tRNA Gln Gene Research Articles

A novel pathogenic mitochondrial DNA variant m.4344T>C in tRNAGln causes developmental delay.

Mitochondrial diseases are a group of genetic diseases caused by mutations in mitochondrial DNA and nuclear DNA. However, the genetic spectrum of this disease is not yet complete. In this study, we identified a novel variant m.4344T>C in mitochondrial tRNAGln from a patient with developmental delay. The mutant loads of m.4344T>C were 95% and 89% in the patient's blood and oral epithelial cells, respectively. Multialignment analysis showed high evolutionary conservation of this nucleotide. TrRosettaRNA predicted that m.4344T>C variant would introduce an additional hydrogen bond and alter the conformation of the T-loop. The transmitochondrial cybrid-based study demonstrated that m.4344T>C variant impaired the steady-state level of mitochondrial tRNAGln and decreased the contents of mitochondrial OXPHOS complexes I, III, and IV, resulting in defective mitochondrial respiration, elevated mitochondrial ROS production, reduced mitochondrial membrane potential and decreased mitochondrial ATP levels. Altogether, this is the first report in patient carrying the m.4344T>C variant. Our data uncover the pathogenesis of the m.4344T>C variant and expand the genetic mutation spectrum of mitochondrial diseases, thus contributing to the clinical diagnosis of mitochondrial tRNAGln gene variants-associated mitochondrial diseases.

Novel Gene Rearrangement and the Complete Mitochondrial Genome of Cynoglossus monopus: Insights into the Envolution of the Family Cynoglossidae (Pleuronectiformes).

Cynoglossus monopus, a small benthic fish, belongs to the Cynoglossidae, Pleuronectiformes. It was rarely studied due to its low abundance and cryptical lifestyle. In order to understand the mitochondrial genome and the phylogeny in Cynoglossidae, the complete mitogenome of C. monopus has been sequenced and analyzed for the first time. The total length is 16,425 bp, typically containing 37 genes with novel gene rearrangements. The tRNA-Gln gene is inverted from the light to the heavy strand and translocated from the downstream of tRNA-Ile gene to its upstream. The control region (CR) translocated downstream to the 3’-end of ND1 gene adjoining to inverted to tRNA-Gln and left a 24 bp trace fragment in the original position. The phylogenetic trees were reconstructed by Bayesian inference (BI) and maximum likelihood (ML) methods based on the mitogenomic data of 32 tonguefish species and two outgroups. The results support the idea that Cynoglossidae is a monophyletic group and indicate that C. monopus has the closest phylogenetic relationship with C. puncticeps. By combining fossil records and mitogenome data, the time-calibrated evolutionary tree of families Cynoglossidae and Soleidae was firstly presented, and it was indicated that Cynoglossidae and Soleidae were differentiated from each other during Paleogene, and the evolutionary process of family Cynoglossidae covered the Quaternary, Neogene and Paleogene periods.

Characterization of the complete mitochondrial genome of Cynoglossus nanhaiensis (Pleuronectiformes: Cynoglossidae)

In this study, the complete mitochondrial genome of Cynoglossus nanhaiensis was determined. The total length of the mitochondrial genome is 17,130 bp, including 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and two noncoding regions. The gene rearrangement of tRNAGln gene and control region (CR) were detected, forming a unique gene order of CR-Ile-Gln-Met. Maximum-likelihood and Bayesian inference method are used to perform the phylogenetic analysis, and the result reveals a close relationship between C. nanhaiensis and Cynoglossus itinus.

Associations of mitochondrial DNA 3777\u20134679 region mutations with maternally inherited essential hypertensive subjects in China

BackgroundNuclear genome or family mitochondrial screening system has become the hot focus of studies into essential hypertension. The role of mitochondrial DNA (mtDNA) in sporadic Chinese patients with hypertension has not been fully understood. The study was to evaluate the associations of mtDNA mutations with maternally inherited essential hypertensive subjects in China.MethodsFrom June 2009 to June 2016, a total of 800 gender-matched Chinese patients with maternally inherited essential hypertension (MIEH) and control group were 1:1 enrolled in this case-control study. Genomic DNA was extracted from each person’s peripheral blood cells. The main mtDNA locations for MIEH were screened with oligodeoxynucleotides 3777-4679 bp, analyzed and compared with the updated consensus Cambridge Sequence. Pathogenic mtDNA mutations were identified from the mitochondrial map.ResultsMIEH subjects presented significantly higher values than those of control group in abdominal circumference (AC), waist circumference (WC), body mass index (BMI), fasting blood glucose (FBG), triglyceride (TG), low-density lipoprotein cholesterol (LDL) and renal function (P < 0.05). MIEH subjects carried more amino acid changes and coding sequence variants (P < 0.01) than control group. The allele frequencies of the eight single nucleotide polymorphisms (SNPs) were significantly different between the two groups, including m.3970 C > T, m.4048G > A, m.4071C > T, m.4086C > T, m. 4164A > G and m.4248 T > C in ND1 gene, and m.4386 T > C and m.4394C > T in tRNAGln gene(P < 0.001). Fifty-five homoplasmic or heteroplasmic mutations were detected in 5 genes: ND1, tRNAIle, tRNAMet, tRNAGln and ND2 gene. The ND1 gene was the main mutation site, where the most mtDNA mutation was m.3970 C > T.ConclusionsThe mtDNA mutations were involved in the process of MIEH. We identified mitochondrial genetic characteristics in MIEH patients in China. The present research serves as a solid foundation for further detailed research on the association between MIEH and mitochondrial dysfunction, and their causal relationship in Chinese and other populations with a similar lifestyle.

The complete mitochondrial genome of Cynoglossus roulei (Pleuronectiformes: Cynoglossidae): novel rearrangement and phylogenetic position analysis

The complete mitochondrial genome of Cynoglossus roulei was determined in this study. The 16,565-bp-long genome is made of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and two noncoding regions. We detected the rearrangement of tRNA-Gln and control region in C. roulei. Additionally, tRNA-Gln gene was inverted from the L-strand position to the H-strand position. Phylogenetic analysis showed that C. roulei is most closely related to C. semilaevis.

Novel gene rearrangement pattern in Cynoglossus melampetalus mitochondrial genome: New gene order in genus Cynoglossus (Pleuronectiformes: Cynoglossidae)

Mitochondrial genome (mitogenome) structure and gene order are generally considered conserved in vertebrates. However, the flatfish (Pleuronectiformes) mitogenomes exhibit the most diversified gene rearrangement patterns. Here, we report a newly sequenced mitogenome of Cynoglossus melampetalus (Pleuronectiformes: Cynoglossidae). The total length of the C. melampetalus mitogenome is 16,651 bp, containing 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, a putative control region, and an L-strand replication origin. Like all previously reported tongue sole (Cynoglossinae) mitogenomes, the C. melampetalus tRNA-Gln gene is inverted from the light to the heavy strand (Q inversion), accompanied by the translocation of CR, which is downstream to the 3'-end of ND1. In addition, we observed a unique tRNA-Ile-Met-Glu (IMQ) gene order that differed from the tRNA-Glu-Ile-Met (QIM) order previously reported for other 14 Cynoglossinae mitogenomes. To our knowledge, it is the first report of two different patterns of mitogenomic gene-arrangement within the same genus in teleost. According to the Q inversion, Met pseudogene (ψMet) and long intergenic gap (186 bp) between M and Q genes, the observed gene rearrangement pattern were presumably supported by mitochondrial recombination and tandem duplication/random loss models. The reduced trend of the intergenic gap between Q and I also suggests that the event of gene rearrangement can be traced back to early Cynoglossinae differentiation.

Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.

Mitochondrial tRNA processing defects were associated with human diseases but their pathophysiology remains elusively. The hypertension-associated m.4401A>G mutation resided at a spacer between mitochondrial tRNAMet and tRNAGln genes. An in vitro processing experiment revealed that the m.4401A>G mutation caused 59% and 69% decreases in the 5′ end processing efficiency of tRNAGln and tRNAMet precursors, catalyzed by RNase P, respectively. Using human umbilical vein endothelial cells-derived cybrids, we demonstrated that the m.4401A>G mutation caused the decreases of all 8 tRNAs and ND6 and increases of longer and uncleaved precursors from the Light-strand transcript. Conversely, the m.4401A>G mutation yielded the reduced levels of tRNAMet level but did not change the levels of other 13 tRNAs, 12 mRNAs including ND1, 12S rRNA and 16S rRNA from the Heavy-strand transcript. These implicated the asymmetrical processing mechanisms of H-strand and L-strand polycistronic transcripts. The tRNA processing defects play the determined roles in the impairing mitochondrial translation, respiratory deficiency, diminishing membrane potential, increasing production of reactive oxygen species and altering autophagy. Furthermore, the m.4401A>G mutation altered the angiogenesis, evidenced by aberrant wound regeneration and weaken tube formation in mutant cybrids. Our findings provide new insights into the pathophysiology of hypertension arising from mitochondrial tRNA processing defects.

The complete mitochondrial genome of Pyxicephalus adspersus: high gene rearrangement and phylogenetics of one of the world's largest frogs.

The family Pyxicephalidae including two subfamilies (Cacosterninae and Pyxicephalinae) is an ecologically important group of frogs distributed in sub-Saharan Africa. However, its phylogenetic position among the Anura has remained uncertain. The present study determined the complete mitochondrial genome sequence of Pyxicephalus adspersus, the first representative mitochondrial genome from the Pyxicephalinae, and reconstructed the phylogenetic relationships within Ranoidae using 10 mitochondrial protein-coding genes of 59 frog species. The P. adspersus mitochondrial genome showed major gene rearrangement and an exceptionally long length that is not shared with other Ranoidae species. The genome is 24,317 bp in length, and contains 15 protein-coding genes (including extra COX3 and Cyt b genes), four rRNA genes (including extra 12S rRNA and 16S rRNA genes), 29 tRNA genes (including extra tRNALeu (UAG), tRNALeu (UUR), tRNAThr, tRNAPro, tRNAPhe, tRNAVal, tRNAGln genes) and two control regions (CRs). The Dimer-Mitogenome and Tandem duplication and random loss models were used to explain these gene arrangements. Finally, both Bayesian inference and maximum likelihood analyses supported the conclusion that Pyxicephalidae was monophyletic and that Pyxicephalidae was the sister clade of (Petropedetidae + Ptychadenidae).

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5′-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5′-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3′-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

The complete mitochondrial genome of hawthorn trunk borer, Platypus contaminatus (Coleoptera: Curculionidae: Platypodinae)

Platypus contaminatus attacked healthy hawthorn trees in China. The complete mitogenome of P. contaminatus was 15,712 bp in length. It contained 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes and a 1038-bp long non-coding AT-rich region. This species showed rearrangements in mitochondrial genome. These changes included: (1) the reversal of the order of tRNATrp and tRNAGln genes; (2) tRNASer moved from its ancestral position (between cob and nad1) to the nad2–tRNAGln gene junction. Phylogenetic analysis using nucleotide sequences of 13 PCGs showed that P. contaminatus was closely related to Euplatypus parallelus, which was similar to the previous research.

Association of mitochondrial variants A4336G of the tRNAGln gene and 8701G/A of the MT-ATP6 gene in Mexicans Mestizos with Parkinson disease.

Sporadic Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. In recent years, it has been established that a genetic component underlies different forms of the disease. For instance, mitochondrial genome variants have been implicated in the pathogenesis of the PD. To determine the association of tRNA(Gln) 4336 and 8701A>G (ATP6: Thr59Ala) mitochondrial DNA polymorphisms with the presence of PD in Mexican mestizo patients. This was a cross-sectional study in which patients were recruited from four tertiary-care level hospitals in Mexico. Genotyping was performed using real-time PCR with TaqMan genotyping assays. Genotypes were confirmed by automated sequencing. The 4336C allele of the tRNAGln gene was present at a low frequency, and the 8701G allele of the MT-ATP6 gene was not associated with PD. The 4336C variant of the tRNAGln gene was uncommon in the study population, and 8701A/G of MT-ATP6 was not associated with PD in Mexican Mestizos.

Characterization of the mitogenome of Cynoglossus Senegalensis (Pleuronectiformes: Cynoglossidae)

The complete mitogenome of the Senegalese tonguesole, Cynoglossus senegalensis was determined by Illumina MiSeq platform. The complete mitochondrial genome of C. senegalensis was 16,519 bp in length. The mitochondrial genome of C. senegalensis showed a cynoglosidae-characteristic gene organization, in which translocation of control region to the position between ND1 and tRNA-Gln gene, and also inversions in tRNA-Gln gene from L-strand position to H-strand position. Phylogenetic analysis showed that C. senegalensis is most closely related to C. sinicus and S.bilineatus, which supports the previous result that genus Cynoglossidae is evolutionary paraphyletic.

The complete mitochondrial genome of Cynoglossus joyneri and its novel rearrangement

The complete mitochondrial genome of Cynoglossus joyneri was determined. The length of the C. joyneri complete mitochondrial DNA sequence is 16,428 bp. The tRNA Gln gene is translocated from the light to the heavy strand (Q inversion), which is accompanied by shuffling of the tRNA Ile gene and long-range translocation of the putative control region downstream to a site between ND1 and the tRNA Gln gene, which is also seen in the closely related Cynoglossus semilaevis. C. joyneri has a closer relationship to Cynoglossus lineolatus and C. semilaevis, but had a distant relationship to Phoxinus percnurus and Sternula albifrons.

Discovery and Complete Genome Sequence of a Bacteriophage from an Obligate Intracellular Symbiont of a Cellulolytic Protist in the Termite Gut.

Termites depend nutritionally on their gut microbes, and protistan, bacterial, and archaeal gut communities have been extensively studied. However, limited information is available on viruses in the termite gut. We herein report the complete genome sequence (99,517 bp) of a phage obtained during a genome analysis of “Candidatus Azobacteroides pseudotrichonymphae” phylotype ProJPt-1, which is an obligate intracellular symbiont of the cellulolytic protist Pseudotrichonympha sp. in the gut of the termite Prorhinotermes japonicus. The genome of the phage, designated ProJPt-Bp1, was circular or circularly permuted, and was not integrated into the two circular chromosomes or five circular plasmids composing the host ProJPt-1 genome. The phage was putatively affiliated with the order Caudovirales based on sequence similarities with several phage-related genes; however, most of the 52 protein-coding sequences had no significant homology to sequences in the databases. The phage genome contained a tRNA-Gln (CAG) gene, which showed the highest sequence similarity to the tRNA-Gln (CAA) gene of the host “Ca. A. pseudotrichonymphae” phylotype ProJPt-1. Since the host genome lacked a tRNA-Gln (CAG) gene, the phage tRNA gene may compensate for differences in codon usage bias between the phage and host genomes. The phage genome also contained a non-coding region with high nucleotide sequence similarity to a region in one of the host plasmids. No other phage-related sequences were found in the host ProJPt-1 genome. To the best of our knowledge, this is the first report of a phage from an obligate, mutualistic endosymbiont permanently associated with eukaryotic cells.

Characterization of the complete mitochondrial genome of Cynoglossus gracilis and a comparative analysis with other Cynoglossinae fishes

Mitochondrial genomes can provide basic information for phylogenetic analysis and evolutionary studies. We present here the mitochondrial genome of Cynoglossus gracilis, which is 16,565bp in length. Numerous distinct regions were identified, including 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, a light-strand replication origin, and a control region. Interestingly, we detected rearrangement of genes in C. gracilis, including a control region translocation, tRNAGln gene inversion, and tRNAIle gene shuffling. Additionally, a phylogenetic analysis based on the nucleotide sequences of the 13 PCGs using maximum likelihood and Bayesian inference methods reveals that C. gracilis is closely related to Cynoglossus semilaevis. This study provides important mitogenomic data for analyzing phylogenetic relationships in the Cynoglossinae.

The complete mitochondrial genome sequence of Cynoglossus abbreviatus (Pleuronectiformes: Cynoglossidae) with control region translocation and tRNA-Gln gene inversion

Cynoglossus abbreviatus (Cynoglossidae, Soleoidei) is characterized by a bilaterally asymmetrical with both eyes on the left side. In this study, the complete mitogenome of this tongue sole has been reported for the first time. The gene order in C. abbreviatus mitogenome possesses a novel rearrangement like other tonguefish. The tRNA-Gln gene moves from the light strand to the heavy strand, accompanied by tRNA-Ile gene shuffling, leaving a large non-coding region (88 bp) between these two tRNAs. Additionally, the control region translocates to the place between ND1 and tRNA-Gln genes. The total length is 16,417 bp, with 30.9%, 29.5%, 24.9% and 14.7% for A, T, C and G, respectively (60.4% for AT content). These molecular data will provide useful information about the mechanism of gene reorganization in Cynoglossidae mitogenome and further phylogenetic study on Pleuronectiformes.

The complete mitochondrial genome of Cynoglossus zanzibarensis (Pleuronectiformes: Cynoglossidae)

Cynoglossus zanzibarensis (Cynoglossidae, Soleoidei) is characterized by both eyes on the left side of the body. Here we report the mitogenome of this tonguesole for the first time, which is 16,569 bp in length, with gene order reorganized. The tRNA-Gln gene is translocated from the light strand (L-strand) to the heavy strand (H-strand), accompanied by shuffling of tRNA-Ile and tRNA-Met genes with a 160-bp intergenic spacer between tRNA-Gln and tRNA-Ile. The putative control region is translocated downstream to the place between the ND1 and the tRNA-Gln genes, leaving a 25-bp trace fragment in the original position. The rest of the gene arrangement is identical to that of the typical fish.

Gene rearrangements in the mitochondrial genome of Cynoglossus bilineatus (Pleuronectiformes: Cynoglossidae)

Cynoglossus bilineatus (Cynoglossidae, Soleoidei) is characterized by both eyes on the left side of the body and with a rounded snout and a short rostral hook. Here we first report the mitogenome of this tongue sole, which is 16,454 bp in length, and gene rearrangements have been observed. Particularly, the tRNA-Gln gene encoded by the light strand (L-strand) has translocated to the heavy strand (H-strand), along with the tRNA-Ile gene shuffling. In addition, the putative control region has translocated downstream to a position between the ND1 and tRNA-Gln genes, leaving a 26-bp intergenic spacer in its original position. However, the arrangement of the rest genes is identical to that of the typical teleost. This result could contribute to a better understanding the molecular mechanisms of gene rearrangement in fish mitogenome as well as phylogenetic study of Cynoglossidae and Pleuronectiformes.

The complete mitochondrial genome of Cynoglossus puncticeps (Pleuronectiformes: Cynoglossidae)

Cynoglossus puncticeps (Soleoidei Cynoglossidae) is characterized by both eyes on the left side of the body. Here we report the mitogenome of this tonguesole for the first time, which is 17,142 bp in length, and the gene order has been reorganized. The tRNA-Gln gene translocated from the light strand (L-strand) to the heavy strand (H-strand), accompanied by tRNA-Ile gene shuffling. In addition, the putative control region translocated downstream to the position between the ND1 and the tRNA-Gln genes, leaving a 25-bp trace fragment in the original location. In addition, two tandem arrays were found: one was a 17-bp motif with 40.2 copies, and the other was 73-bp with 3.0 copies.

Molecular characterization of a Chinese family carrying a novel C4329A mutation in mitochondrial tRNAIle and tRNAGln genes.

BackgroundHypertension is a very common cardiovascular disease influenced by multiple genetic and environmental factors. More recently, there are some studies showed that mutations in mitochondrial DNA have been involved in its pathogenesis. In this study we did further investigations on this relationship.MethodsEpidemiological research found a Han Chinese family with probable maternally transmitted hypertension. Sequence analysis of the whole mitochondrial DNA was detected from all the family members. And evaluations of the clinical, genetic and molecular characterization were also performed.ResultsMatrilineal relatives within the family exhibited varying degrees of hypertension with an onset age of 48–55 years. Sequence analysis of this pedigree showed a novel homoplasmic 4329C > G mutation located at the 3’ end of the tRNAIle and tRNAGln genes that was absent from 366 Chinese controls. The cytosine (C) at 4329 position was very important in the structural formation and stabilization of functional tRNAs, which was highly conserved in mitochondria of various organisms and also contributed to the high fidelity of the acceptor arm. Cells carrying this mutation were also shown to harbor mitochondrial dysfunctions.ConclusionsThe C4329G point mutation in tRNAIle and tRNAGln was involved in the pathogenesis of hypertension, perhaps in association with other modifying factors.