Displaying publications 1 - 20 of 121 in total

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  1. Fulltext Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA
    Murulitharan K, Yusoff K, Omar AR, Peeters BPH, Molouki A
    Curr Microbiol, 2021 Apr;78(4):1458-1465.
    PMID: 33660046 DOI: 10.1007/s00284-021-02421-z
    Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.
    Matched MeSH terms: DNA, Complementary/genetics
  2. Induced expression of Ganoderma boninense Lanosterol 14α-Demethylase (ERG11) during interaction with oil palm
    Lim FH, Rasid OA, Idris AS, As'wad AWM, Vadamalai G, Parveez GKA, et al.
    Mol Biol Rep, 2023 Mar;50(3):2367-2379.
    PMID: 36580194 DOI: 10.1007/s11033-022-08131-4
    BACKGROUND: The basidiomycete fungus, Ganoderma boninense is the main contributor to oil palm Basal Stem Rot (BSR) in Malaysia and Indonesia. Lanosterol 14α-Demethylase (ERG11) is a key enzyme involved in biosynthesis of ergosterol, which is an important component in the fungal cell membrane. The Azole group fungicides are effective against pathogenic fungi including G. boninense by inhibiting the ERG11 activity. However, the work on molecular characterization of G. boninense ERG11 is still unavailable today.

    METHODS AND RESULTS: This study aimed to isolate and characterize the full-length cDNA encoding ERG11 from G. boninense. The G. boninense ERG11 gene expression during interaction with oil palm was also studied. A full-length 1860 bp cDNA encoding ERG11 was successfully isolated from G. boninense. The G. boninense ERG11 shared 91% similarity to ERG11 from other basidiomycete fungi. The protein structure homology modeling of GbERG11 was analyzed using the SWISS-MODEL workspace. Southern blot and genome data analyses showed that there is only a single copy of ERG11 gene in the G. boninense genome. Based on the in-vitro inoculation study, the ERG11 gene expression in G. boninense has shown almost 2-fold upregulation with the presence of oil palm.

    CONCLUSION: This study provided molecular information and characterization study on the G. boninense ERG11 and this knowledge could be used to design effective control measures to tackle the BSR disease of oil palm.

    Matched MeSH terms: DNA, Complementary/genetics; DNA, Complementary/metabolism
  3. Effects of high stocking density on growth performance and expression of MyD88, and its temporal expression under the challenge of Vibrio parahaemolyticus in the noble scallop Chlamys nobilis
    Feng M, Tan K, Zhang H, Duan X, Li S, Ma H, et al.
    Fish Shellfish Immunol, 2023 Oct;141:109059.
    PMID: 37678479 DOI: 10.1016/j.fsi.2023.109059
    High stocking density has been regarded as an adverse factor in bivalve aquaculture. However, its subsequent molecular response to pathogenic bacteria has been little studied. In order to study the question, a novel MyD88 was first cloned using adult noble scallops Chlamys nobilis (CnMyD88), and its tissue distribution was investigated. Then, 1860 juvenile scallops were divided into two groups with two initial densities of high density (200 individuals/layer, HD) and normal density (110 individuals/layer, ND) and in-situ cultured for three months, in which their growth, survival, and the differential expression of CnMyD88 were examined, respectively. Finally, scallops were injected with the Vibrio parahaemolyticus to assess the temporal expression of CnMyD88. As the results show, CnMyD88 cDNA has a full length of 2241 bp and contains an 1107 bp ORF that encodes a 368-derived protein. It was widely expressed in examined tissues with a significantly higher level in hemolymph, intestine, mantle, and gonad than others. Besides, the HD group showed lower growth (0.39 ± 0.05 mm/day) and survival (37.00 ± 8.49%) than the ND group (0.55 ± 0.02 mm/day and 76.82 ± 5.78%). More importantly, the HD group exhibited significantly lower expression levels of CnMyD88 in their examined tissues than the ND group. After V. parahaemolyticus challenging, CnMyD88 had significantly lower expression levels in the scallops from the HD group than that of the scallops from the ND group at 6th, 24th, and 36th. The present results indicated that high stocking density not only made adverse impacts on growth and survival but also may induce immunosuppression in the noble scallop. Therefore, appropriate low stocking density may be worth considering to adopt in scallop aquaculture.
    Matched MeSH terms: DNA, Complementary/genetics
  4. Fulltext Identification of floral genes for sex determination in Calamus palustris Griff. by using suppression subtractive hybridization
    Ng CY, Wickneswari R, Choong CY
    Genet. Mol. Res., 2014;13(3):6037-49.
    PMID: 25117361 DOI: 10.4238/2014.August.7.18
    Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.
    Matched MeSH terms: DNA, Complementary/genetics
  5. Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda
    Thanh T, Chi VT, Abdullah MP, Omar H, Noroozi M, Ky H, et al.
    Mol Biol Rep, 2011 Jan;38(1):177-82.
    PMID: 20354903 DOI: 10.1007/s11033-010-0092-4
    Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.
    Matched MeSH terms: DNA, Complementary/genetics
  6. A mesocarp-and species-specific cDNA clone from oil palm encodes for sesquiterpene synthase
    Shah FH, Cha TS
    Plant Sci, 2000 May 29;154(2):153-160.
    PMID: 10729614
    The differential display method was used to isolate cDNAs corresponding to transcripts that accumulate during the period of lipid synthesis, 12-20 weeks after anthesis (WAA) in the mesocarp of two oil palms, Elaeis oleifera and Elaeis guineensis, Tenera. DNA-free total RNA from mesocarp and kernel of E. guineensis, Tenera and E. oleifera (15 WAA) were used to obtain differential gene expression patterns between these tissues from the two species. In this report, we describe the isolation and characterization of a specific cDNA clone, MO1 (434 bp) which was shown to be mesocarp-specific as well as species-specific for E. oleifera Sequencing of this fragment showed homology to the enzyme sesquiterpene synthase. Its longer cDNA clone, pMO1 (1072 bp), isolated from a 15-week E. oleifera mesocarp cDNA library confirmed that it encodes for sesquiterpene synthase. The complete sequence of 1976 bp was obtained using 5'RACE method. Northern hybridization showed that MO1 and pMO1 mRNA transcripts are highly expressed only in the mesocarp of E. oleifera from 5 to 20 WAA. No expression was detected in the kernel (12-17 WAA) and vegetative tissues of both species nor in the mesocarp of E. guineensis. This is the first communication to document on the isolation and characterisation of a mesocarp-and species-specific cDNA clone from oil palm.
    Matched MeSH terms: DNA, Complementary
  7. Transformasi gen metalotionin (eiMT1) daripada eleusine indica ke dalam pokok tembakau, nicotiana tabacum berperantaraan agrobacterium tumefaciens
    Nik Marzuki Sidik, Roslina Mat Yazid, Che Radziah Che Mohd. Zain, Ismanizan Ismail
    Sains Malaysiana, 2010;39.
    Metalotionin (MT ) merupakan protein pengikat logam berberat molekul rendah dan kaya dengan sistein yang hadir dalam pelbagai jenis organisma termasuklah bakteria, kulat, tumbuhan dan haiwan. MT tumbuhan dipercayai mengambil bahagian dalam metabolisme dan penyahtoksikan logam dengan cara pengkelatan ion-ion logam berat. Fungsinya yang unik ini telah mendorong minat untuk memencilkan gen MT daripada rumput sambau, Eleusine indica. DNA pelengkap (cDNA) eiMT 1 telah diklonkan ke dalam vektor binari pBI121 untuk ditransformasikan ke dalam pokok tembakau melalui perantaraan Agrobacterium tumefaciens. Penyaringan pokok tembakau transgenik dengan PCR dilakukan menggunakan 3 pasang pencetus yang direka khas iaitu pasangan CMT F dan CMT R, 35SF dan PMT R, dan pasangan pencetus khusus-gen MT FS2 dan MT RS2. Ketiga-tiga pasangan pencetus ini berjaya menghasilkan saiz serpihan DNA jangkaan iaitu masing-masing 270 pb, 1.1 kb dan 170 pb. Penjujukan terhadap serpihan bersaiz 170 pb dan analisis jujukan menunjukkan persamaan 100 % dengan eiMT 1. Kajian pengekspresan gen melalui pendekatan transkripsi berbalik-PCR membuktikan bahawa transgen eiMT 1 telah berjaya diekspreskan dalam 11 daripada 19 pokok transgenik yang dikaji.
    Matched MeSH terms: DNA, Complementary
  8. Pengklonan dan pencirian klon CUKMCS yang mengekodkan kapsaisin sintase (Cs)
    Nurulhikma Md. Isa, Ismanizan Ismail, Zamri Zainal
    Sains Malaysiana, 2010;39.
    Kapsaisinoid merupakan alkaloid yang memberikan ciri kepedasan pada cili serta khusus pada genus Capsicum. Sebatian kapsaisinoid terdiri daripada dua komponen utama iaitu kapsaisin dan dihidrokapsaisin. Dalam kajian ini, pengklonan cDNA Kapsaisin sintase (Cs) telah berjaya dilakukan menerusi kaedah transkripsi berbalik PCR (RT-PCR) dan klon cDNA tersebut dinamakan CUKMCS yang bersaiz 981 pb. Pencarian homologi menggunakan program blastx dan blastp yang terdapat pada pangkalan data NCBI mendapati CUKMCS mempunyai persamaan yang sangat tinggi terhadap Cs pada Capsicum frutescens, Capsicum annuum dan Capsicum chacoense. Saiz ramalan protein CUKMCS diangggarkan sekitar 36 kDa. Penentuan pengekspresan transkrip Cs pada 5 tisu yang berbeza mendapati transkrip dikesan pada tisu plasenta, mesokarp dan biji manakala tiada transkrip Cs dikesan pada daun dan akar
    Matched MeSH terms: DNA, Complementary
  9. Fulltext Nucleotide sequence of Phaseolus vulgaris L. alcohol dehydrogenase encoding cDNA and three-dimensional structure prediction of the deduced protein
    Amelia K, Khor CY, Shah FH, Bhore SJ
    Pharmacognosy Res, 2015 Apr-Jun;7(2):203-8.
    PMID: 25829796 DOI: 10.4103/0974-8490.150532
    Common beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken.
    Matched MeSH terms: DNA, Complementary
  10. Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense
    Tan YC, Yeoh KA, Wong MY, Ho CL
    J Plant Physiol, 2013 Nov 01;170(16):1455-60.
    PMID: 23769496 DOI: 10.1016/j.jplph.2013.05.009
    Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection.
    Matched MeSH terms: DNA, Complementary/genetics; DNA, Complementary/metabolism
  11. From Plant Extract to a cDNA Encoding a Glucosyltransferase Candidate: Proteomics and Transcriptomics as Tools to Help Elucidate Saponin Biosynthesis in Centella asiatica
    de Costa F, Barber CJ, Reed DW, Covello PS
    Methods Mol Biol, 2016;1405:43-8.
    PMID: 26843164 DOI: 10.1007/978-1-4939-3393-8_5
    Centella asiatica (L.) Urban (Apiaceae), a small annual plant that grows in India, Sri Lanka, Malaysia, and other parts of Asia, is well-known as a medicinal herb with a long history of therapeutic uses. The bioactive compounds present in C. asiatica leaves include ursane-type triterpene sapogenins and saponins-asiatic acid, madecassic acid, asiaticoside, and madecassoside. Various bioactivities have been shown for these compounds, although most of the steps in the biosynthesis of triterpene saponins, including glycosylation, remain uncharacterized at the molecular level. This chapter describes an approach that integrates partial enzyme purification, proteomics methods, and transcriptomics, with the aim of reducing the number of cDNA candidates encoding for a glucosyltransferase involved in saponin biosynthesis and facilitating the elucidation of the pathway in this medicinal plant.
    Matched MeSH terms: DNA, Complementary
  12. Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs
    Jeyaseelan K, Armugam A, Lachumanan R, Tan CH, Tan NH
    Biochim. Biophys. Acta, 1998 Apr 10;1380(2):209-22.
    PMID: 9565688
    Cardiotoxins are the most abundant toxin components of cobra venom. Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported. In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N. n. sputatrix by cDNA cloning. This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra. The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli. The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing. These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells. Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N. n. sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins. The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N. n. atra and N. n. naja, indicating that it may be universally present in all Naja naja subspecies. Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4). We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts.
    Matched MeSH terms: DNA, Complementary/analysis; DNA, Complementary/genetics; DNA, Complementary/chemistry
  13. Pengenalpastian dan profil pengekspresan gen biosintesis asid amino yis psikrofil, glaciozyma antarctica
    Izwan Bharudin, Radziah Zolkefli, Shazilah Kamaruddin, Farah Diba Abu Bakar, Abdul Munir Abdul Murad, Mohd Faizal Abu Bakar, et al.
    Sains Malaysiana, 2018;47:1675-1684.
    Mekanisme pengambilan dan penghasilan asid amino bagi mikroorganisma psikrofil yang bermandiri dan berpoliferasi
    pada persekitaran sejuk melampau masih belum difahami sepenuhnya. Objektif kajian ini ialah untuk mengenal pasti
    gen yang terlibat dalam penjanaan asid amino bagi yis psikrofil, Glaciozyma antarctica serta menentukan pengekspresan
    gen tersebut semasa kehadiran dan kekurangan asid amino dalam medium pertumbuhan. Pengenalpastian gen telah
    dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) daripada dua perpustakaan cDNA yang dibina daripada
    sel yang dikultur dalam medium pertumbuhan kompleks dan medium pertumbuhan minimum tanpa asid amino. Sebanyak
    3552 klon cDNA daripada setiap perpustakaan dipilih secara rawak untuk dijujuk menghasilkan 1492 transkrip unik
    (medium kompleks) dan 1928 transkrip unik (medium minimum). Analisis pemadanan telah mengenl pasti gen mengekod
    protein yang terlibat di dalam pengambilan asid amino bebas, biosintesis asid amino serta gen yang terlibat dengan
    kitar semula asid amino berdasarkan tapak jalan yang digunakan oleh yis model, Saccharomyces cerevisiae. Analisis
    pengekspresan gen menggunakan kaedah RT-qPCR menunjukkan pengekspresan gen mengekod protein yang terlibat di
    dalam pengambilan asid amino bebas iaitu permease adalah tinggi pada medium kompleks manakala pengekspresan
    kebanyakan gen mengekod protein yang terlibat dalam kitar semula dan biosintesis asid amino adalah tinggi di dalam
    medium minimum. Kesimpulannya, gen yang terlibat dalam penjanaan dan pengambilan asid amino bagi mikroorganisma
    psikrofil adalah terpulihara seperti mikroorganisma mesofil dan pengekspresan gen-gen ini adalah diaruh oleh kehadiran
    atau ketiadaan asid amino bebas pada persekitaran.
    Matched MeSH terms: DNA, Complementary
  14. Differential gene expression during flowering in the oil palm (Elaeis guineensis)
    Singh R, Cheah SC
    Plant Cell Rep, 2000 Jul;19(8):804-809.
    PMID: 30754873 DOI: 10.1007/s002999900179
    The technique of mRNA fingerprinting was used to isolate flower-specific cDNAs in the oil palm. Differences in the RNA populations between vegetative tissue (leaf) and inflorescences at various stages of flower development were examined using 18 primer combinations. A total of 16 flower-specific cDNAs were identified, of which 15 were successfully re-amplified. Reverse Northern analysis confirmed that 8 of the 15 cDNAs appeared to truly represent differentially expressed mRNAs in flowering tissues. Northern blot analysis subsequently showed that 5 of the clones are preferentially or exclusively expressed in the flowering tissues of oil palm.
    Matched MeSH terms: DNA, Complementary
  15. Fulltext Data on degradome sequencing and analysis from mock-inoculated and Fusarium oxysporum treated leaves samples in Persicaria minor
    Samad AFA, Sajad M, Jani J, Murad AMA, Ismail I
    Data Brief, 2018 Oct;20:555-557.
    PMID: 30197911 DOI: 10.1016/j.dib.2018.08.034
    Degradome sequencing referred as parallel analysis of RNA ends (PARE) by modifying 5'-rapid amplification of cDNA ends (RACE) with deep sequencing method. Deep sequencing of 5' products allow the determination of cleavage sites through the mapping of degradome fragments against small RNAs (miRNA or siRNA) on a large scale. Here, we carried out degradome sequencing in medicinal plant, Persicaria minor, to identify cleavage sites in small RNA libraries in control (mock-inoculated) and Fusarium oxysporum treated plants. The degradome library consisted of both control and treated samples which were pooled together during library preparation and named as D4. The D4 dataset have been deposited at GenBank under accession number SRX3921398, https://www.ncbi.nlm.nih.gov/sra/SRX3921398.
    Matched MeSH terms: DNA, Complementary
  16. Pengenalpastian dan pencirian gen trichoderma virens ukm1 mengekod enzim terlibat dalam pencuraian kitin krustasea
    Abdul Munir Abdul Murad, Rafidah Badrun, Sakina Shahabudin, Shazilah Kamaruddin, Madihah Ahmad Zairun, Farahayu Khairuddin, et al.
    Sains Malaysiana, 2013;42:715-724.
    Kitin merupakan polisakarida struktur yang dapat dicurai oleh enzim kitinolisis kepada pelbagai terbitan yang boleh digunakan dalam bidang perubatan, pertanian dan rawatan air. Pengenalpastian dan pencirian gen-gen Trichoderma virens UKM1 mengekod enzim terlibat dalam pencuraian kitin krustasea telah dilakukan melalui penjanaan penanda jujukan terekspres (ESTs) dan analisis pengekspresan gen menggunakan mikroatur DNA. Sebanyak tiga perpustakaan cDNA T. virens UKM1 yang masing-masing diaruh oleh kitin, glukosamina dan kitosan telah dibina. Sejumlah 1536 klon cDNA telah dijujuk dan sebanyak 1033 ESTs berkualiti telah dijana. Seterusnya, perbezaan pengekspresan gen apabila pertumbuhan kulat diaruh dengan kehadiran kitin krustasea dan tanpa kitin pada hari ketiga dan kelima telah ditentukan. Sebanyak 1824 klon cDNA telah dititik ke atas slaid kaca dan dihibrid bersama dengan cDNA terlabel Cy3 atau Cy5 yang disintesis daripada mRNA yang dipencil daripada kulat yang ditumbuhkan dalam medium mengandungi kitin krustasea atau glukosa (kawalan). Sebanyak 91 dan 61 gen, masing-masing bagi hari ketiga dan kelima didapati terekspres melebihi dua gandaan apabila kulat menggunakan kitin krustasea sebagai sumber karbon. Beberapa gen mengekod kitinase seperti ech1 dan cht3 (endokitinase), nag1 (eksokitinase) dan nagB (glukosamina 6-P-deaminase) didapati terekspres dengan tinggi pada kedua-dua hari. Selain daripada itu, gen mengekod protein hidrofobin, protease serina dan beberapa protein hipotetik juga terekspres dengan tinggi dengan kehadiran kitin krustasea. Protein-protein ini dijangka memainkan peranan penting dalam membantu pencuraian kitin krustasea.
    Matched MeSH terms: DNA, Complementary
  17. Nucleotide sequencing, cloning, and expression of Capra hircus Heme Oxygenase-1 in caprine islets to promote insulin secretion in vitro
    Vakhshiteh F, Allaudin ZN, Lila MA, Abbasiliasi S, Ajdari Z
    Mol Biotechnol, 2015 Jan;57(1):75-83.
    PMID: 25218408 DOI: 10.1007/s12033-014-9803-8
    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.
    Matched MeSH terms: DNA, Complementary/genetics
  18. Fulltext Efficient oil palm total RNA extraction with a total RNA extraction kit
    Habib SH, Saud HM, Kausar H
    Genet. Mol. Res., 2014;13(2):2359-67.
    PMID: 24781991 DOI: 10.4238/2014.April.3.8
    Oil palm tissues are rich in polyphenols, polysaccharides and secondary metabolites; these can co-precipitate with RNA, causing problems for downstream applications. We compared two different methods (one conventional and a kit-based method - Easy-Blue(TM) Total RNA Extraction Kit) to isolate total RNA from leaves, roots and shoot apical meristems of tissue culture derived truncated leaf syndrome somaclonal oil palm seedlings. The quality and quantity of total RNA were compared through spectrophotometry and formaldehyde gel electrophoresis. The specificity and applicability of the protocols were evaluated for downstream applications, including cDNA synthesis and RT-PCR analysis. We found that the conventional method gave higher yields of RNA but took longer, and it was contaminated with genomic DNA. This method required extra genomic DNA removal steps that further reduced the RNA yield. The kit-based method, on the other hand, produced good yields as well as well as good quality RNA, within a very short period of time from a small amount of starting material. Moreover, the RNA from the kit-based method was more suitable for synthesizing cDNA and RT-PCR amplification than the conventional method. Therefore, we conclude that the Easy-BlueTM Total RNA Extraction Kit method is suitable and superior for isolation of total RNA from oil palm leaf, root and shoot apical meristem.
    Matched MeSH terms: DNA, Complementary/genetics*
  19. Granulometric analysis of spots in DNA microarray images
    Latha B, Venkatesh B
    Genomics Proteomics Bioinformatics, 2004 Nov;2(4):222-36.
    PMID: 15901251
    As the topological properties of each spot in DNA microarray images may vary from one another, we employed granulometries to understand the shape-size content contributed due to a significant intensity value within a spot. Analysis was performed on the microarray image that consisted of 240 spots by using concepts from mathematical morphology. In order to find out indices for each spot and to further classify them, we adopted morphological multiscale openings, which provided microarrays at multiple scales. Successive opened microarrays were subtracted to identify the protrusions that were smaller than the size of structuring element. Spot-wise details, in terms of probability of these observed protrusions, were computed by placing a regularly spaced grid on microarray such that each spot was centered in each grid. Based on the probability of size distribution functions of these protrusions isolated at each level, we estimated the mean size and texture index for each spot. With these characteristics, we classified the spots in a microarray image into bright and dull categories through pattern spectrum and shape-size complexity measures. These segregated spots can be compared with those of hybridization levels.
    Matched MeSH terms: DNA, Complementary/genetics
  20. Fulltext Top-Down Nanofabrication and Characterization of 20 nm Silicon Nanowires for Biosensing Applications
    M Nuzaihan MN, Hashim U, Md Arshad MK, Rahim Ruslinda A, Rahman SF, Fathil MF, et al.
    PLoS One, 2016;11(3):e0152318.
    PMID: 27022732 DOI: 10.1371/journal.pone.0152318
    A top-down nanofabrication approach is used to develop silicon nanowires from silicon-on-insulator (SOI) wafers and involves direct-write electron beam lithography (EBL), inductively coupled plasma-reactive ion etching (ICP-RIE) and a size reduction process. To achieve nanometer scale size, the crucial factors contributing to the EBL and size reduction processes are highlighted. The resulting silicon nanowires, which are 20 nm in width and 30 nm in height (with a triangular shape) and have a straight structure over the length of 400 μm, are fabricated precisely at the designed location on the device. The device is applied in biomolecule detection based on the changes in drain current (Ids), electrical resistance and conductance of the silicon nanowires upon hybridization to complementary target deoxyribonucleic acid (DNA). In this context, the scaled-down device exhibited superior performances in terms of good specificity and high sensitivity, with a limit of detection (LOD) of 10 fM, enables for efficient label-free, direct and higher-accuracy DNA molecules detection. Thus, this silicon nanowire can be used as an improved transducer and serves as novel biosensor for future biomedical diagnostic applications.
    Matched MeSH terms: DNA, Complementary/analysis
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