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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.

Pilhofer M, Bauer AP, Schrallhammer M, Richter L, Ludwig W, Schleifer KH, Petroni G - Nucleic Acids Res. (2007)

Bottom Line: The genomic environments of the characterized btub-operons are always different.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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Technical University Munich, Am Hochanger 4, D-85354 Freising, Germany.

ABSTRACT
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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Related in: MedlinePlus

Basic principle of the Two-Step Gene Walking procedure. Known sequence stretch is shown in gray; unknown sequence stretch is shown in white; Walking-PCR primer is shown in black; specific nested sequencing primer is shown striped. In first 30 cycles, the PCR primer binds at stringent conditions; specific ssDNA of different length (caused by different drop off sites of polymerase) is produced. One subsequent cycle at low annealing temperature allows unspecific binding of primer at different sites on ssDNA as reverse primer. dsDNA of different length with primer sequence incorporated at each 5-prime end is produced. Thirty cycles at stringent conditions specifically and exponentially amplify dsDNA. PCR product is sequenced directly by using a specific nested primer.
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Figure 1: Basic principle of the Two-Step Gene Walking procedure. Known sequence stretch is shown in gray; unknown sequence stretch is shown in white; Walking-PCR primer is shown in black; specific nested sequencing primer is shown striped. In first 30 cycles, the PCR primer binds at stringent conditions; specific ssDNA of different length (caused by different drop off sites of polymerase) is produced. One subsequent cycle at low annealing temperature allows unspecific binding of primer at different sites on ssDNA as reverse primer. dsDNA of different length with primer sequence incorporated at each 5-prime end is produced. Thirty cycles at stringent conditions specifically and exponentially amplify dsDNA. PCR product is sequenced directly by using a specific nested primer.

Mentions: The Two-Step Gene Walking method consists of a Walking-PCR (step 1) and direct sequencing of the PCR product (step 2). The basic principle is outlined in Figure 1. Primers were obtained from MWG Biotech AG (Germany) and their sequences are listed in supporting material. Walking-PCRs were performed using 0.25 µl (100 µM) of one specific primer, 0.25 µl Ex-Taq (Takara Bio Inc., Japan), 5 µl Ex-Taq buffer (10×), 5 µl dNTP mixture (2.5 mM each), 38.5 µl ultra pure water and 1 µl template DNA (50 ng). PCRs were performed using a Primus 96 Plus (MWG Biotech AG, Germany) and the cycling program shown in Table 1.Figure 1.


Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method.

Pilhofer M, Bauer AP, Schrallhammer M, Richter L, Ludwig W, Schleifer KH, Petroni G - Nucleic Acids Res. (2007)

Basic principle of the Two-Step Gene Walking procedure. Known sequence stretch is shown in gray; unknown sequence stretch is shown in white; Walking-PCR primer is shown in black; specific nested sequencing primer is shown striped. In first 30 cycles, the PCR primer binds at stringent conditions; specific ssDNA of different length (caused by different drop off sites of polymerase) is produced. One subsequent cycle at low annealing temperature allows unspecific binding of primer at different sites on ssDNA as reverse primer. dsDNA of different length with primer sequence incorporated at each 5-prime end is produced. Thirty cycles at stringent conditions specifically and exponentially amplify dsDNA. PCR product is sequenced directly by using a specific nested primer.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175320&req=5

Figure 1: Basic principle of the Two-Step Gene Walking procedure. Known sequence stretch is shown in gray; unknown sequence stretch is shown in white; Walking-PCR primer is shown in black; specific nested sequencing primer is shown striped. In first 30 cycles, the PCR primer binds at stringent conditions; specific ssDNA of different length (caused by different drop off sites of polymerase) is produced. One subsequent cycle at low annealing temperature allows unspecific binding of primer at different sites on ssDNA as reverse primer. dsDNA of different length with primer sequence incorporated at each 5-prime end is produced. Thirty cycles at stringent conditions specifically and exponentially amplify dsDNA. PCR product is sequenced directly by using a specific nested primer.
Mentions: The Two-Step Gene Walking method consists of a Walking-PCR (step 1) and direct sequencing of the PCR product (step 2). The basic principle is outlined in Figure 1. Primers were obtained from MWG Biotech AG (Germany) and their sequences are listed in supporting material. Walking-PCRs were performed using 0.25 µl (100 µM) of one specific primer, 0.25 µl Ex-Taq (Takara Bio Inc., Japan), 5 µl Ex-Taq buffer (10×), 5 µl dNTP mixture (2.5 mM each), 38.5 µl ultra pure water and 1 µl template DNA (50 ng). PCRs were performed using a Primus 96 Plus (MWG Biotech AG, Germany) and the cycling program shown in Table 1.Figure 1.

Bottom Line: The genomic environments of the characterized btub-operons are always different.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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Technical University Munich, Am Hochanger 4, D-85354 Freising, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus