| Summary: | 博士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 96 === Abstract
Streptomyces are highly abundant soil bacteria that possess linear chromosomes (and linear plasmids). The 5' ends of these linear DNA molecules are covalently bound by terminal proteins (TPs), which are important for integrity and replication of the telomeres. While most typical TPs (Tpg) are conserved in sequence and size (about 185 aa), there are atypical TPs with heterologous sequences and size, such as Tpc that caps SCP1 plasmid.
Interactions between the TPs have been suggested by electrophoretic immobility of the TP-DNA and the observed circular genetic maps from different Streptomyces species with linear chromosomes. Applying disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) to Streptomyces cultures that harbored linear plasmids capped by Tpg and/or Tpc, I discovered in vivo interactions between like and different telomeres both intermolecularly and intermolecularly. The intramolecular telomere interactions generate negative superhelicity in the plasmid DNA. Intermolecular (but not intramolecular) interactions involve non-covalently proteins and presumably would associate the telomeres of the linear plasmids and chromosomes in vivo. These discoveries indicate that in vivo the linear Streptomyces plasmids (and presumably the chromosomes also) form a supercoiling circular configuration, and these replicons are associated with one another at their telomeres.
There are at least two heterologous types of TPs, both of which contain a DNA binding domain and a classical eukaryotic nuclear localization signal (NLS). Here I show that the NLS motifs on these TPs are highly efficient in targeting fused green fluorescence protein as well as covalently-bound plasmid DNA into the nuclei of human cells. The NLSs are dispensable for replication of linear plasmids, and NLS of Tpc is required for conjugal transfer of linear plasmids SCP1. The discovery of the nuclear localization functions of the Streptomyces TPs not only suggests possible inter-kingdom gene exchange between Streptomyces and eukaryotes in soil but also provides a novel strategy for gene delivery in humans and other eukaryotes.
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