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Single Polypeptide (single + polypeptide)
Selected AbstractsA GyrB-GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase IIGENES TO CELLS, Issue 3 2002Sonia Trigueros Background: Type II topoisomerases are a highly conserved class of enzymes which transport one double-stranded DNA segment through a transient break in another. Whereas the eukaryotic enzymes are homodimers of a single polypeptide, their bacterial homologues are homodimers of two independently coded protein subunits. Unlike eukaryotic topoisomerase II and bacterial topoisomerase IV, DNA gyrase is a bacterial type II topoisomerase which specializes in intramolecular DNA transport. Results: We have fused the Escherichia coli coding sequences for the proteins GyrB and GyrA, which comprise DNA gyrase. This fusion was expressed in yeast cells and yielded the expected full-length protein product. When it was expressed in ,top1- top2-4 yeast cells, the fusion protein compensated their slow growth and reverted their elevated chromosomal excision of ribosomal genes. Furthermore, it removed DNA positive supercoils. The fusion protein, however, was unable to complement the temperature-dependent lethality of top2-4 cells. Conclusion: Fusion of the E. coli GyrB and GyrA proteins leads to a catalytically active topoisomerase which compensates several phenotypic traits attributed to unconstrained DNA supercoiling in topoisomerase-deficient cells. However, since the fusion protein cannot substitute for topoisomerase II, it may be efficient in intramolecular but not intermolecular DNA passage, resembling the catalytic properties of DNA gyrase. [source] Identification of isopeptidase activity in the midgut of insects: Purification, properties and nutritional ecology of a Hofmannophila pseudospretella (Lepidoptera: Oecophoridae) larval enzymeINSECT SCIENCE, Issue 4 2010Robert M. Simpson Abstract, A ,-glutamyl transpeptidase (isopeptidase) has been purified 580-fold to homogeneity from the midgut of keratinophagous larvae of Hofmannophila pseudospretella. The enzyme is a single polypeptide of molecular mass 80 kDa. The enzyme was identified by its hydrolytic activity against the synthetic substrate, ,-glutamyl-AMC, its molecular mass and inhibition profile compared to other ,-glutamyl transpeptidases. The enzyme is low or absent from most other insect digestive systems apart from other keratinophagous lepidopteran larvae and predatory carabids. While isopeptide bonds are present in high levels of the proteins in the diet of keratinophages, their presence in the diet of predatory beetles has not been established. [source] Extracytoplasmic prosthetic group ligation to apoproteins: maturation of c -type cytochromesMOLECULAR MICROBIOLOGY, Issue 3 2006Serdar Turkarslan Summary In all organisms, haem is post-translationally and covalently attached to c apocytochromes to produce c holocytochromes via a process called c -type cytochromes maturation, which involves numerous components. In bacteria it was not clear which of these components catalyses the extracytoplasmic haem,apocytochrome ligation per se. In this issue of Molecular Microbiology, Feissner and colleagues report that a single polypeptide from Helicobacter pylori, corresponding to the fusion of two proteins found in other organisms, performs haem ligation to a coexpressed Bordetella pertussis apocytochrome c in an Escherichia coli mutant lacking its own cytochrome c maturation proteins. This simple experimental system pinpoints the components catalysing extracytoplasmic covalent haem ligation and raises intriguing issues about the requirements for delivery of haem and apocytochrome c substrates to produce c holocytochromes. [source] Heritable targeted mutagenesis in maize using a designed endonucleaseTHE PLANT JOURNAL, Issue 1 2010Huirong Gao Summary The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I- CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant. [source] Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urineELECTROPHORESIS, Issue 14 2005Dan Theodorescu Dr. Abstract We describe the use of capillary eletrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47,patients who underwent prostate biopsy. Of this group, 21,patients had benign pathology and 26 with,CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47,urine samples were compared to that of 41,young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474,samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP. [source] | ||||||||||