The phylogenetic relationships of long-tailed macaque (Macaca fascicularis fascicularis) populations distributed in Peninsular Malaysia in relation to other regions remain unknown. The aim of this study was to reveal the phylogeography and population genetics of Peninsular Malaysia's M. f. fascicularis based on the D-loop region of mitochondrial DNA. Sixty-five haplotypes were detected in all populations, with only Vietnam and Cambodia sharing four haplotypes. The minimum-spanning network projected a distant relationship between Peninsular Malaysian and insular populations. Genetic differentiation (F(ST), Nst) results suggested that the gene flow among Peninsular Malaysian and the other populations is very low. Phylogenetic tree reconstructions indicated a monophyletic clade of Malaysia's population with continental populations (NJ = 97%, MP = 76%, and Bayesian = 1.00 posterior probabilities). The results demonstrate that Peninsular Malaysia's M. f. fascicularis belonged to Indochinese populations as opposed to the previously claimed Sundaic populations. M. f. fascicularis groups are estimated to have colonized Peninsular Malaysia ~0.47 million years ago (MYA) directly from Indochina through seaways, by means of natural sea rafting, or through terrestrial radiation during continental shelf emersion. Here, the Isthmus of Kra played a central part as biogeographical barriers that then separated it from the remaining continental populations.
A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.
The activities of lipase produced by five lipases-producing thermophilic bacteria strains (SY1, SY5, SY6, SY7 and SY9) isolated from Selayang Hot Spring in the western part of Peninsular Malaysia were analyzed and compared. SY7 and SY9 had considerably higher lipolytic activities than those of SY1, SY5 and SY6. Thermostabilities of lipase produced by all strains were determined after heating at 80°C for 30 minutes. Strain SY7 retained the highest lipolytic activity of 77%, while others had infinitesimally low thermostability (retaining less than 34% of their original activity) at the same temperature and time. SY7 was chosen for further characterization because it showed exceptionally high lipase activity and thermostability. It was identified as belonging to Bacillus species by the conventional Gram-staining technique, Biochemical tests and Biolog Microstation system. By using 16S rRNA gene sequencing, strain SY7 generated the same expected PCR product with molecular weight of 1500 base pair. It displayed 98% sequence similarity to Bacillus cereus strain J-1 16S rRNA gene partial sequence with accession number: AY305275 and has been deposited in the database of Genbank.
The secondary structure of RNA pseudoknots has been extensively inferred and scrutinized by computational approaches. Experimental methods for determining RNA structure are time consuming and tedious; therefore, predictive computational approaches are required. Predicting the most accurate and energy-stable pseudoknot RNA secondary structure has been proven to be an NP-hard problem. In this paper, a new RNA folding approach, termed MSeeker, is presented; it includes KnotSeeker (a heuristic method) and Mfold (a thermodynamic algorithm). The global optimization of this thermodynamic heuristic approach was further enhanced by using a case-based reasoning technique as a local optimization method. MSeeker is a proposed algorithm for predicting RNA pseudoknot structure from individual sequences, especially long ones. This research demonstrates that MSeeker improves the sensitivity and specificity of existing RNA pseudoknot structure predictions. The performance and structural results from this proposed method were evaluated against seven other state-of-the-art pseudoknot prediction methods. The MSeeker method had better sensitivity than the DotKnot, FlexStem, HotKnots, pknotsRG, ILM, NUPACK and pknotsRE methods, with 79% of the predicted pseudoknot base-pairs being correct.
Nanoparticles have been investigated as flagging tests for the sensitive DNA recognition that can be utilized as a part of field applications to defeat restrictions. Gold nanoparticles (AuNPs) have been widely utilized due to its optical property and capacity to get functionalized with a mixed bag of biomolecules. This study exhibits the utilization of AuNPs functionalized with single-stranded oligonucleotide (AuNP-oligo test) for fast the identification of Human Papillomavirus (HPV). This test is displayed on interdigitated electrode sensor and supported by colorimetric assay. DNA conjugated AuNP has optical property that can be controlled for the applications in diagnostics. With its identification abilities, this methodology incorporates minimal effort, strong reagents and basic identification of HPV.
The intra- and inter-specific variation of Acetes shrimps were evaluated based on samples collected from in-shore catches and off-shore trawling around the west coast of Peninsular Malaysia. Species captured were identified as Acetes indicus, A. serrulatus, A. japonicus and A. sibogae. A region of the mitochondrial cytochrome c oxidase subunit I (COI) gene comprising 552 base pairs (bp) was amplified from 159 Acetes specimens. The sequence alignment analysis generated phylogenetic trees which depicted the four major clades that were consistent with the species identified morphologically. These four species varied considerably for haplotype and nucleotide diversity, with A. indicus and A. serrulatus showing different demographic histories. Furthermore, the observation of two clades in the A. indicus and A. sibogae lineages, with relatively high levels of intraspecific divergence, suggests that cryptic diversity is possibly present in these two taxa. This study has contributed to the knowledge of the distribution patterns and molecular phylogenetics of four Acetes spp. in the Straits of Malacca.
Yardlong bean (Vigna unguiculata subsp. sesquipedalis) is extensively cultivated in Indonesia for consumption as a green vegetable. During the 2008 season, a severe outbreak of a virus-like disease occurred in yardlong beans grown in farmers' fields in Bogor, Bekasi, Subang, Indramayu, and Cirebon of West Java, Tanggerang of Banten, and Pekalongan and Muntilan of Central Java. Leaves of infected plants showed severe mosaic to bright yellow mosaic and vein-clearing symptoms, and pods were deformed and also showed mosaic symptoms on the surface. In cv. 777, vein-clearing was observed, resulting in a netting pattern on symptomatic leaves followed by death of the plants as the season advanced. Disease incidence in the Bogor region was approximately 80%, resulting in 100% yield loss. Symptomatic leaf samples from five representative plants tested positive in antigen-coated plate-ELISA with potyvirus group-specific antibodies (AS-573/1; DSMZ, German Resource Center for Biological Material, Braunschweig, Germany) and antibodies to Cucumber mosaic virus (CMV; AS-0929). To confirm these results, viral nucleic acids eluted from FTA classic cards (FTA Classic Card, Whatman International Ltd., Maidstone, UK) were subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (CIFor: 5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3' and CIRev: 5'-ACICCRTTYTCDATDATRTTIGTIGC-3') (3) and degenerate primers (CMV-1F: 5'-ACCGCGGGTCTTATTATGGT-3' and CMV-1R: 5' ACGGATTCAAACTGGGAGCA-3') specific for CMV subgroup I (1). A single DNA product of approximately 683 base pairs (bp) with the potyvirus-specific primers and a 382-bp fragment with the CMV-specific primers were amplified from ELISA-positive samples. These results indicated the presence of a potyvirus and CMV as mixed infections in all five samples. The amplified fragments specific to potyvirus (four samples) and CMV (three samples) were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 93 to 100% identity among the cloned amplicons produced using the potyvirus-specific primers (GenBank Accessions Nos. FJ653916, FJ653917, FJ653918, FJ653919, FJ653920, FJ653921, FJ653922, FJ653923, FJ653924, FJ653925, and FJ653926) and 92 to 97% with a corresponding nucleotide sequence of Bean common mosaic virus (BCMV) from Taiwan (No. AY575773) and 88 to 90% with BCMV sequences from China (No. AJ312438) and the United States (No. AY863025). The sequence analysis indicated that BCMV isolates from yardlong bean are more closely related to an isolate from Taiwan than with isolates from China and the United States. The CMV isolates (GenBank No. FJ687054) each were 100% identical and 96% identical with corresponding sequences of CMV subgroup I isolates from Thailand (No. AJ810264) and Malaysia (No. DQ195082). Both BCMV and CMV have been documented in soybean, mungbean, and peanut in East Java of Indonesia (2). Previously, BCMV, but not CMV, was documented on yardlong beans in Guam (4). To our knowledge, this study represents the first confirmed report of CMV in yardlong bean in Indonesia and is further evidence that BCMV is becoming established in Indonesia. References: (1) J. Aramburu et al. J. Phytopathol. 155:513, 2007. (2) S. K. Green et al. Plant Dis. 72:994, 1988. (3) C. Ha et al. Arch. Virol. 153:25, 2008. (4) G. C. Wall et al. Micronesica 29:101, 1996.
Extrachromosomal (ec) DNA in eukaryotic cells has been known for decades. The structures described range from linear double stranded (ds) DNA to circular dsDNA, distinct from mitochondrial (mt) DNA. The sizes of circular forms are described from some hundred base pairs (bp) up to more than 150 kbp. The number of molecules per cell ranges from several hundred to a thousand. Semi-quantitative determinations of circular dsDNA show proportions as high as several percentages of the total DNA per cell. These ecDNA fractions harbor sequences that are known to be present in chromosomal DNA (chrDNA) too. Sequencing projects on, for example the human genome, have to take into account the ecDNA sequences which are simultaneously ascertained; corrections cannot be performed retrospectively. Concerning the results of sequencings derived from extracted whole DNA: if the ecDNA fractions contained therein are not taken into account, erroneous conclusions at the chromosomal level may result.
The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53-63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.
The mitogenome of the Australian freshwater blackfish, Gadopsis marmoratus was recovered coverage by genome skimming using the MiSeq sequencer (GenBank Accession Number: NC_024436). The blackfish mitogenome has 16,407 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 819 bp non-coding AT-rich region. This is the 5th mitogenome sequence to be reported for the family Percichthyidae.
The mitochondrial genome sequence of the Australian tadpole shrimp, Triops australiensis is presented (GenBank Accession Number: NC_024439) and compared with other Triops species. Triops australiensis has a mitochondrial genome of 15,125 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The T. australiensis mitogenome is composed of 36.4% A, 16.1% C, 12.3% G and 35.1% T. The mitogenome gene order conforms to the primitive arrangement for Branchiopod crustaceans, which is also conserved within the Pancrustacean.
The mitochondrial genome sequence of the Australian crayfish, Euastacus yarraensis, is documented and compared with other Australian crayfish genera. Euastacus yarraensis has a mitogenome of 15,548 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The base composition of E. yarraensis mitogenome is 32.39% for T, 22.45% for C, 34.43% for A, and 10.73% for G, with an AT bias of 66.82%. The mitogenome gene order conforms to what is considered the primitive arrangement for parastacid crayfish.
The aim of the present study was to determine the genetic aberrations and novel transcripts, particularly the fusion transcripts, involved in the pathogenesis of low-grade and anaplastic oligodendroglioma. In the present study, tissue samples were obtained from patients with oligodendroglioma and additionally from archived tissue samples from the Brain Tumor Tissue Bank of the Brain Tumor Foundation of Canada. Six samples were obtained, three of which were low-grade oligodendroglioma and the other three anaplastic oligodendroglioma. DNA and RNA were extracted from each tissue sample. The resulting genomic DNA was then hybridized using the Agilent CytoSure 4×180K oligonucleotide array. Human reference DNA and samples were labeled using Cy3 cytidine 5'-triphosphate (CTP) and Cy5 CTP, respectively, while human Cot-1 DNA was used to reduce non-specific binding. Microarray-based comparative genomic hybridization data was then analyzed for genetic aberrations using the Agilent Cytosure Interpret software v3.4.2. The total RNA isolated from each sample was mixed with oligo dT magnetic beads to enrich for poly(A) mRNA. cDNAs were then synthesized and subjected to end-repair, poly(A) addition and connected using sequencing adapters using the Illumina TruSeq RNA Sample Preparation kit. The fragments were then purified and selected as templates for polymerase chain reaction amplification. The final library was constructed with fragments between 350-450 base pairs and sequenced using deep transcriptome sequencing on an Illumina HiSeq 2500 sequencer. The array comparative genomic hybridization revealed numerous amplifications and deletions on several chromosomes in all samples. However, the most interesting result was from the next generation sequencing, where one anaplastic oligodendroglioma sample was demonstrated to have five novel fusion genes that may potentially serve a critical role in tumor pathogenesis and progression.
Betulinic acid (BA) is a pentacyclic triterpene found in several botanical sources that has been shown to cause apoptosis in a number of cell lines. This study was undertaken to determine the in vitro cytotoxic properties of BA towards the human mammary carcinoma cell line MDA-MB-231 and the human promyelocytic leukaemia cell line HL-60 and the mode of the induced cell death. The cytotoxicity and mode of cell death of BA were determined using the MTT assay and DNA fragmentation analysis, respectively. In our study, the compound was found to be cytotoxic to MDA-MB-231 and HL-60 cells with IC50 values of 58 μg/mL and 134 μg/mL, respectively. Cells treated with high concentrations of BA exhibited features characteristic of apoptosis such as blebbing, shrinking and a number of small cytoplasm body masses when viewed under an inverted light microscope after 24h. The incidence of apoptosis in MDA-MB-231 was further confirmed by the DNA fragmentation analysis, with the formation of DNA fragments of oligonucleosomal size (180-200 base pairs), giving a ladder-like pattern on agarose gel electrophoresis. BA was more cytotoxic towards MDA-MB-231 than HL-60 cells, and induced apoptosis in MDA-MB-231 cells.
DNA barcoding of the cytochrome oxidase subunit I (COI) gene was utilized to assess the species diversity of the freshwater halfbeak genus Hemirhamphodon. A total of 201 individuals from 46 locations in Peninsular Malaysia, north Borneo (Sarawak) and Sumatra were successfully amplified for 616 base pairs of the COI gene revealing 231 variable and 213 parsimony informative sites. COI gene trees showed that most recognized species form monophyletic clades with high bootstrap support. Pairwise within species comparisons exhibited a wide range of intraspecific diversity from 0.0% to 14.8%, suggesting presence of cryptic diversity. This finding was further supported by barcode gap analysis, ABGD and the constructed COI gene trees. In particular, H. pogonognathus from Kelantan (northeast Peninsular Malaysia) diverged from the other H. pogonognathus groups with distances ranging from 7.8 to 11.8%, exceeding the nearest neighbor taxon. High intraspecific diversity was also observed in H. byssus and H. kuekanthali, but of a lower magnitude. This study also provides insights into endemism and phylogeographic structuring, and limited support for the Paleo-drainage divergence hypothesis as a driver of speciation in the genus Hemirhamphodon.
This article contains data of the sequence variation in the mitochondrial DNA D-loop region of the Malayan gaur (Bos gaurus hubbacki), locally known as the seladang, from two captive centers. Thirty fecal samples of Malayan gaur were collected from Jenderak Selatan Wildlife Conservation Center (Pahang) and the Sungkai Wildlife Reserve (Perak) for DNA extraction and amplification with polymerase chain reactions. DNA sequences were then analyzed using neighbor joining (NJ) and maximum parsimony (MP) methods. Based on the 652 base pairs obtained, we found seven variable characters with a value of 1%. The genetic distance between the two captive centers was 0.001. Haplotype analyses detected only four haplotypes between these two captive centers. Both NJ and MP trees demonstrate that all individuals in the Jenderak and Sungkai captive centers are in the same clade. Genetic variation of the Malayan gaur in these centers is considered low, possibly because individuals share the same common parent. This sequence variation data are of paramount importance for designing a proper breeding and management program of the Malayan gaur in the future.
MicroRNAs (miRNAs) are short, endogenous, non-coding RNAs that post-transcriptionally regulate gene expression by base pairing with mRNA targets. Altered miRNA expression profiles have been observed in several diseases, including neurodegeneration. Multiple studies have reported altered expressions of miRNAs in the brains of individuals with Alzheimer's disease (AD) as compared to those of healthy elderly adults. Some of the miRNAs found to be dysregulated in AD have been reported to correlate with neuropathological changes, including plaque and tangle accumulation, as well as altered expressions of species that are known to be involved in AD pathology. To examine the potentially pathogenic functions of several dysregulated miRNAs in AD, we review the current literature with a focus on the activities of ten miRNAs in biological pathways involved in AD pathogenesis. Comprehensive understandings of the expression profiles and activities of these miRNAs will illuminate their roles as potential therapeutic targets in AD brain and may lead to the discovery of breakthrough treatment strategies for AD.
Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm.
Present study investigates the genetic diversity and genetic distribution of the longtail tuna Thunnus tonggol collected from east Malaysia (Borneo states of Sabah and Sarawak) based on mitochondrial DNA D-loop sequence analysis. 58 fish samples were obtained, specifically from Kota Kinabalu, KK (n = 22), Miri, MR (n=20) and Bintulu, BT (n = 17). DNA template was isolated using the salt extraction method. Final length of 404 base pair (bp) D-loop sequences revealed 52 haplotypes that comprise of 77 variable sites (38 of parsimony informative and 39 singleton). A total of 20 haplotypes were found in KK, 19 haplotypes in MR and 16 haplotypes in BT. Molecular diversity indices revealed high haplotype diversity and low nucleotide diversity in all populations; KK (h = 0.9913 ± 0.0165, π = 0.00239 ± 0.0127), MR (h = 0.9942 ± 0.0193, π = 0.0226 ± 0.0121) and BT (h = 0.9926 ± 0.0230, π = 0.0196 ± 0.0171). Population comparison pairwise FST show that KK and BT were significantly genetically differentiated. The result from this study will be beneficial for fisheries management and also to provide information on the population genetics of T. tonggol in East Malaysian waters.
A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.