Browsing by Subject "RT-PCR"
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Item Effect of heat shock on hilA expression in Salmonella Typhimurium(Texas A&M University, 2005-02-17) Churi, Asawari ShreeniwasThe effect of heat shock was observed on the expression of hilA in Salmonella Typhimurium by creating a fluorescence-based reporter strain of Salmonella and by realtime reverse transcriptase polymerase chain reaction (RT-PCR). The hilA gene in Salmonella is known to play an important role in its pathogenesis. hilA is known to be activated when the bacteria encounter stress-inducing conditions. A number of factors have been identified that affect hilA expression, such as, pH, osmolarity, oxygen tension. When Salmonella enter their warm-blooded hosts, they encounter an increase in temperature. Therefore, heat is another stressor that is encountered by Salmonella during infection of their hosts. A fluorescence-based strain of Salmonella was created to study the effect of heat shock. The gene for green fluorescent protein (gfp) was placed under the control of the promoter of hilA on a plasmid. This plasmid was used to transform Salmonella cells to create a fluorescent strain. In this strain, when the hilA promoter is activated, gfp is transcribed, which encodes the green fluorescent protein. This protein can be measured by a fluorescence assay. The results of this study indicated that at 45?C, hilA is activated. RT-PCR was used to look at hilA expression at different temperature. The results of this study indicated that, compared to 37?C, higher temperatures like 45?C and 55?C significantly activate hilA.Item Expression of defense genes in sorghum grain mold and tagging and mapping a sorghum anthracnose resistance gene(2009-05-15) Katile, Seriba OusmaneSorghum grain mold and anthracnose are two major diseases of sorghum (Sorghum bicolor) that constrain sorghum production worldwide. Grain mold is caused by several species of fungi, but the two most common are Curvularia lunata and Fusarium thapsinum. Isolates of these two species were used to inoculate panicles of selected sorghum cultivars in green house and field experimentations. Panicles were sprayed at the time of anthesis with conidial suspensions of the two fungal species individually or in a mixture and with water to serve as a control. Samples were collected 48 hours after inoculation for RNA extraction. In greenhouse studies, four cultivars (Tx2911, Sureno, SC170 and RTx430) were used while thirteen cultivars were grown in the field experiments. Gene expression was measured for the following genes using real time polymerase chain reactions (rt-PCR): PR10, ?-glucanase, chitinase, thaumatin, sormatin, phenyalanine ammonia lyase (PAL), obtusifoliol 14?-demethylase (Obtus), antifungal protein (AFP), apoptosis related protein (Apop) and leucine rich repeat (LRR). Seed germination tests in field grown cultivars indicated that germination rates for SC279-14E, SC660 and Sureno were not greatly influenced by grain mold. Covering the panicles with bags served to protect them against grain mold pathogens. The seed mycoflora test showed that Fusarium thapsinum was the most frequently recovered species and there were more species present in non-covered panicles. The response of sorghum cultivars to grain mold infection involves multiple defense genes. Real time PCR used to study the expression of sorghum defense in greenhouse grown plants showed that mRNA encoding PR-10, a small 10 kDa protein, was highly expressed in the glumes and spikelets of resistant cultivars Tx2911 and Sureno and constitutively in leaves. The expression of some other defense genes like beta-glucanase, chitinase and AFP was variable. Sormatin was not expressed. Expression of ?-glucanase, chitinase, and PR10 was higher in field than in greenhouse experiments. A second area of research involved tagging of a resistance gene for sorghum anthracnose. Three AFLP markers (Xtxa607, Xtxa3181 and Xtxa4327) and three SSRs (Xtxp3, Xtxp55 and Xtxp72) were identified. These markers were loosely linked to the resistance genes. The markers are located on linkage group B. The results suggest that markers located 20-30 cM on one side or the other of those tested should provide useful tags for the resistance gene.Item Growth and virulence response of Salmonella typhimurium to soluble Maillard reaction products(Texas A&M University, 2004-09-30) Kundinger, Megan MaryIn order to determine the effects that Maillard Reaction Products (MRP) have on Salmonella Typhimurium, growth rates and virulence expression, in the presence of Maillard reaction products, were observed, using the ??galactosidase Miller Assay and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The presence of MRP compounds in liquid media caused no negative effect on the growth rate of Salmonella cells. However, the addition of MRP compounds at a 1% level in the media caused a significant increase in hilA expression in Salmonella Typhimurium, and the highest induction levels were observed in media supplemented with arginine and histidine-MRP compounds. There was no effect on the induction of hilA with the 0.5% addition of the MRP compounds in the amended media as shown by the Miller Assay. However, there was an effect seen when using the Real Time RT-PCR assay that resulted in the same levels of significance seen at 1.0% additions of MRPs being seen at the 0.5% level as well. Since rsmC was shown to be a constitutive gene that had continuous levels of expression in Salmonella based on cell number, Real-Time PCR was then used to assess the hilA expression of Salmonella Typhimurium under different oxygen, pH levels, and osmolarity conditions. The results under low oxygen indicate that the combination of low osmolarity and high pH have the highest inducing effect on hilA expression. The hilA response under the same media conditions and a high oxygen environment showed the same pattern of expression as those bacteria grown under a non-aerobic environment. The media with a pH of 8 and low osmolarity conditions had the greatest effect on the induction of hilA with none of the other media showing any significant effect. The relative expression of hilA did decrease for those bacteria grown under aerobic conditions versus those grown under low oxygen conditions.Item MicroRNA expression in canine mammary cancer(Texas A&M University, 2008-10-10) Boggs, Rene' MichelleMicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan? Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.