Hemagglutinin (HA) protein plays an important role in binding the influenza virus to infected cells and therefore mediates infection. Deposited HA sequences of 86 Asian strains of influenza A (H1N1) viruses during the first outbreak were obtained from the NCBI database and compared. Interaction of the HA protein of influenza A (H1N1) virus with the human sialic acid receptor was also studied using bioinformatics. Overall, not more than three single-point amino acid variants/changes were observed in the HA protein region of influenza A (H1N1) virus from Asian countries when a selected group sequence comparison was made. The bioinformatics study showed that the HA protein of influenza A (H1N1) binds to the sialic acid receptor in human airway receptors, possibly key to air-borne infection in humans.
Inheritance of polymorphisms in the interleukin (IL)-10 promoter and IL-12B genes, which influence cytokine production and activities, may define the balance in T helper response in infection and autoimmune diseases. In the present study, we investigated the distribution of the IL-10 promoter and IL-12B gene polymorphisms in a multiethnic Malaysian population. Overall, our findings suggest that the IL-12B and IL-10 -592 genotypes were distributed homogenously across all major ethnic groups, including Malays, Chinese, and Indians, except for polymorphisms at IL-10 -1082. At this gene locus, the ethnic Chinese showed a significantly lower allele frequency of -1082G (2.1%) compared to the Malay (12.2%) and Indian (15.3%) populations. Results for the IL-12B and IL-10 gene polymorphisms were consistent with those reported for the Asian population, but markedly different from those of the African and Caucasian populations. Our findings suggest that there are specific genetic variations between different ethnic groups, which should be examined in all gene population-based association studies.
The diversity of specific bacteria taxa, such as the actinomycetes, has not been reported from the Antarctic island of Barrientos. The diversity of actinomycetes was estimated with two different strategies that use PCR-denaturing gradient gel electrophoresis. First, a PCR was applied, using a group-specific primer that allows selective amplification of actinomycete sequences. Second, a nested-PCR approach was used that allows the estimation of the relative abundance of actinomycetes within the bacterial community. Molecular identification, which was based on 16S rDNA sequence analysis, revealed eight genera of actinomycetes, Actinobacterium, Actinomyces, an uncultured Actinomycete, Streptomyces, Leifsonia, Frankineae, Rhodococcus, and Mycobacterium. The uncultured Actinomyces sp and Rhodococcus sp appear to be the prominent genera of actinomycetes in Barrientos Island soil. PCR-denaturing gradient gel electrophoresis patterns were used to look for correlations between actinomycete abundance and environmental characteristics, such as type of rookery and vegetation. There was a significant positive correlation between type of rookery and abundance of actinomycetes; soil samples collected from active chinstrap penguin rookeries had the highest actinomycete abundance. Vegetation type, such as moss, which could provide a microhabitat for bacteria, did not correlate significantly with actinomycete abundance.
Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.
A limited backcross procedure was utilized to introgress genes associated with grain quality traits from Oryza rufipogon (Accession No. IRGC 105491), a wild rice from Malaysia, to the cultivated rice O. sativa cv. MR219, a popular high yielding Malaysian rice cultivar. A set of 10 BC(2)F(7) progenies were selected based on the field performance and phenotypic appearance in BC(2)F(5) and BC(2)F(6) generations, which initially started with 266 progenies in the BC(2)F(2) generation. These 10 advanced breeding lines are similar to each other but differ in several important grain quality traits, which can be traced to O. rufipogon introgressions. Phenotyping and genotyping of BC(2)F(7) variants were considered for QTL analysis. The introgressed lines did not show any significant changes compared to the recurrent parent MR219 for the traits grain density and milled rice percentage. All 10 progenies showed significantly higher head rice percentages (70-88%) than the recurrent parent MR219. Variants G13 and G15 had higher amylose contents than MR219. All variants were analyzed using polymorphic SSR markers. Of the 34 SSR markers, only 18 showed introgression from O. rufipogon for chromosomes 1, 2, 3, 5, 6, 8, 10, and 11. Graphical genotypes were prepared for each variant, and association between the introgression regions and the traits that increased grain quality was visualized. Based on marker trait association, some of the QTLs are stable across environments and genetic backgrounds and could be used universally.
Advanced backcross families derived from Oryza sativa cv MR219/O. rufipogon IRGC105491 were utilized for identification of quantitative trait loci (QTL) for blast resistance using simple sequence repeat markers. Two hundred and sixty-one BC(2)F(3) families were used to construct a linkage map, using 87 markers, which covered 2375.2 cM of 12 rice chromosomes, with a mean density of 27.3 cM. The families were evaluated in a greenhouse for resistance to blast disease caused by pathotypes P7.2 and P5.0 of Magnaporthe oryzae. Five QTLs (qBL5.1, qBL5.2, qBL6.1, qBL8.1, and qBL10.1) for pathotype P5.0 and four QTLs (qBL5.3, qBL5.4, qBL7.1, and qBL8.2) for pathotype P7.2 were identified using the BC(2)F(3) families. Another linkage map was also constructed based on 31 BC(2)F(5) families, using 63 SSR markers, which covered 474.9 cM of 9 rice chromosomes, with a mean density of 8.01 cM. Five suggestive QTLs (qBL11.2, qBL11.3, qBL12.1, qBL12.2, qBL12.3) and one putative QTL (qBL2.1) were identified for pathotype P7.2. Also, seven suggestive QTLs (qBL1.1, qBL2.2, qBL4.1, qBL4.2, qBL5.3, qBL8.3, and qBL11.1) were detected for pathotype P5.0. We conclude that there is a non-race-specific resistance spectrum of O. rufipogon against M. oryzae pathotypes.
Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.
Mutations in the PAX6 gene that cause aniridia have been identified in various ethnicities but not in the Malaysian population. Therefore, the objective of this study was to investigate the PAX6 mutation in a Malaysian family with congenital aniridia. In this study, a complete ophthalmic examination was performed on a Dusun ethnic family with aniridia. Genomic DNA was extracted from the peripheral blood of the subjects and screened for the PAX6 gene mutation using polymerase chain reaction amplification high-resolution melting curve analysis (PCR-HRM) followed by confirmation via direct DNA sequencing. A heterozygous G deletion (c.857delG) in exon 7 causing a frame shift in PAX6 was identified in all affected family members. Genotype-phenotype correlation analysis revealed congenital cataract and all affected family members showed a similar spectrum of aniridia with no phenotypic variability but with differences in severity that were age-dependent. In summary, by using a PCR-HRM approach, this study is the first to report a PAX6 mutation in a Malaysian family. This mutation is the cause of the aniridia spectra observed in this family and of congenital cataract.
For centuries, morphology-based fish identification has been applied without molecular evaluation. Many studies showed that specimens with a similar morphology are frequently found to be quite genetically distinct. One of the fish species that still remains taxonomically problematic is a commercial snapper species, Lutjanus johnii. Because of morphological ambiguities among local fish taxonomists in Malaysia, we examined the ability of the cytochrome oxidase I (COI) gene to genetically examine the taxonomic status of L. johnii. A 626-base pair COI region was successfully amplified and aligned with conspecific sequences that were retrieved from GenBank. The phylogenetic tree obtained showed two major clusters; the first cluster consists of L. johnii from Straits of Malacca, Thailand, Australia, and China while the second cluster comprises L. johnii from China and India. The latter group showed sequence divergence greater than 3.5%. After observing this, we suspected that there might be a cryptic species between the South China Sea and Indian Ocean. This is the first molecular report concerning the commercial species of snapper, L. johnii, in Malaysia, which had only gained provisional recognition from morphological examination.
This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.
Malaysian arowana (dragonfish; Scleropages formosus) is an ancient osteoglossid fish from southeast Asia. Due to the high demand of the ornamental fish trade and because of habitat loss, the species is close to extinction. We isolated and characterized 10 polymorphic microsatellites of this species, using 5'-anchored PCR. The number of alleles at the 10 microsatellite loci ranged from 2 to 28, with a mean of 7.8/locus. The observed heterozygosity ranged from 0.03 to 0.93 (mean: 0.39), whereas the expected heterozygosity ranged from 0.03 to 0.94 (mean: 0.46). Seven microsatellites deviated from Hardy-Weinberg equilibrium, and three conformed to Hardy-Weinberg equilibrium and were in linkage equilibrium. These 10 novel microsatellites should facilitate studies of genetic diversity and population structure of arowana to help plan actions for the conservation of the indigenous Malaysian arowana.
Diabetic retinopathy is the most common diabetic eye disease, occurring in about 60% of type 2 diabetic patients. Other than known clinical risk factors, the influence of genes has been suggested as part of the development of diabetic retinopathy. We investigated the association of Gly82Ser, 1704G/T and 2184A/G polymorphisms in the RAGE gene with retinopathy in type 2 diabetic patients in Malaysia. Ninety-eight unrelated retinopathy patients and 185 unrelated healthy controls from all over Malaysia were recruited in this study. The allele and genotype frequencies of the three gene polymorphisms were investigated using PCR-RFLP. The allele frequency of the three polymorphisms did not differ significantly between the control and the retinopathy group (P > 0.05). Analysis of the frequency of GA+AA, GT+TT and AG+GG in the retinopathy group did not reveal significant differences (P > 0.05) compared to the control group. We conclude that RAGE gene Gly82Ser, 1704G/T and 2184A/G polymorphisms are not associated with retinopathy development in the Malaysian population.
Regulated on activation, normal T-cell expressed and secreted (RANTES) and stromal cell-derived factor 1 (SDF-1) are members of the CC- and CXC-chemokine families, respectively. Both genes have been postulated to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We analyzed position 28 of the RANTES gene promoter region, as well as the SNP observed in the 3' UTR of the SDF-1 gene at position 801, in 130 patients presenting SLE at the Malaya University Medical Centre. Screening of 130 healthy volunteer controls using RFLP was also performed. RANTES-28 polymorphism analysis showed no significant (P = 0.3520) relationship, even though homozygous C/C was more frequent in SLE patients (OR = 1.4183) and heterozygous C/G was more frequent in healthy controls (OR = 0.7051). There were no significant (P = 0.2650) associations between A/A (OR = 0.783), G/G (OR = 1.5914) and G/A (OR = 0.8289) genotypes in the SDF-1 gene polymorphism with SLE. We conclude that there is no significant association of RANTES-28 and SDF-1 gene polymorphisms and occurrence of SLE in Malaysia.
This study aimed to investigate the potential association of TYK2 and STAT3 genes with the susceptibility to Crohn's disease (CD) among Malaysians. DNA samples were obtained from 80 CD patients and 100 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism methods were employed for genotyping, followed by statistical analysis. In our current study, none of the single nucleotide polymorphisms of either TYK2 or STAT3 was statistically associated with the susceptibility to CD in our local population (P > 0.05). In contrast, there was a statistically significant association between the G/G homozygotes of the STAT3 rs2293152 and the healthy control group (χ(2) = 6.229, P < 0.05). In conclusion, our study does not support the role of the TYK2 and STAT3 genes influencing CD susceptibility.
The mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) gene has been universally and successfully utilized as a barcoding gene, mainly because it can be amplified easily, applied across a wide range of taxa, and results can be obtained cheaply and quickly. However, in rare cases, the gene can fail to distinguish between species, particularly when exposed to highly sensitive methods of data analysis, such as the Bayesian method, or when taxa have undergone introgressive hybridization, over-splitting, or incomplete lineage sorting. Such cases require the use of alternative markers, and nuclear DNA markers are commonly used. In this study, a dendrogram produced by Bayesian analysis of an mtDNA COI dataset was compared with that of a nuclear DNA ATPS-α dataset, in order to evaluate the efficiency of COI in barcoding Malaysian nerites (Neritidae). In the COI dendrogram, most of the species were in individual clusters, except for two species: Nerita chamaeleon and N. histrio. These two species were placed in the same subcluster, whereas in the ATPS-α dendrogram they were in their own subclusters. Analysis of the ATPS-α gene also placed the two genera of nerites (Nerita and Neritina) in separate clusters, whereas COI gene analysis placed both genera in the same cluster. Therefore, in the case of the Neritidae, the ATPS-α gene is a better barcoding gene than the COI gene.
The serotoninergic system has been implicated in the etiology of schizophrenia and other behavioral disorders. Association studies have focused on the tryptophan hydroxylase 2 gene (TPH2) and the 5-hydroxytryptamine receptor 2A gene (5-HTR2A). We genotyped two single-nucleotide polymorphisms, A1438G of 5-HTR2A and intronic rs1386494 of TPH2 in the Malay population, using a sample size of 289 schizophrenic patients and 130 healthy controls. We found a significant association of A1438G of 5-HTR2A with schizophrenia in Malays. On the other hand, TPH2 polymorphism was not associated with schizophrenia. This is the first genetic association study concerning schizophrenia in the Malay population.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal recessive disorder associated with mutations in the thymidine phosphorylase (TYMP) gene. The main objective of this study was to characterize the genetic profiles of the deceased proband's family members (N = 4) using DNA sequencing and to determine miRNA deregulation in MNGIE using miRNA microarray profiling and bioinformatic analysis. We found that the genetic profile of the younger sister showed similar TYMP gene mutations as that of the proband with the exception of a heterozygous mutation in exon 10. The miRNA microarray revealed 55 significantly up-regulated and 65 significantly down-regulated miRNAs. These miRNAs have been implicated in various mitochondrial dynamics such as energy metabolism, Krebs cycle, mitochondria-associated apoptosis, and mitophagy. In conclusion, we demonstrate that blood miRNAs are deregulated in the pathogenesis of MNGIE and these changes may have therapeutic implications. Further experimental studies will be required to elucidate the functional miRNA-mRNA interactions in MNGIE.
Plants have been used throughout human history for food and medicine. However, many plants are toxic, and cannot easily be morphologically distinguished from non-toxic plants. DNA identification solves this problem and is widely used. Nonetheless, plant DNA barcode identification faces a number of challenges, and many studies have been conducted to find suitable barcodes. The present study was conducted to test the efficiency of commonly used primers, namely ITS2, rpoC1, and trnH-psbA, in order to find the best DNA barcode markers for the identification of medicinal plants in Malaysia. Fresh leaves from 12 medicinal plants that are commonly used by Malay traditional healers were collected from the Tropical Spice Garden, Pulau Pinang, and subjected to polymerase chain reaction amplification using ITS2, rpoC1, and trnH-psbA DNA markers. We found that trnH-psbA is the best DNA marker for the species-level identification of medicinal plants in Malaysia.
Mackerel (Scombridae; Rastrelliger) are small commercially important pelagic fish found in tropical regions. They serve as a cheap source of animal protein and are commonly used as live bait. By using a truss morphometrics protocol and RAPD analysis, we examined morphological and genetic variation among 77 individual mackerel that were caught using long lines and gillnets at 11 locations along the west coast of Peninsular Malaysia. Nineteen morphometric traits were evaluated and genetic information was estimated using five 10-base RAPD random primers. Total DNA was extracted from muscle tissue. Morphometric discriminant function analysis revealed that two morphologically distinct groups of Rastrelliger kanagurta and a single group of R. brachysoma can be found along the west coast of Peninsular Malaysia. We also found that the head-related characters and those from the anterior part of the body of Rastrelliger spp significantly contribute to stock assessment of this population. RAPD analysis showed a trend similar to that of the morphometric analysis, suggesting a genetic component to the observed phenotypic differentiation. These data will be useful for developing conservation strategies for these species.
Colorectal cancer (CRC) is one of the most common types of cancer in both developed and developing countries. This disease is triggered by and progresses via the sequential accumulation of multiple genetic alterations. In addition, the interaction between low-penetrance genes and environmental factors can also increase the risk of developing CRC. Since inflammatory bowel diseases (IBDs) are one of the predisposing factors for CRC, IBD-related genes might, to a certain extent, be associated with cancer initiation. The nucleotide oligomerization domain 2/caspase activating recruitment domain 15 gene (NOD2/CARD15) is the most well-established gene to be associated with increased susceptibility to Crohn's disease. Thus, various studies have been performed to investigate the potential contribution of this gene to CRC risk. In this study, we aimed to determine the frequency of the Arg702Trp, Gly908Arg, 3020insC, Pro268Ser, and JW1 variants of NOD2/CARD15, and to investigate their association with CRC susceptibility. A total of 130 CRC patients and 212 healthy controls were recruited for this study. Subsequently, real-time polymerase chain reaction with TaqMan was performed for the genotyping of these NOD2/ CARD15 variants. None of the NOD2/CARD15 variants was statistically associated to CRC susceptibility in our Malaysian population. Our findings were remarkably similar to those of other Asian cohorts, which indicated that these NOD2/CARD15 variants exhibit genetic heterogeneity between Caucasian and Asian populations.