DNA typing of the human small intestinal protozoan parasite Giardia lamblia

PhDAt present there is no satisfactory means of typing strains of Giardia lamblia which can explain the broad range of clinical symptoms seen in giardiasis or which can identify genotypes in epidemiological studies. This thesis attempts to address these problems by developing DNA based typing systems sensitive enough to be able to identify many different Giardia genotypes and which may be applied to the organisms found in clinical samples. Four different techniques were assessed for their ability to identify multiple polymorphic loci in the Giardia genome which may be used to genotype and identify isolates of Giardia and upon which the future development of PCR-typing protocols may be based. These techniques included RFLP analysis, random amplified polymorphic DNA (RAPD) analysis, M13 DNA fingerprinting and minisatellite DNA fingerprinting. Minisatellite DNA fingerprinting proved to be the most discriminatory, recognising many hypervariable loci within the Giardia genome which proved useful for in vitro studies on genotypic heterogeneity within Giardia isolates. This approach would require further development in order to be used on in vivo infections where it could directly assess the relationship between genotype and pathogenicity. Therefore the variable repeats recognised on Giardia fingerprints were sought by constructing and screening a Giardia genomic DNA cosmid library. Once cloned these repeats would form the basis of sensitive and specific PCR-based fingerprinting protocols ideal for typing large numbers of infections. The repeat sequences cloned in this way turned out to be Giardia variable surface protein genes with short, imperfect tandem repeats in their 3' flanking DNA. This work has important implications for the future development and use of fingerprinting techniques on Giardia and may be useful in the study of chromosome rearrangement in Giardia which is likely to be involved in surface antigen switching

Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and <or=12.5 pg/microg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305 pg/microg of membrane protein 25 daf and is not expressed 20 daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization