Browsing by Subject "Polymerase chain reaction (PCR)"
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Item Analysis of sodium pump gene expression in microdissected nephrons using competitive RT-PCR and a novel HPLC technique(Texas Tech University, 1996-12) Hayward-Lester, Amanda LHypertension is determined by the interaction of multiple genetic and environmental factors, making it difficult to elucidate any single genetic determinant. Biochemical markers such as intraerythrocytic sodium concentration, erythrocytic ouabain binding-site density and passive sodium leak indicate that abnormal membrane cation flux segregates with some hypertension subtypes. The sodium pump, Na+, K+ ATP-ase (NKA) may therefore participate in the development of hypertension. It is a multi-subunit cell membrane protein which translocates sodium and potassium ions with hydrolysis of ATP. It is inhibited by ouabain and regulated by phosphorylation. The alpha subunit (of which there are four isoforms encoded by different genes) is currently ascribed all catalytic function, while the beta and gamma subunits may have regulatory roles. We examined NBCA alpha and gamma subunit gene expression in spontaneously hypertensive rats (SHR) and Wistar Kyoto controls (WKY). Both prehypertensive and adult SHR exhibit abnormal renal sodium retention. Solution hybridization studies in adult SHR revealed a decrease in alpha 1 expression in kidney. Kidney is a heterogeneous tissue whose functional unit, the nephron, may be divided into 12 distinct segments. To examine NKA expression in individual segments requires an assay allowing quantitation of NKA alpha and gamma isoform RNA in microdissected tissue samples. We combined competitive RT-PCR with a novel ion-paired reversed phase HPLC to produce rapid, accurate and precise measurement of gene expression in a single-tube assay. The ability of HPLC to resolve heteroduplex molecules formed between native and competitor products proved essential. Assay validation confirmed absolute quantification is possible if competitors have identical reverse-transcription efficiency to the native RNA. We used the assay to examine qualitative and quantitative expression of NKA subunits in normotensive Sprague-Dawley, prehypertensive and adult SHR and WKY. Qualitative analysis revealed alpha 1 and gamma expression in all segments examined. Expression of the other alpha isoforms was not detected. Quantitative analysis in the prehypertensive SHR revealed that a selective alteration in alpha 1 expression in proximal convoluted tubule may explain the results obtained in whole kidney and suggest an attempted feedback response by SHR to reduce sodium reabsorption.Item Culturability and dynamics of bacterial communities along the Pine Canyon Watershed of Big Bend National Park, TX(2007-12) Clark, Jeb; Zak, John; Francisco, Michael J. D. S.; Jeter, Randall M.Soil bacterial communities are known to play a significant role in ecosystems as they are responsible for, among other things, nutrient cycling, and plant and rhizosphere interactions. Those bacterial communities which persist in arid environments have gained special attention over the past several years as life in these environments is often believed to be limited due to a lack of available water. Since soil bacteria play such a significant role in the environment, understanding how seasonal changes in soil moisture and temperature affect bacterial community structure and function is critical for understanding how soil bacteria contribute to the functioning of arid ecosystems. However, the study of soil bacterial communities has been limited due to difficulties experienced in culturing organisms from the environment. Furthermore, PCR-based, culture-independent techniques have become increasingly popular amongst researchers, but these techniques which impose a bias limiting observations to only the most DNA-dominant bacterial species, species which are not believed to play a large functional role in the ecosystem. Hence, a large and possibly important proportion of the bacterial population could be commonly overlooked. A year-long, two-part research project on the environmental effects on soil bacterial communities in three sites along the Pine Canyon Watershed of Big Bend National Park began in August 2005. The first part examined the DNA-dominant bacterial community structure using PCR and Denaturing Gradient Gel Electrophoresis along with Fatty Acid Methyl Ester analysis. These techniques revealed a stable DNA-dominant bacterial community structure throughout the summer and winter seasons, and no significant changes in bacterial structure were recorded during a large precipitation event during August 2006. The second study utilized a culture-based approach to examine the functional bacterial communities along the three sites. The culture-based approach revealed that two different assemblages of soil bacteria exist, a culturable-dominant and a culturable-non-dominant. The culturable-dominant assemblage remained relatively stable throughout the seasonal changes and during the precipitation event of August 2006 and consisted largely of bacterial species of the Arthrobacter and Burkholderia genus. However, the culturable-non-dominant assemblage responded to seasonal changes by either increasing or decreasing in population density and increased in density during the precipitation event. The responses observed in the culturable-non-dominant assemblage may provide an explanation towards the difficulty many researchers experience in culturing organisms from the environment. Furthermore, culture-dependant techniques proved to be more sensitive in detecting changes in the bacterial community structure than culture-independent based techniques. Also, to aide in the collection of data for the second study, computer software was developed using the C# computer language and has been made available at: http://www.biol.ttu.edu/faculty/jzak/dgge_soft.