Displaying publications 1 - 20 of 28 in total

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  1. A robust test based on bootstrapping for the two-sample scale problem
    Padmanabhan A, Abdul Rahman Othman, Teh SY
    Sains Malaysiana, 2011;40:1123-1127.
    For testing the homogeneity of variances, modifications of well-known tests are known which combine rigorous theory with resampling (bootstrap). We propose versions of these tests, which are computationally simpler (although asymptotically equivalent). The earlier procedures used the smooth bootstrap with two thousand bootstrap replications per sample whereas our proposals use only the classical bootstrap (or percentile method) with just one thousand bootstrap replications per sample, and also required much less computing time. Our proposals cover the Ansari-Bradley-, Mood- and Klotz-tests. We explain their superiority over the existing methodologies available in textbooks and packages.
    Matched MeSH terms: DNA Replication
  2. DNA barcodes and citizen science provoke a diversity reappraisal for the "ring" butterflies of Peninsular Malaysia (Ypthima: Satyrinae: Nymphalidae: Lepidoptera)
    Jisming-See SW, Sing KW, Wilson JJ
    Genome, 2016 Oct;59(10):879-888.
    PMID: 27333330 DOI: 10.1139/gen-2015-0156
    The "rings" belonging to the genus Ypthima are amongst the most common butterflies in Peninsular Malaysia. However, the species can be difficult to tell apart, with keys relying on minor and often non-discrete ring characters found on the hindwing. Seven species have been reported from Peninsular Malaysia, but this is thought to be an underestimate of diversity. DNA barcodes of 165 individuals, and wing and genital morphology, were examined to reappraise species diversity of this genus in Peninsular Malaysia. DNA barcodes collected during citizen science projects-School Butterfly Project and Peninsular Malaysia Butterfly Count-recently conducted in Peninsular Malaysia were included. The new DNA barcodes formed six groups with different Barcode Index Numbers (BINs) representing four species reported in Peninsular Malaysia. When combined with public DNA barcodes from the Barcode Of Life Datasystems, several taxonomic issues arose. We consider the taxon Y. newboldi, formerly treated as a subspecies of Y. baldus, as a distinct species. DNA barcodes also supported an earlier suggestion that Y. nebulosa is a synonym under Y. horsfieldii humei. Two BINs of the genus Ypthima comprising DNA barcodes collected during citizen science projects did not correspond to any species previously reported in Peninsular Malaysia.
    Matched MeSH terms: DNA Replication
  3. Some Studies into the Effects of Metaldehyde on Cultured Cells
    Salmijah S., Anita Mohd. Zubir, Maimon A.
    Sains Malaysiana, 1997;26.
    Metaldehyde is used widely in Malaysia for the control of molluscs. This communication reports the cytotoxic effects of this chemical on cultured cells as assessed by cell morphology and the DNA synthesising capability as well as its transport into cells. After 15 days of exposure with 20.0 ppm of the compound, the DNA synthesising capability was shown to be unaffected. The IC50 for Vero cells was 276.0 ppm. Transport of thymidine across cells was found to be not significantly affected even at high metaldehyde concentrations (up to 320.0 ppm) suggesting integrity of cells were not significantly affected. The present cellular studies have therefore shown that the cytotoxic effects of this chemical is rather low.
    Metaldehida digunakan dengan meluas di Malaysia untuk mengawal perosak moluska. Kesan sitotoksik bahan kimia ini di peringkat sel dari segi ciri-ciri perubahan moifologi dan keupayaan mensintesis DNA serta kajian awal kesannya terhadap proses kemasukan ke dalam sel dilaporkan di sini. Keupayaan mensintesis DNA didapati tidak terjejas secara signifikan selepas diberikan 20.0 ppm metaldehida secara berterusan selama 15 hari. Nilai IC50 bagi sel Vero adalah 276.0 ppm. Kemasukan timidina ke dalam sel tidak terjejas secara signifikan apabila sel diperlakukan dengan metaldehida, walaupun pada kepekatan yang agak tinggi iaitu sehingga 320.0 ppm. Kajian telah menunjukkan bahawa kesan sitotoksik oleh metaldehida adalah rendah.
    Matched MeSH terms: DNA Replication
  4. Performance comparison between bootstrap and multiscale bootstrap for assessing phylogenetic tree for RNA polymerase
    Safinah Sharuddin, Nora Muda
    Sains Malaysiana, 2015;44:1643-1651.
    Phylogenetic inference refers to the reconstruction of evolutionary relationships among various species that is usually
    presented in the form of a tree. This study constructs the phylogenetic tree by using a novel distance-based method known
    as Modified one step M-estimator (MOM) method. The branches of the phylogenetic tree constructed were then evaluated
    to see their reliability. The performance of the reliability was then compared between the p-value of multiscale bootstrap
    (AU value) and bootstrap p-value (BP value). The aim of this study was to compare the performance between the AU value
    and BP value for assessing phylogenetic tree of RNA polymerase. The results have shown that multiscale bootstrap analysis
    can detect high sampling errors but not in bootstrap analysis. To overcome this problem, the multiscale bootstrap analysis
    has reduced the sampling error by increasing the number of replications. The clusters were indicated as significant if AU
    values or BP values were 95% or higher. From the analysis, the results showed that the BP and AU values differ at 11th
    and 15th branch of the phylogenetic tree. The BP values at both branches were 72 and 85%, respectively, thereby making
    the cluster not significant but by looking at the AU values, the two branches were more than 95% and the clusters were
    significant. This was due to the biasness in calculation of the probability of bootstrap analysis, therefore, the multiscale
    bootstrap analysis has improved the calculation of the probability value compared to the bootstrap analysis.
    Matched MeSH terms: DNA Replication
  5. Fulltext DNA polymerase δ: A single Pol31 polymorphism suppresses the strain background-specific lethality of Pol32 inactivation in Saccharomyces cerevisiae
    Loeillet S, Nicolas A
    DNA Repair (Amst), 2023 Jul;127:103514.
    PMID: 37244009 DOI: 10.1016/j.dnarep.2023.103514
    The evolutionarily conserved DNA polymerase delta (Polδ) plays several essential roles in eukaryotic DNA replication and repair, responsible for the synthesis of the lagging-strand, lower replicative mutagenesis via its proof-reading exonuclease activity and synthetizes both strands during break-induced replication. In Saccharomyces cerevisiae, the Polδ protein complex consists of three subunits encoded by the POL3, POL31 and POL32 genes. Surprisingly, in contrast to POL3 and POL31, the POL32 gene deletion was found to be viable but lethal in all other eukaryotes, raising the question to which extent the viability of the POL32 deletion in S. cerevisiae was species specific. To address this issue, we inactivated the POL32 gene in 10 evolutionary close or distant S. cerevisiae strains and found that POL32 was either essential (3 strains including SK1), non-essential (5 strains including the reference S288C strain) or confers a slow-growth phenotype (2 strains). Whole-genome sequencing of S288C/SK1 pol32∆ meiotic segregants identified the lethal/suppressor effect of the single Pol31-C43Y polymorphism. Consistently, the introduction of the Pol31-43C allele in the SK1 and West African (WA) pol32∆ mutants was sufficient to restore cell viability and wild-type growth upon introduction of two copies of POL31-43C in the SK1 haploid strain. Reciprocally, introduction of the SK1 POL31-43Y allele in the S288C pol32∆ mutant was lethal. Sequence analyses of the POL31 polymorphisms in the 1,011 yeasts genome dataset correlates with the strict occurrence of the POL31-43Y allele in the yeast African palm wine clade. Differently, the single Pol31-E400G polymorphism confers pol32∆ lethality in the Malaysian strain. In the yeast two-hybrid assay, we observed a weakened interaction between Pol3 and Pol31-43Y versus Pol31-43C suggesting an insufficient level of the Polδ holoenzyme stability/activity. Thus, the enigmatic non-essentiality of Pol32 in S. cerevisiae results from single Pol31 amino acid polymorphism and is clade rather than species specific.
    Matched MeSH terms: DNA Replication
  6. In-silico discovery of inhibitors against human papillomavirus E1 protein
    Gunasinghe J, Hwang SS, Yam WK, Rahman T, Wezen XC
    J Biomol Struct Dyn, 2023;41(12):5583-5596.
    PMID: 35751129 DOI: 10.1080/07391102.2022.2091659
    High-risk (HR) Human papillomavirus (e.g. HPV16 and HPV18) causes approximately two-thirds of all cervical cancers in women. Although the first and second-generation vaccines confer some protection against individuals, there are no approved drugs to treat HR-HPV infections to-date. The HPV E1 protein is an attractive drug target because the protein is highly conserved across all HPV types and is crucial for the regulation of viral DNA replication. Hence, we used the Random Forest algorithm to construct a Quantitative-Structure Activity Relationship (QSAR) model to predict the potential inhibitors against the HPV E1 protein. Our QSAR classification model achieved an accuracy of 87.5%, area under the receiver operating characteristic curve of 1.00, and F-measure of 0.87 when evaluated using an external test set. We conducted a drug repurposing campaign by deploying the model to screen the Drugbank database. The top three compounds, namely Cinalukast, Lobeglitazone, and Efatutazone were analyzed for their cell membrane permeability, toxicity, and carcinogenicity. Finally, these three compounds were subjected to molecular docking and 200 ns-long Molecular Dynamics (MD) simulations. The predicted binding free energies for the candidates were calculated using the MM-GBSA method. The binding free energies for Cinalukast, Lobeglitazone, and Efatutazone were -37.84 kcal/mol, -25.30 kcal/mol, and -29.89 kcal/mol respectively. Therefore, we propose their chemical scaffolds for future rational design of E1 inhibitors.Communicated by Ramaswamy H. Sarma.
    Matched MeSH terms: DNA Replication
  7. Fulltext The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness
    Sugrue E, Wickenhagen A, Mollentze N, Aziz MA, Sreenu VB, Truxa S, et al.
    PLoS Pathog, 2022 Nov;18(11):e1010973.
    PMID: 36399512 DOI: 10.1371/journal.ppat.1010973
    HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be.
    Matched MeSH terms: DNA Replication
  8. Fulltext Scaling-up recombinant plasmid DNA for clinical trial: current concern, solution and status
    Ismail R, Allaudin ZN, Lila MA
    Vaccine, 2012 Sep 7;30(41):5914-20.
    PMID: 22406276 DOI: 10.1016/j.vaccine.2012.02.061
    Gene therapy and vaccines are rapidly developing field in which recombinant nucleic acids are introduced in mammalian cells for enhancement, restoration, initiation or silencing biochemical function. Beside simplicity in manipulation and rapid manufacture process, plasmid DNA-based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases. This present review focuses on the safety concern of the genetic elements of plasmid such as propagation and expression units as well as their host genome for the production of recombinant plasmid DNA. The highlighted issues will be beneficial in characterizing and manufacturing plasmid DNA for save clinical use. Manipulation of regulatory units of plasmid will have impact towards addressing the safety concerns raised in human vaccine applications. The gene revolution with plasmid DNA by alteration of their plasmid and production host genetics will be promising for safe delivery and obtaining efficient outcomes.
    Matched MeSH terms: DNA Replication*
  9. Fulltext Antiviral Potential of Algae Polysaccharides Isolated from Marine Sources: A Review
    Ahmadi A, Zorofchian Moghadamtousi S, Abubakar S, Zandi K
    Biomed Res Int, 2015;2015:825203.
    PMID: 26484353 DOI: 10.1155/2015/825203
    From food to fertilizer, algal derived products are largely employed in assorted industries, including agricultural, biomedical, food, and pharmaceutical industries. Among different chemical compositions isolated from algae, polysaccharides are the most well-established compounds, which were subjected to a variety of studies due to extensive bioactivities. Over the past few decades, the promising results for antiviral potential of algae-derived polysaccharides have advocated them as inordinate candidates for pharmaceutical research. Numerous studies have isolated various algal polysaccharides possessing antiviral activities, including carrageenan, alginate, fucan, laminaran, and naviculan. In addition, different mechanisms of action have been reported for these polysaccharides, such as inhibiting the binding or internalization of virus into the host cells or suppressing DNA replication and protein synthesis. This review strives for compiling previous antiviral studies of algae-derived polysaccharides and their mechanism of action towards their development as natural antiviral agents for future investigations.
    Matched MeSH terms: DNA Replication/drug effects*
  10. Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells
    Liew PS, Chen Q, Ng AWR, Chew YC, Ravin NV, Sim EUH, et al.
    Anal Biochem, 2019 10 15;583:113361.
    PMID: 31306622 DOI: 10.1016/j.ab.2019.113361
    Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
    Matched MeSH terms: DNA Replication/physiology*
  11. Screening for microsatellite instability in colorectal carcinoma: Practical utility of immunohistochemistry and PCR with fragment analysis in a diagnostic histopathology setting
    Cheah PL, Li J, Looi LM, Koh CC, Lau TP, Chang SW, et al.
    Malays J Pathol, 2019 Aug;41(2):91-100.
    PMID: 31427545
    Since 2014, the National Comprehensive Cancer Network (NCCN) has recommended that colorectal carcinoma (CRC) be universally tested for high microsatellite instability (MSI-H) which is present in 15% of such cancers. Fidelity of resultant microsatellites during DNA replication is contingent upon an intact mismatch repair (MMR) system and lack of fidelity can result in tumourigenesis. Prior to commencing routine screening for MSI-H, we assessed two commonly used methods, immunohistochemical (IHC) determination of loss of MMR gene products viz MLH1, MSH2, MSH6 and PMS2 against PCR amplification and subsequent fragment analysis of microsatellite markers, BAT25, BAT26, D2S123, D5S346 and D17S250 (Bethesda markers) in 73 unselected primary CRC. 15.1% (11/73) were categorized as MSI-H while deficient MMR (dMMR) was detected in 16.4% (12/73). Of the dMMR, 66.7% (8/12) were classified MSI-H, while 33.3% (4/12) were microsatellite stable/low microsatellite instability (MSS/MSI-L). Of the proficient MMR (pMMR), 95.1% (58/61) were MSS/MSI-L and 4.9% (3/61) were MSI-H. The κ value of 0.639 (standard error =0.125; p = 0.000) indicated substantial agreement between detection of loss of DNA mismatch repair using immunohistochemistry and the detection of downstream microsatellite instability using PCR. After consideration of advantages and shortcomings of both methods, it is our opinion that the choice of preferred technique for MSI analysis would depend on the type of laboratory carrying out the testing.
    Matched MeSH terms: DNA Replication
  12. Ultrastructural aspects of sylvatic dengue virus infection in Vero cell
    Fish-Low CY, Abubakar S, Othman F, Chee HY
    Malays J Pathol, 2019 Apr;41(1):41-46.
    PMID: 31025636
    INTRODUCTION: Dengue virus (DENV), the causative agent of dengue disease exists in sylvatic and endemic ecotypes. The cell morphological changes and viral morphogenesis of two dengue ecotypes were examined at the ultrastructural level to identify potential similarities and differences in the surrogate model of enzootic host.

    MATERIALS AND METHODS: Vero cells were inoculated with virus at a multiplicity of infection (MOI) of 0.1. Cell cultures were harvested over a time course and processed for transmission electron microscopic imaging.

    RESULTS: The filopodia protrusions on cell periphery preceded virus entry. Additionally, sylvatic DENV infection was found spreading slower than the endemic DENV. Morphogenesis of both dengue ecotypes was alike but at different level of efficiency in the permissive cells.

    CONCLUSIONS: This is the first ultrastructural study on sylvatic DENV and this comparative study revealed the similarities and differences of cellular responses and morphogenesis of two dengue ecotypes in vitro. The study revealed the weaker infectivity of sylvatic DENV in the surrogate model of enzootic host, which supposed to support better replication of enzootic DENV than endemic DENV.

    Matched MeSH terms: DNA Replication
  13. Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA Polymerase β's activity
    Homouz D, Joyce-Tan KH, Shahir Shamsir M, Moustafa IM, Idriss H
    J Mol Graph Model, 2018 01;79:192.
    PMID: 29223917 DOI: 10.1016/j.jmgm.2017.11.002
    DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity.
    Matched MeSH terms: DNA Replication
  14. Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells
    Wong YC, Ng AWR, Chen Q, Liew PS, Lee CW, Sim EUH, et al.
    ACS Synth Biol, 2023 Apr 21;12(4):909-921.
    PMID: 37026178 DOI: 10.1021/acssynbio.2c00580
    Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.
    Matched MeSH terms: DNA Replication
  15. A Self-Replicating Linear DNA
    Liew PS, Tan TH, Wong YC, Sim EUH, Lee CW, Narayanan K
    ACS Synth Biol, 2020 04 17;9(4):804-813.
    PMID: 32196315 DOI: 10.1021/acssynbio.9b00478
    TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.
    Matched MeSH terms: DNA Replication/genetics*
  16. Characterization of pR18, a novel rolling-circle replication plasmid from Lactobacillus plantarum
    Jalilsood T, Baradaran A, Ling FH, Mustafa S, Yusof K, Rahim RA
    Plasmid, 2014 May;73:1-9.
    PMID: 24785193 DOI: 10.1016/j.plasmid.2014.04.004
    Lactobacillus plantarum PA18, a strain originally isolated from the leaves of Pandanus amaryllifolius, contains a pR18 plasmid. The pR18 plasmid is a 3211bp circular molecule with a G+C content of 35.8%. Nucleotide sequence analysis revealed two putative open reading frames, ORF1 and ORF2, in which ORF2 was predicted (317 amino acids) to be a replication protein and shared 99% similarity with the Rep proteins of pLR1, pLD1, pC30il, and pLP2000, which belong to the RCR pC194/pUB110 family. Sequence analysis also indicated that ORF1 was predicted to encode linA, an enzyme that enzymatically inactivates lincomycin. The result of Southern hybridization and mung bean nuclease treatment confirmed that pR18 replicated via the RCR mechanism. Phylogenetic tree analysis of pR18 plasmid proteins suggested that horizontal transfer of antibiotic resistance determinants without genes encoding mobilization has not only occurred between Bacillus and Lactobacillus but also between unrelated bacteria. Understanding this type of transfer could possibly play a key role in facilitating the study of the origin and evolution of lactobacillus plasmids. Quantitative PCR showed that the relative copy number of pR18 was approximately 39 copies per chromosome equivalent.
    Matched MeSH terms: DNA Replication*
  17. Fulltext Somatic mitochondrial DNA D-loop mutations in meningioma discovered: A preliminary data
    Mohamed Yusoff AA, Mohd Khair SZN, Wan Abdullah WS, Abd Radzak SM, Abdullah JM
    J Cancer Res Ther, 2020 12 22;16(6):1517-1521.
    PMID: 33342822 DOI: 10.4103/jcrt.JCRT_1132_16
    Background and Objective: Meningiomas are among the most common intracranial tumors of the central nervous system. It is widely accepted that the initiation and progression of meningiomas involve the accumulation of nucleus genetic alterations, but little is known about the implication of mitochondrial genomic alterations during development of these tumors. The human mitochondrial DNA (mtDNA) contains a short hypervariable, noncoding displacement loop control region known as the D-Loop. Alterations in the mtDNA D-loop have been reported to occur in most types of human cancers. The purpose of this study was to assess the mtDNA D-loop mutations in Malaysian meningioma patients.

    Materials and Methods: Genomic DNA was extracted from 21 fresh-frozen tumor tissues and blood samples of the same meningioma patients. The entire mtDNA D-loop region (positions 16024-576) was polymerase chain reaction amplified using designed primers, and then amplification products were purified before the direct DNA sequencing proceeds.

    Results: Overall, 10 (47.6%) patients were detected to harbor a total of 27 somatic mtDNA D-loop mutations. Most of these mtDNA mutations were identified in the hypervariable segment II (40.7%), with 33.3% being located mainly in the conserved sequence block II of the D310 sequence. Furthermore, 58 different germline variations were observed at 21 nucleotide positions.

    Conclusion: Our results suggest that mtDNA alterations in the D-loop region may be an important and early event in developing meningioma. Further studies are needed, including validation in a larger patient cohort, to verify the clinicopathological outcomes of mtDNA mutation biomarkers in meningiomas.

    Matched MeSH terms: DNA Replication/genetics
  18. Fulltext Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells
    Tan GM, Looi CY, Fernandez KC, Vadivelu J, Loke MF, Wong WF
    Sci Rep, 2015;5:11046.
    PMID: 26078204 DOI: 10.1038/srep11046
    Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.
    Matched MeSH terms: DNA Replication/genetics; DNA Replication/immunology
  19. Recombineering linear BACs
    Chen Q, Narayanan K
    Methods Mol Biol, 2015;1227:27-54.
    PMID: 25239740 DOI: 10.1007/978-1-4939-1652-8_2
    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
    Matched MeSH terms: DNA Replication*
  20. Replication of 13 obesity loci among Singaporean Chinese, Malay and Asian-Indian populations
    Dorajoo R, Blakemore AI, Sim X, Ong RT, Ng DP, Seielstad M, et al.
    Int J Obes (Lond), 2012 Jan;36(1):159-63.
    PMID: 21544081 DOI: 10.1038/ijo.2011.86
    Recent genome-wide association studies (GWAS) have identified 38 obesity-associated loci among European populations. However, their contribution to obesity in other ethnicities is largely unknown.
    Matched MeSH terms: DNA Replication*
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