The translational accuracy in protein synthesis is contributed to by several mechanisms in the ribosome, generally called kinetic proofreading. This process in the ribosome inhibits the non-cognate codon-anticodon interaction. However, it is not sufficient for fidelity of protein synthesis since a wrong amino acid can easily be added to the growing polypeptide chain if a tRNA while cognate to the mRNA, carries a non-cognate amino acid. Therefore, additional to the kinetic proofreading, there must be some hitherto unknown characteristic in misacylated-tRNAs to stop the process of protein synthesis if such misacylated-tRNA is accommodated in the ribosomal A-site. In order to understand this characteristic, we have performed computational quantum chemistry analysis on five different tRNA molecules, each one attached to five different amino acids with one being cognate to the tRNA and the other four non-cognate. This study shows the importance of aminoacyl-tRNA binding energy in ensuring fidelity of protein synthesis.
This study describes the development of a rapid and sensitive Loop-mediated isothermal
amplification assay for detection of swine DNA in adulterated meat and meat products. The
need to protect consumer’s right to eat foods of their choices, has made it imperative for
researchers to develop efficient means of screening and certification of food products. Six sets
of LAMP primers designed based on porcine tRNA lysine gene and ATPase subunit 8 genes
were used for the assay. Amplification was carried out under constant temperature (630C), using
a simple laboratory water bath. Average time spent in amplification and detection of results was
25 min. All results were visually detected and confirmed by electrophoresis. Detection limit of
the assay was 0.03 femtogram (fg) much high than the PCR assay, and detection probability of
the assay was 100%. Detection of 0.5% of pork spiked with 99.5% of cattle beef is indicative
of the sensitivity and robustness of the assay. This could serve as a prototype for development
of a sensitive and inexpensive Swine DNA LAMP detection kit.
Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) are established subgroups of mitochondrial encephalomyopathy. m.3243A>G a common point mutation is detected in tRNA in majority of patients with MELAS phenotype whereas m.8344A>G point mutation in tRNA is observed, in MERRF phenotype. Adrenal insufficiency has not been reported in mitochondrial disease, except in Kearns-Sayre Syndrome (KSS), which is a mitochondrial deletion syndrome. We report an unusual presentation in a five year old boy who presented with clinical phenotype of MELAS and was found to have m.8344A>G mutation in tRNA. Addison disease was identified due to hyperpigmentation of lips and gums present from early childhood. This is the first report describing adrenal insufficiency in a child with MELAS phenotype.
Mycobacterium abscessus is a species of rapidly growing nontuberculous mycobacteria that is frequently associated with opportunistic infections in humans. Here, we report the annotated genome sequence of M. abscessus strain M94, which showed an unusual cluster of tRNAs.
We describe the characteristics of complete mitogenome of C. brachyotis in this article. The complete mitogenome of C. brachyotis is 16,701 bp long with a total base composition of 32.4% A, 25.7% T, 27.7% C and 14.2% G. The mitogenome consists of 13 protein-coding genes (11,408 bp), (KM659865) two rRNA (12S rRNA and 16S rRNA) genes (2,539 bp), 22 tRNA genes (1518 bp) and one control region (1239 bp).
The complete mitochondrial genome sequence of Atergatis integerrimus from China has been amplified and sequenced in this study. The mitogenome assembly was found to be 15,924 bp in length with base composition of A (32.88%), G (10.58%), C (20.87%), T (35.66%), A + T (68.54%), and G + C (31.46%). It contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a control region. The phylogenetic position was constructed and the A. integerrimus was closely clustered with Pseudocarcinus gigas and Leptodius sanguineus. The complete mitochondrial genome sequence would be useful for further understanding the evolution of A. integerrimus.
Two mitogenomes of long-tailed giant rat, Leopoldamys sabanus (Thomas, 1887), which belongs to the family Muridae were sequenced and assembled in this study. Both mitogenomes have a length of 15,973 bp and encode 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes and one control region. The circular molecule of L. sabanus has a typical vertebrate gene arrangement. Phylogenetic and BLASTn analysis using 10 Leopoldamys species mitogenomes revealed sequence variation occurred within species from different time zones. Along with the taxonomic issues, this suggests a landscape change might influence genetic connectivity.
In order to authenticate the genomic information of Barleriacristata L., B. lupulina Lindl., B. repens Nees, B. siamensis Craib, and B. strigosa Willd, cp genomes were investigated. They revealed a general structure with a total size of 151,997-152,324 bp. The genomes encoded a total of 131 genes, including 86 CDS, 37 tRNA, and 8 rRNA genes. Other details found were as follows: different numbers and types of SSRs; identical gene content, which is adjacent to the border regions, except for B. strigosa, that revealed a shorter ndhF gene sequence and lacked the ycf1 gene; slightly different genetic distance values, which can be used for species identification; three distinct gaps of nucleotide variations between the species located at the intergenic spacer regions of the LSC and CDS of the SSC; three effective molecular markers derived from divergent hotspot regions, including the ccsA-ndhD, ndhA-ndhH-rps15, and ycf1. The genetic relationships derived from the cp genome and the CDS phylogenetic trees of Barleria and the 13 genera in Acanthaceae and different families, Scrophulariaceae and Phrymaceae, showed similar results. The six Barleria species as monophyletic groups with inner and outer outgroups were found to have perfect discrimination. These results have helped to authenticate the five Barleria species and the six genera in Acanthaceae.
Syzygium malaccense is native to Malaysia. It is sometimes called the malay apple, malay rose-apple, mountain rose-apple, mountain apple, water apple, or French cashew. The tree is very popular in many tropical and subtropical regions for its fruit and traditional medicine. The first complete chloroplast genome of Syzygium malaccense has been reported in this study. The complete chloroplast genome of Syzygium malaccense is 158,954 bp, composed of four regions: a large single-copy region with a size of 87,991 bp, a small single copy region with a size of 18,793 bp, and two inverted repeat regions with a size of 26,085 bp. The GC content is 36.97%. A total of 132 genes were annotated, including 84 encoding proteins, eight encoding rRNA genes, 37 encoding tRNA genes, and three encoding pseudo genes. Phylogenetic analysis showed that Syzygium aromaticum, Syzygium cumini, and Syzygium forrestii are closely related to Syzygium malaccense.
Gigantochloa verticillata is produced in Mengla and Jinghong, Yunnan Province, China, and cultivated in Hong Kong. Vietnam, Thailand, India, Indonesia, and Malaysia are distributed and cultivated. We determined the complete chloroplast genome sequence for G. verticillata using Illumina sequencing data. The complete chloroplast sequence is 139,489 bp, including large single-copy (LSC) region of 83,062 bp, small single-copy (SSC) region of 12,877 bp, and a pair of invert repeats (IR) regions of 21,775 bp. Plastid genome contain 132 genes, 85 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 23 chloroplast genomes indicates that G. verticillata is closely related to Dendrocalamus latiflorus in Bambusodae.
Angiostrongylus malaysiensis is a nematode parasite of various rat species. When first documented in Malaysia, it was referred to as A. cantonensis. Unlike A. cantonensis, the complete mitochondrial genome of A. malaysiensis has not been documented. We report here its complete mitogenome, its differentiation from A. cantonensis, and the phylogenetic relationships with its congeners and other Metastrongyloid taxa. The whole mitogenome of A. malaysiensis had a total length of 13,516bp, comprising 36 genes (12 PCGs, 2 rRNA and 22 tRNA genes) and a control region. It is longer than that of A. cantonensis (13,509bp). Its control region had a long poly T-stretch of 12bp which was not present in A. cantonensis. A. malaysiensis and A. cantonensis had identical start codon for the 12 PCGs, but four PCGs (atp6, cob, nad2, nad6) had different stop codon. The cloverleaf structure for the 22 tRNAs was similar in A. malaysiensis and A. cantonensis except the TΨC-arm was absent in trnV for A. malaysiensis but present in A. cantonensis. The Angiostrongylus genus was monophyletic, with A. malaysiensis and A. cantonensis forming a distinct lineage from that of A. costaricensis and A. vasorum. The genetic distance between A. malaysiensis and A. cantonensis was p=11.9% based on 12 PCGs, p=9.5% based on 2 rRNA genes, and p=11.6% based on 14 mt-genes. The mitogenome will prove useful for studies on phylogenetics and systematics of Angiostrongylus lungworms and other Metastrongyloid nematodes.
The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general.
This is the first documentation of the complete mitochondrial genome sequence of the Malaysian Mahseer, Tor tambroides. The 16,690 bp mitogenome with GenBank accession number JX444718 contains 13 protein genes, 22 tRNAs, two rRNAs, and a noncoding control region (D-loop) as is typical of most vertebrates. The phylogenomic reconstruction of this newly generated data with 21 Cypriniformes GenBank accession ID concurs with the recognized status of T. tambroides within the subfamily Cyprininae. This is in agreement with previous hypotheses based on morphological and partial mitochondrial analyses.
We report here the complete mitochondrial (mt) genomes of six individuals of Cheilinus undulatus (Napoleon Wrasse), an endangered marine fish species. The six mt DNA sequences had an average size of 17,000 kb and encoded 22 tRNA, two sRNA, 13 highly conserved protein coding genes and a control region. The polymorphic variation (control region) in these six individuals suggests their potential use as a specific marker for phylogeographic conservation. Moreover, the sequence polymorphism within the control region (D-loop) suggests that this locus can be applied for phylogenetic studies.
We sequenced the complete mitochondrial genome of the Tioman Island rock gecko, Cnemaspis limi, which is known as an endemic species to Malaysia. The complete mitogenome is 16,680 bp in size, consisting of 37 genes coding for 13 proteins, 22 transfer RNAs, two ribosomal RNAs and one control region. The A + T content of the overall base composition of H-strand is 53.09% (T: 23.20%, C: 32.48%, A: 29.89% and G: 14.43%). The major non-coding region (control region) is 1254 bp in length with the A + T content of 55.09% and four replicates of a 76-bp repeat within this region.
The black-winged fly, Felderimyia fuscipennis (Diptera: Tephritidae), is an insect pest of bamboo shoot, mainly distributed in Thailand, Malaysia and Yunnan Province and Guangxi Autonomous Region, China. The complete sequence of the mitogenome of F. fuscipennis has been determined in this study. The whole mitogenome sequence is 16,536 bp in length, which totally contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a non-coding region (putative control region, CR). The phylogeny indicates that F. fuscipennis of subfamily Trypetinae was monophyletic and clearly separated from both Dacinae and Tephritinae with high bootstrap value supported.
In this study, the complete mitogenome sequence of two moray eels of Gymnothorax formosus and Scuticaria tigrina (Anguilliformes: Muraenidae) has been sequenced by the next-generation sequencing method. The assembled mitogenome, with the length of 16,558 bp for G. formosus and 16,521 bp for S. tigrina, shows 78% identity to each other. Both mitogenomes follow the typical vertebrate arrangement, including 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs genes, and a non-coding control region of D-loop. The length of D-loop is 927 bp (G. formosus) and 850 bp (S. tigrina), which is located between tRNA-Pro and tRNA-Phe. The overall GC content is 45.5% for G. formosus and 47.9% for S. tigrina. Complete mitogenomes of G. formosus and S. tigrina provide essential and important DNA molecular data for further phylogenetic and evolutionary analysis for moray eel.
Vatica mangachapoi is a tree up to 20 m tall with white resinous. It is distributed in China (Hainan province), Indonesia, Malaysia (N Borneo), Philippines, Thailand, and Vietnam. It grows in forests on hills and mountain slopes below 700 metres. Its durable wood is used for making boats and building bridges and houses. It has been ranked as a VU (Vulnerable) species in China. Here we report and characterize the complete plastid genome sequence of V. mangachapoi in an effort to provide genomic resources useful for promoting its conservation and phylogenetic research. The complete plastome is 151,538 bp in length and contains the typical structure and gene content of angiosperm plastome, including two Inverted Repeat (IR) regions of 23,921 bp, a Large Single-Copy (LSC) region of 83,587 bp and a Small Single-Copy (SSC) region of 20,109 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA gene, and 4 unique rRNA genes. The overall A/T content in the plastome of V. mangachapoi is 62.80%. The phylogenetic analysis indicated that V. mangachapoi and V. odorata is closely related and as an independent branch in Malvales in our study. The complete plastome sequence of V. mangachapoi will provide a useful resource for the conservation genetics of this species and for the phylogenetic studies for Vatica.
Finding a proper transition structure for the peptide bond formation process can lead to a better understanding of the role of the ribosome in catalyzing this reaction. A potential energy surface scan was performed on the ester bond dissociation of the P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragment of initiator tRNAi met attached to both cognate (met) and non-cognate (ala) amino acids as the P-site substrate and the methionine as the A-site amino acid was used in this study. Due to the large size of tRNA, ONIOM calculations were used to reduce the computational cost. This study illustrated that the rate of peptide bond formation was reduced for misacylated tRNA without the presence of ribosomal bases. This demonstrated that there were indeed specific structural interactions involving the amino acid side chain within the tRNAi met.
Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.