Towards the construction of protein-based biological sensors, chimeric proteins comprised of an antibody single chain antigen-binding protein (scFv) and the green fluorescent protein (GFP) were constructed. Although correct folding of the scFv domain typically requires the oxidizing conditions of extracellular compartments, such as the periplasmic space of E. coli, GFP is unable to mature under these conditions. Using DNA recombinant technology, fusion constructs were made in the cytoplasm under control of the araBAD promoter. Weak fluorescence of the GFP domain and antigen binding activity of the sFv domain were obtained in the cytoplasm of E. coli BL21, but improved expression and activities of both domains were obtained by using a trxB- mutant of E. coli, as well as by modifying physical and genetic conditions for expression of the fusion proteins. Assessment of the fluorescence and antigen binding activity of the fusion proteins indicates that GFP fluorescence can serve as an indicator of correct folding of fusion proteins.

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Leishmania chagasi is the causative agent of visceral leishmaniasis in South America. The most abundant glycoprotein on the surface of L. chagasi promastigotes is the glycosylphosphatidylinositol (GPI) anchored protease MSP (major surface protease), also called GP63. MSP is encoded by more than 18 tandem MSP genes on a single chromosome. MSP genes are classified according to unique sequences at their 3' ends and distinct expression patterns. The five MSPS genes (MSPS1, MSPS2, etc.) express 3.0 kb RNAs in stationary phase of promastigote growth in vitro in culture. The > twelve MSPL genes express 2.7 kb RNAs in logarithmic phase of promastigote growth, and the single MSPC RNA is constitutively expressed as two RNA species (2.6 and 3.1 kb) throughout promastigote growth. The progression from logarithmic to stationary phase is accompanied by an increase in parasite virulence for a mammalian host, and a 16-fold increase in the total MSP protein associated with the cell. As such, MSP has been called a virulence factor of leishmania. Little is known about the differences between isoforms of MSP proteins encoded by the three MSP gene classes, because they have a very similar amino acid sequences. The purpose of this thesis was to study the protein expression and localization of MSPS, MSPL, and MSPC in the promastigote and amastigote stages of the L. chagasi. We took three approaches to this problem. First, we produced constructs in which the fluorescent marker GFP was flanked by putative targeting sequences of the MSPs. Second, we generated Leishmania transfectants expressing Myc-tagged full-length MSPs and studied their localization in promastigote cells. Third, we generated antibodies to immunogenic peptides in the few regions with unique sequences that allowed us to distinguish between some of the MSP classes. One monoclonal anti-peptide antibody, named C51, recognized only MSPS1 and MSPL1. Data indicated that the product of the MSPC gene runs at a higher molecular size than products of the MSPL and MSPS genes, both of which localize to the promastigote surface. Overall the data set the stage for future studies of the properties and functions of specific MSP gene products.

Animals carry lipids such as hydrocarbons, fats, and sterols throughout their circulatory systems bound to a carrier protein known as lipophorin. The lipophorin receptor has been characterized in locusts, mosquitoes and cockroaches yet little is known about it in Drosophila melanogaster. An antibody against the eleven variants of the lipophorin receptor was developed and tested. Although this was the main feature of the work, several preliminary experiments using RNA interference were conducted to determine the effects of lipophorin receptor. Flies whose lipophorin receptor proteins were knocked down were found to have no major differences in locomotor activity in total darkness suggesting that their circadian rhythms are unaffected. The same flies were found to have extensive differences in their cuticular hydrocarbon profiles as compared with wild‐type flies. Whole‐mount tissue staining of Drosophila adult brains revealed that several cells in the central nervous system are immunoreactive with the anti‐Lipophorin receptor antibody.

Animals carry lipids such as hydrocarbons, fats, and sterols throughout their circulatory systems bound to a carrier protein known as lipophorin. The lipophorin receptor has been characterized in locusts, mosquitoes and cockroaches yet little is known about it in Drosophila melanogaster. An antibody against the eleven variants of the lipophorin receptor was developed and tested. Although this was the main feature of the work, several preliminary experiments using RNA interference were conducted to determine the effects of lipophorin receptor. Flies whose lipophorin receptor proteins were knocked down were found to have no major differences in locomotor activity in total darkness suggesting that their circadian rhythms are unaffected. The same flies were found to have extensive differences in their cuticular hydrocarbon profiles as compared with wild‐type flies. Whole‐mount tissue staining of Drosophila adult brains revealed that several cells in the central nervous system are immunoreactive with the anti‐Lipophorin receptor antibody.

ROGDI is a novel gene which has unknown function. According to GenBank, the gene is located on chromosome 16p13.3 and the size of coding region is 864 bp which encodes 287 amino acids. It was a novel gene isolated from primary human renal epithelial cells in NEDO human cDNA sequencing project (AK026039). The definition of its reference sequence (RefSeq NM_024589) described as Homo sapiens rogdi homolog (Drosophila), ROGDI. By bioinformatic analysis, the gene was predicted as a hydrophilic protein with leucine zipper domain and located in cytoplasm. A partial cDNA of this gene was cloned in our laboratory. For further study of the biological function of the gene, the coding region of this gene was cloned into pGEX-6p and pET-28a vector and expressed in E. coli BL21 (DE3). The fusion protein was partially purified for preparation of polyclonal antibody. Northern blot analysis revealed that the gene was not expressed in all tissues. From the results of RT-PCR and western blot analysis, it can be concluded that the products, both mRNA and protein, of this gene were found in many cancer cell lines, but protein level expression of the gene was much less in normal cell lines. By immunocytochemistry analysis and subcellular localization analysis of GFP-tagged ROGDI, the gene was expressed both in the nucleus and cytoplasm, but expressed more in the nucleus than cytoplasm. In addition, ROGDI was up-regulated in early stages of liver fibrosis of TAA-treated mouse livers. This novel gene may play roles in tumorigenesis and liver fibrosis.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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