Browsing by Subject "Genetic markers"
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Item An examination of the crocodilian mitochondrial control region: structural and functional units and utility in phylogenetic and phylogeographic analysis(Texas Tech University, 2002-12) Ray, David AThe use of molecular markers for elucidating crocodilian phylogenetic relationships has been ongoing for several decades now. While significant progress has been made in understanding how each of the three major morphological groups of crocodilians are related to one another and to the remainder of Archosauria, issues involving the relationships of the two gharial species to the remainder of the order Crocodylia and understanding phylogeny within the family Crocodylidae remain. In this dissertation, I describe the development of a new marker for examining each of these issues as well as problems involving population level relationships. For representatives of each major crocodilian lineage, the mitochondrial control region is characterized from structural and functional perspectives and then utilized as a marker for the determination of phylogenetic relationships within the Crocodylia and for examining the population dynamics of one species, Crocodylus moreletii. I found that the control region of crocodilians contains several features in common with other vertebrate groups as well as several features that deserve special interest, including a series of heteroplasmic tandem repeats. I also found that the control region appears to be useful in resolving relationships at several levels within the order Crocodylia. Finally, while control region sequences reveal little with regard to phylogeography in C. moreletii, the inclusion of these new data have significantly affected our interpretation of other studies with regard to population structure in this species.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 Characterization of the mitochondrial DNA control region of Clethrionomys and its use as a genotoxicological marker(Texas Tech University, 1999-12) Matson, Cole WesleyThe purpose of this study was to develop a maternal marker which could be utilized in genotoxicological and systematic studies. The mtDNA control region was chosen as the area of primary interest because of its high rate of evolution, and the fact that this region has been shown to be useful in previous gene diversity studies (Bickham et al., 1998). To understand which portion of the mtDNA control region would be appropriate to use to address various aspects of population genetics use, I analyzed the sequence variation across various regions of the control region of the genus Clethrionomys. This work is presented in Chapter II, and has been submitted to the journal. Molecular Biology and Evolution. The authors on this manuscript were: Robert J. Baker and Cole W. Matson. The maternal marker that was chosen after consideration of the results presented in Chapter II was employed to examine the consequences of long-term chronic radiation exposure. To study the patterns of variation across time and space in exposed and unexposed populations of Clethrionomys glareolus inhabiting highly contaminated sites in the Chomobyl region, I examined three populations. The results of this study are presented in Chapter III. This chapter has been submitted for publication in the journal. Environmental Toxicology and Chemistry. The authors on this manuscript were: Cole W. Matson, Brenda E. Rodgers, Ronald K. Chesser, and Robert J. Baker.Item Development of species- and genome-specific genetic markers by representational difference analysis: application in systematic and evolutionary research(Texas Tech University, 1999-08) Nekrutenko, AntonIn the study of biodiversity, it is important to have a reliable system for identification of various genetically distinct units (species, subspecies, etc). One of the most efficient tools available today is the polymerase chain reaction (PCR) with diagnostic primers, that yield a detectable product for one taxon but not for other taxa. Critical to this method is the identification of diagnostic DNA fragments from which primers can be designed. Representational difference analysis (RDA) can reliably isolate DNA fragments that are unique to a specific taxon. In this report, we demonstrate the utility of the technique by development of binary markers that distinguish between two cryptic species of voles (genus Microtus).Item Localization of chromosomal regions influencing the phenotypes of the metabolic syndrome(2004) Cai, Guowen; Freeland-Graves, Jeanne H.The goal of this project was to study the genetic structure of the metabolic syndrome. The first objective was to locate chromosomal regions influencing insulin resistance in Mexican Americans of the San Antonio Family Heart Study (SAFHS). Two studies were performed to achieve this objective using a genomewide scan. In the first study using data from the first visit of the SAFHS, we detected significant linkage evidence on chromosome 8p between marker D8S1130 and D8S1106 and on chromosome 13q between marker D13S787 and D13S252. In the second study that used data from the second visit of the SAFHS, markers D1S1663 on chromosome 1 and D2S436 on chromosome 2 were found to be linked to insulin sensitivity indices. Candidate genes on detected locations were proposed. The significant findings in both studies duplicate those of previous investigations. The second objective of this project was to identify the genetic locations related to the quantitative traits that constitute the metabolic syndrome in the same population of Mexican Americans. Principal component factor analysis (PCFA) was conducted, and significant and suggestive evidence for linkage of lipid (factor 4) and body size/adiposity (factor 1) were found on chromosome 4 near marker D4S403 and chromosome 1 near marker D1S1597, respectively. The third objective of this project was to explore the genetic pleiotropy between insulin resistance and adiposity, especially visceral obesity using the baboon as a model. The present study is the first to use omental tissue to investigate gene pleiotropy between visceral fat and insulin resistance. The results from the baboon study in this thesis, coupled with research in humans, suggest that a common set of genes contribute to insulin resistance and obesity in both species. It is also plausible that those two groups of genes completely overlap each other. At present, the variance decomposition based, multipoint linkage analysis is a mathematical model that can provide useful information for susceptible gene mapping. Future finemapping and the positional candidate gene approach will be helpful to further our understanding of the genetic structure of this complex disease.Item Microsatellite evolution in mice (Apodemus): origin of alleles, multiple paternity, and mutation rate at Chernobyl(Texas Tech University, 1999-05) Makova, Kateryna D.The studies of fauna at Chemobyl are very complex. They involve obtaining permits to the restricted zone, intensive field work with exposure to radiation, precise cataloging of skins and tissues in the museum, laboratory molecular biology work and sophisticated data analysis. This requires a collaborative effort. Several papers were outlined as a function of my dissertation. Their status and correspondence to the chapters of the dissertation are outlined in Table 1.1. The author line reflects the complexity of work at Chernobyl and the need to have both field and laboratory analyses. The choice of mice of the genus Apodemus was dictated by their abundance in both control and zone locations (Baker et al. 1996). In addition, Apodemus is closely related to Mus and Rattus: all three genera belong to the same subfamily (Murinae). The original idea was to test microsatellite primer pairs that were shown to successfully amplify both Mus and Rattus DNA on DNA from Apodemus. Four primer pairs specific to both Mus and Rattus microsatellites in genes encoding tumor necrosis factor, nerve growth hormone, insulin-like growth factor II and c-myc oncogene (Moore ef al. 1991) were used for amplification of Apodemus DNA. Consistently clean amplification products could not be obtained. This is in agreement with Kondo etal. (1993), who used Rattus microsatellite primers to amplify mouse Mus DNA and vice versa. Less than 10% of primer pairs they tested amplified DNA from the other species and gave polymorphic PCR products. Thus, I was faced with the challenge of developing microsatellites specifically for Apodemus.