Binninger, David

It has been suggested that 60 Hz electromagnetic fields (EMF) from power lines might present a health hazard to humans. Toward understanding the molecular mechanisms, two genes--EMF-A and EMF-B--had been tentatively identified in Saccharomyces cerevisiae as being responsive to EMF. The aim of this thesis was to confirm that EMF-A and EMF-B were convincingly and reproducibly inducible by 60 Hz EMF. The results confirmed that expression of both genes was induced following exposure to 60 Hz EMF. DNA sequence analysis of these genes showed a nearly perfect match to two Saccharomyces cerevisiae genes in the GenBank DNA database. The available data indicates that while both genes have been previously identified in other laboratories, precise identification of their biological functions has not been established. The cloned genes will provide valuable molecular probes for the ongoing research into the biological effects of power line EMF.

The position of the cactus family, Cactaceae, within the order Caryophyllales was examined by outgroup analysis of chloroplast rbcL gene sequence data. Comparative data came from 28 outgroup species in 15 families and six ingroup species. Phylogenetic Analysis with PAUP 3.1 produced twelve equally parsimonious trees; these were used to generate a strict consensus tree. MacClade 3.06 was used to refine the polytomies in the consensus tree. Analysis indicates that the Cactaceae is a monophyletic clade; moreover, these results support the work of other analyses that the sister family to Cactaceae is the Portulacaceae. Other relationships within the order are not completely consistent with previous studies. For example Molluginaceae and Caryophyllaceae occupy a central position in the cladogram; Achatocarpaceae aligns with the Amaranthaceae and Chenopodiaceae; and Basellaceae is presented as the basal family within the order. Lastly, the Jamaican cactus Opuntia spinosissima was derived from a common ancestor with the Florida Keys endemic, Opuntia corallicola.

Pseudopterosin C is a novel diterpene glycoside isolated from the Caribbean gorgonian coral, Pseudopterogorgia elisabethae. Pseudopterosin C is non-steroidal and has been shown to exhibit promising anti-inflammatory and analgesic properties. The in vitro production of pseudopterosin C through biosynthetic methods has been examined. A cell-free system as well as an enzyme precipitate capable of transforming terpene precursors to the product has been developed. The parameters toward optimizing the enzymatic production through cofactor requirements, differing terpene precursors and sugars were investigated.

Despite the many epidemiological studies which have shown some effects of EMF on biological systems, there has yet to be any data that indicates the molecular mechanisms by which this effect takes place. My goal was to genetically engineer a yeast strain that would have a clear biological effect to the EMF's. The strategy involved using a yeast strain which requires histone function from a plasmid, and the plasmid construct that has a Gal1 promoter controlling the histone function. The plasmid construct could then be engineered to contain a promoter sequence for a known EMF-repressed gene in yeast, which would control the histone production. Without a functional histone gene, the cells will die, and the effects will be easily visualized. Although the genetic screening for the desired transformants appeared to work, the molecular analysis of those transformants did not show the promoter insertion. There are a few possible reasons for why this happened, including possible reversions from one of the original mutations of the chromosomal histone H4 genes, or the mutation of the Gal1 promoter which would no longer repress the histone H4 gene and allow the cells to grow on glucose.

The population decline in the Florida Semaphore cactus, Opuntia corallicola, due to grazing by the moth, Cactoblastus cactorum, has placed this plant species on the verge of extinction in the Florida Keys. Although this semaphore cactus is endemic to Florida, it was mis-named as an almost uncommon Jamaican species (Opuntia spinosissima) in the 1970s. Our goal was to provide genetic data to distinguish these cacti with nuclear and chloropast genes previously used in other plant phylogenetic analyses. Phylogenetic relationships were inferred from nucleotide sequence variation in both the Leucine-Phenylalanine transfer RNA intergenic spacer (trnL-trnF) and ribulose bisphosphate carboxylase-oxygenase (rbcL) genes of cactus chloropast DNA. While the trnL-trnF gene sequence data could only group cacti at the genus level, it was the data from the rbcL gene which show speciation and differences between the Florida Semaphore cactus, Opuntia corallicola, and the Jamaican species, Opuntia spinosissima. Sequence data from the internal transcribed spacer (ITS) regions of nuclear DNA were not available due to the divergence of the cactus ITS region from the universal primers sequences used to initiate the polymerase chain reaction (PCR).

Epidemiological studies have suggested that 60 Hz AC electromagnetic fields (EMF) generated by power lines might play a role in the increased incidence of certain human cancers, especially in children. Previous studies with human cells indicate that one molecular mechanism may involve differential effects on protein phosphorylation. The aim of this thesis was to determine whether 60 HZ EMF also affect phosphorylation in the yeast Saccharomyces cerevisiae which has been developed into a paradigm for molecular biologists. The data presented in this thesis show that protein phosphorylation is indeed altered in yeast in response to 60 Hz EMF. This work validates yeast as an experimental model for more in depth investigation of the molecular and genetic basis of this response. Insight gained from these studies should shed light on the interaction of power line EMF with human cells and its possible role in development of human cancers.