The presence or absence of two DNA modification systems, XorI and XorII, in 195 strains of Xanthomonas oryzae pv. oryzae collected from different major rice-growing countries of Asia was assessed. All four possible phenotypes (XorI+ XorII+, XorI+ XorII-, XorI- XorII+ and XorI- XorII-) were detected in the population at a ratio of approximately 1:2:2:2. The XorI+ XorII+ and XorI- XorII+ phenotypes were observed predominantly in strains from southeast Asia (Philippines, Malaysia, and Indonesia), whereas strains with the phenotypes XorI- XorII- and XorI+ XorII- were distributed in south Asia (India and Nepal) and northeast Asia (China, Korea, and Japan), respectively. Based on the prevalence and geographic distribution of the XorI and XorII systems, we suggest that the XorI modification system originated in northeast Asia and was later introduced to southeast Asia, while the XorII system originated in southeast Asia and moved to northeast Asia and south Asia. Genomic DNA from all tested strains of X. oryzae pv. oryzae that were resistant to digestion by endonuclease XorII or its isoschizomer PvuI also hybridized with a 7.0-kb clone that contained the XorII modification system, whereas strains that were digested by XorII or PvuI lacked DNA that hybridized with the clone. Size polymorphisms were observed in fragments that hybridized with the 7.0-kb clone. However, a single hybridization pattern generally was found in XorII+ strains within a country, indicating clonal maintenance of the XorII methyl-transferase gene locus. The locus was monomorphic for X. oryzae pv. oryzae strains from the Philippines and all strains from Indonesia and Korea.
Matched MeSH terms: DNA Restriction Enzymes/metabolism*
The modelling of splicing systems is simulated by the process of cleaving and recombining DNA molecules with the presence of a ligase and restriction enzymes which are biologically called as endodeoxyribonucleases. The molecules resulting from DNA splicing systems are known as splicing languages. Palindrome is a sequence of strings that reads the same forward and backward. In this research, the splicing languages resulting from DNA splicing systems with one non-palindromic restriction enzyme are determined using the notation from Head splicing system. The generalisations of splicing languages for DNA splicing systems involving a cutting site and two non-overlapping cutting sites of one non-palindromic restriction enzyme are presented in the first and second theorems, respectively, which are proved using direct and induction methods. The result from the first theorem shows a trivial string which is the initial DNA molecule; while the second theorem determines a splicing language consisting of a set of resulting DNA molecules from the respective DNA splicing system.
In DNA splicing system, the potential effect of sets of restriction enzymes and
a ligase that allow DNA molecules to be cleaved and re-associated to produce further
molecules is modelled mathematically. This modelling is done in the framework of formal
language theory, in which the nitrogen bases, nucleotides and restriction sites are modelled
as alphabets, strings and rules respectively. The molecules resulting from a splicing system
is depicted as the splicing language. In this research, the splicing language resulting from
DNA splicing systems with one palindromic restriction enzyme for one and two (nonoverlapping)
cutting sites are generalised as regular expressions.
Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.
Matched MeSH terms: DNA Restriction Enzymes/economics; DNA Restriction Enzymes/chemistry*
The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.
Matched MeSH terms: DNA Restriction Enzymes/metabolism
The genome of the human gastric pathogen Helicobacter pylori encodes a large number of DNA methyltransferases (MTases), some of which are shared among many strains, and others of which are unique to a given strain. The MTases have potential roles in the survival of the bacterium. In this study, we sequenced a Malaysian H. pylori clinical strain, designated UM032, by using a combination of PacBio Single Molecule, Real-Time (SMRT) and Illumina MiSeq next generation sequencing platforms, and used the SMRT data to characterize the set of methylated bases (the methylome).
Matched MeSH terms: DNA Restriction Enzymes/metabolism
Hematological and clinical data are presented for a young Malay patient with a homozygous (delta beta)zero-thalassemic condition. His red blood cells contained 100% fetal hemoglobin with alpha and G gamma chains only. Detailed gene mapping defined a large deletion with a 5' end between the Aha III and Apa I sites, some 200-400 bp 5' to the A gamma globin gene and a 3' end beyond sequences 17-18 kb 3' to the beta globin gene. This G gamma (A gamma delta beta)zero-type of thalassemia is different from all the other six types described before. Comparison of the hematological data of this patient with those of homozygotes for either the Sicilian or Spanish types of G gamma A gamma (delta beta)zero-thalassemia showed no differences; all homozygotes have a moderate anemia which is accentuated by the relatively high oxygen affinity of the Hb F containing erythrocytes.
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.
Determination of feline meat in food products is an important issue for social, health, economic and religious concern. Hence this paper documented the application of species specific polymerase chain reaction-restriction fragment length polymorphism (SP-PCR-RFLP) assay targeting a short-fragments (69 bp) of mitochondrial cytochrome b (cytb) gene to screen feline meat in commercial meat products using lab-on-a-chip. The SP-PCR assay proved its specificity theoretically and experimentally while testing with different common animal, aquatic and plant species of DNA. The feline specific (69 bp, 43- and 26-bp) characteristic molecular DNA pattern was observed by SP-PCR and RFLP analysis. For assay performance, it was tested in three different types of commercial dummy meat products such as frankfurters, nuggets and meatballs and digested with AluI-restriction enzyme. The highest sensitivity of the assay using lab-on-a-chip was as low as 0.1 pg or 0.01 % (w/w) in commercial dummy meat products. We have also applied this assay to screen three important commercial meat products of six different brand from six supermarket chains located at three different states of Malaysia. Thus total 378 samples were tested to validate the specificity, sensitivity, stability of the assay and utilization of it for commercial meat product screening.
Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins. Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms.
The need to detect genetic variation has fueled the development of novel marker systems in fisheries biology. In this study, a simple, fast and cost effective method was used to differentiate between species of freshwater fishes focusing on Malaysian freshwater fishes by employing
Restriction Fragment Length Polymorphisms (RFLPs) analysis of a 470-bp cytochrome b mtDNA segment. RFLP analysis using six restriction enzymes (AluI, BamHI, BsuRI, Csp61, HpaII and SalI) found variations in the digestion profile among most of the fish samples analyzed. Diagnostic digestion profiles were observed among the Hampala fishes, especially between H. macrolepidota and the other Hampala species/forms (using BsuRI and Csp61). Diagnostic digestion profiles were also detected between H.
bimaculata Type A and Type B (using AluI, BamHI, BsuRI and SalI), supporting their status as distinct species. Additionally, unique digestion profiles were observed in other species such as Leptobarbus hosii (Csp61), Osteocheilus hasseltii (Csp61), Osteocheilus sp. (Csp61), Puntioplites bulu (Csp61), Puntius bramoides (AluI), P. sealei (AluI) and Helostoma temmincki (AluI and Csp61), which can be used as genetic markers for discriminating these species. Overall, the RFLP analysis of the cytochrome
b mtDNA segment has proven to be a considerably effective, fast and non-expensive technique to discriminate among several freshwater fish species in Malaysia.
A method of PCR-restriction fragment length polymorphism (RFLP) has been utilized to differentiate the mitochondrial genes of pork and wild boar meat (Sus scrofa). The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of these two meats. The amplification product of pork and wild boar using mt-12S rRNA gene successfully produced a single band with molecular size of 456 bp. Three restriction endonucleases (AluI, HindIII and BsaJI) were used to restrict the amplification products of the mitochondrial genes. The restriction enzymes of AluI and BsaJI were identified as potential restriction endonucleases to differentiate those meats. HindIII enzyme was unable to restrict the PCR product of both meats. The genetic differences within the cyt b gene among the two meats were successfully confirmed by PCR-RFLP analysis.
A small plasmid designated pAR141 was isolated from Lactococcus lactis subsp. lactis M14 and its complete 1,594 base pair nucleotide sequence was determined. Analysis of the sequence indicated that this plasmid does not carry any industrially important determinants besides the elements involved in plasmid replication and control. The transcriptional repressor CopG and replication initiation protein RepB appeared as a single operon. A small countertranscribed RNA (ctRNA) coding region was found between the copG and repB genes. The double strand origin (dso) and single strand origin (sso) of rolling circle replicating (RCR) plasmids were also identified in pAR141, suggesting that this plasmid replicates by rolling circle (RC) mode. This observation was supported by S1 nuclease and Southern hybridization analyses.
Matched MeSH terms: DNA Restriction Enzymes/pharmacology
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
Matched MeSH terms: DNA Restriction Enzymes/metabolism
A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.
Matched MeSH terms: DNA Restriction Enzymes/metabolism
Molecular analysis of chromosomal DNA from 193 multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates from 1990 to 1995 from Pakistan, Kuwait, Malaysia, Bangladesh, and India produced a total of five major different pulsed-field gel electrophoresis (PFGE) patterns. Even within a particular country MDR S. enterica serovar Typhi DNA was found to be in different PFGE groups. Similar self-transferable 98-MDa plasmids belonging to either incompatibility group incHI1 or incHI1/FIIA were implicated in the MDR phenotype in S. enterica serovar Typhi isolates from all the locations except Quetta, Pakistan, where the majority were of incFIA. A total of five different PFGE genotypes with six different plasmids, based on incompatibility and restriction endonuclease analysis groups, were found among these MDR S. enterica serovar Typhi isolates.
Matched MeSH terms: DNA Restriction Enzymes/metabolism
Fusarium species section Liseola namely F. fujikuroi, F. proliferatum, F. andiyazi, F. verticillioides, and F. sacchari are well-known plant pathogens on rice, sugarcane and maize. In the present study, restriction analysis of the intergenic spacer regions (IGS) was used to characterize the five Fusarium species isolated from rice, sugarcane and maize collected from various locations in Peninsular Malaysia. From the analysis, and based on restriction patterns generated by the six restriction enzymes, Bsu151, BsuRI, EcoRI, Hin6I, HinfI, and MspI, 53 haplotypes were recorded among 74 isolates. HinfI showed the most variable restriction patterns (with 11 patterns), while EcoRI showed only three patterns. Although a high level of variation was observed, it was possible to characterize closely related species and isolates from different species. UPGMA cluster analysis showed that the isolates of Fusarium from the same species were grouped together regardless of the hosts. We conclude that restriction analysis of the IGS regions can be used to characterize Fusarium species section Liseola and to discriminate closely related species as well as to clarify their taxonomic position.
A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.
Most of the population in certain areas of Melanesia have one alpha-globin gene deletion (alpha thal2). It is thought that the high frequencies of alpha thal2 in this population is due to a selective advantage given by malaria infection to carriers of alpha thal2. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to 32P-labeled alpha-globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated alpha-globin loci type 1, while none had alpha thal2. On digestion with Bgl II the third alpha-globin gene was found in an additional 3.7 kb fragment in all seven Samoans with triplicated alpha-globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying alpha-globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had alpha thal2 or alpha thal1. Only two of the Malaysians had triplicated alpha-globin loci.
In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.