Novel Proteins And Novel Functions

Overview

Trypanosoma brucei is a eukaryotic parasite that has attracted research interest because of its disease significance as well as its unique biology. For instance, each trypanosome has only a single mitochondrion. The genome of this mitochondrion is organized into a unique structure called kinetoplast DNA (kDNA), that is a network of topologically interlocked DNA circles. The first protein to have a demonstrated role in kDNA replication was a mitochondrial topoisomerase II (TbTOP2mt). Here, we propose a second ...
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More About This Book

Overview

Trypanosoma brucei is a eukaryotic parasite that has attracted research interest because of its disease significance as well as its unique biology. For instance, each trypanosome has only a single mitochondrion. The genome of this mitochondrion is organized into a unique structure called kinetoplast DNA (kDNA), that is a network of topologically interlocked DNA circles. The first protein to have a demonstrated role in kDNA replication was a mitochondrial topoisomerase II (TbTOP2mt). Here, we propose a second function for TbTOP2mt. Circles must be individually released from the network in order to be replicated. Since they are all topologically interlocked, this releasing activity must create a small hole in the network. When we knocked down the TbTOP2mt transcript by RNA interference (RNAi) we found by electron microscopy (EM) of isolated networks that these holes persisted and grew, until eventually the network fragmented. Using thin-section EM we found that these network fragments adopted an unusual conformation in the mitochondrial matrix. In addition to studying known proteins involved in kDNA replication, we are also interested in finding novel proteins. For this, we have developed a forward genetic approach using an RNAi library to screen for loss of kDNA, a typical result of knockdown of kDNA replication proteins. During one screen we identified a protein that we called Lok1. While RNAi of Lok1 caused the kDNA to shrink and disappear, the effect was not very dramatic. However, there was a striking effect on cell growth. After using fluorescence microscopy and Southern blotting to study the effect of RNAi on kDNA in more detail, we concluded that the effect on kDNA was secondary. Instead, we found that RNAi had the most dramatic effect on the shape of the mitochondrion itself, converting it from a highly branched tubule into a single straight tubule. Nothing is currently known about how the shape of the trypanosome mitochondrion is established and maintained. We presented evidence that Lok1 is found in punctate assemblies along the outer membrane of the mitochondrion, and that this protein could be the first component of the machinery that controls mitochondrial shape in these organisms.
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Product Details

  • ISBN-13: 9781243576521
  • Publisher: BiblioLabsII
  • Publication date: 9/3/2011
  • Pages: 130
  • Product dimensions: 7.44 (w) x 9.69 (h) x 0.28 (d)

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