The increasing interest in understanding the evolutionary relationship between members of the Pteropodidae family has been greatly aided by genomic data from the Old World fruit bats. Here we present the complete mitogenome of Geoffroy's rousette, Rousettus amplexicaudatus found in Peninsular Malaysia . The mitogenome constructed is 16,511bp in length containing 37 genes; 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a D-loop region. The overall base composition is estimated to be 32.28% for A, 25.64% for T, 14.09% for G and 27.98% for C, indicating a slightly AT rich feature (57.93%). A phylogenetic and BLASTn analysis against other available mitogenomes showed Malaysian R. amplexicaudatus matched 98% similarity to the same species in Cambodia and Vietnam. However, it differed considerably (92.53% similarity) with the same species in the Philippines. This suggests flexibility in Rousettus sp. with regards to adapting to mesic and dry habitats, ability for long-distance dispersal and remarkably precise lingual echolocation thus supporting its wide-range distribution and colonization. Further taxonomical and mitogenomic comparatives are required in resolving the evolutionary relationship between Rousettus spp.
The mitogenome of a plantain squirrel, Callosciurus notatus, collected from Bukit Tarek Forest Reserve (Extension), Selangor, Malaysia was sequenced using BGISEQ-500RS technology. The 16,582 bp mitogenome consists of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region. A phylogenetic and BLASTn analysis against other available datasets showed that the mitogenome matched with 99.49% similarity to a previously published C. notatus mitogenome from Peninsular Malaysia. However, it also diverged by nearly 8% (92.24% match) from a second previously published mitogenome for the same species, sampled in East Kalimantan, Indonesia. This suggests a difference in landscape features between both localities might affect its genetic connectivity.
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.
GCN5-related N-acetyltransferase (GNAT) is a huge superfamily of proteins spanning the prokaryotic and eukaryotic domains of life. GNAT proteins usually transfer an acetyl group from acetyl-CoA to a wide variety of substrates ranging from aminoglycoside antibiotics to large macromolecules. Type II toxin-antitoxin (TA) modules are typically bicistronic and widespread in bacterial and archael genomes with diverse cellular functions. Recently, a novel family of type II TA toxins was described, which presents a GNAT-fold and functions by acetylating charged tRNA thereby precluding translation. These GNAT toxins are usually associated with a corresponding ribbon-helix-helix-fold (RHH) antitoxin. In this issue, Qian et al. describes a unique GNAT-RHH TA system, designated KacAT, from a multidrug resistant strain of the pathogen, Klebsiella pneumoniae. As most type II TA loci, kacAT is transcriptionally autoregulated with the KacAT complex binding to the operator site via the N-terminus region of KacA to repress kacAT transcription. The crystal structure of the KacT toxin is also presented giving a structural basis for KacT toxicity. These findings expand our knowledge on this newly discovered family of TA toxins and the potential role that they may play in antibiotic tolerance and persistence of bacterial pathogens.
Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.
The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA(i) (met) compared to the one for the elongator tRNAs indicates that only the initiator tRNA(i) (met) specifies the amino acid side chain.
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.
Background. The gram-negative Neisseria is associated with two of the most potent human epidemic diseases: meningococcal meningitis and gonorrhoea. In both cases, disease is caused by bacteria colonizing human mucosal membrane surfaces. Overall, the genus shows great diversity and genetic variation mainly due to its ability to acquire and incorporate genetic material from a diverse range of sources through horizontal gene transfer. Although a number of databases exist for the Neisseria genomes, they are mostly focused on the pathogenic species. In this present study we present the freely available NeisseriaBase, a database dedicated to the genus Neisseria encompassing the complete and draft genomes of 15 pathogenic and commensal Neisseria species. Methods. The genomic data were retrieved from National Center for Biotechnology Information (NCBI) and annotated using the RAST server which were then stored into the MySQL database. The protein-coding genes were further analyzed to obtain information such as calculation of GC content (%), predicted hydrophobicity and molecular weight (Da) using in-house Perl scripts. The web application was developed following the secure four-tier web application architecture: (1) client workstation, (2) web server, (3) application server, and (4) database server. The web interface was constructed using PHP, JavaScript, jQuery, AJAX and CSS, utilizing the model-view-controller (MVC) framework. The in-house developed bioinformatics tools implemented in NeisseraBase were developed using Python, Perl, BioPerl and R languages. Results. Currently, NeisseriaBase houses 603,500 Coding Sequences (CDSs), 16,071 RNAs and 13,119 tRNA genes from 227 Neisseria genomes. The database is equipped with interactive web interfaces. Incorporation of the JBrowse genome browser in the database enables fast and smooth browsing of Neisseria genomes. NeisseriaBase includes the standard BLAST program to facilitate homology searching, and for Virulence Factor Database (VFDB) specific homology searches, the VFDB BLAST is also incorporated into the database. In addition, NeisseriaBase is equipped with in-house designed tools such as the Pairwise Genome Comparison tool (PGC) for comparative genomic analysis and the Pathogenomics Profiling Tool (PathoProT) for the comparative pathogenomics analysis of Neisseria strains. Discussion. This user-friendly database not only provides access to a host of genomic resources on Neisseria but also enables high-quality comparative genome analysis, which is crucial for the expanding scientific community interested in Neisseria research. This database is freely available at http://neisseria.um.edu.my.
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.
Gymnothorax minor is a moray eel of the family Muraenidae found in the Western Pacific Ocean. We report here
its complete mitogenome as determined by Illumina next-generation sequencing and the phylogenetic relationship
with its congeners and other taxa of the family Muraenidae. The whole mitogenome of G. minor had a total length
of 16,574 bp, comprising 37 genes - 13 protein-coding genes (PCGs), two ribosomal ribonucleic acid (rRNA) and 22
transfer ribonucleic acid (tRNA) genes - and a control region. Excepting cox1 with GTG, the other 12 PCGs had ATG
start codon. Seven of its PCGs had incomplete stop codon - five (nad2; cox1; cox2; nad3 and nad4) with T and two
(atp6 and cox3) with TA. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs
and 2 rRNA genes). The subfamily Muraeninae as well as the subfamily Uropterygiinae were monophyletic. However,
the genus Gymnothorax was paraphyletic, with G. minor forming a sister group with Rhinomuraena quaesita in the
lineage containing also G. kidako and G. formosus forming a sister group with Enchelynassa canina. The phylogenetic
relationship of the genus Gymnothorax and related taxa of the family Muraenidae, based on the mitochondrial cob
gene, was in general similar to that based on 15 mt-genes. The mitogenome is useful for future studies on phylogenetics
and systematics of eels of the family Muraenidae and other taxa of the order Anguilliformes.
This study was conducted to detect the presence of chicken and porcine DNA in meatballs using mitochondria DNA (mtDNA) of cytochrome b (cyt b) and nuclear DNA (nDNA) short interspersed nuclear element (SINE) species-specific primers, respectively. While, the mtDNA primers targeted transfer RNA-ATP8 (tRNA-ATP8) gene was used for 1 and 5% (w/w) chicken meatball spiked with commercial porcine blood plasm. Chicken meatballs spiked with 1% and 5% (v/w) fresh and commercial porcine blood plasma, respectively were prepared and heat-treated using five (n = 5) cooking methods: boiling, pan-frying, roasting, microwaving and autoclaving. Two pairs of mtDNA and nDNA primers used, produced 129 and 161 bp amplicons, respectively. Whereas, tRNA-ATP8 primers produced 212 bp of amplicon. Electrophoresis analysis showed positive results for porcine DNA at 1% and 5% (w/w or v/v) for all of the different cooking techniques, either for fresh or commercial blood plasma using SINE primers but not for tRNA-ATP8 primers. The present study has highlighted the useful of species-specific primers of SINE primers in PCR analysis for detecting porcine DNA blood plasma in heat-treated chicken meatballs.
Mycobacterium abscessus (Ma) is an emerging human pathogen that causes both soft tissue infections and systemic disease. We present the first comparative whole-genome study of Ma strains isolated from patients of wide geographical origin. We found a high proportion of accessory strain-specific genes indicating an open, non-conservative pan-genome structure, and clear evidence of rapid phage-mediated evolution. Although we found fewer virulence factors in Ma compared to M. tuberculosis, our data indicated that Ma evolves rapidly and therefore should be monitored closely for the acquisition of more pathogenic traits. This comparative study provides a better understanding of Ma and forms the basis for future functional work on this important pathogen.
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.
Forensic entomology applies knowledge about insects associated with decedent in crime scene investigation. It is possible to calculate a minimum postmortem interval (PMI) by determining the age and species of the oldest blow fly larvae feeding on decedent. This study was conducted in Malaysia to identify maggot specimens collected during crime scene investigations. The usefulness of the molecular and morphological approach in species identifications was evaluated in 10 morphologically identified blow fly larvae sampled from 10 different crime scenes in Malaysia. The molecular identification method involved the sequencing of a total length of 2.2 kilo base pairs encompassing the 'barcode' fragments of the mitochondrial cytochrome oxidase I (COI), cytochrome oxidase II (COII) and t-RNA leucine genes. Phylogenetic analyses confirmed the presence of Chrysomya megacephala, Chrysomya rufifacies and Chrysomya nigripes. In addition, one unidentified blow fly species was found based on phylogenetic tree analysis.
The mitochondiral DNA region encompassing the cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII) genes of two Malaysian blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) were studied. This region, which spans 2303bp and includes the COI, tRNA leucine and partial COII was sequenced from adult fly and larval specimens, and compared. Intraspecific variations were observed at 0.26% for Ch. megacephala and 0.17% for Ch. rufifacies, while sequence divergence between the two species was recorded at a minimum of 141 out of 2303 sites (6.12%). Results obtained in this study are comparable to published data, and thus support the use of DNA sequence to facilitate and complement morphology-based species identification.
Examination of types and recently collected specimens revealed that Ansonia anotis Inger, Tan, and Yambun, 2001 and Pedostibes maculatus (Mocquard, 1890), both described from Kinabalu, Sabah, Malaysia, are hardly differentiated morphologically. Analyses of a total of 2,427 bp of the 12S rRNA, tRNA(val), and 16S mitochondrial rRNA genes revealed that the two species are very close genetically. Thus A. anotis is regarded as conspecific and is synonymized with P. maculatus. Genetically, this species proved to form a lineage distinct from other bufonids from Southeast Asia, including species of Ansonia and Pedostibes. Because the species has also some unique morphological traits different from known bufonid genera, we propose to establish a new genus for Nectophryne maculata Mocquard, 1890.
Phylogenetic analyses based on the mitochondrial gene ND2 and its flanking tRNAs indicate the diminutive upland and insular species Sphenomorphus bukitensis, S. butleri, S. langkawiensis, S. perhentianensis, and S. temengorensis form a monophyletic group that is phylogenetically embedded within the Southeast Asian genus Tytthoscincus. The analyses also indicate that a new swamp-dwelling skink from the Bukit Panchor State Park, Pulau Pinang, Peninsular Malaysia is the sister species to the swamp-dwelling species S. sibuensis from Pulau Sibu, Johor and Singapore and that these two are also embedded in the genus Tytthoscincus. By transferring the two Peninsular Malaysian clades of Sphenomorphus into the genus Tytthoscincus, the monophyly of the latter is maintained. The new species T. panchorensis sp. nov. can be distinguished from all other species of Tytthoscincus by having a unique combination of morphological and color pattern characteristics.
We present here the first observation of Bipalium admarginatum de Beauchamp, 1933 since its original description 90 years ago. Three specimens were found on Perhentian Kecil Island, off Terengganu State, Malaysia and photographed in the field, and two were collected. This report thus includes the first colour photographs published for this species, from a locality close to the type-locality, Tioman Island (which is ca. 200 km south of the locality in this study, on the east coast of Peninsula Malaysia). We describe the external morphology and colour pattern of the species, which correspond well to the original description, itself based only on two preserved specimens. We performed an in-depth molecular characterisation of the species, including its complete mitochondrial genome, the 18S sequence and elongation 1-alpha (EF1-α) sequence. In addition, EF1-α sequences were also retrieved for 5 additional geoplanid species. No tRNA-Thr could be detected in the mitogenome of B. admarginatum, a lack already reported in several species of geoplanids, but we found a 13 bp sequence that contains the anticodon loop and seems to be conserved among geoplanids and might thus possibly represent a non-canonical undetected tRNA. We discuss the difficulties encountered in trying to reconstruct the cluster of nuclear ribosomal genes, a problem already mentioned for other Triclads. Three phylogenies, based respectively on all mitochondrial proteins, 18S, and EF1-α, were computed; the position of B. admarginatum within the Bipaliinae was confirmed in each tree, as sister-group to various bipaliine species according to the sequences available for each tree. In the mitochondrial proteins tree, which had high support, B. admarginatum was sister to Bipalium kewense and Diversibipalium multilineatum.