• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.223-228
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• 2008

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.410-417
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• 2013

In 2011-2012, sixty nine samples were collected from alfalfa plants showing viral infection symptoms in Riyadh region. Mechanical inoculation with sap prepared from two collected samples out of twenty five possitive for Alfalfa mosaic virus (AMV) by ELISA were produced systemic mosaic on Vigna unguiculata and Nicotiana tabacum, local lesion on Chenopodium amaranticolor and C. quinoa. Vicia faba indicator plants that induce mosaic and mottle with AMV-Sagir isolate and no infection with AMV-Wadi aldawasser isolate. Approximately 700-bp was formed by RT-PCR using AMV coat protein specific primer. Samples from infected alfalfa gave positive results, while healthy plant gave negative result using dot blot hybridization assay. The nucleotide sequences of the Saudi isolates were compared with corresponding viral nucleotide sequences reported in GenBank. The obtained results showed that the AMV from Australia, Brazil, Puglia and China had the highest similarity with AMV-Sajer isolate. While, the AMV from Spain and New Zealaland had the lowest similarity with AMV-Sajer and Wadi aldawasser isolates. The data obtained in this study has been deposited in the GenBank under the accession numbers KC434083 and KC434084 for AMV-Sajer and AMV-Wadialdawasser respectively. This is the first report regarding the gnetic make up of AMV in Saudi Arabia.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.6
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• pp.676-681
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• 2002

Different types of transcripts encoding growth hormone (GH) were identified from cDNA libraries constructed with pituitaries of a marine fish species, greenling (Hexagrammos otakii). GH-homologous cDNA clones were isolated using the high-density filter hybridization and the expressed sequence tag techniques. Of 39 full-length positive cDNA clones, 31 clones ($79\%$) displayed an identical sequence, however, remaining 8 clones exhibited several polymorphisms in their sequences including (1) the length and sequence variability in the 5' upstream region, (2) insertional sequences in open reading frame, and (3) deletion and/or single nucleotide polymorphism in the untranslated 3' region. Based on RT-PCT and RNA dot blot analyses, these transcripts were proven to be expressed in a pituitary-specific manner.

• Molecular & Cellular Toxicology
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• v.2 no.1
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• pp.60-66
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• 2006

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. This study, using of suppression subtractive hybridization (SSH) method, was peformed to identify differentially expressed genes by MeHg in SH-SY5Y human neuroblastoma cell line. We prepared to total RNA from SH-SY5Y cells treated with solvent (DMSO) and $6.25\;{\mu}M\;(IC_{50})$ MeHg and performed forward and reverse SSH. Differentially expressed cDNA clones were screened by dot blot, sequenced and confirmed that individual clones indeed represent differentially expressed genes with real time RT-PCR. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

• Research in Plant Disease
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• v.17 no.3
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• pp.358-363
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• 2011

Dapple fruits of plum cv. Oiishiwase (Prunus salicina L.) were occurred at Gyeonggi-do and Gyeongsangbukdo. The symptoms resembled the dapple fruit disease caused by Hop stunt viroid (HSVd). To identify the causal disease agents, RT-PCR was performed with the specific primers of HSVd. RT-PCR analysis showed that HSVd variants (DP1, DP2) were detected from dapple fruits. HSVd detection was also confirmed by the dot blot hybridization using a DIG-probe specific to HSVd. Nucleotide sequences of DP1 and DP2 had the identities of 94-100% with those of other 7 variants of HSVd in Genbank database. DP1 and DP2 were different in two nucleotides of CG and AA at position of 59 and 60, orderly. Based on nucleotide sequences at position of 59 and 60, HSVd variants associated with plum dapple fruits could be divided mainly into three groups as CG, AA and TG.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.132-137
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• 1999

This study was carried out to develop the molecular marker for the detection of Pseudomonas tolaasii, a causative agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). When several primers designed from repetitive sequences and pectin lyase genes of bacteria were used to produce DNA polymorphism from different Pseudomonas spp. isolated from edible mushrooms, PEU1 primer derived from pectin lyase gene produced polymorphic bands differentiating P. tolaasii strains from other Pseudomonas species. Two bands, 1.0kb and 0.4kb, found commonly in 6 isolates of P. tolaasii were cloned into pGEM-T vector which were designated as pPTOP1 and pPTOP2, respectively, to use as probe. The 0.4 kb insert of pPTOP2 hybridized to only 6 isolates of P. tolaasii, but did not to the other Pseudomonas species. As few as $1.5{\times}10^3$ colony forming unit (cfu) of P. tolaasii could be detected by dot blot hybridization with the cloned 0.4kb DNA in pPTOP2.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.1
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• pp.17-24
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• 2010

In the antral follicle phase, several layers of cumulus cells surround the oocyte and play an important support and regulation role in oocyte development and maturation via intercellular communications and interactions between oocytes and cumulus cells. However, information on stage specific gene expression in swine during the phase is not well understood. To investigate the function of cumulus cells during in vitro maturation of porcine oocytes and gene expression, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed between cumulus-oocyte complexes (COCs) and naked oocytes (NOs). Utilizing mRNAs from in vitro maturation oocytes, a SSH cDNA library from COCs as the tester and NOs as the driver was constructed. The SSH cDNA library was then screened using dot blot analysis. Results showed that a total of 70 clones randomly selected from the library were differentially expressed. Among these, 41 exhibited high homology to known genes and 11 were novel expressed sequences tags (ESTs). Four differentially expressed genes, including bfgf, sprouty 2, egr and btc, were further studied by real time quantitative PCR; results confirmed an increased expression of respective mRNA in COCs compared with NOs, which suggests that these factors may play an important role in oocyte development and maturation.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.10a
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• pp.177-177
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• 2003

Methylmercury (MeHg) is a well-known neurotoxicant that causes severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, suppressive subtractive hybridization (SSH) was performed to identify differentially expressed genes on human neuroblastoma cell line, SH-SY5Y treated with DMSO and MeHg (6.25 uM) for 6 hr. Differentially expressed cDNA clones were sequenced and were screened by dot blot to eliminate false positive clones. 13 of 35 screened genes were confirmed using real time RT-PCR. These genes include EB1,90-kDa heat-shock protein, chromosome condensation-related SMC-associated protein and brain peptide Al, etc. Analysis of these genes may provide an insight into the neurotoxic effects of MeHg in human neuronal cells and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

• The Plant Pathology Journal
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• v.34 no.5
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• pp.426-434
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• 2018

During the spring season of 2014, a total of 148 melon and watermelon leaf samples were collected from symptomatic and asymptomatic plants in the western and southwestern regions of Saudi Arabia and were tested for the presence of Watermelon chlorotic stunt virus (WmCSV) and other suspected cucurbit viruses by double antibody sandwich enzyme-linked immunosorbent assays. Ninety-eight samples were found to be positive for the presence of WmCSV, nine samples were positive for the presence of Cucurbit yellow stunting disorder virus (CYSDV), and 22 showed a mixed infection with both WmCSV and CYSDV. No other cucurbit viruses were detected in any of the samples. Host range experiments revealed that eight out of fourteen tested plant species were susceptible to WmCSV. PCR products of approximately 1.2 kb were obtained after amplification using primers specifically targeting the coat protein region of WmCSV. Positive PCR results were confirmed by dot blot hybridization. Coat protein gene sequences from eleven WmCSV isolates indicated that the highest identity was between the 104WMA-SA isolate from the Wadi Baish location and a previously reported isolate from the AL-Lith location in Saudi Arabia. The lowest identity was observed between the 42WMA-SA isolate and an isolate from Palestine.

• Journal of Aquaculture
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• v.19 no.3
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• pp.157-165
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• 2006

Expression of major antioxidant enzyme (AOE) including Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione-S-transferase (GST) and 3 glutathione peroxidase isotypes (GPXs) at mRNA levels during heat stress was examined in mud loach (Misgurnus mizolepis) liver. Based on the semi-quantitative RT-PCR, real-time RT-PCR and/or northern dot blot hybridization, the antioxidant enzyme genes were generally up-regulated during elevation of water temperature from $23^{\circ}C$ up to $32^{\circ}C$. GPXs and SOD displayed the most significant elevation of mRNA levels (up to 3 and 2 folds, respectively) while CAT showed the steady-state expression irrespective of thermal conditions. GST represented the relatively moderate response (1.3-fold increase) in its transcription to thermal stress. The transcriptional activation of AOE genes was not significant at the treatment temperature lower than $29^{\circ}C$. Increased mRNA levels of GPX (extracellular form) and SOD genes in the fish exposed to $32^{\circ}C$ was readily detectable 1 day after exposure to heat stress.