The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA m1G37 methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, m1G37 modification was reported to take place on three conserved tRNA subsets (tRNA(Arg), tRNA(Leu), tRNA(Pro)); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the m1G37 modification. The present study reveals Gm18, m1G37 modification, and positions of m1G that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the m1G and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs (tRNA(Met), tRNA(Pro), tRNA(Val)). Whereas the m1G37 modification base G is formed only on tRNA(Arg), tRNA(Leu), tRNA(Pro), and tRNA(His), the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and m1G modification occur irrespective of a G residue in tRNAs.
Adenine ; Anticodon ; Archaea ; Bacteria ; Eukaryota ; Guanosine ; Methylation ; Ribose ; RNA, Transfer* ; RNA, Transfer, Arg ; RNA, Transfer, His ; RNA, Transfer, Leu ; RNA, Transfer, Pro

Adult ; DNA, Mitochondrial ; genetics ; Electroencephalography ; Humans ; MERRF Syndrome ; genetics ; Male ; Mitochondrial Myopathies ; genetics ; Mutation ; RNA, Transfer, Asn ; genetics

OBJECTIVETo examine mitochondrial DNA mutations in mitochondrial myopathy.

METHODSThree suspected cases of mitochondrial myopathy were examined by HE staining, histochemical staining methods and electron microscopy. The mutations in all 22 tRNA genes of mitochondrial genome were screened by polymerase chain reaction-single strand conformation polymorphism and DNA sequencing.

RESULTSThe three cases were diagnosed as mitochondrial myopathy. The examinations revealed that patient 1 had a homoplasmic A1627G mutation in tRNA-Val gene, and patient 2 had a heteroplasmic A1627G/A mutation in tRNA-Val gene, and patient 3 had two mutationsuone was homoplasmic T5554C mutation in tRNA-Trp gene, the other was heteroplasmic A10412C/A mutation in tRNA-Arg gene.

CONCLUSIONtRNA genes mutations of mtDNA might be one of the etiologies of mitochondrial myopathy.

Adult ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Female ; Humans ; Male ; Microscopy, Electron, Transmission ; Mitochondrial Myopathies ; genetics ; pathology ; Muscle Fibers, Skeletal ; metabolism ; pathology ; ultrastructure ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; RNA, Transfer, Val ; genetics ; Young Adult

OBJECTIVETo identify secondary mutations associated with deafness in a Chinese family affected with deafness.

METHODSThe family has been subjected to clinical and molecular analyses, in addition with measurement of reactive oxygen species and doubling time after establishment of immortalized lymphocyte cell lines.

RESULTSThe results showed that the hearing loss level and audiometric configuration were discrepant among the family members with maternally transmitted hearing loss. The penetrance of hearing loss in this family was respectively 66.7% and 44.4% when aminoglycoside-induced hearing loss was included or excluded. Analysis of whole mitochondrial genome has found 33 variants as previously reported polymorphisms, except for a 12s rRNA A1555G mutation and a tRNA(Thr)T15943C mutation. Haplotype evolutionary tree has verified that this family belonged to East-Asian haplogroup F. 15943 position was located on the T-stem of the tRNA(Thr), which has destroyed the extremely conserved T-A base pair when T changed to C at this position. However, functional experiments indicated that the population doubling time in special galactose and glucose were longer, whilst the level of reactive oxygen species has increased. Compared with the control cell line groups and a family only carrying the 12s rRNA A1555G mutation, all of the three groups belonged to the same haplogroup.

CONCLUSIONMitochondrial tRNA(Thr)T15943C mutation may act as a potential modifying factor and interact with 12s rRNA A1555G mutation, and thereby enhance the penetrance and expression of deafness.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; China ; DNA, Mitochondrial ; genetics ; Deafness ; genetics ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Phenotype ; Point Mutation ; RNA, Ribosomal ; genetics ; RNA, Transfer, Thr ; genetics ; Young Adult

OBJECTIVETo screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).

METHODSBased on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.

RESULTCo-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.

CONCLUSIONThe expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

Base Sequence ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Embryo, Mammalian ; Green Fluorescent Proteins ; genetics ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B e Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Kidney ; cytology ; Liver Neoplasms ; pathology ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; RNA, Small Interfering ; RNA, Transfer, Val ; genetics ; RNA, Viral ; genetics ; Transfection

OBJECTIVE: To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms. METHODS: A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People's Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level. RESULTS: This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m.4420A>T variant and a m.10463T>C variant, both of which were highly conserved among various species. Structurally, the m.4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m.10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m.4420A>T and m.10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P < 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively. CONCLUSION Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.
Humans ; Female ; Mutation ; Pedigree ; RNA, Transfer, Met ; East Asian People ; RNA, Transfer, Arg ; DNA, Mitochondrial/genetics* ; Coronary Disease/genetics* ; Adenosine Triphosphate

OBJECTIVETo explore clinical, genetic and molecular features of two Chinese Han families with Leber's hereditary optic neuropathy (LHON).

METHODSOphthalmologic examinations revealed variable severity and age-at-onset of visual loss among probands and other matrilineal relatives of both families. The families exhibited extremely low penetrance of visual impairment. The entire mitochondrial genome of two probands was amplified by PCR in 24 overlapping fragments using sets of oligonucleotide primers.

RESULTSSequence analysis of complete mitochondrial genome in the pedigrees excluded three common LHON associated mutations G11778A, G3460A and T14484C, but revealed the presence of a known homoplasmic tRNA(Thr) A15951G mutation. It also showed distinct sets of mtDNA polymorphisms belonging to Eastern Asian haplogroup D4b1. The A15951G mutation is located at the extremely conserved nucleotide (conventional position 71) of tRNA(Thr). Thus, this mutation may alter the structure and stability of mitochondrial tRNA(Thr), thereby leading to a failure in the tRNA metabolism and mitochondrial dysfunction, causing visual impairment.

CONCLUSIONThe results suggested that the A15951G mutation might be involved in the pathogenesis of Leber's hereditary optic neuropathy in the two families.

Adolescent ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Humans ; Male ; Mitochondria ; genetics ; Molecular Sequence Data ; Mutation ; genetics ; Optic Atrophy, Hereditary, Leber ; genetics ; Pedigree ; RNA, Transfer, Thr ; genetics ; Sequence Alignment

OBJECTIVETo establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella, and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs. The appearance of Bartonella infectious diseases calls for genus and species detection and tools for identification in order to make clinical diagnosis and carry on epidemiological studies.

METHODSOne pair of primer TIle.455p-TAla.885n was designed based on the fact that tRNA(Ile)-tRNA(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer (ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes. 16S-23S rRNA was longer than that which had been described in other bacteria. Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis.

RESULTSAmplification products of the three pairs of primers had the same predicted size of those in Bartonella spp. According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control. Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle.455p-TAla.885n was identical to the Bartonella isolated from Yunnan in China.

CONCLUSIONSPCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters. This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China.

Animals ; Bartonella ; genetics ; isolation & purification ; Bartonella Infections ; diagnosis ; microbiology ; Base Sequence ; Cat-Scratch Disease ; diagnosis ; microbiology ; Cats ; China ; Diagnosis, Differential ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; RNA, Transfer, Ala ; chemistry ; genetics ; RNA, Transfer, Ile ; chemistry ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid

As conserved enzymes with important functions, aminoacyl-tRNA synthetase are expressed ubiquitously in cells. These include cytoplasmic aminoacyl-tRNA synthetase, mitochondrial aminoacyl-tRNA synthetase and bifunctional aminoacyl-tRNA synthetase. Mitochondrial aminoacyl-tRNA synthetases catalyze the binding of amino acids with its corresponding tRNA in the mitochondria and participate in the translation of 13 subunits of oxidative phosphorylation enzyme complexes encoded by the mitochondrial genome. Mutations in genes encoding mitochondrial aminoacyl-tRNA synthase may cause a variety of genetic disorders. This review has summarized the clinical characteristics, molecular pathogenesis and treatment of genetic diseases caused by mutations of such genes.
Humans ; RNA, Transfer, Amino Acyl ; Genes, Mitochondrial ; Amino Acyl-tRNA Synthetases/genetics* ; Genome, Mitochondrial ; Mitochondria/genetics*

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.
Amino Acids ; genetics ; Amino Acyl-tRNA Synthetases ; Genetic Engineering ; Protein Engineering ; RNA, Transfer ; Viral Vaccines