Structural determinations by analytical analysis of 7-phosphanorbornadiene derivatives and amino acid enantiomers

The purpose of Chapter 1 is to create a bicyclic, two-coordinate phosphorus derivative in the form of a 2,3-benzo-l,4,5,6-tetraphenyl-7-phosphanorbomadiene anion and measure its experimental 3,P NMR chemical shift. This value is compared to a theoretical NMR shift of 8 +1085 ppm calculated by D.B. Chesnut at Duke University using ab initio quantum mechanics. This is the largest downfield chemical shift ever predicted for an organophosphorus compound. Diphenylacetylene was reacted with solid Li to produce l,4-dilithio-l,2,3,4-tetraphenylbutadiene (LTPB). Then, LTPB was reacted with dichlorophenylphosphine to produce pentaphenylphosphole, which was oxidized with 30% hydrogen peroxide to pentaphenylphosphole oxide. Next, this oxide was reacted with benzyne to synthesize 2,3-benzo-l,4,5,6,7- pentaphenyl-7- phosphabicyclo[2.2. l]hept-5-ene oxide which has a 31P NMR chemical shift of 8 + 96 ppm. Reduction of this product using trichlorosilane afforded products with 31P NMR chemical shifts o f 8 +57 and +58 ppm which could possibly be the two isomers of 2,3-benzo-l,4,5,6,7- pentaphenyl-7- phosphabicyclo[2.2. l]hept-5-ene. Further reduction of these crude products was attempted using elemental sodium but with no success. Because of a time restraint and the difficulty in obtaining a stable 7-phosphanorbomadiene, the bicyclic, two- coordinate anionic phosphorus derivative could not be synthesized and proven to have a 31P NMR chemical shift of 8+1085 ppm. Amino acid racemization occurs according to the environment to which the amino acids are exposed. Racemization occurs when laevorotary-forms of amino acids are converted to dextrorotary-forms of amino acids by exposure to weak acids or bases, over time. This conversion in ancient samples was found to take place at the same rate as degradation of DNA. It was found [1] that if the D/L ratio of aspartic acid was lower than 0.08 in a bone or tissue sample, viable DNA could be extracted from it In Chapter 2 this research attempted to derivatize enantiomeric amino acids with L-Marfey’s Reagent to produce diastereomers that could be separated using capillary electrophoresis, gas chromatography, and high pressure liquid chromatography. Problems with the equipment, contamination, unrepeatable results and a variety of unknown factors plagued this portion of the research. This mode of testing for the presence of viable DNA was deemed a non-viable process

Successful Genotyping of Microsatellites in the Woolly Mammoth

Genetic analyses using ancient DNA from Pleistocene and early Holocene fossils have largely relied on mitochondrial DNA (mtDNA) sequences. Among woolly mammoths, Mammuthus primigenius, mtDNA analyses have identified 2 distinct clades (I and II) that diverged 1-2 Ma. Here, we establish that microsatellite markers can be effective on Pleistocene samples, successfully genotyping woolly mammoth specimens at 2 loci. Although significant differentiation at the 2 microsatellite loci was not detected between 16 clade I and 4 clade II woolly mammoths, our results demonstrate that the nuclear population structure of Pleistocene species can be examined using fast-evolving nuclear microsatellite markers

Genetic variation at hair length candidate genes in elephants and the extinct woolly mammoth

<p>Abstract</p> <p>Background</p> <p>Like humans, the living elephants are unusual among mammals in being sparsely covered with hair. Relative to extant elephants, the extinct woolly mammoth, <it>Mammuthus primigenius</it>, had a dense hair cover and extremely long hair, which likely were adaptations to its subarctic habitat. The fibroblast growth factor 5 (<it>FGF5</it>) gene affects hair length in a diverse set of mammalian species. Mutations in <it>FGF5 </it>lead to recessive long hair phenotypes in mice, dogs, and cats; and the gene has been implicated in hair length variation in rabbits. Thus, <it>FGF5 </it>represents a leading candidate gene for the phenotypic differences in hair length notable between extant elephants and the woolly mammoth. We therefore sequenced the three exons (except for the 3' UTR) and a portion of the promoter of <it>FGF5 </it>from the living elephantid species (Asian, African savanna and African forest elephants) and, using protocols for ancient DNA, from a woolly mammoth.</p> <p>Results</p> <p>Between the extant elephants and the mammoth, two single base substitutions were observed in <it>FGF5</it>, neither of which alters the amino acid sequence. Modeling of the protein structure suggests that the elephantid proteins fold similarly to the human FGF5 protein. Bioinformatics analyses and DNA sequencing of another locus that has been implicated in hair cover in humans, type I hair keratin pseudogene (<it>KRTHAP1</it>), also yielded negative results. Interestingly, <it>KRTHAP1 </it>is a pseudogene in elephantids as in humans (although fully functional in non-human primates).</p> <p>Conclusion</p> <p>The data suggest that the coding sequence of the <it>FGF5 </it>gene is not the critical determinant of hair length differences among elephantids. The results are discussed in the context of hairlessness among mammals and in terms of the potential impact of large body size, subarctic conditions, and an aquatic ancestor on hair cover in the Proboscidea.</p