Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Thesis (M. S.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

The cytoplasmic region (c-fragment) of the E. coli aspartate receptor plays an important role in modulating intracellular signaling cascade and adaptation. This study focuses on understanding the structural organization of the receptor and characterizing the interactions between the receptor and the methyltransferase. Monitored by circular dichroism (CD), thermal denaturation of the c-fragment was found to be reversible. The c-fragment derived from signaling mutants displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in gel filtration chromatography (GFC). An analysis of secondary structure indicated that the oligomers were more folded than monomers. A nondenaturing detergent, octylglucoside (OG), and an $\alpha$-helix promoting reagent, trifluoroethanol (TFE), were used to reveal perturbing influences on the secondary and tertiary structure of the c-fragment and were interpreted to influence the extent of coiled-coil in the c-fragment. OG was found to diminish the cooperativity of the thermal denaturation, which could be restored by the addition of glycerol and phospholipid. These conditions where the cooperatlve transition was observed correlated with the conditions where methylation of the intact receptor had been observed. Binding between the receptor and the methyltransferase was characterized using isothermal titration calorimetry (ITC), giving a complex of 1:1 stoichiometry. Both dimeric and monomeric protein behaved in the same way, suggesting that the interaction between the receptor and the methyltransferase was not influenced by an equilibrium between the monomer and dimer of the cytoplasmic domain. The c-fragment of the receptor proved to have thie same binding activity as did the full-length receptor; a deletion of either 36 or 16 amino acids from the carboxyl terminus led to a csmplete loss of the binding activity. Finally, a synthetic peptide corresponding to the last five amino acids (NWETF) was found to have all binding activity of the full-length receptor. Evidence that the NWETF binding site for the transferase, which is strictly conserved among the serine and aspartate receptors, has physiological significance was obtained in assays of the transferase activity in which excess peptide was able to completely block the receptor methylation. The binding site was thus identified with the carboxyl "tail" of the receptor, indicating the mechanism by which the transferase can modify all the sites of methylation which it is bound to the receptor. Also, methylation between receptors was deduced as a possible consequence of this mode of binding, by comparing the data obtained in this study with numerous observations on receptor methylation and adaptation found in literature.

Ribosomes are large macromolecules responsible for protein synthesisand they consist of both RNA and proteins. Each ribosome is made of one large and one small subunit. Even though the ribosome is one of the most studied machineries in the cell there is a gap in our understanding of how this macromolecule functions in vivo. In this project we aimed to develop a method for tracking a specific subset of ribosomes using super-resolution fluorescence microscopy. This was achieved by using the MS2 coat protein (MS2CP) fused to a fluorescent marker and by modifying ribosomes to have the RNA loop to which theMS2CP binds with high affinity. We were able to obtain the most promising results when the MS2CP was fused to a Halotag with the dye JF549 attached to it. The JF549 has good cell permeability which allows simple and efficient labelling of ribosomes. To be able to observe translation of specific mRNAs we used this labelling strategy to track orthogonal ribosomes which do not recognise mRNA normally produced in cells but can translate mRNAs with a modified 5’-UTR.Orthogonal ribosomes were tested with several different 5´-UTRs. With the construct for which we obtained the highest expression level we observed that up to 43% of the labelled orthogonal ribosomes were engaged in translation of the specific mRNA. The system will make it possible to determine how the sequence of a particular mRNA will affect its in vivo translation.

No description available.

1. Abstrakt:Bakgrund: Malaria är en av världens viktigaste infektionssjukdomar och det beräknas vara ca300 miljoner drabbade varje år, därför är metabolismen av läkemedel som används för attbehandla malaria såsom kinin och artemisinin värt att lägga fokus på. Cytokrom P450enzymerna har en viktig roll i metabolism av malarialäkemedel och dessa uppvisar variationinterindividuellt samt mellan olika populationer på grund av polymorfism.Syfte: Denna studie har fokuserat på att undersöka tre polymorfa gener (CYP3A4*1B,CYP3A5*3 och CYP2B6*6) hos både friska och malariasmittade barn i Uganda för attjämföra resultaten med en population i Mwanza, Tanzania. Dessa polymorfa alleler påverkarmetabolismen av artemisinin och kinin, vilket i sin tur kan förorsaka minskad/ökad ellermisslyckad klinisk effekt.Metod: Genotypning av individernas blodprov gällande dessa genvarianter undersöktesgenom laboratoriestudier med PCR som huvudsaklig metod. DNA sekvensering utfördes vidUppsala Genome Center.Resultat: Resultaten visade att allelfrekvensen i Mwanza för CYP3A4*1B var 78%respektive 16% för CYP3A5*3, medan de var 72 % respektive 50% hos populationen iUganda. Allelfrekvensen för CYP2B6*6 i Uganda var 72 % och 36 % i Mwanza, Tanzania.Sammanfattning: De flesta malariamediciner uppvisar skillnader i kinetik och dynamikvilket kräver terapeutisk läkemedelsmonitorering. Sammanfattningsvis finns det skillnaderoch variationer bland de studerade polymorfa CYP enzymer interindividuellt och mellanolikaetniska grupper.Resultatet av denna studie visade både små skillnader mellan de två studerade populationernamen även skillnader mellan individer i den studerade gruppen i Uganda.

The use of molecular chaperones can increase the yield of correctly folded proteins. This is especially needed in the expression of proteins non-native to the host organism. This study set out to investigate the function of the chaperone GroES; a component in the GroE-system. The function of this chaperone has only been studied alone in vitro. Here we lay ground to further studies on GroES and its ability to act alone in vivo. GroES was expressed from a plasmid and characterized through its potential to increase the amount of correctly folded proteins. Characterization was mainly done by fluorescence spectroscopy with hard-to-fold proteins linked to fluorescent probes. Results show a very clear increase in fluorescence for most of the substrate proteins tested, indicating that GroES has a significant role in the GroE-system and perhaps outside of it.

Approximately 30 million years ago, ancestors of fungus-growing termites started an obligate mutualistic relationship with a Basidiomycete fungus Termitomyces. The success of this obligate relation is the division of labour and reliance on termite caste gut microbial symbionts. Termites workers maintain Termitomyces fungal garden with their workforce and dual gut passage while the soldier caste protects the colony from predators. The fungal garden concurrently provides enough food for the colony members. Royal pair (a king and a queen) is the centralised caste in the colony, and they control the colony population by their massive reproduction, but their gut community composition remains unexplored. This project aimed to characterise the gut microbes associated with royal pairs of a fungus‐growing termite species Macrotermes natalensis. Four colonies were explored using high throughput sequencing of 16S rRNA gene amplicon dataset. The high-throughput sequence result showed that royal gut microbiotas were comprised of a lower number of bacterial taxa than sterile caste (workers and soldiers). This less number of bacterial taxa suggested that the royal pair gut was completely decoupled from the sterile castes gut, which indicates that the royal pair were possibly provided with a unique diet. The study also showed diversity in bacterial genus-level OTUs of royal pairs in all four colonies which indicated that there is a diet variation between the king and queen. The media predicting strategy could facilitate future cultivation efforts for targeted royal pair gut bacterial strains.

Many regions of the world are facing the problem with arsenic toxicity. Arsenic contamination has become a considerable threat to the environment triggering various big health issues for every life in that contaminated environment. Lysinibacillus sphaericus (B1-CDA) is an arsenic tolerant strain of bacteria that has been reported and characterized before by the researchers of the University of Skövde, Sweden. The bacteria were found to contain many arsenic responsive genes such as arsB, arsC, and arsR which are responsible for arsenic tolerance in the bacterium. The main focus of the current study was to characterize one of the arsB genes (gene-4251) of Lysinibacillus sphaericus B1-CDA by in silico and in vitro analyses in order to determine the molecular function of this gene. The in silico studies conducted by using the Iterative Threading Assembly and Refinement (I-TASSER) server predicted the tertiary structure of the ArsB protein and suggested that this protein is an intrinsic component of the membrane which primarily helps in the binding of metal ions and liberation of metabolic energy. To validate this predictive results, several in vitro experiments were performed. For complementation studies, the arsB gene was cloned from L. sphaericus B1-CDA and transferred to an arsB knock-out mutant of Escherichia coli JW3469-1. Both, the transgenic and mutant strains were grown under the arsenic stress of 50 mM for 96 hrs followed by measuring their growth and arsenic tolerance after every 24 hrs. Statistical analysis confirmed that there was a significant difference in growth between the transgenic and the mutant E. coli strains. The ICP-MS (Inductive Coupled Plasma-Mass Spectroscopy) analysis revealed that after 24 hrs of culture, the arsenic content in the cell-free broth of transgenic strain was reduced from 50 mM to 9.10 mM (81.8%), whereas the reduction in arsenic content by the mutant strain was from 50 mM to 9.80 mM (80.2%). These results suggest that the arsB gene is partly involved in the accumulation of arsenic inside the cells and this feature could be used for a large scale removal of arsenic from the contaminated environment.