 3' ECRs could be discriminated from the 5' ECR-mediated effects by transcriptional analysis of two chimeric constructs derived from pHtV1 and pHtV3. Surprisingly, the pHtV1 3' ECR causes an eight-fold increase of transcription efficiency, although it has only minor influence on stable preinitiation complex formation. Instead, this ECR stimulates transcription by promoting the transition of the preinitiation complex into an activity synthesizing transcription complex. This novel function of a 3' ECR contributes an additional regulatory level for tRNA gene expression.

Base Sequence↗
PubMed2003

Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells.

The post-transcriptional gene silencing in animals and plants is called RNA interference (RNAi). Guides for the sequence-specific degradation of mRNA are 21-nt small interfering RNAs (siRNAs) that are generated by Dicer-dependent cleavage from longer double-stranded RNAs (dsRNAs). To examine the relationship between the localization of dsRNA and the target cleavage of RNAi in human cells, we constructed five kinds of dsRNA expression vector that were controlled by tRNA(Val) or U6 promoter. Transcripts of tRNA-dsRNA were consistently localized in the cytoplasm and were efficiently processed by Dicer. In contrast, transcripts of tRNA-dsRNA were not processed in cells that expressed Dicer-directed ribozymes. In addition, transcripts of U6-dsRNA were basically localized in the nucleus and were not significantly processed, unless the transcripts of U6-dsRNAs possessed a microRNA-based loop motif: in the latter case, U6-dsRNAs with a microRNA-based loop were transported to the cytoplasm and were effectively processed. More over, tRNA-dsRNA directed against a mutant k-ras transcript cleaved its target mRNA efficiently in assays of RNAi not only in vitro with a cytoplasmic extract but also in vivo. Therefore, it appears that RNAi in human cells occur in the cytoplasm. Importantly, the same tRNA-dsRNA did not affect the degradation of the normal k-ras mRNA in vitro and in vivo. Our tRNA-dsRNA technology should be a powerful tool for studies of the mechanism of RNAi and the functions of various genes in mammalian cells with potential utility as a therapeutic agent.

Cell Division↗
PubMed1991

Control regions of an archaeal gene. A TATA box and an initiator element promote cell-free transcription of the tRNA(Val) gene of Methanococcus vannielii.

To identify the DNA sequences required for initiation of transcription in archaea, the 5'-flanking region of the tRNA(Val) gene of Methanococcus vannielii was modified by deletions, restructuring and site-directed mutagenesis, and the tRNA encoding sequence was replaced by a fortuitous Escherichia coli sequence. The effects of these mutations on promoter function were tested in an homologous cell-free transcription system. The DNA region from position -35 to +9 relative to the transcription start site was sufficient for maximal initiation of cell-free transcription. Removal of the DNA region between -35 and -30 reduced initiation by a factor of 2. Deletions extending to position -24 almost completely abolished specific transcription. Analysis of 16 site-specific mutations in the region from -33 to +2 provided evidence that a conserved A + T-rich sequence (TATA box), centered at -25, is essential for initiation of transcription. Single point mutations in six positions of the TATA box reduced initiation of transcription from 0.2 to 0.01 of wild-type levels. A second conserved motif at the transcription start site (consensus ATGC) could be replaced by some sequences containing a pyrimidine-purine dinucleotide but appeared necessary for a maximal rate of gene transcription. Mutations altering the spacing between the two conserved elements demonstrated that initiation occurs at a strictly defined distance of 22 to 27 base-pairs downstream from the TATA box. Our results support the conclusion that the TATA box is the major DNA region mediating promoter recognition, influencing the efficiency of transcription and specifying the site of transcription initiation. This Methanococcus promoter element closely resembles in structure and function the TATA box of promoters of eukaryotic protein-encoding genes transcribed by RNA polymerase II.

Base Sequence↗
PubMedDate not supplied

[Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods].

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.

Escherichia coli↗
PubMedDate not supplied

Single nucleotide modulation of uridine to pseudouridine rearrangement in transfer RNA catalyzed by pseudouridine synthase I.

E. coli pseudouridine synthase I (PSUI) catalyzes the rearrangement of uridine residues in positions 38, 39 and 40 of tRNA transcripts to pseudouridine. These positions are located in the anticodon stem-loop of the tRNA molecule. Fourteen different E. coli tRNAs are substrates for the enzyme, whereas four other tRNAs which contain uridine in position 38 are not. Investigations were focused on the basis of enzyme differentiation between substrate and non-substrate tRNAs. Comparison of modification reactions with mutant and wild-type tRNA transcripts demonstrates that the presence of a G36 residue modulates modification by PSUI at position 38. In addition to local sequence effects, steady-state kinetic analyses suggest the existence of other recognition elements distinct from the immediate vicinity of modification.

Base Sequence↗
PubMed1996

Novel autoantibodies directed against the common tertiary configuration of transfer RNA in a patient with interstitial lung disease.

OBJECTIVE: To identify and characterize a novel autoantibody, anti-WS, that binds total transfer RNA (tRNA). METHODS: Serum from patient WS, who had polyarthritis, Sjögren's syndrome, Raynaud's phenomenon, and interstitial pulmonary fibrosis, was used in this study. Characteristics of anti-WS and antibody-reactive determinants of tRNA were investigated by 32P immunoprecipitation using HeLa cell RNA and deletion mutants of tRNA transcribed in vitro. RESULTS: WS serum produced nucleolar and cytoplasmic staining on indirect immunofluorescence. 32P immunoprecipitation assays demonstrated that this serum immunoprecipitated total tRNAs and 5.8S and 5S ribosomal RNAs from 32P-labeled HeLa cell extract. When deproteinized RNA was used as antigen source, total tRNAs were still precipitated by WS serum. An immunoprecipitation study, using various deletion mutants of Escherichia coli tRNA, demonstrated that both D and T psi C loops were needed for antibody binding. Substitution of nucleotide 18G with 18A of E coli tRNA(Trp), which is essential in the formation of the tertiary "L" shape of tRNA, inhibited binding by anti-WS antibodies. CONCLUSION: Anti-WS antibodies are novel autoantibodies directed against tRNAs. The antibody binding site is the common L-shaped tertiary structure conformed by the D loop and T psi C loop of tRNA, suggesting that the antibodies are induced by a conserved sequence among all species. Furthermore, these antibodies could be a marker for a newly recognized subset of connective tissue disease.

Autoantibodies↗
PubMed1989

tRNA hopping: enhancement by an expanded anticodon.

At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons. Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences. We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing. Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site. The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine). In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues. It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site.

Alleles↗
PubMed1993

Functional tRNAs with altered 3' ends.

The CCA trinucleotide is a universally conserved feature of the 3' end of tRNAs, where it serves as the site of amino acid attachment. Despite this extreme conservation, we have isolated functional mutants of tRNA(His) and tRNA(Val1) with altered CCA ends. A mutant that leads to de-repression of the histidine biosynthetic operon in Salmonella typhimurium has been characterized and found to have the CCA end of the sole tRNA(His) species mutated to UCA. However, constructed mutants of tRNA(His) with ACA or GCA ends appeared to be nonfunctional in vivo. Mutants of Escherichia coli tRNA(Val1) with GCA or ACA ends were isolated on the basis of their ability to promote frameshifting at a specific sequence. These same tRNA(Val1) mutants also caused read-through of stop codons that were one, or in some instances two, codons downstream of the valine codon decoded by the mutant tRNA. A startling implication of these data is that disruption of interactions between the CCA end of the tRNA and the large ribosomal subunit promotes these aberrant codon-anticodon interactions on the small ribosomal subunit.

Amino Acid Sequence↗
PubMed2004

LIM kinase-2 targeting as a possible anti-metastasis therapy.

BACKGROUND: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. METHODS: We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. RESULTS: Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. CONCLUSIONS: Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells.

Base Sequence↗
PubMed1994

Analysis of an 11.7 kb DNA fragment of chromosome XI reveals a new tRNA gene and four new open reading frames including a leucine zipper protein and a homologue to the yeast mitochondrial regulator ABF2.

We report the nucleotide sequence of an 11.7 kb fragment from the left arm of Saccharomyces cerevisiae chromosome XI. Analysis reveals a new tRNA for valine and four unknown open reading frames among which YKL245 shows homology with a yeast mitochondrial regulatory protein and YKL244, YKL246 and YKL247 are unknown.

Amino Acid Sequence↗
PubMed1999

Pseudouridine synthetase Pus1 of Saccharomyces cerevisiae: kinetic characterisation, tRNA structural requirement and real-time analysis of its complex with tRNA.

Pseudouridine synthetase Pus1 from Saccharomyces cerevisiae is a multisite-specific enzyme that catalyses the formation of pseudouridine residues at different positions in several tRNA transcripts. Recombinant Pus1, tagged with six histidine residues at its N terminus was expressed in Escherichia coli and purified. Transcripts of yeast tRNAValand intronless yeast tRNAIlewere used as substrates to measure pseudouridine formation at position 27. The catalytic parameters Kmand kcatfor tRNAValand tRNAIlewere 420(+/-100) nM and 0.4(+/-0.1) min-1, 740(+/-100) nM and 0.5(+/-0.1) min-1, respectively. Pus1 possesses a general affinity for tRNA, irrespective of whether they are substrates. Its equilibrium dissociation constant ranges from 15 nM for the substrate yeast tRNAValand non-substrate yeast intronless tRNAPhe, to 150 nM for the substrate yeast intronless tRNAIle. The difference in the affinity for the different tRNA species is not reflected in the specific activity of the enzyme, indicating that the binding of Pus1 to tRNA is not the kinetically limiting step. The importance of tertiary base-pairs was investigated with several variants of yeast tRNAs. Although dispensable for activity, both the presence of a D-stem-loop and the presence of a G26.A44 base-pair, near the target uridine U27, are important elements for Pus1 tRNA high affinity recognition. The presence of a G26.A44 base-pair in tRNA increases its association constant rate with Pus1 (ka) by a factor of approximately 100, resulting in a decrease of the overall equilibrium dissociation constant (Kd). The dissociation rate (kd) is the same, independent of the presence of a G26.A44 base-pair in the tRNA. A model describing the interaction of Pus1 with tRNA is proposed.

Base Pairing↗
PubMed1999

Origin of the unisexual lizard Gymnophthalmus underwoodi (Gymnophthalmidae) inferred from mitochondrial DNA nucleotide sequences.

Analysis of variation in nucleotide sequences in mitochondrial DNA genes among species of Gymnophthalmus supports three major conclusions. First, samples of G. underwoodi, a unisexual species of hybrid origin, have identical nucleotide sequences, supporting the hypothesis that all individuals from localities in the Guianan Region of South America and the Lesser Antilles descended from a single hybridization event. Second, G. underwoodi and G. cryptus share derived similarities in nucleotide sequences of the maternally inherited mitochondrial DNA, suggesting that the female participant in the hybridization event that produced the hybrid species was either G. cryptus or most closely related to it. Last, phylogenetic analyses recovered historical structure among population samples of G. speciosus supporting previous claims that there is unrecognized diversity under that binomial, and some analyses suggest that G. speciosus does not comprise a natural group.

Animals↗
PubMed2001

Genetically distinct populations in an Asian soldier-producing aphid, Pseudoregma bambucicola (Homoptera: Aphididae), identified by DNA fingerprinting and molecular phylogenetic analysis.

To estimate genetic structure of a soldier-producing aphid, Pseudoregma bambucicola, samples from natural populations throughout southeastern Asia were analyzed by a DNA fingerprinting technique. We unexpectedly found that P. bambucicola comprises two geographic groups, the northern group and the southern group, which are genetically distinct from each other but morphologically almost indistinguishable. Molecular phylogenetic and statistical analyses based on mitochondrial ribosomal DNA sequences demonstrated that the northern and southern groups of P. bambucicola are not closely related but constitute distinct lineages in the genus Pseudoregma. Detailed morphological reexamination revealed that the two groups could be distinguished by the number of setae on the 8th abdominal tergite of 1st instar nymphs and soldiers. From these results, it was suggested that P. bambucicola should be divided into two species. The northern group from Japan, Taiwan, Hong Kong, and northern Vietnam retains the name P. bambucicola, whereas we suggest that the name P. carolinensis (R. Takahashi, 1941, Tenthredo 3, 208-220) should be used for the southern group from Thailand, Malay Peninsula, Java, Irian Jaya, and Micronesia. The morphological resemblance between P. bambucicola and P. carolinensis might be due to shared ancestral characters of the genus Pseudoregma.

Animals↗
PubMed2001

Phylogenetic relationships of major clades of Catostomidae (Teleostei: Cypriniformes) as inferred from mitochondrial SSU and LSU rDNA sequences.

Suckers (Family Catostomidae) are holarctic in distribution and include 76 recent species in 14 genera, with 13 genera and 75 species occurring in North and Central America and Siberia. Although this group constitutes a significant component of many aquatic ecosystems, most historic systematic effort has been either alpha- or limited beta-level studies focusing on the two largest tribes within the family, the Catostomini and the Moxostomatini. A recent phylogenetic study based on morphological, biochemical, and early life history characters has advanced current understanding of relationships among catostomid fishes. To further examine phylogenetic relationships among basal lineages of catostomids, we sequenced the entire mitochondrial (mt) SSU and LSU rRNA genes from genera representing all subfamilies and tribes within Catostomidae. Phylogenetic analysis of gene sequences yielded monophyletic Catostomidae, Ictiobinae, and Catostominae and para- or polyphyletic Cycleptinae, with Myxocyprinus as the basal-most taxon and Cycleptus as either the next most-basal taxon or the taxon basal to the Catostominae. Relationships within the Catostominae were generally consistent with those proposed in the above-noted recent phylogenetic study although Thoburnia and Hypentelium were either a clade sister to or a grade group relative to Moxostoma and Scartomyzon. In all trees, Scartomyzon was paraphyletic and embedded within Moxostoma. Phylogenetic affinities of Erimyzon and Minytrema varied depending on data set and character weighting scheme employed. To better reflect phylogenetic relationships resolved in this extensive analysis, we propose the following changes to the classification of catostomids: formation of the new subfamily Myxocyprininae, containing Myxocyprinus from China; restriction of the Cycleptinae to the two species of Cycleptus from North America; restriction of the tribe Moxostomatini to Moxostoma and Scartomyzon; Erimyzon and Minytrema are incertae sedis within Catostominae; and resurrection of the tribe Thoburniini, containing Thoburnia and expanded to include Hypentelium.

Animals↗
PubMed2001

Phylogenetics of the lizard genus Tropidurus (Squamata: Tropiduridae: Tropidurinae): direct optimization, descriptive efficiency, and sensitivity analysis of congruence between molecular data and morphology.

By use of the technique of direct optimization the phylogenetics of the cis-Andean lizard genus Tropidurus were examined on the basis of both molecular (ca. 1.04 kb of sequences from 12S rDNA, valine tDNA, and 16S rDNA) and morphological (93 characters) data. Although equal weighting of all parsimony cost functions logically must maximize descriptive efficiency and explanatory power of all evidence, a sensitivity analysis demonstrated that equal weighting of indels, transitions, transversions, and morphological change provided the most congruent solution between the molecular and the morphological data partitions. The position of Uranoscodon is resolved as the sister taxon of the remaining members of the Tropidurinae. Plica, Uracentron, and Strobilurus, previously considered synonyms of Tropidurus, are resurrected; the group of these three genera form the sister taxon of the former Tropidurus nanuzae group (herein named Eurolophosaurus) plus Tropidurus sensu stricto (composed of the T. bogerti, T. semitaeniatus, T. spinulosus, and T. torquatus groups, herein diagnosed).

Animals↗
PubMed2002

Molecular evidence for the monophyly of tenrecidae (mammalia) and the timing of the colonization of Madagascar by Malagasy Tenrecs.

Tenrecs are a diverse family of insectivores, with an Afro-Malagasian biogeographic distribution. Three subfamilies (Geogalinae, Oryzorictinae, Tenrecinae) are restricted to Madagascar and one subfamily, the otter shrews (Potamogalinae), occurs on the mainland. Morphological studies have generated conflicting hypotheses according to which both tenrecids and Malagassy tenrecs are either monophyletic or paraphyletic. Competing hypotheses have different implications for the biogeographic history of Tenrecidae. At present, there are no molecular studies that address these hypotheses. The present study provides sequences of a nuclear protein-coding gene (vWF) and the mitochondrial 12S rRNA, tRNA valine, and 16S rRNA genes from a potamogaline (Micropotamogale). New sequences of these genes are also reported for the tenrecine, Tenrec ecaudatus. The 12S sequences from these taxa were combined with data already available for this locus from two other tenrecids (Echinops telfairi, subfamily Tenrecinae and Oryzorictes talpoides, subfamily Oryzorictinae). Phylogenetic analyses provided strong bootstrap support for the monophyly of Tenrecidae and Malagasy tenrecs. The majority of statistical tests rejected morphological claims for both a Tenrecinae--Chrysochloridae clade and an Oryzorictinae--Potamogalinae clade. Molecular clock estimates suggest a split of otter shrews and Malagasy tenrecs at approximately 53 MYA. We estimate that the ancestor of Malagasy tenrecs dispersed to Madagascar subsequent to this split but prior to about 37 MYA.

Animals↗
PubMed2002

Molecular phylogeny of a circum-global, diverse gastropod superfamily (Cerithioidea: Mollusca: Caenogastropoda): pushing the deepest phylogenetic limits of mitochondrial LSU rDNA sequences.

The Cerithioidea is a very diverse group of gastropods with ca. 14 extant families and more than 200 genera occupying, and often dominating, marine, estuarine, and freshwater habitats. While the composition of Cerithioidea is now better understood due to recent anatomical and ultrastructural studies, the phylogenetic relationships among families remain chaotic. Morphology-based studies have provided conflicting views of relationships among families. We generated a phylogeny of cerithioideans based on mitochondrial large subunit rRNA and flanking tRNA gene sequences (total aligned data set 1873 bp). Nucleotide evidence and the presence of a unique pair of tRNA genes (i.e., threonine + glycine) between valine-mtLSU and the mtSSU rRNA gene support conclusions based on ultrastructural data that Vermetidae and Campanilidae are not Cerithioidea, certain anatomical similarities being due to convergent evolution. The molecular phylogeny shows support for the monophyly of the marine families Cerithiidae [corrected], Turritellidae, Batillariidae, Potamididae, and Scaliolidae as currently recognized. The phylogenetic data reveal that freshwater taxa evolved on three separate occasions; however, all three recognized freshwater families (Pleuroceridae, Melanopsidae, and Thiaridae) are polyphyletic. Mitochondrial rDNA sequences provide valuable data for testing the monophyly of cerithioidean [corrected] families and relationships within families, but fail to provide strong evidence for resolving relationships among families. It appears that the deepest phylogenetic limits for resolving caenogastropod relationships is less than about 245--241 mya, based on estimates of divergence derived from the fossil record.

Animals↗