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Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

BACKGROUND Familial exudative vitreoretinopathy (FEVR) is a group of inherited eye diseases characterized by premature arrest of retinal vessel development. The purpose of our study was to characterize the genetic causes and clinical features in eight Chinese families with FEVR using next-generation sequencing (NGS) technology. MATERIALS AND METHODS Eight families with FEVR were included in genetic and clinical analyses. We screened the proband and the parents in eight pedigrees with FEVR using targeted NGS approach and in silico analysis to determine the causative mutation for their family's phenotype. RESULTS Four cases (4/8, 50.0%) were confirmed to harbor mutations in known genes, including 3 novel mutations and one previously reported mutation. Among the detected mutations, TSPAN12 accounted for 75% (3/4). We identified a novel stop codon of TSPAN12, a heterozygous missense mutation NM_012338.4:c.633T>A, NP_036470.1:p.Tyr211Ter involved in highly conserved residues in the proband. Retrospective analysis of its clinical manifestation showed that the mutant carrier presented mild clinical features. CONCLUSIONS We found the novel stop codon mutation p.Tyr211Ter in the TSPAN12, which creates a milder phenotype. Discovery of this novel mutation expands the mutation spectrum of TSPAN12, and would be valuable for future genetic disease diagnosis.

Selenoproteins are a unique class of proteins that contain the 21st amino acid, selenocysteine (Sec). Addition of Sec into a protein is achieved by recoding of the UGA stop codon. All 25 mammalian selenoprotein mRNAs possess a 3′ UTR stem-loop structure, the Selenocysteine Insertion Sequence (SECIS), which is required for Sec incorporation. It is widely believed that the SECIS is the major RNA element that controls Sec insertion, however recent findings in our lab suggest otherwise for Selenoprotein S (SelS). Here we report that the first 91 nucleotides of the SelS 3′ UTR contain a proximal stem loop (PSL) and a conserved sequence we have named the SelS Positive UGA Recoding (SPUR) element. We developed a SelS-V5/UGA surrogate assay for UGA recoding, which was validated by mass spectrometry to be an accurate measure of Sec incorporation in cells. Using this assay, we show that point mutations in the SPUR element greatly reduce recoding in the reporter; thus, the SPUR is required for readthrough of the UGA-Sec codon. In contrast, deletion of the PSL increased Sec incorporation. This effect was reversed when the PSL was replaced with other stem-loops or an unstructured sequence, suggesting that the PSL does not play an active role in Sec insertion. Additional studies revealed that the position of the SPUR relative to the UGA-Sec codon is important for optimal UGA recoding. Our identification of the SPUR element in the SelS 3′ UTR reveals a more complex regulation of Sec incorporation than previously realized.

Abnormal hemoglobins (Hbs) are one of the most common hemoglobinopathies worldwide. Some Hb gene mutations may produce unstable, abnormal Hbs causing macrocytic hemolysis. We identified a novel, de novo deletion/frameshift mutation at nucleotide position 408 in exon 3 of the β-globin gene (HBB: c.408delT) compound with an Hb F-associated regulatory single nucleotide polymorphism (rSNP) (rs368698783) through next generation sequencing (NGS). This β-globin gene variant was identified in a 5-year-old Chinese girl with splenomegaly, jaundice and macrocytic, hemolytic anemia. This variant causes a new stop codon to be formed in the 3' untranslated region (3'UTR) of the HBB gene at amino acid position 158, consequently leading to a β-sheet disruption of the last α helix of this abnormal β-globin chain. We named this variant Hb Urumqi after the proband's current city of residence.

Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNA(Sec)) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNA(Sec) requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins. Several methods are currently available for the synthesis selenoproteins. Here we focus on strategies for in vivo Sec insertion at any position(s) within a recombinant protein in a SECIS-independent manner: (i) engineering of tRNA(Sec) for use by EF-Tu without the SECIS requirement, and (ii) design of a SECIS-independent SelB route.

As a genetic disease, cancer is caused by the activation of oncogenes and the inhibition of tumor suppressor genes via genetic and epigenetic mechanisms. Given the important role of energy metabolism in tumors, we analyzed the cancer-derived mutations occurring in the DNA of the mitochondrion. Mutations in the mitochondrial DNA (mtDNA) compared to nuclear DNA are 62% decreased relative to the coding length per chromosome. We find that the majority of these mutations affects highly conserved nucleotides - significantly exceeding the conservation of the mtDNA - and are devoid of single nucleotide polymorphisms (SNPs). Surprisingly, the leading resources for tumor genetics information universally use the standard genetic code for translation of nucleotide into amino acid sequences in their online resources. However, the nuclear and mitochondrial genetic codes differ for four codons and the usage of incomplete STOP codons. Hence, we analyze and curate the consequences for all mutations in the mtDNA and comprehensively reclassify missense, nonsense and synonymous mutations accordingly. In total, 10% of the mutations are incorrectly translated leading to significant changes in the distribution of mutation types with tripling of nonsense and 69% loss of nonstop extension mutations. Lastly, we provide a curated dataset of coding and non-coding mitochondrial mutations in cancer merged, standardized, duplicate-free and aggregated from two databases as a resource including orthogonal data on their high conservation and SNPs. This study generally highlights the need to universally regard the important differences between the standard and mitochondrial genetic code in life science research.

Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.

Pyrrolysine is the 22nd proteinogenic amino acid encoded into proteins in response to amber (TAG) codons in a small number of archaea and bacteria. The incorporation of pyrrolysine is facilitated by a specialized aminoacyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNAPyl). The secondary structure of tRNAPyl contains several unique features not found in canonical tRNAs. Numerous studies have demonstrated that the PylRS/tRNAPyl pair from archaea is orthogonal in E. coli and eukaryotic hosts, which has led to the widespread use of this pair for the genetic incorporation of non-canonical amino acids. In this brief review we examine the work that has been done to elucidate the structure of tRNAPyl, its interaction with PylRS, and survey recent progress on the use of tRNAPyl as a tool for genetic code expansion.

mRNA decoding by tRNAs and tRNA charging by aminoacyl-tRNA synthetases are biochemically separated processes that nevertheless in general involve the same nucleotides. The combination of charging and decoding determines the genetic code. Codon reassignment happens when a differently charged tRNA replaces a former cognate tRNA. The recent discovery of the polyphyly of the yeast CUG sense codon reassignment challenged previous mechanistic considerations and led to the proposal of the so-called tRNA loss driven codon reassignment hypothesis. Accordingly, codon capture is caused by loss of a tRNA or by mutations in the translation termination factor, subsequent reduction of the codon frequency through reduced translation fidelity and final appearance of a new cognate tRNA. Critical for codon capture are sequence and structure of the new tRNA, which must be compatible with recognition regions of aminoacyl-tRNA synthetases. The proposed hypothesis applies to all reported nuclear and organellar codon reassignments.

β-Thalassemia (β-thal) is one of the most common genetic disorders worldwide. Molecular characterization of β-thal is essential for prevention and understanding the biology of the disease. More and more rare and novel mutations are being reported. Here, we report a novel 7 bp deletion at codons 63-65 (HBB: c.189_195delTCATGGC) in exon 2 of the β-globin gene in a family from Guangxi Province, China. This novel mutation causes a shift in the normal reading frame of the β-globin coding sequence and created a stop codon at codon 87 in exon 2, which leads to a β(0)-thal phenotype.

The complete mitochondrial genome of the small-toothed forest hedgehog Mesechinus miodon (Eulipotyphla: Erinaceidae) has been reconstructed from Illumina sequencing data. The circular genome is 16 842 bp long, comprising 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), and one control region. All PCGs are initiated with ATR (ATA/ATG) codons, except for nad4 with GTG as its initiation codon. Two PCGs (cox3 and nad4) harbor an incomplete termination codon T, while the others are terminated with TAA (atp6, cox1, cox2, nad1, nad2, nad3, nad4l, nad5, and nad6), TAG (atp8) or AGA (cytb). The base composition is highly biased (34.9% A, 19.7% C, 11.9% G, and 33.5% T for the light strand) with an overall A + T content of 68.4%. Phylogenetic analysis indicated that M. miodon is more closely related to the consubfamilial long-eared hedgehog Hemiechinus auritus than to those within the order Eulipotyphla.

The complete mitochondrial genomes of Red-billed Chough (Pyrrhocorax pyrrhocorax) and Yellow-billed Chough (Pyrrhocorax graculus) were sequenced using the Ion Torrent PGM platform. These mitogenomes contain 16,889 bp (Red-billed Chough) and 16,905 bp (Yellowbilled Chough), including 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region (D-loop). The gene content, orientation, and structure are similar to a wide range of other vertebrate species and the nucleotide composition is very similar to other Passeriformes. All PCGs start with ATG, except for COX1 that starts with GTG, and four stop codons and one incomplete stop codon are used (TAA, TAG, AGG, AGA, and T-). The size of PCGs is the same in both mitogenomes, except for ND6 that has one codon less in the Yellow-billed Chough. All the tRNAs can fold into a typical cloverleaf secondary structure. These mitogenomic data can be of great value in complementing forthcoming approaches on molecular ecology, comparative and functional genomics., This work was partially supported by the Ph.D. Grant SFRH/BD/77872/2011 of the Portuguese Foundation for Science and Technology (FCT).

Selenium, a micronutrient, is primarily incorporated into human physiology as selenocysteine (Sec). The 25 Sec-containing proteins in humans are known as selenoproteins. Their synthesis depends on the translational recoding of the UGA stop codon to allow Sec insertion. This requires a stem-loop structure in the 3’ untranslated region of eukaryotic mRNAs known as the Selenocysteine Insertion Sequence (SECIS). The SECIS is recognized by SECIS-binding protein 2 (SBP2) and this RNA:protein interaction is essential for UGA recoding to occur. Genetic mutations cause SBP2 deficiency in humans, resulting in a broad set of symptoms due to differential effects on individual selenoproteins. Progress on understanding the different phenotypes requires developing robust tools to investigate SBP2 structure and function. In this study we demonstrate that SBP2 protein produced by in vitro translation discriminates among SECIS elements in a competitive UGA recoding assay and has a much higher specific activity than bacterially expressed protein. We also show that a purified recombinant protein encompassing amino acids 517-777 of SBP2 binds to SECIS elements with high affinity and selectivity. The affinity of the SBP2:SECIS interaction correlated with the ability of a SECIS to compete for UGA recoding activity in vitro. The identification of a 250 amino acid sequence that mediates specific, selective SECIS-binding will facilitate future structural studies of the SBP2:SECIS complex. Finally, we identify an evolutionarily conserved core cysteine signature in SBP2 sequences from the vertebrate lineage. Mutation of multiple, but not single, cysteines impaired SECIS-binding but did not affect protein localization in cells.

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

To establish that newborn Ser447Stop and Asn291Ser may have interactive effects with maternal genotypes on the plasma lipoprotein levels, risk of preeclampsia as well as on the prognosis of preeclampsia.Seventy preeclamptic women and 94 normotensive pregnant women, and their newborns were genotyped using PCR-RFLP methods.The risk of mild and severe preeclampsia was 4 (p = 0.004) and 5.18 (p = 0.001), respectively, if both the mother and newborn were carriers of the Ser447/Ser477 genotype. If both the mother and newborn were carriers of the Asn291Ser variant, the risk to develop severe preeclampsia was 6.07 (p = 0.03). Women with mild and severe preeclampsia had higher TG (p 0.001; p 0.001) and LDL-C levels (p = 0.008; p 0.001) if both the mother and newborn were carriers of the Ser447/Ser447 genotype. Women with severe preeclampsia had significantly higher TG (p = 0.03) and LDL-C levels (p = 0.037) if both the mother and newborn were carriers of Asn291Ser. Newborn/maternal LpL interaction had no statistically significant influence on pregnancy outcome.The newborn/maternal LpL interaction influences the severity of preeclampsia and modulates the lipid profile particularly in severe preeclampsia.

We have determined the complete mitochondrial genome of Gekko japonicus, whose status as an endemic or invasive species is currently under debate in Korea. The total genome size is 16 544 bp and consists of 13 protein-coding genes, 2 rRNA (12S and 16S RNA) genes, 22 tRNAs and 2 non-coding regions. The A + T content of the genome is 55.8% (A, 31.2%; C, 29.4%; T, 24.6%; G, 14.9%). Phylogenetic analysis shows that G. japonicus has a close phylogenetic relationship with both G. swinhonis and G. chinensis. Our result will facilitate further genetic studies of this species to ascertain its species status.

Here, we report the mitochondrial genome sequence of the multicolored Asian lady beetle Harmonia axyridis (Pallas, 1773) (Coleoptera: Coccinellidae) (GenBank accession No. KR108208). This is the first species with sequenced mitochondrial genome from the genus Harmonia. The current length with partitial A + T-rich region of this mitochondrial genome is 16,387 bp. All the typical genes were sequenced except the trnI and trnQ. As in most other sequenced mitochondrial genomes of Coleoptera, there is no re-arrangement in the sequenced region compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN codons. Five, five and three protein-coding genes stop with termination codon TAA, TA and T, respectively. Phylogenetic analysis using Bayesian method based on the first and second codon positions of the protein-coding genes supported that the Scirtidae is a basal lineage of Polyphaga. The Harmonia and the Coccinella form a sister lineage. The monophyly of Staphyliniformia, Scarabaeiformia and Cucujiformia was supported. The Buprestidae was found to be a sister group to the Bostrichiformia.

The complete mitochondrial genome (mitogenome) of the butterfly, Euripus nyctelius, was determined in the present study. The mitogenome is a typical circular DNA molecule of 15,417 bp, containing 37 genes and a putative control region. Thirteen protein-coding genes all initiate with ATN codons and mostly terminate with TAA or TAG codons except for COII, ND4 and ND5 use a single T residue as the termination codon. All tRNAs have the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. Both maximum likelihood and Bayesian analyses support the monophyly of butterflies and recover high supports for the following family level relationships: (Papilionidae + (Hesperioidea +(Pieridae (Lycaenidae + Nymphalidae)))). Euripus nycteliusis is placed as sister to the genus Sasakia within Nymphalidae.

Sebastes is a diverse genus of fish in the family Scorpaenidae. The larvae and adults in this genus have similar morphological characteristics. To obtain basic information for their identification, the mitogenome of S. steindachneri was sequenced using next-generation sequencing. The mitogenome was 16,450 bp in length, and comprised of 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs, and a control region. The mitogenome is 27.4% A, 21.1% T, 17.5% G, and 28.0% C, showing a slight AT bias (54.5%). ATG was the start codon in all of the PCGs, except COX1 (GTG). The termination codon in the PCGs was TAA, except in ND1 and ND3 (TAG), ND4 (AGA), and Cytb (incomplete termination codon, T). All of the tRNAs had a typical cloverleaf shape, except tRNASer(AGY), which lacked the DHU arm. The mitogenome of S. steindachneri should be useful for identifying larvae and constructing a phylogenetic structure for the Scorpaenidae.

To determine the cause of Leber congenital amaurosis (LCA) in a consanguineous Emirati family.The clinical diagnosis was made on the basis of medical history, ophthalmoscopy and standard ERG. The diagnosis was confirmed by molecular genetic analysis of known LCA genes by Next-Generation Sequencing (NGS). The latter was performed by Bioscientia Institut, Germany (as a clinical service for Latifa Hospital, Dubai).The next generation sequencing of known LCA genes revealed a homozygous 1bp-insertion c.2608_2609insA in exon 16 of the RPGRIP1 gene. This mutation, which was confirmed by conventional Sanger sequencing, leads to a frameshift, resulting in a premature stop codon (p.Leu870TyrfsX7) and subsequently in a degradation of the m-RNA or in a truncation of the RPGRIP1 protein. The segregation analysis of the identified mutation was performed for the parental samples. Both parents carry the frameshift mutation in a heterozygous state.We report a novel RPGRIP1 mutation causing LCA in a consanguineous Emirati family. To the best of our knowledge, this alteration has not been described in the literature so far.

The complete mitochondrial genome of the giant electric ray is 17,081 bp long (GenBank accession KM386678) and includes 2 ribosomal RNA, 22 transfer RNA, 13 protein-coding genes, an origin of replication, 2 non-coding regions. The mitochondrial gene arrangement is similar to that found in other batoids. The control region possessed a set of tandem repeats. Start codon ATG and stop codon TAA/T were found in most protein-coding genes. The base composition of the genome is 36.2% A, 29.9% T, 21.9% C, and 11.9% G.

In this study, the complete mitochondrial genome of Paramisgurnus dabryanus was obtained by PCR base on 18 pairs of primers. Among the 18 primers, the 14 primers were from the previously published universal primers for Cyprinus carpio L. mitogenome amplification. The remaining 4 primers were designed on the basis of related species mtDNA sequences. The genome is 16,570 bp in length, including 2 ribosomal RNA genes. 13 proteins-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of which was similar to most reported from other vertebrates. Sequence analysis showed that the overall base composition of Paramisgurnus dabryanus is T 27.3%, C 26.9%, A 28.5%, and G 17.4%. The sequence is a slight A + T bias of 55.8%, which is similar to other fishes. Mitochondrial genome is widely used in phylogenetic analysis, evolutionary genomics, species identification and related research of fish.

In the present study, we amplified and sequenced the complete mitochondrial genome from a Vietnamese domestic goat (Capra hircus). The data were compared with mtDNA sequences available in the nucleotide databases.The results revealed many problems in the goat mitochondrial reference genome (GenBank accession number NC_005044). Firstly, the authors did not sequence the complete genome, simply 44.5% of its total length. Secondly, two fragments (representing 1201 and 2384 nt) contained an unusually high percentage of sequencing errors. Thirdly, a segment of 1881 nt, covering most of nd5 and the 5' part of nd6, was shown to be a nuclear sequence of mitochondrial origin (Numt). Surprisingly, a similar Numt was also detected in four other goat mitochondrial genomes available in GenBank (GU22978-81). Two primers were designed specially to amplify approximately 960 nt of the Numt identified in goat mtDNA genomes. After cloning, two Numts were detected for C. hircus. Several Numts, most of them with stop codon or frameshift mutations, were also found in Hemitragus jemlahicus (Himalayan tahr) and Pseudois nayaur (bharal). Phylogenetic analyses suggest that a nuclear integration occurred in the common ancestor of Ammotragus, Arabitragus, Capra, Hemitragus and Pseudois, followed by several subsequent duplication events.As poor-quality sequences can produce misleading interpretations of both phylogeny and molecular evolution, we propose including a new link to each accession number in the nucleotide databases, named "external expertise", which could be openly and continually updated by non-anonymous searchers in order to validate good-quality data, or, conversely, to indicate possible problems in the sequence, such as DNA contamination or sequencing errors. This information could prove very useful over time to select good-quality sequences for in silico analyses.

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup+ strains for selection and sup0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to lambda phage display vector.

The complete Grey-backed Shrike mitochondrial genome has been sequenced to be 16,820 bp in length, consisting of 37 encode genes: 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. In addition, a single control region was also observed. Compared with other reported Passeriformes mtgenome sequences, three bases CAA were detected at the end of Lanius tephronotus cox2 gene with the downstream adjacent base T. The first base of CAA probably occurred C to U transcript editing event resulting in a normal stop codon UAA.

Cell fate mapping techniques which can label clonal cell lineages are of importance because they allow one to investigate the distribution and types of daughter cells arising from single precursor cells. Thus, the potential of precursor cells to generate various types of descendent cells can be studied at the single-cell level. The stop-EGFP transgenic mouse carries a premature stop codon-containing enhanced green fluorescent protein (EGFP) gene as a target gene for mutations. A cell having undergone a mutation at the premature stop codon and its descendant cell lineage will express EGFP, thus a clonal cell lineage can be traced in vivo using a fluorescent microscope. Using the stop-EGFP mouse, stem cell clonal lineages in the mouse dorsal epidermis can be investigated in vivo and repeated analyses of the same cell lineages can be performed over time. In vivo imaging studies possible with the stop-EGFP mouse provide new insights into the structure of epidermal proliferative units (EPUs). The stop-EGFP system provides a novel tool for investigating clonal cell lineages in developmental studies as well as in stem cell biology.

The pharmacokinetics of YM-64227 (4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]-pyrimidine-2-(1H)-one), a novel and selective phosphodiesterase type 4 inhibitor, was characterized in beagle dogs. Based on the plasma parent drug to major hydroxylated metabolite ratio, 21 dogs were phenotyped as 16 extensive metabolizers (EM) and five poor metabolizers (PM). Nucleotide sequences of CYPs 1A2, 2B11, 2C21, 2D15, 2E1 and 3A12 were investigated in the EM and PM dogs. A CYP1A2 1117 C>T single nucleotide polymorphism was found, which resulted in an amino acid change from an Arg codon to a stop codon at position 373. All dogs phenotyped as PM were T/T homozygous, whereas EMs were C/C homozygous and C/T heterozygous. In Western blotting of liver microsomes, CYP1A protein expression was detected in the C/C and C/T types, but not in the T/T type. Of 65 dogs genotyped using genome DNA, the frequencies of the C and T alleles were 0.61 and 0.39, respectively, suggesting approximately 15% of the dogs would not express the CYP1A2 protein. The findings provide a coherent explanation for the inter-individual variability in the pharmacokinetics of CYP1A2 substrate drugs in dogs.

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

The great cormorant (Phalacrocorax carbo) is widely distributed in the world. Here, we report the complete mitogenome sequence of P. carbo, which was 18,995 bp long and composed of 15 protein-coding genes, 25 tRNA genes, 2 rRNA genes and 2 control regions. Most genes were located on H-strand except for 10 tRNAs and 2 ND6 genes. In protein-coding genes, ATG, ATC and GTG were used as initiation codons, and TAA, AGA, AGG and TAG were used as stop codons. The 25 tRNA genes ranged in size from 67 to 75 bp. And 12S and 16S rRNA were respectively 983 and 1517 bp. Except for a tRNA-Ala in P. carbo which was different from the tRNA-Lys annotated in P. chalconotus, both species of Phalacrocorax had the same order of other genes. In addition, a phylogenetic analysis may help us understand the evolutionary status of this widespread species better.

PaLi yak is a very important local breed in China; as a year-round grazing animal, it plays a very important role for the economic and native herdsmen. The PaLi yak complete mitochondrial DNA is sequenced in this study, the total length is 16,324 bp, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a non-coding control region (D-loop region). The order and composition are similar to most of the other vertebrates. The base contents are: 33.72% A, 25.80% C, 13.21% G and 27.27% T; A + T (60.99%) was higher than G + C (39.01%). The phylogenetic relationships were analyzed using the complete mitogenome sequence, results showed that the genetic relationship between yak and cattle is distinct. These information provides useful data for further study on protection of genetic resources and the taxonomy of Bovinae.

The white-tailed tropicbird, Phaethon lepturus (Pelecaniformes, Phaethontidae) is a tropicbird, smallest of three closely related seabirds of the tropical oceans and smallest member of the order Pelecaniformes. Here, we first determined the complete mitochondrial genome of while-tailed tropicbird. The mitogenome (17,773 bp) was composed of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 putative control region. Most protein-coding genes started with a traditional ATG codon except for COX1, COX2, and ND3, which initiated with non-typical start codon GTG, GTG, and ATA instead, respectively, and terminated with the mitochondria stop codon (TAA/TCC/AGG/AGA). The mitogenome structural organization was identical to the same genus species Phaethon rubricauda. The overall AT content was 52.04%, which was higher than GC. To obtain the phylogenetic status of Phaethon lepturus, we constructed the species phylogenetic tree together with the 12 protein-coding genes of nine other closely species. We expected that the complete mitogenome of while-tailed tropicbird would contribute to address taxonomic issues and study the related evolution events.

Apolipoprotein AII (ApoAII) amyloidosis, first reported in 2001 in a family with renal amyloidosis, is associated with mutations in the stop codon of the apolipoprotein AII gene resulting in a carboxyl terminal peptide extension of 21 amino acid residues in the protein. Since death from this form of amyloidosis is due to renal failure, kidney dialysis and renal transplantation are presently the only two therapeutic options. We report the case of a Caucasian man who developed proteinuria in his late 20's, had renal biopsy at the age of 33 which gave the diagnosis of renal amyloidosis, and required continuous ambulatory peritoneal dialysis by age 45. He received a cadaver renal transplant at age 47 and has maintained stable renal function for nine years without other evidence for organ system dysfunction from amyloidosis. Laboratory studies confirmed persistence of the ApoAII variant in the patient's plasma in addition to the normal ApoAII protein. This is in agreement with the DNA analysis which showed the patient to be heterozygous for the ApoAII stop78Gly mutation. These results indicate that renal transplantation is an effective therapy for apolipoprotein AII amyloidosis since recurrence of amyloid in the graft and progression of other organ involvement may be very slow.

The complete mitochondrial genome of the codling moth Cydia pomonella (Lepidoptera: Tortricidae) was determined. The genome is 15,253 bp long with 37 typical animal mitochondrial genes and an A+T-rich region. All genes are arranged in their conserved positions compared with the pupative ancestral arrangement of insects except for trnM, which was translocated to the upstream of the transfer RNA cluster trnI-trnQ as in all previously reported lepidopteran mitochondiral genomes. Seven portein-coding genes use ATG start codon and five use ATT. However, the cox1 gene uses the CGA start codon as it is found in all previous reported mitochondrial genomes of Lepidoptera. Nine protein-coding genes stop with termination codon TAA. Four protein-coding genes use incomplete stop codons TA or T. The A+T region is located between rrnS and trnM with a length of 331 bp.

The complete mitochondrial genome of the beet armyworm Spodoptera exigua (Hübner) (Lepodiptera: Noctuidae) was determined (GenBank accession number JX316220). The genome is 15,365 bp long with 37 typical animal mitochondrial genes and an A+T-rich region. As in other sequenced mitochondrial genomes of Lepidoptera, trnM was rearranged to the upstream of trnI-trnQ-trnM cluster compared with the pupative ancestral arrangement of insects. All protein-coding genes start with ATN start codon except for the gene cox1, which uses CGA as in other lepidopteran species. Eight protein-coding genes stop with termination codon TAA. Five protein-coding genes use incomplete stop codons TA or T. The A+T-rich region is located between rrnS and trnM with a length of 331 bp. This is the third completely sequenced mitochondrial genome from the family Noctuidae of Lepidoptera.

Liobagrus obesus (Siluriformes, Amblycipitidae) is an endemic, endangered species in South Korea. Throughout this study, complete mitochondrial genome of L. obesus was sequenced, which is 16,506 bp in length. Its detail characteristics were described in this study.

The complete mitochondrial genome of Hormaphis betulae has been sequenced and annotated, which is the first representation from the aphid subfamily Hormaphidinae. This mitogenome is 15 088 bp long with an A + T content of 82.2%, containing 37 genes arranged in the same order as the putative ancestral arrangement of insects and a control region. All protein-coding genes start with an ATN codon and terminate with a TAA codon or a single T residue. All the 22 tRNAs, ranging from 61 to 78 bp, have the typical clover-leaf structure except for trnS (AGN). The lengths of rrnL and rrnS genes are 1275 and 776 bp, respectively. The control region is 509 bp long and located between rrnS and trnI, including three domains: an AT-rich zone, a poly-thymidine stretch, and a stem-loop region. The phylogenetic tree supports H. betulae as the basal lineage within Aphididae.

The ghost moth Endoclita signifer is a new wood-boring pest of eucalyptus in south of China. It infests dozens of native plant species, and causes severe damage to the plantations of exotic eucalyptus. We sequenced the complete mitochondrial genome (mitogenome) of E. signifer, which has the typical 37 mitochondrial genes of insects. Contrary to most of the Lepidoptera, the E. signifer mitogenome has the putative ancestral insect gene order. Atypical start codon (TTG) and incomplete stop codon (one T-nucleotide) were found for COX2. The E. signifer control region is 389 bp without tandem repeats, and two (TA)n stretches were observed.

In the present study, the complete mitochondrial genome sequence of Labeo gonius is reported using PGM sequencer (Ion Torrent). The complete mitogenome of L. gonius is obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP) which is 16 614 bp in length. The mitogenome of L. gonius comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and D-loop as control region along with gene order and organization, being similar to most of other fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of Labeo fimbriatus, as reported earlier. The phylogenetic analysis of Cypriniformes depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of L. gonius would be helpful in understanding the population genetics, phylogenetics, and evolution of Indian Carps.

In this paper, the circular mitochondrial genome of Amphioctopus aegina (Cephalopoda: Octopodidae) was sequenced. The whole mitogenome of A. aegina was 15 545 base pairs (bp) in length with a base composition of 42.53% A, 33.26% T, 16.70% C, and 7.51% G. The complete mitogenome contained 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and a major non-coding region. The gene arrangements of A. aegina showed remarkable similarity to other Octopodidae species reported. The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, and illustrated that A. aegina had the closest genetic relatives to A. fangsiao.

The complete mitochondrial genome of the white-browed laughingthrush Garrulax sannio was assembled using next-generation sequencing (NGS) technology. The double-stranded circular genome is 17 848 bp in length, including 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), and 2 putative control region. All PCGs are initiated with the ATG codon except for COX1 with GTG as its start codon. Five distinct types of stop codons are present, i.e., AGA (ND5), AGG (COX1 and ND1), TAA (ATP6, ATP8, COX2, CYTB, and ND4L), TAG (ND6), and the incomplete codon T/TA (COX3, ND2, ND3, and ND4). The nucleotide composition is moderately asymmetric (28.94% A, 32.82% C, 15.00% G, and 23.24% T) with an overall GC content of 47.82% ("light strand"). Phylogenetic analysis indicated a close genetic relationship between this species and its congeners G. perspicillatus, G. cineraceus, and G. canorus.

The giant Asian mantis Hierodula formosana (Mantodea: Mantidae) is widely distributed in Taiwan. In the present study, we investigated the complete mitochondrial genome of H. formosana and the mitogenome is 16 266 bp in length. The circular molecule consists of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region, with an AT content of 75.8%. An extra non-coding region is inserted between trnM and ND2, similar to the other Mantidae species T. tamolana. A preliminary phylogenetic analysis has been carried out with 11 related species and the status of Hierodula formosana is further confirmed.

Ptychobarbus kaznakovi is endemic to the Qinghai-Tibet Plateau in China. The complete mitochondrial genome of P. kaznakovi was sequenced and characterized. The genome is 16 700 bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The gene arrangement and nucleotide composition of the mitochondrial genome are similar to those of Cyprinidae fish. A 2 bp tandem repeat was identified in the control region. We conclude that the control region is variable in length between and within species. The complete mitochondrial DNA in this study should be used in the studies on population genetics and phylogeny of P. kaznakovi.

We report two unrelated cases of Hb H-Constant Spring (Hb H-CS) disease caused by a compound heterozygosity for alpha0-thalassemia (--THAI deletion) and Hb CS (alpha142, TAA--CAA) in Thai patients. Hematological and clinical observations in the probands are more severe than those of deletional type of Hb H disease (- -/-alpha), owing to an early onset of anemia and recurrent episode of anemic crises. These cases also address the importance of the - - THAI deletion which causes a severe clinical phenotype, and that could be missed by routine screening. We suggest that testing for this mutation should be included in the screening program for the prevention and control of thalassemia in Thailand, and possibly in other countries in Southeast Asia, where alpha0-thalassemias are highly prevalent.

We determined the complete mitochondrial (mt) genome sequences of the striped field mice Apodemus agrarius coreae and Apodemus agrarius chejuensis. The mt genomes of A. a. coreae and A. a. chejuensis are 16,260 and 16,261 base pairs in length, respectively. The general features of the 13 protein-coding genes of the two species are similar to those of other rodents. The TAG termination codon for NADH dehydrogenase subunit (ND) 3 is unique to Apodemus in the Muroidea. The L-strand replication origin has the potential to form a stable stem-loop structure. Within the control region, a termination-associated sequence and several conserved sequence blocks were observed. The diversity of the 13 protein-coding genes, 2 rRNAs, and 1 control region between the two species ranged between 0.005 (ATP8) and 0.027 (ND4L).

Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth. This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved. Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity.

hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins.

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

The complete nucleotide sequence of Gymnodiptychus dybowskii mitogenome (16 677 bp) has been determined, containing 13 protein-coding genes, two ribosomal RNA genes, 22 tRNA genes, and one non-coding control region. The base composition is 28.04% A, 26.92% T, 18.94% G, 26.10% C, with an AT bias of 54.97%. The origin of light-strand replication (OL) is found between tRNAAsn and tRNACys, which has the potential to fold in a step-loop secondary structure with a stem formed by 11 pairs of nucleotides and a loop of 14 nucleotides. The phylogenetic analysis indicates close relationship between genus Gymnodiptychus and Diptychus.