The ligand activated transcription factor retinoid X receptor (RXR) forms a DNA binding heterodimer with pregnane X rseceptor (PXR) in response to foreign xenobiotics. In addition to RXR and PXR there are other proteins involved in the RXR/PXR signaling pathway. Many proteins involved in this pathway are still unknown. This study documents the production of RXR and PXR in a bacterial recombinant fusion system. These proteins were expressed in a system that allowed purification with six histidine residues. Once the proteins were expressed and purified from E. coli, they were solublized and tested for function. Different strategies were employed including temperature and inducer studies and denaturing and renaturing techniques to solublize PXR. Following the solubilzation of each protein, all proteins were subjected to a method of functional analysis. RXR function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with PXR. These studies involving RXR and PXR demonstrate that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system. In addition, this study documents various strategies for combating "inclusion body" formation in the overexpression ofPXR. Also, it describes the production of plasmid pCMV-RXR for transfection into the HepG2 cell line to monitor the levels of cellular RXR in various tissue types.

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No description available.

Mammary cells from lactating rats and dairy cows were cultured in Eagle's minimal essential medium (MEM) with added amino acids. Changes in free intracellular amino acid pools and milk protein synthesis in response to amino acid additions to the medium were measured. Increases in free intracellular amino acid pools are associated with increased protein synthesis and the rate of their change in response to extracellular amino acids would partially reflect the cell's amino acid requirement. The intracellular pools from medium amino acids (except methionine, tryptophan and glutamine) increased with extracellular amino acids but at their own characteristic rate. The ratio of medium amino acids to nonmedium amino acids inside the mammary cell increased with the concentration of amino acids in the medium. Methionine and tryptophan did not have measurable pools in the rat mammary cell and only very small pools in the bovine mammary cell which did not increase with extracellular amino acids. Culturing rat mammary cells with labeled methionine showed only 42% of the intracellular radioactivity was still associated with labeled methionine indicating significant conversion of this amino acid after it entered the cell. A small linear increase in intracellular cystine was observed with elevated cystine in the medium. The responses in cystine, tryptophan and methionine intracellular pools to extracellular amino acids suggest the concentration of these amino acids in the medium are insufficient to meet the bovine mammary cells requirement. Increasing the concentration of amino acids in MEM 1-, 3-, 5- and 7 fold significantly (P < .05) increased β-casein and to a lesser degree β-lactoglobulin synthesis by bovine mammary cells in culture. Individually increasing each of the 13 amino acids in MEM 3-fold showed cystine followed by threonine and then methionine significantly (P < .05) increased the combined synthesis of β-casein and β-lactoglobulin. The correlation between intracellular pool size change and the response in milk protein synthesis to increased individual amino acids was -.51 which was not significant at the .05 level. / Ph. D.

Methionine- and lysine-containing di- to octapeptides were evaluated for their ability to serve as methionine and lysine sources respectively for the synthesis of secreted proteins. Mammary tissue explants from lactating (10 to 11 d) CD-1 mice and cultured bovine mammary epithelial cells (MAC-T) were used as experimental models. Explants and cultured cells were incubated at 37°C in a humidified atmosphere of 90% air/10% CO₂ and 95% air/5% CO₂, respectively, for 1 to 24 h in Dulbecco's modified Eagle's medium containing hormones, ³H-leucine, and methionine or lysine substrate in either free or peptide-bound form. The ability of methionine and lysine substrates to promote incorporation of ³H-leucine into secreted proteins was quantified. Mouse mammary explants were able to utilize methionine and lysine from all peptides tested except the lysyl octapeptide. All the methionyl peptides were at least as effective as free methionine in promoting ³H-leucine incorporation into secreted proteins. Most methionyl di- and tri-peptides promoted 15 to 76% greater (P < .05) ³H-leucine incorporation than did free methionine. A negative correlation (r = -.89, P < .01) was detected between the rate of ³H-leucine incorporation and the number of amino acid residues in the peptides. The incorporation of ³H-leucine promoted by some methiony] dipeptides was reduced (P < .05) in the presence of a 200-fold higher concentration of glycylsarcosine or carnosine. Incorporation of ³H-leucine promoted by lysyl peptides ranged from 91 to 117% of the incorporation promoted by free lysine. MAC-T cells were also able to utilize methionine from all di- and tri-peptides studied. The ability of the peptides to promote ³H-leucine incorporation varied with experimental conditions. For cells allowed to grow/differentiate for 3 or 8 d, incorporation of ³H-leucine promoted by peptides ranged from 67 to 85% and 86 to 110% of the incorporation promoted by free methionine, respectively. The effect of extracellular matrix on the utilization of peptide-bound methionine by MAC-T cells was also examined. Generally, there was no difference in ³H-leucine incorporation/DNA promoted by methionyl dipeptides in MAC-T cells cultured on matrigel, collagen, laminin, or fibronectin coated or uncoated plates. These results suggest that peptides can serve as sources of amino acids for the synthesis of secreted proteins by both lactating mammary explants and cultured mammary epithelial cells. Mouse mammary explants appear to have a greater ability to utilize peptide-bound methionine than to utilize peptide-bound lysine. Mediated transport of some methiony] peptides may be involved in the peptide utilization by the mammary explants. More extensive utilization of peptides by MAC-T cells following a longer (3 vs 8 d) growth/differentiation period may indicate that some maturation process(s) in the cells was necessary for the most effective utilization of peptides. / Ph. D.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of these viruses in planting material namely seed potato stocks are routinely diagnosed by enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South Africa are obtained from Europe. These kits have produced false positive and false negative results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and specific for the detection of South African strains of the two respective viruses. In this study the viral coat protein genes were amplified by RT-PCR from a South African source of infected plant material. The PVY and PLRV coat protein genes were subsequently cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were aligned and compared to corresponding viral coat protein gene sequences obtained from Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV were sub-cloned into an expression system (pET-14b) to induce and express the respective recombinant viral coat proteins. The induction of the cloned coat protein genes yielded successful production of the recombinant PVY coat protein but the induction and expression of the recombinant PLRV coat protein was unsuccessful. The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The ELISA kit was subsequently used in preliminary trials to determine if the kit could detect PVY infected plant material. The initial results of the ELISA trials using PVY infected material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA) toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid- Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar, goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is. In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal. Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol. Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.

The zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S-transferase (GST)-ZAD domain chimeric proteins for use in pull down protein binding assays,and GST-Zinc finger (ZnF) array domain chimera for electrophoretic mobility shift assays (EMSA). Protein binding assays indicated two putative conserved interactors, similar to the analogous KRAB system in mammals. ... Competitive bindings were carried out to show a specificity of binding conferred by the identified conserved positions. While the consensus binding sites show relatively few similarities, the predicted target genes identified by the consensus binding sites show significant overlap. The nature of this overlap conforms to the known characteristics of the ZAD family but points to a more positive selection to maintain conservation of function. / by Joseph Krystel. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Longitudinals lacking gene (LOLA) is a transcription factor that is involved in a variety of axon guidance decisions in Drosophila melanogaster nervous system. Besides having a role as an epigenetic silencer and in the programmed cell death in Drosophila's ovary, this gene is also an example of complex transcription unit. LOLA is a transcription repressor and can generate 17 DNA - binding isoforms, through alternative splicing, each containing distinct zinc-finger proteins. This unique DNAbinding binding sequence to which LOLA-ZFP binds has been determined for four of the lola isoforms F, J, P and K. Also, bioinformatics' tool approach has been taken to identify the target genes that are regulated by these four LOLA splice variants. Future work will be done for the five other LOLA isoforms to categorize their putative DNA-binding sequences and subsequently their protein interactions. / by Bazila Qureshi. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins. / by Joseph Krystel. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Synthetic biology, which aims to enable the design and assembly of customized biological systems, holds great promise for delivering solutions to numerous modern day challenges in agriculture, sustainable energy production, and medicine. However, at its current stage, synthetic biology is not yet equipped with the necessary tools and understanding to reprogram the immensely complex molecular environment of the cell beyond simple proof of concept demonstrations. One current objective within synthetic biology is to create robust tools that can be used to manipulate biological systems in a predictable and reliable manner. While many transcription-based control devices have been reported, little consideration has been given to the eukaryotic protein translation apparatus as a target for engineering gene-regulatory tools. In this work, we explore the potential for reprogramming the protein synthesis machinery for cell engineering. We begin in Chapter 1 by reviewing canonical protein synthesis and survey the assortment of translation reprogramming mechanisms that exist in nature, focusing on the role of RNA in these processes. We then cover previous efforts to engineer the protein synthesis machinery and discuss their methodological approaches. Lastly, we examine potential opportunities for engineering protein synthesis that have not yet been explored. RNA’s prominent role in protein synthesis and its amenability to high-throughput in vitro selection approaches raises the possibility that the translation apparatus could be engineered through in vitro directed evolution of its RNA components. In Chapter 2, we develop an experimental framework for identifying mRNA sequence elements that reprogram protein synthesis, focusing on stop codon readthrough. By adapting a previously developed in vitro selection technology called mRNA display, we demonstrate that molecules of RNA derived from expansive libraries of random sequences can be enriched as a result of their translation reprogramming activity. We then analyze these stop codon readthrough signals and propose the use of these sequences for enhanced unnatural amino acid incorporation technologies. In Chapter 3, we apply this very same selection principle for the in vitro directed evolution of RNA sequences that stimulate -1 programmed ribosomal frameshifting. Then, using previously reported RNA aptamers, we rationally engineer RNA switches that regulate translation reading frame in response to small molecule inputs. To further optimize switch performance, an in vivo directed evolution platform was established. We explore the utility of these RNA switches, particularly their ability to regulate multi-protein stoichiometry, for performing cellular logic operations and controlling cell fate. A major focus of translation engineering has been the incorporation of unnatural amino acids for fluorescent labeling of proteins in living cells. The successful achievement of this goal will require small molecule fluorophores with desirable biological properties, as well as robust synthetic methods for their production. In Chapter 4, we present a scalable approach to oxazine and xanthene fluorophores that utilizes a general diaryl ether synthetic intermediate. Finally, in Chapter 5, we describe a photoactivatable oxazine fluorophore and demonstrate its utility as a live-cell imaging reagent with applicability to advanced microscopy techniques.