Epigenetic Natural Variation in Arabidopsis thaliana

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F2 families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means

Metabolic engineering of central carbon metabolism in Escherichia coli : improving the production of biomass and metabolites

The pathway for central carbon metabolism provides precursors for cell biosynthesis and metabolite synthesis along with ATP and NADH. We investigated the metabolic engineering of one of the branches of the central carbon pathways: the pathway of glycogen synthesis and degradation. We were motivated in selecting the glycogen pathway for genetic manipulation by the literature on acetate production in E. coli. The literature indicates that in aerobic cultures the uptake of nutrients occurred faster than the utilization of the precursors, formed from the nutrients, in making biomass and energy. We decided to sequester the excess carbon in glycogen which is a storage polymer. We also devised vectors to degrade the sequestered glycogen. The effects, possible causes of the effects, and potential applications of the sequestering of carbon in the form of glycogen, sometimes combined with engineered degradation of the sequestered glycogen, have been the subject of this thesis. This manipulation of the glycogen pathway yielded practically useful results. The metabolic engineering was done in an Escherichia coli mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. The sequestering of glycogen was achieved by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under the control of the IPTG-inducible tac promoter. If glycogen overproduction in the ack pta strain grown in complex medium was induced during late log-phase, biomass production increased by 15 - 20% relative to uninduced controls. When glycogen was sequestered and then degraded in E. coli cultures grown in minimal medium, by overamplifying the genes for glycogen synthesis and degradation, then glutamate production was increased almost 3-fold compared to the plasmid-free strain. When glycogen was sequestered, we observed changes in some of the secreted end-products. We observed that, after overproduction of glycogen, uptake of the previously secreted pyruvate was increased with respect to the control strain, and the CO2 production rate was also increased. These dual observations suggest an increased activity of the gluconeogenic pathways or the TCA cycle. The increase in glutamate, when glycogen sequestering was combined with degradation, also indicate an increase in TCA flux. Comparison of cAMP levels with and without glycogen overproduction indicate a higher level in cAMP after glycogen is overproduced. There appears to be a tentative link, though not conclusive, between cAMP synthesis and glycogen synthesis pathway. cAMP is a global regulator of central carbon metabolism including many genes of the TCA cycle enzymes. By affecting the TCA flux, cAMP may be one of the causes behind the pleiotropic effects of glycogen overproduction and degradation

Kaleidaseq: A Web-Based Tool to Monitor Data Flow in a High Throughput Sequencing Facility

Tracking data flow in high throughput sequencing is important in maintaining a consistent number of successfully sequenced samples, making decisions on scheduling the flow of sequencing steps, resolving problems at various steps and tracking the status of different projects. This is especially critical when the laboratory is handling a multitude of projects. We have built a Web-based data flow tracking package, called Kaleidaseq, which allows us to monitor the flow and quality of sequencing samples through the steps of preparation of library plates, plaque-picking, preparation of templates, conducting sequencing reactions, loading of samples on gels, base-calling the traces, and calculating the quality of the sequenced samples. Kaleidaseq’s suite of displays allows for outstanding monitoring of the production sequencing process. The online display of current information that Kaleidaseq provides on both project status and process queues sorted by project enables accurate real-time assessment of the necessary samples that must be processed to complete the project. This information allows the process manager to allocate future resources optimally and schedule tasks according to scientific priorities. Quality of the sequenced samples can be tracked on a daily basis, which allows the sequencing laboratory to maintain a steady performance level and quickly resolve dips in quality. Kaleidaseq has a simple easy-to-use interface that allows access to all major functions and process queues from one Web page. This software package is modular and designed to allow additional processing steps and new monitoring variables to be added and tracked with ease. Access to the underlying relational database is through the Perl DBI interface, which allows for the use of different relational databases. Kaleidaseq is available for free use by the academic community from http://www.cshl.org/kaleidaseq

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana

The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions. Here we report the analysis of the genomic sequence of Arabidopsis. The sequenced regions cover 115.4 megabases of the 125-megabase genome and extend into centromeric regions. The evolution of Arabidopsis involved a whole-genome duplication, followed by subsequent gene loss and extensive local gene duplications, giving rise to a dynamic genome enriched by lateral gene transfer from a cyanobacterial-like ancestor of the plastid. The genome contains 25,498 genes encoding proteins from 11,000 families, similar to the functional diversity of Drosophila and Caenorhabditis elegans - the other sequenced multicellular eukaryotes. Arabidopsis has many families of new proteins but also lacks several common protein families, indicating that the sets of common proteins have undergone differential expansion and contraction in the three multicellular eukaryotes. This is the first complete genome sequence of a plant and provides the foundations for more comprehensive comparison of conserved processes in all eukaryotes, identifying a wide range of plant-specific gene functions and establishing rapid systematic ways to identify genes for crop improvement

Initial sequencing and analysis of the human genome

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