Browsing by Subject "Ligation"
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Item Design and evolution of synthetic biological systems(2006-08) Tabor, Jeffrey Jay; Ellington, Andrew D.The study of biology has undergone a fundamental change due to advancements in genetic engineering, DNA synthesis and DNA sequencing technologies. As opposed to the traditional dissective mentality of discovering genes via genetics, describing genetic behaviors through biochemistry, and then drawing diagrams of functional networks, researchers now have the potential (albeit limited) to construct novel biological molecules, networks, and even whole organisms with user-defined specifications. We have engineered novel catalytic DNAs (deoxyribozymes) with the ability to 'read' an input DNA sequence and then 'write' (by ligation) a separate DNA sequence which can in turn be detected sensitively. In addition, the deoxyribozymes can read unnatural (synthetic) nucleotides and write natural sequence information. Such simple nanomachines could find use in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of difficult to detect (short) nucleic acids such as microRNAs. As an extension of in vitro biological engineering efforts, we aimed to construct novel signal transduction systems in vivo. To this end, we used directed evolution to generate a catalytic RNA (ribozyme) capable of creating genetic memory in E. coli. In the end we evolved an RNA which satisfied the conditions of our genetic screen. Rather than maintaining genetic memory, however, the RNA increased relative cellular gene expression by minimizing the translational burden it imposed on the host cell. Interestingly, detailed mutational analysis of the evolved RNA led us to new studies on the relationship between ribosome availability and stochasticity in cellular gene expression, an effect that had frequently been alluded to in the literature, yet never examined. We have also taken a more canonical approach to the forward engineering of biological systems with unnatural behaviors. To this end, we designed a protein-based synthetic genetic circuit that allows a community of E. coli to function as biological film, capable of capturing and recapitulating a projected light pattern at high resolution (theoretically 100 mexapixels). The ability to control bacterial gene expression at high resolution could be used to ‘print’ complex bio-materials or deconvolute signaling pathways through precise spatial and temporal control of regulatory states.Item Telomere Dynamics and End Processing in Mammalian Cells(2006-05-15) Sfeir, Agnel J.; Shay, Jerry W.Telomeres are repetitive DNA sequences that end in single-stranded 3' overhangs. With each cell division, normal human cells lose a small amount of telomeric DNA due to the end-replication problem and the action of an unidentified nuclease. In order for tumor cells to divide indefinitely, they maintain telomere length by expressing the enzyme telomerase. The end structure of mammalian telomeres is not very well understood. Two assays were developed using ligation and PCR amplification to identify the terminal nucleotides of both the C-rich and G-rich telomeric strands in human cells. The results showed that ~ 80 % of the C-strands terminate precisely in ATC-5', demonstrating that the nuclease resection of the C-strand post replication is specific for a single nucleotide. In contrast, the last base of the G-strand in normal human cells was less precise with 70% of the ends being TAG-3', TTA-3' or GTT-3'. An enrichment for the TAG-3' end was noted in cells that express telomerase. A series of nucleases were tested for their involvement in specifying the last base of C-strands and the results indicated that none of those nucleases were responsible for telomere-end resection. Inhibiting the normal function of most telomere binding proteins altered normal telomere function, however only one protein (POT-1) influenced last base specificity. Knocking down POT-1 in normal and tumor cells randomized the last base of the C-strand. These finding have important implications for the processing events that act on the telomere ends and they will help identify the nuclease that resects the chromosome ends. In the second part of this study, the dynamics of telomerase action in mammalian cells was examined. Using a PCR-based, single telomere-length measurement assay (STELA) we showed that telomerase adds an average of 250-nucleotides per end in one replication cycle. Cell cycle studies showed that while the telomeres on the Xp chromosome replicated in early S-phase their elongation by telomerase took place during late S/early G2 phase. Therefore, in mammalian cells telomerase action is not coupled to DNA replication. These studies will provide much needed information for exploiting our knowledge of telomere biology for telomerase-based therapeutic purposes.