![]() ![]() Despite several methods have been proposed, the characterization of a sequence adjacent to a known region (gene walking or chromosome walking) still remains a laborious and time-consuming task. The main limit of this approach is that both extremities of the investigated gene remain unknown. This approach, which is nowadays a routine procedure, rapidly provides sequence data on yet uncharacterized genes belonging to known gene families. The intervening region is then amplified, cloned and sequenced. If the target-DNA is represented by a specific gene, the typical approach to accomplish its characterization is to design two degenerated primers, targeting highly conserved regions of the gene. The standard methods to shift the ratio towards target-DNA are either to amplify the target-DNA using the PCR technique ( 1–3) or to clone the target-DNA into a vector (phage or plasmid), which is highly amplified in a bacterial host and subsequently specifically isolated. Any kind of non-target DNA can lead to unspecific binding of the sequencing primer. Sequencing of DNA using a specific primer requires a template, which must consist of a maximum of target-DNA and a minimum of non-target DNA. 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. 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). The newly developed Two-Step Gene Walking method is based on randomly primed polymerase chain reaction (PCR). This supports the hypothesis that this group of genes represents an independent functional unit, which was acquired by Prosthecobacter via horizontal gene transfer. The genomic environments of the characterized btub-operons are always different. Strikingly, all btub-operons comprise a third gene with similarities to kinesin light chain sequences. In all investigated Prosthecobacters, btubAB are organized in a typical bacterial operon. 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. However, more recently they have been found in the unusual bacteria Prosthecobacter ( btubAB). ![]() Tubulins are still considered as typical proteins of Eukaryotes. ![]()
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