Displaying publications 21 - 40 of 94 in total

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  1. Molecular Characterization of Avian Infectious Bronchitis Virus Isolated in Malaysia during 2014-2016
    Leow BL, Syamsiah Aini S, Faizul Fikri MY, Muhammad Redzwan S, Khoo CK, Ong GH, et al.
    Trop Biomed, 2018 Dec 01;35(4):1092-1106.
    PMID: 33601856
    Avian Infectious Bronchitis (IB) is a highly contagious disease which can cause huge economic losses to the poultry industry. Forty five IB viruses (IBV) were isolated from poultry in Malaysia during 2014-2016. Phylogenetic analysis of the spike glycoprotein 1 (S1) gene revealed that all isolates were clustered into five distinct groups. The predominant type of IBV isolated was QX strains (47%), second was 4/91 type (27%), followed by Malaysian strain MH5365/95 (13%), Massachusetts type (11%) and finally Taiwanese strains (2%). Four types of S1 protein cleavage recognition motifs were found among the isolates which includes HRRRR, RRSRR, RRFRR and RRVRR. To our knowledge, this is the first report describing the motif RRVRR and are unique to Malaysian strains. Six IBVs were grouped in Malaysian MH5365/95 strains. Among these, one isolate was different from others where it only shared 82% identity with MH5365/95 and to others. It formed its own branch in the Malaysian cluster suggesting it may be a variant unique to Malaysia. Alignment analysis of the S1 amino acid sequences indicated that point mutations, insertions and deletions contribute to the divergence of IB variants. This study indicated at least five groups of IBV are circulating in Malaysia with most of the isolates belonged to QX strains. As new IBV variants continue to emerge, further study need to be carried out to determine whether the current available vaccine is able to give protection against the circulating virus.
    Matched MeSH terms: Mutagenesis, Insertional
  2. Fulltext Effects of acute and chronic gamma irradiation on in vitro growth of stevia rebaudiana bertoni
    Norazlina Noordin, Rusli Ibrahim, Nur Hidayah Mohd Sajahan, Salmah Moosa, Sobri Hussein
    Jurnal Sains Nuklear Malaysia, 2014;26(2):17-25.
    MyJurnal
    Stevia rebaudiana Bertoni is a perennial herb that belongs to the family of Asteraceae. It is a
    natural sweetener plant known as sweet leaf, which is estimated to be 300 times sweeter than cane
    sugar. In this study, micropropagation and in vitro mutagenesis of this natural herb was
    successfully conducted. It was found that shoot tips on MS medium supplemented with 1 mg/l
    Kinetin showed the highest shoot induction and multiplication after 3 weeks of culture
    (5.50±1.95a). Radiosensitivity test was conducted to identify the dose that killed 50% of the
    irradiated explants (LD50) for in vitro stevia shoots and to select effective doses to be used for the in
    vitro mutagenesis. Shoot tips were irradiated with acute and chronic gamma radiation at 0, 10, 20,
    30, 40, 60, and 80Gy. At 60 Gy and 80 Gy, all treated shoot tips were not survived. In this study,
    LD50 for the stevia was estimated at 29 Gy for acute irradiation and 45 Gy for chronic irradiation.
    The effective doses were selected at 10, 20, 30 and 40 Gy. These selected doses were applied for the
    in vitro mutagenesis of the stevia shoots
    Matched MeSH terms: Mutagenesis
  3. Fulltext Enhancement of a protocol purifying T1 lipase through molecular approach
    Che Hussian CHA, Raja Abd Rahman RNZ, Thean Chor AL, Salleh AB, Mohamad Ali MS
    PeerJ, 2018;6:e5833.
    PMID: 30479887 DOI: 10.7717/peerj.5833
    T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.
    Matched MeSH terms: Mutagenesis, Site-Directed
  4. Fulltext Mutagenicity and antimutagenic activities of lactic acid bacteria (LAB) isolated from fermented durian (tempoyak)
    Asmariah Ahmad, Safura Salik, Yap Wei Boon, Ahmad Rohi Ghazali, Noorhisham Tan Kofli
    Jurnal Sains Kesihatan Malaysia, 2018;16(101):23-26.
    MyJurnal
    Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer.
    Matched MeSH terms: Mutagenesis
  5. An in vitro study to evaluate the genotoxicity of value added hydroxyapatite as a bone replacement material
    Akram Hassan, Swaminathan D
    Sains Malaysiana, 2011;40.
    Hydroxyapatite (HA) used for bone replacement is one of the most active areas of ceramic biomaterials research currently. It has been used clinically for the last 20 years due to its excellent biocompatibility, osseoconduction and osseointegration. Many modifications have been done to develop a stronger, tougher and biocompatible ceramic biomaterial because pure HA is brittle. Researchers in Universiti Sains Malaysia had developed this value added HA that is stronger and less brittle compared to pure HA. The objective of this in vitro study was to evaluate the genotoxic characteristic of the value added HA based material by using Bacterial Reverse Mutation Assay (Ames test). The Bacterial Reverse Mutation Assay of HA was performed on Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA using the preincubation method in the presence and absence of an exogenous metabolic activation system. All the bacterial tester strains treated with and without S9 Mix showed no increase of revertant colonies with increase in concentration of test substance for both the dose finding test and the main test. The number of revertant colonies was less than twice that of the solvent control for all the five bacterial strains and this was reproducible for both the dose finding test and the main test. The numbers of revertant colonies in the negative and positive controls were within the background data of our laboratory. In conclusion the results of the tests showed that the value added HA was considered to have no reverse mutagenic potential under the present test conditions.
    Matched MeSH terms: Mutagenesis
  6. A review of in vitro stochastic and non-stochastic affinity maturation strategies for phage display derived monoclonal antibodies
    Nur A, Lai JY, Ch'ng ACW, Choong YS, Wan Isa WYH, Lim TS
    Int J Biol Macromol, 2024 Oct;277(Pt 2):134217.
    PMID: 39069045 DOI: 10.1016/j.ijbiomac.2024.134217
    Monoclonal antibodies identified using display technologies like phage display occasionally suffers from a lack of affinity making it unsuitable for application. This drawback is circumvented with the application of affinity maturation. Affinity maturation is an essential step in the natural evolution of antibodies in the immune system. The evolution of molecular based methods has seen the development of various mutagenesis approaches. This allows for the natural evolutionary process during somatic hypermutation to be replicated in the laboratories for affinity maturation to fine-tune the affinity and selectivity of antibodies. In this review, we will discuss affinity maturation strategies for mAbs generated through phage display systems. The review will highlight various in vitro stochastic and non-stochastic affinity maturation approaches that includes but are not limited to random mutagenesis, site-directed mutagenesis, and gene synthesis.
    Matched MeSH terms: Mutagenesis
  7. Fulltext Combination of oxyanion Gln114 mutation and medium engineering to influence the enantioselectivity of thermophilic lipase from Geobacillus zalihae
    Wahab RA, Basri M, Rahman MB, Rahman RN, Salleh AB, Leow TC
    Int J Mol Sci, 2012;13(9):11666-80.
    PMID: 23109876 DOI: 10.3390/ijms130911666
    The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  8. Fulltext Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli
    Jonet MA, Mahadi NM, Murad AM, Rabu A, Bakar FD, Rahim RA, et al.
    J. Mol. Microbiol. Biotechnol., 2012;22(1):48-58.
    PMID: 22456489 DOI: 10.1159/000336524
    A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  9. Manipulation of the conformation and enzymatic properties of T1 lipase by site-directed mutagenesis of the protein core
    Wahab RA, Basri M, Rahman RN, Salleh AB, Rahman MB, Chor LT
    Appl Biochem Biotechnol, 2012 Jun;167(3):612-20.
    PMID: 22581079 DOI: 10.1007/s12010-012-9728-2
    In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  10. Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase
    Ismail A, Illias RM
    J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
    PMID: 28921081 DOI: 10.1007/s10295-017-1980-6
    The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  11. Genes associated with amyloid-beta-induced inflammasome-mediated neuronal death identified using functional gene trap mutagenesis approach
    Yap JKY, Pickard BS, Gan SY, Chan EWL
    Int J Biochem Cell Biol, 2021 07;136:106014.
    PMID: 34022435 DOI: 10.1016/j.biocel.2021.106014
    Alzheimer's disease is an irreversible neurodegenerative disease, which accounts for most dementia cases. Neuroinflammation is increasingly recognised for its roles in Alzheimer's disease pathogenesis which, in part, links amyloid-beta to neuronal death. Neuroinflammatory signalling can be exhibited by neurons themselves, potentially leading to widespread neuronal cell death, although neuroinflammation is commonly associated with glial cells. The presence of the inflammasomes such as nucleotide-binding leucine-rich repeat receptors protein 1 in neurons accelerates amyloid-beta -induced neuroinflammation and has been shown to trigger neuronal pyroptosis in murine Alzheimer's disease models. However, the pathways involved in amyloid-beta activation of inflammasomes have yet to be elucidated. In this study, a gene trap mutagenesis approach was utilised to resolve the genes functionally involved in inflammasome signalling within neurons, and the mechanism behind amyloid-beta-induced neuronal death. The results indicate that amyloid-beta significantly accelerated neuroinflammatory cell death in the presence of a primed inflammasome (the NLR family pyrin domain-containing 1). The mutagenesis screen discovered the atypical mitochondrial Ras homolog family member T1 as a significant contributor to amyloid-beta-induced inflammasome -mediated neuronal death. The mutagenesis screen also identified two genes involved in transforming growth factor beta signalling, namely Transforming Growth Factor Beta Receptor 1 and SNW domain containing 1. Additionally, a gene associated with cytoskeletal reorganisation, SLIT-ROBO Rho GTPase Activating Protein 3 was found to be neuroprotective. In conclusion, these genes could play important roles in inflammasome signalling in neurons, which makes them promising therapeutic targets for future drug development against neuroinflammation in Alzheimer's disease.
    Matched MeSH terms: Mutagenesis*
  12. Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function
    Goh KM, Liew KJ, Chai KP, Illias RM
    Methods Mol Biol, 2017;1498:385-396.
    PMID: 27709591
    Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods
  13. Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified
    Rasineni GK, Loh PC, Lim BH
    Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
    PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027
    BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.

    METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.

    RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.

    CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.

    GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

    Matched MeSH terms: Mutagenesis, Site-Directed/methods
  14. Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants
    Dong AN, Pan Y, Palanisamy UD, Yiap BC, Ahemad N, Ong CE
    Appl Biochem Biotechnol, 2018 Sep;186(1):132-144.
    PMID: 29524040 DOI: 10.1007/s12010-018-2728-0
    Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  15. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture
    Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, et al.
    Nat Biotechnol, 2016 Jun;34(6):652-5.
    PMID: 27111722 DOI: 10.1038/nbt.3543
    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  16. Fulltext Genome-Wide Discovery of Genes Required for Capsule Production by Uropathogenic Escherichia coli
    Goh KGK, Phan MD, Forde BM, Chong TM, Yin WF, Chan KG, et al.
    mBio, 2017 10 24;8(5).
    PMID: 29066548 DOI: 10.1128/mBio.01558-17
    Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
    Matched MeSH terms: Mutagenesis, Insertional; Mutagenesis
  17. Fulltext Molecular characterization of putative virulence determinants in Burkholderia pseudomallei
    Puah SM, Puthucheary SD, Wang JT, Pan YJ, Chua KH
    ScientificWorldJournal, 2014;2014:590803.
    PMID: 25215325 DOI: 10.1155/2014/590803
    The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.
    Matched MeSH terms: Mutagenesis, Insertional
  18. Fulltext Increasing leaf vein density by mutagenesis: laying the foundations for C4 rice
    Feldman AB, Murchie EH, Leung H, Baraoidan M, Coe R, Yu SM, et al.
    PLoS One, 2014;9(4):e94947.
    PMID: 24760084 DOI: 10.1371/journal.pone.0094947
    A high leaf vein density is both an essential feature of C4 photosynthesis and a foundation trait to C4 evolution, ensuring the optimal proportion and proximity of mesophyll and bundle sheath cells for permitting the rapid exchange of photosynthates. Two rice mutant populations, a deletion mutant library with a cv. IR64 background (12,470 lines) and a T-DNA insertion mutant library with a cv. Tainung 67 background (10,830 lines), were screened for increases in vein density. A high throughput method with handheld microscopes was developed and its accuracy was supported by more rigorous microscopy analysis. Eight lines with significantly increased leaf vein densities were identified to be used as genetic stock for the global C4 Rice Consortium. The candidate population was shown to include both shared and independent mutations and so more than one gene controlled the high vein density phenotype. The high vein density trait was found to be linked to a narrow leaf width trait but the linkage was incomplete. The more genetically robust narrow leaf width trait was proposed to be used as a reliable phenotypic marker for finding high vein density variants in rice in future screens.
    Matched MeSH terms: Mutagenesis
  19. Fulltext Enzymatic properties and mutational studies of chalcone synthase from Physcomitrella patens
    Rahman RN, Zakaria II, Salleh AB, Basri M
    Int J Mol Sci, 2012;13(8):9673-91.
    PMID: 22949824 DOI: 10.3390/ijms13089673
    PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.
    Matched MeSH terms: Mutagenesis, Site-Directed
  20. Effect of ion pair on thermostability of F1 protease: integration of computational and experimental approaches
    Rahman RN, Muhd Noor ND, Ibrahim NA, Salleh AB, Basri M
    J Microbiol Biotechnol, 2012 Jan;22(1):34-45.
    PMID: 22297217
    A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
    Matched MeSH terms: Mutagenesis, Site-Directed
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