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.
We evaluated the possible influence of glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genes on genetic damage due to occupational exposure, which contributes to accelerate ageing. This study was conducted on 120 car auto repair workshop workers exposed to occupational hazards and 120 controls without this kind of exposure. The null and non-null genotypes of GSTM1 and GSTT1 genes were determined by multiplex PCR. Micronucleus frequency, Comet tail length and relative telomere length differences between the null and non-null genotypes of the GSTM1 gene were significantly greater in the exposed group. Lack of GSTT1 did not affect the damage biomarkers significantly (P > 0.05), while lack of GSTM1 was associated with greater susceptibility to genomic damage due to occupational exposure. It was concluded that early ageing is under the influence of these genes and the environmental and socio-demographic factors. Duration of working time was significantly associated with micronucleus frequency, Comet tail length and relative telomere length.
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.
The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.
Studies of genetic mutations that have been used in predicting glioma prognosis have revealed a complex relationship between clinical and genetic factors. Epidermal growth factor (EGF) and the NAT2 gene play a central role in carcinogenesis. An adenine (A) to guanine (G) single nucleotide polymorphism at position 61 in the 5'-untranslated region (5'-UTR) of the EGF gene has been found to be associated with levels of EGF production, and the mutations in the NAT2 gene have been postulated as a risk factor for cancer. We investigated EGF and the NAT2 gene in 13 glioma tissue samples and 12 normal controls. In the EGF 5'-UTR 61G polymorphism, the heterozygote GA was the most common genotype in the glioma patients. In the NAT2 polymorphism at nucleotide position 857G/A, the G allele and the GG genotype were the most prevalent forms in both the glioma and normal samples. We did not find any homozygous AA genotypes in the glioma patients. Based on this preliminary evidence, the EGF 5'-UTR at position 61 and the NAT2 SNP at position 857 polymorphisms are associated with increased risk for glioma.
Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.
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.
DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.
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.
Malaysia remains as a crossroad of different cultures and peoples, and it has long been recognized that studying its population history can provide crucial insight into the prehistory of Southeast Asia as a whole. The earliest inhabitants were the Orang Asli in Peninsular Malaysia and the indigenous groups in Sabah and Sarawak. Although they were the earliest migrants in this region, these tribes are divided geographically by the South China Sea. We analyzed DNA sequences of 18 Orang Asli using mitochondrial DNA extracted from blood samples, each representing one sub-tribe, and from five Sarawakian Iban. Mitochondrial DNA was extracted from hair samples in order to examine relationships with the main ethnic groups in Malaysia. The D-loop region and cytochrome b genes were used as the candidate loci. Phylogenetic relationships were investigated using maximum parsimony and neighbor joining algorithms, and each tree was subjected to bootstrap analysis with 1000 replicates. Analyses of the HVS I region showed that the Iban are not a distinct group from the Orang Asli; they form a sub-clade within the Orang Asli. Based on the cytochrome b gene, the Iban clustered with the Orang Asli in the same clade. We found evidence for considerable gene flow between Orang Asli and Iban. We concluded that the Orang Asli, Iban and the main ethnic groups of Malaysia are probably derived from a common ancestor. This is in agreement with a single-route migration theory, but it does not dismiss a two-route migration theory.
Contamination of insect DNA for RAPD-PCR analysis can be a problem because many primers are non-specific and DNA from parasites or gut contents may be simultaneously extracted along with that of the insect. We measured the quantity of food ingested and assimilated by two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice and the other from Leersia hexandra (Poaceae), a wetland forage grass, and we also investigated whether host plant DNA contaminates that of herbivore insects in extractions of whole insects. Ingestion and assimilation of food were reduced significantly when individuals derived from one host plant were caged on the other species. The bands, OPA3 (1.25), OPD3 (1.10), OPD3 (0.80), OPD3 (0.60), pUC/M13F (0.35), pUC/M13F (0.20), BOXAIR (0.50), peh#3 (0.50), and peh#3 (0.17) were found in both rice-infesting populations of brown planthopper and its host plant (rice). Similarly, the bands, OPA4 (1.00), OPB10 (0.70), OPD3 (0.90), OPD3 (0.80), OPD3 (0.60), pUC/ M13F (0.35), pUC/M13F (0.20), and BOXAIR (0.50) were found in both Leersia-infesting populations of brown planthopper and the host plant. So, it is clear that the DNA bands amplified in the host plants were also found in the extracts from the insects feeding on them.
Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.
Molecular components of the dopamine D3 receptor (DRD3) may play an important role in the pathophysiology of schizophrenia. Previous studies have demonstrated an association between DRD3 Ser9Gly and cathechol-o-methyltransferase (COMT, SNP = rs165656) polymorphisms and schizophrenia but the results were inconclusive. We investigated this apparent association between Ser9Gly (A/G) polymorphism and an intronic SNP (dbSNP or rs165656) in 261 Malay patients diagnosed with schizophrenia and 216 controls, using PCR-RFLP. The genotype distribution of the polymorphism DRD3 Ser9Gly was in Hardy-Weinberg equilibrium (HWE) for patients (P = 0.1251) and out of HWE for controls (P = 0.0137). However, both healthy controls and schizophrenia patients were out of HWE for the polymorphism COMT rs165656. Based on allele and genotype frequencies in both groups, we found no significant association of DRD3 Ser9Gly polymorphisms and COMT (rs165656) with schizophrenia in Malays. Further studies should examine the association between other dopamine-related genes and the behavioral phenotypes of schizophrenia.
Blood cockles are among the most economically important brackish water invertebrates found in Malaysia. However, our knowledge of blood cockle phylogeny and systematics is rudimentary, especially for the species Tegillarca granosa. It is unclear, for instance, whether the cockles occurring on the west coast of peninsular Malaysia constitute a single species, or multiple, phylogenetically distinct species. We performed the first DNA molecular phylogenetic analysis of T. granosa to distinguish it from other related species found in other parts of the world and to create a DNA database for the species. An approximately 585-nucleotide fragment of the mitochondrial DNA (cytochrome oxidase I, COI) was sequenced for 150 individual cockles, representing 10 populations: three from the north, four from the central part and three from the southern part of peninsular Malaysia. Phylogenetic analyses of the resulting dataset yielded tree topologies that not only showed the relationship between T. granosa and its closest relatives but its position in the evolutionary tree. Three mitochondrial clades were evident, each containing an individual genus. Using the mutation rate of the COI gene, the divergence time between T. granosa and its closest related species was estimated to be 460 thousand years ago. This study provides a phylogenetic framework for this ecologically prominent and commercially important cockle species.
Allele frequencies of 15 short tandem repeat (STR) loci, namely D5S818, D7S820, D13S317, D16S539, TH01, TPOX, Penta D, Penta E, D3S1358, D8S1179, D18S51, D21S11, CSF1PO, vWA, and FGA, were determined for 154 individuals from the Kadazan-Dusun tribe, an indigenous population of East Malaysia. All loci were amplified by polymerase chain reaction, using the Powerplex 16 system. Alleles were typed using a gene analyzer and the Genemapper ID software. Various statistical parameters were calculated and the combined power of discrimination for the 15 loci in the population was calculated as 0.999999999999999. These loci are thus, informative and can be used effectively in forensic and genetic studies of this indigenous population.
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.
The white-bellied sea eagle, Haliaeetus leucogaster, displays reversed sexual size dimorphism and is monomorphic for adult plumage coloration. Early attempts to identify sex in sexually monomorphic birds were based on morphological or chromosomal characters, but since avian W-specific DNA sequences were identified, PCR amplification has become commonly used for molecular sexing. We used a PCR test employing primers that amplify two homologous fragments of both the CHD-W gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with high reproducibility. We conclude that this PCR-based test with feathers as the DNA source is a reliable sexing method for H. leucogaster. This sexing technique is objective and non-invasive and could be used to test sex ratio theories, as well as to help improve conservation and management actions for captive breeding program of this species in Malaysia.
Variable number of tandem repeats (VNTR) polymorphism in the interleukin 4 (IL-4) gene has been associated with end-stage renal disease (ESRD) subjects in many different populations, although with conflicting results. We determined the 70 bp of VNTR polymorphism at intron 3 of the IL-4 gene in Malaysian ESRD subjects. Buccal cells were collected from 160 case and 160 control subjects; genomic DNA was amplified using PCR, followed by agarose gel electrophoresis. There were significant differences in genotypes and alleles of the IL-4 gene. We conclude that VNTR polymorphism of the IL-4 gene is a risk factor for the development of ESRD among Malaysians.
Epinephelus fuscoguttatus is a commercially important marine fish species in southeast Asia. Due to overfishing and water pollution, this species has been declared as near-threatened. Thus, to provide information to help maintain and preserve the species, microsatellites were developed, using an enriched genomic library method. Thirty individuals were collected from the hatchery of the Fishery Research Institute, Terengganu, Malaysia. These individuals, from four to six years old, originated from Sabah and are maintained in captive culture as broodstock. Genomic DNA was extracted from the fins of selected individuals that weighed 3-8 kg. Ten microsatellite loci were found to be polymorphic in this population, with 5 to 21 alleles per locus. Observed and expected heterozygosities ranged from 0.53 to 0.97 and 0.59 to 0.95, respectively. Only one locus deviated significantly from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found among the pairs of loci. These polymorphic microsatellite loci will be used by the Malaysian Fishery Research Institute for investigating genetic diversity and for developing breeding strategies.