Effective and rapid methods for RNA extraction from conidia, germinating conidia and appressoria of the fungal plant pathogen, Colletotrichum gloeosporioides is reported in this study. The procedure for the RNA extraction from conidia and germinating conidia was carried out using TRI REAGENT® solution (Molecular Research Center, USA) and can be completed in less than one and a half hours. The procedure for RNA extraction from appressoria was carried out using a modified protocol employing guanidine isothiocyanate and mechanical cell disruption by glass beads. The efficiency of the RNA extraction procedures was evaluated by several measures to determine RNA integrity, purity and applicability in RT-PCR. RNA integrity was assessed by observing the integrity of the major RNA species (18S and 28S rRNA) on denaturing agarose gel electrophoresis. The ethidium bromide-staining pattern of intact total RNA extracted from the three fungal morphogenetic cells showed clearly defined 28S and 18S rRNA bands and no genomic DNA contamination. Spectrophotometric assessment of RNA from each sample indicated relatively high purity and absence of carbohydrate contamination. Finally, we have demonstrated that the methods used for RNA extraction of conidia, germinating conidia and appressoria produced RNA of sufficient quality suitable for RT-PCR in detecting the expression of protein kinase A regulatory subunit gene in C. gloeosporioides.
Teknologi DNA mikroatur merupakan salah satu teknologi penting dalam penyelidikan biologi kini. Teknologi tersebut membolehkan analisis pengekspresan gen pelbagai sistem model pada skala genom dijalankan. Prestasi DNA mikroatur ditentukan oleh parameter seperti kepadatan titik, ciri-ciri titik (morfologi, kepadatan prob dan keamatan isyarat penghibridan), latar belakang, kespesifikan dan kesensitifan. Dengan menggunakan mesin pemegun GeneTac™G3, slaid mikroatur cDNA ikan siakap (Lates calcarifer) yang mengandungi 1920 klon-klon cDNA terpilih daripada perpustakaan cDNA hepar dan limpa yang dipegunkan secara duplikasi telah difabrikasikan. Klon-klon yang dipilih mempunyai fungsi putatif berkaitan dengan keimunan, aruhan tekanan, metabolisme, pengangkutan, transkripsi dan translasi. Kawalan negatif seperti oligonukleotida (A)50 dan beberapa gen daripada Saccharomyces cerevisiae dan Escherichia coli serta kawalan positif seperti beberapa siri pencairan DNA genom dan cDNA L. calcarifer juga dipegunkan ke atas slaid kaca. Diperhatikan bahawa kepekatan prob dalam julat antara 150-250 μg/mL, kesesuaian jenis kawalan positif dan negatif yang digunakan berjaya menghasilkan slaid yang berkualiti. Selain itu, perlakuan pengeringan slaid secara semalaman dan rehidrasi semula slaid menghasilkan morfologi titik DNA yang sekata dan membulat. Kawalan kualiti ke atas slaid yang terhasil membuktikan keberkesanan slaid yang difabrikasi untuk kajian respons transkriptom L. calcarifer terhadap jangkitan Cryptocaryon irritans.
The ability of a locally isolated clinical strain of C. albicans to adapt and response towards oxidative stress were investigated in this study. Treatment of C. albicans with hydrogen peroxide (H2O2) will exert an oxidative stress to C. albicans cells and affect the growth rate of this opportunistic pathogen. Cells that were grown in Yeast Peptone Dextrose (YPD) medium with the presence of 4.0 mM H2O2 gave a doubling time of 51 min/generation compared to 44 min/generation for those grown in 0.4 mM H2O2 and without H2O2. In order to determine the resistance level of C. albicans towards oxidative stress, cells were exposed to different concentration of H2O2. Results showed that percentage of cells viability decreased with the increased concentration of H2O2. These data indicated that C. albicans could overcome oxidative stress to a certain level before they were killed. To determine whether this strain exhibited a stress induce protection, cells were treated with mild oxidative stress for one hour before exposed to a stronger oxidative stress. Data obtained shows that cells that were pre-treated with mild oxidative stress showed higher percentage of survival compared to non pre-treated cells. This strongly suggested that stress induce protection was present in this C. albicans. Finally, in order to determine whether oxidative stress can induce yeast to hyphal morphogenesis in C. albicans, cells were then exposed to 0.4 mM H2O2 at 37 ºC and the number of hyphal formed were compared to hyphal formation when the cells were grown at 37 ºC only. However the results showed that oxidative stress failed to induce yeast to hyphal morphogenesis in this clinically isolated C. albicans strain.
Keywords: Candida albicans; oxidative stress
In this study, the pyrG gene which encodes for orotidine 5-monophosphate decarboxylase (OMP decarboxylase) of Aspergillus oryzae strain S1 was cloned and analysed. This 1.8kb A. oryzae pyrG encompasses the 5’-regulatory flanking region (465 bp), open reading frame (899 bp) and 3’-regulatory region (475 bp). The pyrG contained one intron at position 623-687 bp based on the AUGUSTUS and FGENESH (SoftBerry) analysis corresponding to the intron present in the pyrG of A. oryzae (Accession Number: Y13811). In silico analysis showed that the enzyme encoded by the A. oryzae S1 pyrG gene has a theoretical molecular weight of 30.28 kDa and theoretical pI value of 5.92. This enzyme is hydrophilic, located in a region outside of the transmembrane and it functions in the cytoplasm. Five motives such as N-glycosylation site, protein kinase C (PKC) phosphorylation site, casein kinase II (CK-2) phosphorylation site, N-myristolation site and orotidine 5-monophoshate decarboxylase active site have been identified in the pyrG amino acid sequence. The three dimensional structure of this enzyme generated via protein homology modeling using the bioinformatic software, Swiss Model, shows that OMP decarboxylase is a protein with an α/ß barrel structure possessing 8 ß-strands surrounded by 9 α-helices. The amino acid residues involved in the active site have been identified and it is located on one of the ß-strands. The pyrG DNA sequence will be used for the complementation of a pyrG auxotroph mutant of A. oryzae.
Delta 6-asid lemak desaturase dan delta 12-asid lemak desaturase merupakan enzim yang diperlukan bagi langkah desaturasi semasa proses biosintesis asid gamma-linolenik (GLA) oleh kulat oleaginus. Objektif kajian ini ialah untuk menganalisis profil pengekspresan gen mengekod enzim delta 6-asid lemak desaturase (des6) dan delta 12-asid lemak desaturase (des12) kulat oleaginus Cunninghamella bainieri semasa penghasilan GLA. Jujukan gen separa bersaiz 1372 pb bagi des6 dan 1008 pb bagi des12 telah dipencil daripada C. bainieri. Analisis pengekspresan gen menggunakan kaedah tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan perubahan kadar pengekspresan des6 adalah lebih tinggi berbanding kadar pengekspresan des12 semasa penghasilan GLA. Pengekspresan des6 adalah tertinggi selepas 24 jam dikultur dalam medium penghasilan GLA. Namun, kadar pengekspresannya menurun hingga jam ke-96 pertumbuhan tetapi meningkat semula pada jam ke-120. Bagi des12, kadar pengekspresannya adalah lebih sekata dengan pengekspresan tertinggi dikesan pada jam ke-120. Analisis penghasilan GLA menunjukkan jumlah GLA dalam sel berkolerasi dengan kadar pengekspresan des6. Hasil kajian mencadangkan bahawa aras pengekspresan des6 adalah penting dalam menentukan aras GLA dalam C. bainieri.
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.
DNA microarray technology has permitted large scale parallel analysis of gene expression of several diseases, including cancers. Real-time PCR has become a well-established procedure for
quantifying levels of gene expression. We evaluated Real-Time PCR to validate differentially expressed genes identified by DNA arrays. Gene expression of three different grade of transitional cell carcinoma (TCC) of human bladder cancer was determined using microarray slide containing cancer-related genes. Data obtained were sorted according based on the ratios between Cy3 and Cy5 dyes used and revealed 119, 235 and 183 differentially expressed genes in TCC WHO Grades I, II and III, respectively. Real-time PCR used SYBR Green-1 dye detection to validate relative change in gene expression. Real- Time PCR confirmed the change in gene expression of 17 of 28 (75%) genes identified by microarray. Real-Time PCR also suggest that genes identified by microarray with two or higher fold change cannot be eliminated as false nor be accepted as true without validation. Real-Time PCR based on SYBR Green- 1 is well-suited to validate DNA array results because it is quantitative, rapid and requires less RNA compared to the conventional assays.
Yeasts with GRAS (Generally Regarded as Safe) status are commonly used as hosts for heterologous protein production. Yeasts are suitable expression hosts as they have been extensively characterised genetically. The objective of this project was to isolate yeasts from Malaysian food sources and subsequently to develop these as alternative hosts for heterologous protein production. Yeasts were isolated from Malaysian traditional fermented food namely ‘tapai’, ‘tuak’ and ‘ragi’. A total of 23 isolates were obtained and subjected to molecular identification by amplification and sequencing of the universally conserved ribosomal internal transcribed spacer (ITS), 26S rDNA and 18S rDNA sequences. We identified three species of yeasts, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Pichia anomala, which have a long tradition of usage in food production and have no adverse effects on humans. To test the feasibility of the yeasts as heterologous expression hosts, we have constructed an integrative vector, p1926Zeo containing the yeast 26S rDNA and Zeocin® resistance cassette. The p1926Zeo vector was linearised and transformed into both P. anomala and H. guilliermondii isolates via electroporation. Both hosts were successfully transformed at a relatively high efficiency. The transformants obtained had a growth profile similar to the respective wild type, indicating that integration of the plasmids into the host chromosome did not affect growth. These transformants were stable as they exhibited resistance to Zeocin even after 20 generations. Thus, both P. anomala and H. guilliermondii isolates exhibited the potential to be further developed as alternative heterologous protein expression hosts.
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.