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At least 361 records · Page 20Linked to original sources

PubMed2000

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon.

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42781bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT-PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

Algal Proteins↗

PubMed2026

Ferroptosis as a mediator of gut microbiota-driven inflammatory bowel disease: Evidence from genetic analyses.

Gut microbiota dysbiosis is increasingly recognized as a contributor to inflammatory bowel disease (IBD), yet causal relationships and underlying mechanisms remain unclear. Ferroptosis, an iron-dependent form of regulated cell death, plays a key role in epithelial barrier damage and inflammation. This study aimed to determine whether specific gut microbial taxa are causally associated with IBD and whether ferroptosis-related genes mediate this association using Mendelian randomization (MR). Two-sample MR and mediation MR analyses were performed using genome-wide association study summary data from the FinnGen consortium (IBD), the genome-wide association study catalog (473 gut microbial taxa), and the deCODE database (ferroptosis-related genes). Instrumental variables were selected with thresholds of P < 1 × 10-6 for microbes and P < 5 × 10-8 for traits, and linkage disequilibrium clumping (r2 < 0.001) was applied. Twenty-three microbial taxa showed significant causal associations with IBD (e.g., Chromatiales, OR = 0.51; Acetobacterales, OR = 2.61). Several ferroptosis-related genes were linked to IBD risk (e.g., GPX4, STAT3, IDO1). Mediation MR revealed that genes such as MUC1, IDO1, and ADAM23 partially mediated microbial effects on IBD, with mediation proportions up to 7.6%. This study provides novel genetic evidence supporting a gut microbiota-ferroptosis-IBD axis. Ferroptosis-related pathways may partially mediate microbial effects on IBD pathogenesis and represent promising targets for future therapeutic interventions.

Ferroptosis↗

PubMed1994

Toxicity of a heterologous leucyl-tRNA (anticodon CAG) in the pathogen Candida albicans: in vivo evidence for non-standard decoding of CUG codons.

Plasmids containing derivatives of the Saccharomyces cerevisiae leucyl-tRNA (tRNA(3Leu)) gene that vary in anticodon sequence were constructed and transformed into the pathogen Candida albicans and S. cerevisiae. C. albicans could readily be transformed with plasmids encoding leucyl-tRNA genes with the anticodons CAA and UAA (recognizing the codons UUG and UUA) and expression of the heterologous tRNALeu could be demonstrated by Northern RNA blotting. In contrast, no transformants were obtained if the anticodons were UAG (codons recognized CUN, UUR) and CAG (codon CUG), indicating that the insertion of leucine at CUG codons is toxic for C. albicans. All tRNALeu-encoding plasmids transformed S. cerevisiae with equally high efficiencies. These results provide in vivo evidence that non-standard decoding of CUG codons is essential for the viability of C. albicans.

Anticodon↗

PubMed1998

The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule.

Deviations from the universal genetic code have evolved independently several times in ciliated protozoa. Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine. We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate. It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.

Animals↗

PubMed2005

ATP-sensitive K+ channel channel/enzyme multimer: metabolic gating in the heart.

Cardiac ATP-sensitive K(+) (K(ATP)) channels, gated by cellular metabolism, are formed by association of the inwardly rectifying potassium channel Kir6.2, the potassium conducting subunit, and SUR2A, the ATP-binding cassette protein that serves as the regulatory subunit. Kir6.2 is the principal site of ATP-induced channel inhibition, while SUR2A regulates K(+) flux through adenine nucleotide binding and catalysis. The ATPase-driven conformations within the regulatory SUR2A subunit of the K(ATP) channel complex have determinate linkage with the states of the channel's pore. The probability and life-time of ATPase-induced SUR2A intermediates, rather than competitive nucleotide binding alone, defines nucleotide-dependent K(ATP) channel gating. Cooperative interaction, instead of independent contribution of individual nucleotide binding domains within the SUR2A subunit, serves a decisive role in defining K(ATP) channel behavior. Integration of K(ATP) channels with the cellular energetic network renders these channel/enzyme heteromultimers high-fidelity metabolic sensors. This vital function is facilitated through phosphotransfer enzyme-mediated transmission of controllable energetic signals. By virtue of coupling with cellular energetic networks and the ability to decode metabolic signals, K(ATP) channels set membrane excitability to match demand for homeostatic maintenance. This new paradigm in the operation of an ion channel multimer is essential in providing the basis for K(ATP) channel function in the cardiac cell, and for understanding genetic defects associated with life-threatening diseases that result from the inability of the channel complex to optimally fulfill its physiological role.

ATP-Binding Cassette Transporters↗

PubMed2006

Homologous and heterologous carboxyl terminal peptide (CTP) linker sequences enhance the secretion of bioactive single-chain bovine LH analogs.

Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.

Amino Acid Sequence↗

PubMed2003

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event.

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.

Base Sequence↗

PubMed2005

Four-base codon-mediated incorporation of non-natural amino acids into proteins in a eukaryotic cell-free translation system.

Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.

Amino Acids↗

PubMed1994

Direct recognition of mRNA stop signals by Escherichia coli polypeptide chain release factor two.

The interaction between the translational stop signal and the polypeptide chain release factor protein (RF) within complexes of Escherichia coli ribosomes has been investigated by site-directed photochemical cross-linking experiments. Twelve mRNA analogues containing 4-thiouridine residues as part of stop signals were synthesized. Highly efficient cross-linking to RF-2 from 4-thiouridine (sU) residues of sUGAN-containing mRNAs was observed, and cross-linking from those of sUAAN was observed at a lower efficiency. This indicates that RF-2 is in close physical contact with the stop signal on the ribosome. The yield of the RF-2-mRNA cross-link depended on the identity of the fourth base for the sUGAN set of signals, suggesting that the fourth base of the stop signal affects the interaction between RF-2 and the stop codon. A region previously implicated as part of the decoding site of the small ribosomal subunit, 1385-1420 of the 16 S rRNA, was also cross-linked with these mRNAs. No new cross-links were obtained in the presence of the release factor. The data are consistent with models in which the RF has an anticodon-like domain that contacts the stop signal directly at or near the ribosomal site in which sense codons are decoded.

Bacterial Proteins↗

PubMed1994

The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop.

A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross-linking. From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338. The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site). Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.

Anticodon↗

PubMed1997

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit.

A non-Shine-Dalgamo translational initiator is identified in Escherichia coli. The nucleotide sequence ACCUACUCGAGUUAG, designated as PL, is capable of initiating translation of pokeweed antiviral protein (PAP) and human calcitonin (hCT) mRNAs in E. coli cells. The yield of recombinant protein was double that obtained with the consensus Shine-Dalgarno-sequence-(SD)-driven translation. The PL sequence is composed of two heptanucleotides (ACCUACU, box I and GAGUUAG, box II) which are complementary to nucleotides 1434-1440 and 507-513, respectively, in 16S rRNA. Mutational analysis shows that the translation initiation efficiency with either box alone is much lower than that obtained with the entire PL sequence, indicating that the boxes interact simultaneously with both complementary regions in 16S rRNA during the translation initiation step. Based on these results, we propose that the two widely separated regions 507-513 (part of helical domain 18) and 1434-1440 (belonging to helical domain 44) are organized in close proximity to each other and to the ribosome decoding center on the surface of the E. coli 30S ribosomal subunit.

Base Sequence↗

PubMed1996

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.

Bacteriophage T7↗

PubMed2006

[From population genetics to population genomics of forest trees: integrated population genomics approach].

Early works by Altukhov and his associates on pine and spruce laid the foundation for Russian population genetic studies on tree species with the use of molecular genetic markers. In recent years, these species have become especially popular as nontraditional eukaryotic models for population and evolutionary genomic research. Tree species with large, cross-pollinating native populations, high genetic and phenotypic variation, growing in diverse environments and affected by environmental changes during hundreds of years of their individual development, are an ideal model for studying the molecular genetic basis of adaptation. The great advance in this field is due to the rapid development of population genomics in the last few years. In the broad sense, population genomics is a novel, fast-developing discipline, combining traditional population genetic approaches with the genomic level of analysis. Thousands of genes with known function and sometimes known genomic localization can be simultaneously studied in many individuals. This opens new prospects for obtaining statistical estimates for a great number of genes and segregating elements. Mating system, gene exchange, reproductive population size, population disequilibrium, interaction among populations, and many other traditional problems of population genetics can be now studied using data on variation in many genes. Moreover, population genomic analysis allows one to distinguish factors that affect individual genes, alleles, or nucleotides (such as, for example, natural selection) from factors affecting the entire genome (e.g., demography). This paper presents a brief review of traditional methods of studying genetic variation in forest tree species and introduces a new, integrated population genomics approach. The main stages of the latter are : (1) selection of genes, which are tentatively involved in variation of adaptive traits, by means of a detailed examination of the regulation and the expression of individual genes and genotypes, with subsequent determination of their complete allelic composition by direct nucleotide sequencing; (2) examination of the phenotypic effects of individual alleles by, e.g., association mapping; and (3) determining the frequencies of the selected alleles in natural population for identification of the adaptive variation pattern in the heterogeneous environment. Through decoding the phenotypic effects of individual alleles and identification of adaptive variation patterns at the population level, population genomics in the future will serve as a very helpful, efficient, and economical tool, essential for developing a correct strategy for conserving and increasing forests and other commercially valuable plant and animal species.

Adaptation, Physiological↗

PubMed1994

Characterization of serine and leucine tRNAs in an asporogenic yeast Candida cylindracea and evolutionary implications of genes for tRNA(Ser)CAG responsible for translation of a non-universal genetic code.

Five serine and three leucine isoaceptor tRNAs were purified from the asporogenic yeast Candida cylindracea, in which codon CUG is translated as serine instead of leucine, and their primary structures were determined. From the wobble hypothesis, it was assumed that one of the tRNA(Leu) species (Leu1), with the anticodon CmAA, corresponded to the UUG leucine codon, and that the remaining two leucine tRNAs (Leu2 and Leu3), with the same IAG anticodon sequence would decode the CUU, CUC and CUA codons as leucine, but not the CUG codon; this was clarified by an in vitro translation experiment with C.cylindracea using synthetic mRNAs containing the CUA or CUG codons. One of the serine tRNAs (Ser1) has already been demonstrated to have the anticodon CAG and to be responsible for translation of the codon CUG in C.cylindracea. Three of the other species of tRNA(Ser) (Ser2,3 and 4), with the anticodon sequences cm5UGA, IGA and CGA, can translate all four codons in the UCN codon box, while the remaining species (Ser5), with the anticodon GCU, corresponds to AGU and AGC serine codons. The gene sequences for these five serine and three leucine tRNAs were also determined, with the finding that only tRNA(Ser)CAG (Ser1) has an intron. At least five different types of tRNA(Ser)CAG genes exist in the genome of C.cylindracea. The nucleotide sequences of the flanking regions of these tRNA(Ser)CAG genes indicated that the tRNA(Ser)CAG gene has duplicated at least three times on the genome. The existence of multiple genes for tRNA(Ser)CAG on the genome may account for the observation that codon CUG is used very frequently in C.cylindracea. All of these tRNASerCAG genes contain the CCA sequence in their 3' termini, suggesting the possibility that during their multiplication process in the evolution of the C.cylindracea genome, the tRNA(Ser)CAG molecule was integrated into DNA via reverse transcription.

Base Sequence↗

PubMed1991

Genetic evidence that genes fdhD and fdhE do not control synthesis of formate dehydrogenase-N in Escherichia coli K-12.

Enterobacteria synthesize two formate dehydrogenases, formate dehydrogenase-N (encoded by fdnGHI) and formate dehydrogenase H (encoded by fdhF). Previous work has identified two rha-linked Salmonella typhimurium genes, fdnB and fdnC, which are required primarily for formate dehydrogenase-N activity. Analogous mutants, termed fdhD and fdhE, have been isolated in Escherichia coli. We used gene fusions between fdnG, the structural gene for the large subunit of formate dehydrogenase-N, and lacZ, the structural gene for beta-galactosidase, to examine E. coli fdnGHI operon expression in fdhD and fdhE insertion mutants. Expression of the phi (fdnG-lacZ) gene fusions was little affected by these insertions, suggesting that fdhD and fdhE do not control transcription or UGA decoding of the formate dehydrogenase-N structural genes. Our complementation tests, with cloned E. coli fdhD and fdhE genes, indicate that the S. typhimurium fdnC and fdnB genes are functionally homologous to the E. coli fdhD and fdhE genes, respectively.

Cloning, Molecular↗

PubMed2003

Continuing the search for dyslexia genes on 6p.

This study is a continuation and extension of the work with Orton Developmental Dyslexia (DD) pedigrees [Grigorenko et al., 1997; 2000, Grigorenko et al., 2001]. This study utilized an extended sample (N = 176) and a well-saturated map of chromosome 6p (30 markers). Six phenotypes were constructed to span a range of dyslexia-related cognitive processes. These phenotypes were: (1) Phonemic Awareness (of spoken words); (2) Phonological Decoding (of printed nonwords); (3) Rapid Automatized Naming (of colored squares or object drawings); (4) Single Word Reading (orally, of printed real words); (5) Phonemic Awareness/Decoding/Single-Word Reading pathway; and (6) Phonemic Awareness/Rapid Naming/Single-Word Reading pathway. The study resulted in two major findings. First, considering the distributions of the genetic linkage indicators across all phenotypes examined, there appear to be three regions of interest (around markers D6S109, D6S1261, and in the D6S105-D6S265 region). Any of these regions could serve as a starting point in the search for specific gene candidates contributing to the manifestation of DD, yet they all might be echo peaks of a single peak, the boundary of which is difficult to establish due to the limited power of this sample. Second, the DD-related linkage in 6p21.3 appears to be most closely related to the manifestations of DD through phonemic awareness and single-word reading deficits.

Adolescent↗

PubMed1998

Dependence of the 16S rRNA decoding region structure on Mg2+, subunit association, and temperature.

The effects of Mg2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis. Mg2+ concentrations between 1 and 20 mM and temperatures between 5 and 55 degreesC had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes. In contrast, two cross-links, C967 x C1400 and C1402 x C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg2+, increase in frequency at higher Mg2+ concentration, and are both more frequent when 50S subunits are included in the reactions. In 70S ribosomes, the cross-link C1402 x C1501 increases but the cross-link C967 x C1400 decreases at higher Mg2+ concentrations. One cross-link, C1397 x U1495, is detected only in 70S ribosomes and decreases in frequency as Mg2+ concentration is increased. An additional cross-link, A1093 x C1182, decreases upon subunit association. The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg2+ ions and subunit association.

Base Sequence↗

PubMed2002

The role of neuroscience in the remediation of students with dyslexia.

Dyslexia is a specific learning disability that is neurobiological in origin. It is characterized by difficulties with accurate and/or fluent word recognition, spelling and decoding abilities. Research findings agree that these and other observed behavioral manifestations largely result from a deficit in the phonological component of language. However, conflicting theories on the exact nature of the phonological deficit have given rise to divergent treatment approaches. Recent advances in functional brain imaging and genetics have allowed these theories to be examined more closely. If implemented appropriately, commercial programs can be effective in identifying dyslexia. Treatment of dyslexia has been advanced through neuroscience, yet further study is needed to provide rigorous, reproducible findings that will sustain commercial approaches.

Brain↗