published_or_final_version / Biochemistry / Master / Master of Philosophy

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The objectives of this study were to identify genetic variants, develop the respective haplotypes (combination of alleles) and investigate the association of identified variants with economically important traits in two candidate genes. The first gene was Phosphoenolpyruvate carboxykinase (PEPCK) which is a key regulatory enzyme of gluconeogenesis. The second candidate gene, Ornithine decarboxylase (ODC), is a rate-limiting enzyme in polyamine biosynthesis. It has a significant role in DNA synthesis and cell proliferation. We first analyzed the genetic variability of PEPCK-C, the gene which codes for the cytosolic form of PEPCK. A 3792 by segment of 5'-region of the PEPCK-C gene (pos. -1723 to 2069) was sequenced in 32 White Leghorn chickens (a total of 64 genomes). A total of 19 single nucleotide polymorphisms (SNPs) were identified. We then analyzed the genetic variability of ODC. A 5 kb sequence of 3' end of the gene was sequenced in 20 White Leghorn chickens (a total of 40 genomes). A total of 63 variant sites were identified. The rate of insertion/deletion in ODC was 16%, whereas neither deletions nor insertions were present in PEPCK-C. Gene trees were constructed for both genes assuming maximal parsimony. This led to the delineation of 6 haplotypes in PEPCK-C. Two of the SNPs coincided with RFLP detectable by the restriction enzymes AciI and BstEII, respectively. Three haplotypes in ODC were defined. In the next step, White Leghorn chickens from a non-selected closed population were typed for these two PEPCK-C RFLP. The two RFLP gave rise to three alleles (or haplotype classes), which in turn defined six genotypes. A comparison of genotypes revealed significant differences in feed efficiency (FE) and residual feed consumption (RFC). There was significant interaction between PEPCK-C genotypes and mitochondrial PEPCK (PEPCK-M) genotypes defined by an RFLP. The latter enzyme catalyzes the same reaction, but is located in the matrix of t

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed May 22, 2007). PDF text: 466 p. : ill. ; 17.94Mb. UMI publication number: AAT3237597 . Includes bibliographical references. Also available in microfilm and microfiche formats.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed May 23, 2007). PDF text: 231 p. : ill. ; 7.97Mb UMI publication number: AAT 3237598. Includes bibliographical references. Also available in microfilm and microfiche formats.

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A soluble enzyme system has been isolated from biotin-deficient rat liver acetone powder which catalyzes the synthesis of propionyl holocarboxylase from d-biotin and its apocarboxylase, in the presence of ATP and Mg++ ions. The enzyme system has been partially purified by (NH₄)₂SO₄ precipitation and resolved into two obligatory components by alumina C y gel fractionation. The gel supernatant fraction has been further purified by DEAE cellulose ion-exchange chromatography. Since the active component in the gel supernatant fraction and the endogenous propionyl holocarboxylase have the same elution pattern when chromatographed on DEAE cellulose, it appears likely that the gel supernatant contains the propionyl apocarboxylase. The gel eluate most likely contains an enzyme which catalyzes the covalent bonding or d-biotin to propionyl apocarboxylase and other proteins. In order to measure the activity of the propionyl holocarboxylase synthesizing system, two assay procedures were used. One assay procedure employed the incorporation of biotin-1-c¹⁴ into protein as a measure or the activity of the enzyme system. The other assay was based upon the biotin and ATP dependent increase of propionyl carboxylase activity catalyzed by the enzyme system. Both procedures gave similar results. Propionyl holocarboxylase formation was found to be ATP specific since neither CTP, OTP, ITP, nor UTP could replace ATP. In addition, a mixture or all five nucleoside triphosphates was no more effective than ATP alone. Versene inhibited the reaction and MgCl₂ was able to reverse this inhibition, indicating a Mg++ ion requirement. The ability of various biotin derivatives to replace d-biotin in propionyl holocarboxylase formation was investigated. It was round that if either the valeric acid side chain is altered. as in homo- or nor-biotin, of if the sulfur atom is removed or substituted, as in desthiobiotin or oxybiotin, holocarboxylase formation did not occur. Similarly, neither of these derivatives inhibited the bonding of c¹⁴-biotin to protein. Biocytin has been eliminated as an intermediate in propionyl holocarboxylase formation. Hydroxylamine does not inhibit the reaction. nor is CoA required. These data would appear to eliminate the involvement of free carboxyl activated biotinyl intermediates in the formation of the holocarboxylase from its apocarboxylase and biotin. Although the evidence suggests a concerted mechanism for the reaction, mechanisms involving enzyme bound intermediates are not completely eliminated by these data. In order to determine the nature of the attachment of biotin to propionyl carboxylase, c¹⁴-biotin labeled propionyl carboxylase was prepared. The labeled carboxylase was enzymatically hydrolyzed and chromatographed on Whatman 3MM paper. The biocytin peak contained nearly 100% of the radioactivity recovered. This peak was eluted and rechromatographed by ion-exchange chromatography. The only radioactive component obtained by this procedure was biocytin. The data presented indicate that the propionyl holocarboxylase synthesizing system catalyzes the ATP dependent covalent bonding of d-biotin to the lysyl-(-amino groups or propionyl apocarboxylase. / Ph. D.

Plant aromatic amino acid decarboxylases (AAADs) are a group of economically important enzymes categorically joined through their pyridoxal 5'-phosphate (PLP) dependence and sequence homology. Extensive evolutionary divergence of this enzyme family has resulted in a selection of enzymes with stringent aromatic amino acid substrate specificities. Variations in substrate specificities enable individual enzymes to catalyze key reactions in a diverse set of pathways impacting the synthesis of monoterpenoid indole alkaloids (including the pharmacologically active vinblastine and quinine), benzylisoquinoline alkaloids (including the pharmacologically active papaverine, codeine, morphine, and sanguinarine), and antioxidant and chemotherapeutic amides. Recent studies of plant AAAD proteins demonstrated that in addition to the typical decarboxylation enzymes, some annotated plant AAAD proteins are actually aromatic acetaldehyde synthases (AASs). These AASs catalyze a decarboxylation-oxidative deamination process of aromatic amino acids, leading to the production of aromatic acetaldehydes rather than the AAAD derived arylalkylamines. Research has implicated that plant AAS enzymes are involved in the production of volatile flower scents, floral attractants, and defensive phenolic acetaldehyde secondary metabolites. Historically, the structural elements responsible for differentiating plant AAAD substrate specificity and activity have been difficult to identify due to strong AAAD and AAS inter-enzyme homology. Through extensive bioinformatic analysis and experimental verification of plant AAADs, we have determined some structural elements unique to given types of AAADs. This document highlights structural components apparently responsible for the differentiation of activity and substrate specificity. In addition to producing primary sequence identifiers capable of AAAD activity and substrate specificity differentiation, this work has also demonstrated applications of AAAD enzyme engineering and novel activity identification. / Ph. D.

Abstract not available