No description available.

No description available.

For many years, natural products have been a primary source of new molecules for the treatment of disease, and microorganisms have been a prolific source of these molecules. Recent studies have indicated, however, that many biosynthetic pathways are present in organisms for which no natural product can be associated, and only a small fraction of the microbial life present in the environment can be grown in culture. This indicates that if methods could be developed for the isolation of these pathways and production of their target molecules in heterologous hosts, great numbers of potentially valuable compounds might be discovered. In these investigations, large insert libraries of two microorganisms were constructed, one a bacterial artificial chromosome (BAC) library, the other a fosmid library, and two large insert fosmid libraries were constructed with DNA isolated from marine environmental samples. A mathematical formula was derived to estimate probabilities of cloning intact biosynthetic pathways with large insert genomic libraries and tested with a computer simulation. This indicated that even large pathways could be cloned intact in large insert libraries, provided there was an adequate size difference between the target pathway and the library inserts, and there was a concomitant increase in the size of the library with the targeting of these larger pathways. In addition, an investigation into a mixed marine culture sample lead to the identification of an unusual relationship between two bacteria for which extended co-culture leads to the production of pyocyanin. However, no useful biosynthetic pathways were located within the genomic libraries. It is concluded that significant improvements would be required to make this approach feasible for larger scale investigations. It is further concluded, on the basis of recent developments in the field, including a reduction in the cost of sequencing, improvements in techniques of whole-genome shotgun sequencing, and the development of recombination based cloning, that the employment of mass sequencing efforts and sequence-driven, recombinationbased cloning, might prove to be a more fruitful and efficient alternative to large-insert library construction for the isolation and expression of these pathways. A possible paradigm for the cloning of pathways on the basis of this technology is proposed.

No description available.

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.

Developing biological systems can be approximately described as complex, three dimensional cellular assemblies that change dramatically across time as a consequence of cell proliferation, differentiation and movements. The presented project aims to overcome problems of limited resolution in both space and time of classical analysis by in situ hybridization on fixed tissue. The employment of the newly developed Single Plane Illumination Microscopy (SPIM) combined with new approaches for in vivo data acquisition and processing promise to yield high-resolution four-dimensional data of the complete Drosophila embryogenesis. We developed a toolkit for high-throughput gene engineering in flies, that provides means for creating faithful in vivo reporters of gene expression during Drosophila melanogaster development. The cornerstone of the toolkit is a fosmid genomic library enabling high-throughput recombineering and φC31 mediated site-specific transgenesis. The dominant, 3xP3-dsRed fly selectable marker on the fosmid backbone allows, in principle, transgenesis of the fosmid clones into any non-melanogaster species. In order to extend the capabilities of the gene engineering toolkit to include “evo-devo” studies, we generated genomic fosmid libraries for other sequenced Drosophilidae: D. virilis, D.simulans and D. pseudoobscura. The libraries for these species were constructed in the pFlyFos vector allowing for recombineering modification and φC31 transgenesis of non-melanogaster genomic loci into D. melanogaster. We have developed a PCR pooling strategy to identify clones for a specific gene from the libraries without extensive clone sequencing and mapping. The clones from these libraries will be primarily used for cross-species gene expression studies. As another application, transgenes originating from closely related species can be used to rescue D. melanogaster RNAi phenotypes and establish their specificity. Together with SPIM microscopy, the toolkit will allow to visualize gene expression patterns throughout Drosophila development.

Although the eDNA sequences for the 10 k:Da (hsp 10, hsp 1 0) and the 60 k:Da (hsp60, cpn60) heat shock proteins have been obtained for a number of mammalian species, until very recently information was not available on the functional genes encoding these proteins. The primary objective of this work was to clone and sequence the functional genes for these proteins from CHO, Chinese hamster ovary cells. Screening of a lambda EMBL3 CHO genomic library with the CHO hsp 10 eDNA identified a clone containing the putative hsp 10 functional gene. A -5.5 kb fragment was isolated from one of these clones by enzymatic digestion and -3.3 kb was sequenced. The clone was found to contain consensus regulatory sequences upstream of the putative transcription initiation site, + 1, including two Sp 1 binding sites, a CAAT box, and a single heat shock element, HSE, but lacked a TATA box. The coding region consists of four exons, identical to the hsp10 CHO eDNA sequence, separated by three introns, of 200 bp, 600 bp and 1600 bp in size, containing conserved splice sites. Screening of the same EMBL3 CHO genomic library with the CHO hsp 10 eDNA also resulted in isolation of a full length processed pseudogene with -90 % identity to the eDNA. This pseudogene lacked introns, contained a poly(A) tract, as well as various single bp changes, additions and deletions. The upstream region of this pseudo gene was found to contain similarity to the human LINE sequence, a DNA repetitive element. PCR amplification ofCHO-WT genomic DNA resulted in isolation offive additional processed pseudogenes, corresponding to the central -270 bp of the CHO hsplO eDNA. All the pseudogenes displayed a high degree of similarity to the CHO hsp 10 eDNA sequence despite the presence of numerous mutations. Prior to this report, pseudogenes had not been found associated with hsp 10. The identification of these pseudogenes suggests the presence of a multi gene family for this heat shock protein in the CHO genome. Previously, a semi-processed pseudogene, Gel, was identified for hsp60 from CHO cells which contained a single -87 bp intron near its 3' end (Venner eta/., 1990). From this pseudo gene, a fragment containing the -87 bp intron was isolated for use as a probe to screen a lambda EMBL3 CHO genomic library. This resulted in isolation of several positive clones, two of which were purified, a -1.0 kb fragment amplified by PCR and then sequenced revealing two additional semi-processed pseudogenes, designated .A4 and .AS. These pseudo genes were found to be homologous to the GC 1 clone, containing many similar mutations as well as the -87 bp intron. Utilizing CHO-WT genomic DNA, a separate PCR amplification resulted in isolation of a -2.5 kb fragment which was partially sequenced and found to correspond to the putative hsp60 functional gene. The fragment contained one exon, which was identical to the CHO hsp60 eDNA in the region sequenced, and two introns of800 bp and 1500 bp. This fragment can now provide an ideal probe for isolation ofthe CHO hsp60 functional gene. / Thesis / Master of Science (MSc)

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.