One of the most intriguing questions in evolutionary biology is how biochemical and structural complexity arise through small and incremental changes; however answering this question requires an explicit set of candidate residues and an experimental system in which to test them. This dissertation aims to understand how biochemical complexity evolves and assesses the structure-function relationship in the green fluorescent protein (GFP) protein family using an ancestral reconstruction approach. In the second chapter, I studied the evolution of biochemical complexity in Kaede-type red fluorescent proteins (FPs) from Faviina corals. An increase in biochemical complexity is represented by the emergence of red fluorescence because it necessitates the synthesis of a tri-cyclic chromophore from a precursor bi-cyclic chromophore through an additional autocatalytic reaction step. The autocatalytic reaction is fully enabled by as many as twelve historical mutations. Here, I showed that the red fluorescent chromophore evolved from an ancestral green chromophore by perturbing the ancestral protein stability at multiple levels of protein structure. Moreover, only three historical mutations are sufficient to initiate the selection-accessible evolutionary trajectory leading to emergence of red fluorescence. The third chapter investigates six mutations proximate to the chromophore in the Kaede-type FP that could have facilitated autocatalytic synthesis of the red chromophore by enlarging the chromophore-containing cavity and modifying its microenvironment. Two of these six mutations were found to strongly affect the protein’s stability and oligomeric tendency. Additionally, I showed that the dimeric least divergent Kaede-type FP, R1-2, evolved from the tetrameric green ancestor. Taken together the results of these studies indicate that the step-up in biochemical complexity in the Kaede-type FPs was achieved via disruption of the existing stable interactions at tertiary and quaternary protein structure levels. In the fourth chapter, I resurrected the common ancestor of all FPs cloned from the order Leptothecata (class Hydrozoa), which are characterized by the highest known homo-oligomeric diversity. I showed that the ancestor was a green monomeric FP with a large Stokes shift. The ancestral FP together with the extant Leptothecata FPs could server as a model system to study the evolution of function and homo-oligomerization, and the desirable photophysical characteristics would make this ancestral FP a useful bio-marker in bio-medical research. / text

The LexA repressor of Escherichia coli represses a set of genes that are expressed in response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires RecA protein. Under physiological conditions in vitro, RecA-dependent cleavage also occurs. At alkaline pH, however, the specific cleavage reaction occurs spontaneously without RecA, a reaction which is termed autodigestion. The LexA repressor is, therefore, thought to cleave itself with RecA acting to stimulate autodigestion. A set of lexA (Ind⁻) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function were isolated. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction. Many of the mutant proteins were purified in order to further characterize their properties in both autodigestion and RecA-mediated cleavage. All of these mutant proteins are found to be deficient in both cleavage reactions. A mutant protein, replacing Lys-156 to Arg, requires a higher pH condition than the wild-type protein does for both cleavage reactions. The results suggest that deprotonation of Arg-156, and by inference Lys-156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and that in the latter reaction RecA acts to reduce the pKa of Lys-156, allowing efficient cleavage of wild-type repressor under physiological conditions. Finally, several mutant proteins affecting amino acids around the cleavage site and the proposed nucleophile in the cleavage reaction (Ser-119) could not efficiently act as a competitive inhibitor in the RecA-mediated cleavage of wild-type repressor, presumably because they affect RecA binding.

The LexA protein of E. coli is a repressor of at least 20 genes in the SOS regulon, and by this function plays a major role in regulating the SOS response. Two different genetic approaches have been taken to define the DNA binding domain of LexA repressor. First, several mutant repressors which are defective in DNA binding have been isolated. The mutations generating these repressors were dominant to lexA+, indicating that the mutant proteins can act in trans to interfere with binding of normal repressor to an operator sequence. The repressors may be defective due to elimination or disruption of contacts made between side chain(s) within the protein and the DNA helix but dominant because they can still interact with other monomers of LexA protein. In a second experiment to define the DNA binding domain of LexA protein, a novel genetic selection has been used to isolate DNA binding specificity mutants. The recA operator (CTG TATGA.GCATA CAG), a known lexA binding site, has been altered in a symmetric fashion. This choice was based on the assumption that the dyad symmetry of the operator indicates at least two repressor monomers bind to each operator such that each monomer recognizes one half of the operator. A class of mutant repressors which restored binding to this altered operator but had little affinity for the wild-type recA operator was isolated. This type of mutation allowed the identification of amino acids in the repressor which are likely to make specific contacts with base-pair(s) in the DNA binding site. By examining the effects of a series of amino acid substitutions on repressor specificity, it was possible to show that a glutamic acid residue at position 45 (E45) contacts the first and last base-pair of the consensus recA operator (CTG TATGA.GCATA CAG). Both negative dominant and operator recognition mutations were located in a small region that was previously identified to specify a helix-turn-helix motif based on sequence similarity to other repressors. These studies therefore suggest that LexA protein may bind to DNA by a helix-turn-helix motif similar to these repressors.

No description available.

The use of optical methods for performing non-invasive physiological and biochemical monitoring has been investigated, with particular emphasis on the application of near-infrared spectrophotocetry for following changes in the redox state of cytochrome oxidase. Initial studies of the gross optical properties of in vivo tissue were made using an image intensifier. These demonstrated that some light is transmitted through biological tissues and that such material is very highly scattering. In order to investigate the feasibiity of non-invasively monitoring changes in the redox state of cytochrome oxidase in vivo. spectrophotometric and oxygen measurements were made on solutions containing the pure enzyme and yeast cell suspensions. These demonstrated the high affinity that the enzyme has for oxygen in such preparations, in contrast to the much lower apparent affinities in vivo that have been reported. These results were then modelled mathematically, and a possible-explanation for this anomaly suggested. Potential problems with applying this method are also presented. The interest in cytochrome oxidase is due to its importance in oxidative metabolism. However in performing this role it also assists in the prevention of oxidative damage, whose contribution to various disease states in paediatrics is briefly considered. Two instruments were also constructed, and used, firstly to measure the spectral characteristics of transmitted and reflected light in vivo. ana secondly to study the cardiac synchronous pulsatile component of this light (commonly referred to as the photoplethysmogram).

The primary aim of the work described in this thesis is to establish a foundation for the applicability of a biochemical biomarker, cholinesterase (ChE) activity in food animal species, as an instrument for evaluating exposure to pollutants as well as predicting high-level effects on public health. Secondary aims are to increase the awareness of pesticide users of anti-ChE exposure, to decide whether poisoning episodes involve anti-ChE by measuring residual effects in tissues, and to identify sources of contamination in food animal tissues. The ChE are specialized carboxylic ester hydrolases that break down esters of choline. They are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Both AChE and BChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with EDTA. Of the different tissue tested, the mean of ChE activities was found to be highest in tissue from the liver, followed by lung, muscle, kidney and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle and heart. The effect of freezing on ChE activities in liver and muscle tissues was significant inhibition after 6 months at -80 °C, whereas decreased after 3 months at -20 °C. A technique to improve the purification of AChE in sheep tissue was developed. BW284c51 strongly reduced acetylthiocholine iodide (AcTChI) and propionylthiocholine iodide (PrTChI) hydrolysis and slightly affected that of butyrylthiocholine iodide (BuTChI) in the liver, while iso-OMPA had no significant effect for muscle BuTChI of sheep and pigs. Histochemical study of liver tissue found AChE localised mainly in the cytoplasm of the cell lining in the sinusoids. The optimal pH values of AChE and BChE in liver and muscle ranged between 7.8 and 8.5. Both AChE and BChE activities increased when increase the time course and temperature. The half maximal inhibitory concentration (IC50) was found to be higher for carbaryl than dichlorvos (DDVP) and diazinon (DZN). Very little residual AChE activity was seen in the liver, but more was found in muscles. In general, the rate constants of inhibition (ki) values for liver and muscles were increased in different pHs according to the rank order of 8.5 > 7.5 > 6.5, while in plasma it was decreased in different temperatures as follows: 20 °C > 30 °C > 40 °C. The final experiments were carried out at the rate of spontaneous reactivation (ks) of inhibited AChE by DDVP and DZN from liver and muscle was found to be higher in sheep compared to cattle and pig, while the aging of phosphorylated AChE (ka) was found to be higher in cattle compared to sheep and pig. In addition, this study indicated that the developed bispyridinium symmetric (K048) oxime seems to be promising reactivated to DDVP-inhibited AChE for sheep and pigs while HI-6 was effective in cattle.

Twenty-two different unialgal clonal isolates have been obtained at random for experimental purposes over a period of about one year. Also, during this period, at least 12 other species or strains have been isolated into unialgal cultures which had not yet been identified and/or significantly cleared of heterotrophic contaminants.

Starch gel electrophoresis was used to compare 14 proteins encoded by 15 loci for seven species of the family Icteridae. A close genetic relationship among these species was classified into three groups. The Agelaiine group contained Agelaius phoeniceus, Sturnella magna, and S. neglecta. The Quiscaline group contained Euphagus cyanocephalus, Cassidix mexicanus, and Quiscalus quiscula. Molothrus ater, the most divergent, was placed in a separate group. Divergence times for the seven species were compared to the literature. Heterozygosity of the seven populations of the two species of Sturnella were compared to determine factors influencing their divergence. Two factors proposed were heterosis in S. neglecta and possible hybridization between S. neglect and S. magna.

Recent developments in automation and novel experimental techniques have led to the accumulation of vast amounts of biological data and the emergence of numerous databases to store the wealth of information. Consequentially, bioinformatics have drawn considerable attention, accompanied by the development of a plethora of tools for the analysis of biological data. DNA microarrays constitute a prominent example of a high-throughput experimental technique that has required substantial contribution of bioinformatics tools. Following its popularity there is an on-going effort to integrate gene expression with other types of data in a common analytical approach. Pathway based microarray analysis seeks to facilitate microarray data in conjunction with biochemical pathway data and look for a coordinated change in the expression of genes constituting a pathway. However, it has been observed that genes in a pathway may show variable expression, with some appearing activated while others repressed. This thesis aims to add some contribution to pathway based microarray analysis and assist the interpretation of such observations, based on the fact that in all organisms a substantial number of genes take part in more than one biochemical pathway. It explores the hypothesis that the expression of such genes represents a net effect of their contribution to all their constituent pathways, applying statistical and data mining approaches. A heuristic search methodology is proposed to manipulate the pathway contribution of genes to follow underlying trends and interpret microarray results centred on pathway behaviour. The methodology is further refined to account for distinct genes encoding enzymes that catalyse the same reaction, and applied to modules, shorter chains of reactions forming sub-networks within pathways. Results based on various datasets are discussed, showing that the methodology is promising and may assist a biologist to decipher the biochemical state of an organism, in experiments where pathways exhibit variable expression.

Recent advances in genome editing and metabolic engineering enabled a precise construction of de novo biosynthesis pathways for high-value natural products. One important design decision to make for the engineering of heterologous biosynthesis systems is concerned with which foreign metabolic genes to introduce into a given host organism. Although this decision must be made based on multifaceted factors, a major one is the suitability of pathways for the endogenous metabolism of a host organism, in part because the efficacy of heterologous biosynthesis is affected by competing endogenous pathways. To address this point, we developed an open-access web server called MRE (metabolic route explorer) that systematically searches for promising heterologous pathways by considering competing endogenous reactions in a given host organism. MRE utilizes reaction Gibbs free energy information. However, 25% of the reactions do not have accurate estimations or cannot be estimated. To address this issue, we developed a method called FC (fingerprint contribution) to provide a more accurate and complete estimation of the reaction free energy. To rationally design a productive heterologous biosynthesis system, it is essential to consider the suitability of foreign reactions for the specific endogenous metabolic infrastructure of a host. For a given pair of starting and desired compounds in a given chassis organism, MRE ranks biosynthesis routes from the perspective of the integration of new reactions into the endogenous metabolic system. For each promising heterologous biosynthesis pathway, MRE suggests actual enzymes for foreign metabolic reactions and generates information on competing endogenous reactions for the consumption of metabolites. The URL of MRE is http://www.cbrc.kaust.edu.sa/mre/. Accurate and wide-ranging prediction of thermodynamic parameters for biochemical reactions can facilitate deeper insights into the workings and the design of metabolic systems. Here, we introduce a machine learning method, referred to as fingerprint contribution (FC), with chemical fingerprint-based features for the prediction of the Gibbs free energy of biochemical reactions. From a large pool of 2D fingerprint-based features, this method systematically selects a small number of relevant ones and uses them to construct a regularized linear model. FC is freely available for download at http://sfb.kaust.edu.sa/Pages/Software.aspx.