Browsing by Subject "linkage"
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Item Resolution of the pair-wise allosteric interactions found in phosphofructokinase from Bacillus stearothermophilus(Texas A&M University, 2004-09-30) Ortigosa, Allison DawnPhosphofructokinase from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme with an average of one active site and one allosteric site per subunit. BsPFK is inhibited by phosphoenolpyruvate (PEP) and how this inhibitory signal is propagated throughout the enzyme is the main question we address through this investigation. By possessing a total of eight binding sites, a potential for twenty-eight total pair-wise allosteric interactions result within BsPFK, ten of which are unique. Of these ten interactions, four are heterotropic interactions, or interactions between unlike binding sites, while the remaining six interactions are homotropic interactions, or interactions between like binding sites. Thus, to address the question of how BsPFK is inhibited by PEP, each of these ten interactions needs to be quantified and their roles in the inhibition process assessed. In order to quantify the roles of the 10 allosteric interactions, we created, purified and characterized several different hybrid enzymes by using site-directed mutagenesis to reduce the number of native active sites and native allosteric sites to permit the isolation of specific allosteric interaction(s). Through the creation and isolation of 1:3 hybrid enzymes, in which one native active site and one native allosteric site remain, each of the four heterotropic interactions were characterized. Moreover, through the creation and isolation of the 2:2 hybrid enzymes, in which two native active sites and two native allosteric sites remain, characterization of the remaining six homotropic interactions was performed. Utilizing a linked function approach to quantify the heterotropic and homotropic effects for each hybrid enzyme, we determined that 5 to 6 of the ten pair-wise allosteric interactions found in BsPFK are involved in the inhibition process depending upon pH. More importantly however, our data provides definitive results that the traditional two-state models used to describe an allosteric effect are not sufficient to describe the allosteric effect measured for BsPFK. Rather, our results show that the linked function approach is a more appropriate way to unambiguously measure the nature and magnitude of an allosteric effect. Moreover, this approach can also be used to explain the allosteric behavior of a dimeric enzyme.Item Transmission genetics of pancreatic acinar atrophy in the German Shepherd Dog and development of microsatellite DNA-based tools for canine forensics and linkage analysis(Texas A&M University, 2004-09-30) Clark, Leigh AnneThe domestic dog, Canis lupus familiaris, has emerged as a model system for the study of human hereditary diseases. Of the approximately 450 hereditary diseases described in the dog, half have clinical presentations that are quite similar to specific human diseases. Understanding the genetic bases of canine hereditary diseases will not only complement comparative genetics studies but also facilitate selective breeding practices to reduce incidences in the dog. Whole genome screens have great potential to identify the marker(s) that segregate with canine hereditary diseases for which no reasonable candidate genes exist. The Minimal Screening Set-1 (MSS-1) was the first set of microsatellite markers described for linkage analysis in the dog and was, until recently, the best tool for genome screens. The MSS-2 is the most recently described screening set and offers increased density and more polymorphic markers. The first objective of this work was to develop tools to streamline genomic analyses in the study of canine hereditary diseases. This was achieved through the development of 1) multiplexing strategies for the MSS-1, 2) a multiplex of microsatellite markers for use in canine forensics and parentage assays and 3) chromosome-specific multiplex panels for the MSS-2. Multiplexing is the simultaneous amplification and analysis of markers and significantly reduces the expense and time required to collect genotype information. Pancreatic acinar atrophy (PAA) is a disease characterized by the degeneration of acinar cells of the exocrine pancreas and is the most important cause of exocrine pancreatic insufficiency (EPI) in the German Shepherd Dog (GSD). Although the prognosis for dogs having EPI is typically good with treatment, many dogs are euthanized because the owners are unable to afford the expensive enzyme supplements. The second objective of this work was to determine the mode of transmission of EPI in the GSD and conduct a whole genome screen for linkage. Two extended families of GSDs having PAA were assembled and used to determine the pattern of transmission. The results of this indicate that PAA is an autosomal recessive disease. The multiplexed MSS-1 was used to conduct an initial whole genome screen, although no markers were suggestive of linkage.