Search results for: cDNA

Authors: Chintanu K. Sarmah, Sandhya Samarasinghe, Don Kulasiri, Daniel Catchpoole

Abstract:

Gene expression profiling is rapidly evolving into a powerful technique for investigating tumor malignancies. The researchers are overwhelmed with the microarray-based platforms and methods that confer them the freedom to conduct large-scale gene expression profiling measurements. Simultaneously, investigations into cross-platform integration methods have started gaining momentum due to their underlying potential to help comprehend a myriad of broad biological issues in tumor diagnosis, prognosis, and therapy. However, comparing results from different platforms remains to be a challenging task as various inherent technical differences exist between the microarray platforms. In this paper, we explain a simple ratio-transformation method, which can provide some common ground for cDNA and Affymetrix platform towards cross-platform integration. The method is based on the characteristic data attributes of Affymetrix- and cDNA- platform. In the work, we considered seven childhood leukemia patients and their gene expression levels in either platform. With a dataset of 822 differentially expressed genes from both these platforms, we carried out a specific ratio-treatment to Affymetrix data, which subsequently showed an improvement in the relationship with the cDNA data.

Keywords: Gene expression profiling, microarray, cDNA, Affymetrix, childhood leukaemia.

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Authors: Prasad Senadheera, Younousse Saidi, Frans JM Maathuis

Abstract:

Rice seed expression (cDNA) library in the Lambda Zap 11® phage constructed from the developing grain 10-20 days after flowering was transformed into yeast for functional complementation assays in three salt sensitive yeast mutants S. cerevisiae strain CY162, G19 and Axt3K. Transformed cells of G19 and Axt3K with pYES vector with cDNA inserts showed enhance tolerance than those with empty pYes vector. Sequencing of the cDNA inserts revealed that they encode for the putative proteins with the sequence homologous to rice putative protein PROLM24 (Os06g31070), a prolamin precursor. Expression of this cDNA did not affect yeast growth in absence of salt. Axt3k and G19 strains expressing the PROLM24 were able to grow upto 400 mM and 600 mM of NaCl respectively. Similarly, Axt3k mutant with PROLM24 expression showed comparatively higher growth rate in the medium with excess LiCl (50 mM). The observation that expression of PROLM24 rescued the salt sensitive phenotypes of G19 and Axt3k indicates the existence of a regulatory system that ameliorates the effect of salt stress in the transformed yeast mutants. However, the exact function of the cDNA sequence, which shows partial sequence homology to yeast UTR1 is not clear. Although UTR1 involved in ferrous uptake and iron homeostasis in yeast cells, there is no evidence to prove its role in Na+ homeostasis in yeast cells. Absence of transmembrane regions in Os06g31070 protein indicates that salt tolerance is achieved not through the direct functional complementation of the mutant genes but through an alternative mechanism.

Keywords: Rice seed expression, salt stress, prolamin, salinitytolerance, Oryza sativa

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

Abstract:

Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA microarray. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 NanoLabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring softwares analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with downregulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38-MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: CARD16, CASP10, cDNA microarray, PTPRR, Thymoquinone.

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Authors: JiEun Jeong, Dong Hyun Kim, Bharat Bhusan Patnaik, Se Won Kang, HeeJu Hwang, Yong Hun Jo, Dae-Hyun Seog, YeonSooHan, Yong Seok Lee

Abstract:

An expressed sequence tag (EST) analysis provideus portions of expressed genes. We have constructed cDNA library and determined randomly sequences from cDNA library clones of T. molitor injected with acholeplasma lysate. We identified the homologous to a galectin gene. As the result of cloning and characterization of novel, we found that the protein has an open reading frame (ORF) of 495 bp, with 164 amino acid residues and molecular weight of 18.5 kDa. To characterize the role of novel Tm-galectin in immune system, we quantified the mRNA level of galectin at different times after treatment with immune elicitors. The galectin mRNA was up-regulated about 7-folds within 18 hrs. This suggests that Tm-galectin is a novel member of animal lectins, and has a role in the process of pathogen recognition. Our study would be helpful for the study on immune defense system and signaling cascade.

Keywords: EST, Innate immunity, Tenebrio molitor, Galectin.

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Authors: N. Umasuthan, S. D. N. K. Bathige, W. S. Thulasitha, I. Whang, J. Lee

Abstract:

The MyD88 is an evolutionarily conserved host-expressed adaptor protein that is essential for proper TLR/ IL1R immune-response signaling. A previously identified complete cDNA (1626 bp) of OfMyD88 comprised an ORF of 867 bp encoding a protein of 288 amino acids (32.9 kDa). The gDNA (3761 bp) of OfMyD88 revealed a quinquepartite genome organization composed of 5 exons (with the sizes of 310, 132, 178, 92 and 155 bp) separated by 4 introns. All the introns displayed splice signals consistent with the consensus GT/AG rule. A bipartite domain structure with two domains namely death domain (24-103) coded by 1^st exon, and TIR domain (151-288) coded by last 3 exons were identified through in silico analysis. Moreover, homology modeling of these two domains revealed a similar quaternary folding nature between human and rock bream homologs. A comprehensive comparison of vertebrate MyD88 genes showed that they possess a 5-exonic structure.In this structure, the last three exons were strongly conserved, and this suggests that a rigid structure has been maintained during vertebrate evolution.A cluster of TATA box-like sequences were found 0.25 kb upstream of cDNA starting position. In addition, putative 5'-flanking region of OfMyD88 was predicted to have TFBS implicated with TLR signaling, including copies of NFkB1, APRF/ STAT3, Sp1, IRF1 and 2 and Stat1/2. Using qPCR technique, a ubiquitous mRNA expression was detected in liver and blood. Furthermore, a significantly up-regulated transcriptional expression of OfMyD88 was detected in head kidney (12-24 h; >2-fold), spleen (6 h; 1.5-fold), liver (3 h; 1.9-fold) and intestine (24 h; ~2-fold) post-Fla challenge. These data suggest a crucial role for MyD88 in antibacterial immunity of teleosts.

Keywords: MyD88, Innate immunity, Flagellin, Genomic analysis.

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Authors: Khlopova N.S., Glazko V.I., Glazko T.T.

Abstract:

Using DNA microarrays the comparative analysis of a gene expression profiles is carried out in a liver and kidneys of pigs. The hypothesis of a cross hybridization of one probe with different cDNA sites of the same gene or different genes is checked up, and it is shown, that cross hybridization can be a source of essential errors at revealing of a key genes in organ-specific transcriptome. It is reveald that distinctions in profiles of a gene expression are well coordinated with function, morphology, biochemistry and histology of these organs.

Keywords: Microarray, gene expression profiles, key genes.

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Authors: Yulia Irnidayanti, Win Darmanto, Agus Abadi

Abstract:

research goal was to determine the expression levels cDNA of brain embrio at gestation days 10 (GD-10). The Electroforesis DNA results showed that GAPDH, Fibronectin1, Ncam1, Tenascin, Vimentin, Neurofilament heavy, Neurofilament medium and Neurofilament low were 447 bp, 462 bp, 293 bp. 416 bp, 327 bp, 301 bp, 398 bp and 289 bp. Result of real-time RT-PCR on brain Embryo at gestation days 10 showed that the expression of copy gen Fibronectin 36 copies, Ncam 21,708 copies; Tenascin 24,505 copies; Vimentin 538,554 copies; Neurofilament heavy 2,419 copies; Neurofilament medium 92,928 copies; Neurofilament low 125,809 copies. Vimentin expressed gene copies is very high compared with other gene copies. This condition are caused by Vimentin, that contribute to proliferate of brain development. The vimentin role to cell proliferation of brain.

Keywords: GAPDH, Fibronectin, Ncam, Tenascin, vimentin, Neurofilamen heavy, Neurofilament medium, Neurofilamen low.

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Authors: Yan Ren, Fei Guo, Jun Qiao, Shengwei Hu, Hui Zhang, Yuanzhi Wang, Pengyan Wang, Jinliang Sheng, Xinli Gu, Xiaojun Liu, Chuangfu Chen

Abstract:

Bovine viral diarrhea virus (BVDV) can cause lifelong persistent infection. One reason for the phenomena is attributed to BVDV infection to placenta tissue. However the mechanisms that BVDV invades into placenta tissue remain unclear. To clarify the molecular mechanisms, we investigated the possible means that BVDV entered into bovine trophoblast cells (TPC). Yeast two-hybrid system was used to identify proteins extracted from TPC, which interact with BVDV envelope glycoprotein E2. A PGbkt7-E2 yeast expression vector and TPC cDNA library were constructed. Through two rounds of screening, three positive clones were identified. Sequencing analysis indicated that all the three positive clones encoded the same protein clathrin. Physical interaction between clathrin and BVDV E2 protein was further confirmed by coimmunoprecipitation experiments. This result suggested that the clathrin might play a critical role in the process of BVDV entry into placenta tissue and might be a novel antiviral target for preventing BVDV infection.

Keywords: Bovine viral diarrhea virus, clathrin, glycoprotein E2, yeast two-hybrid system.

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Authors: M. Kaomek, J. R. Ketudat-Cairns

Abstract:

The cDNA encoding the 326 amino acids of a Class I basic chitinase gene from Leucaena leucocephala de Wit (KB3, Genbank accession: AAM49597) was cloned under the control of CaMV35S promoter in pCAMBIA 1300 and transferred to Koshihikari. Calli of Koshihikari rice was transformed with agrobacterium with this construct expressing the chitinase and β- glucouronidase (GUS). The frequencies of calli 90 % has been obtained from rice seedlings cultured on NB medium. The high regeneration frequencies, 74% was obtained from calli cultured on regeneration medium containing 4 mg/l BAP, and 7 g/l phytagel at 25°C. Various factors were studied in order to establish a procedure for the transformation of Koshihikari Agrobacterium tumefaciens. Supplementation of 50 mM acetosyringone to the medium during coculivation was important to enhance the frequency to transient transformation. The 4 week-old scutellum-derived calli were excellent starting materials. Selection medium based on NB medium supplement with 40 mg/l hygromycin and 400 mg/l cefotaxime were an optimized medium for selection of transformed rice calli. The percentage of transformation 70 was obtained. Recombinant calli and regenerated rice plants were checked the expression of chitinase and gus by PCR, northern blot gel, southern blot gel, and gus assay. Chitinase and gus were expressed in all parts of recombinant rice. The rice line expressing the KB3 chiitnase was more resistant to the blast fungus Fusarium monoliforme than control line.

Keywords: chitinase, Leucaena leucocephala de Wit, Koshihikari, transgenic rice.

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Authors: W. Loongyai, N. Saengsawang, W. Danvilai, C. Kridtayopas, P. Sopannarath, C. Bunchasak

Abstract:

Betong chicken is a slow growing and a lean strain of chicken, while the rapid growth of broiler is accompanied by increased fat. We investigated the growth performance, fat accumulations, lipid serum biochemical levels and lipoprotein lipase (LPL) gene expression of female Betong (KU line) at the age of 4 and 6 weeks. A total of 80 female Betong chickens (KU line) and 80 female broiler chickens were reared under open system (each group had 4 replicates of 20 chicks per pen). The results showed that feed intake and average daily gain (ADG) of broiler chicken were significantly higher than Betong (KU line) (P < 0.01), while feed conversion ratio (FCR) of Betong (KU line) at week 6 were significantly lower than broiler chicken (P < 0.01) at 6 weeks. At 4 and 6 weeks, two birds per replicate were randomly selected and slaughtered. Carcass weight did not significantly differ between treatments; the percentage of abdominal fat and subcutaneous fat yield was higher in the broiler (P < 0.01) at 4 and 6 week. Total cholesterol and LDL level of broiler were higher than Betong (KU line) at 4 and 6 weeks (P < 0.05). Abdominal fat samples were collected for total RNA extraction. The cDNA was amplified using primers specific for LPL gene expression and analysed using real-time PCR. The results showed that the expression of LPL gene was not different when compared between Betong (KU line) and broiler chickens at the age of 4 and 6 weeks (P > 0.05). Our results indicated that broiler chickens had high growth rate and fat accumulation when compared with Betong (KU line) chickens, whereas LPL gene expression did not differ between breeds.

Keywords: Lipoprotein lipase gene, Betong (KU line), broiler, abdominal fat, gene expression.

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