Nucleic Acids Research Advance Access originally published online on October 16, 2007
Nucleic Acids Research 2007 35(20):e135; doi:10.1093/nar/gkm836
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Nucleic Acids Research, 2007, Vol. 35, No. 20 e135
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method
1Lehrstuhl für Mikrobiologie, Technical University Munich, Am Hochanger 4, D-85354 Freising, Germany, 2Dipartimento di Biologia, University of Pisa, via A. Volta 4/6, I-56126 Pisa, Italy and 3Machine Learning and Data Mining in Bioinformatics, Technical University Munich, Boltzmannstraße 3, D-85748 Garching b. München, Germany
* To whom correspondence should be addressed. Tel: +39 050 2211384; Fax: +39 050 2211393; Email: gpetroni{at}biologia.unipi.it
Received June 18, 2007. Revised August 24, 2007. Accepted September 25, 2007.
Accession Numbers: AMO41149, AMO41150, EF650087, AM743196
Tubulins are still considered as typical proteins of Eukaryotes. However, more recently they have been found in the unusual bacteria Prosthecobacter (btubAB). In this study, the genomic organization of the btub-genes and their genomic environment were characterized by using the newly developed Two-Step Gene Walking method. In all investigated Prosthecobacters, btubAB are organized in a typical bacterial operon. Strikingly, all btub-operons comprise a third gene with similarities to kinesin light chain sequences. The genomic environments of the characterized btub-operons are always different. This supports the hypothesis that this group of genes represents an independent functional unit, which was acquired by Prosthecobacter via horizontal gene transfer. The newly developed Two-Step Gene Walking method is based on randomly primed polymerase chain reaction (PCR). It presents a simple workflow, which comprises only two major steps—a Walking-PCR with a single specific outward pointing primer (step 1) and the direct sequencing of its product using a nested specific primer (step 2). Two-Step Gene Walking proved to be highly efficient and was successfully used to characterize over 20 kb of sequence not only in pure culture but even in complex non-pure culture samples.
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