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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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Features of sequence raw data. Panels show sequence raw data of three different Walking-PCR products, sequenced directly with specific nested primers. (A) displays high-quality sequence in region 800–830 bp; numbers indicate sequenced base pairs. (B) shows the sequence raw data at the end of a Walking-PCR fragment. Boxed sequence highlights Walking-PCR primer-binding region. Upper sequence displays reverse complement sequence of PCR primer, which is mainly recovered by peaks. Lower sequence displays real genomic sequence obtained through another walking step. (C) shows raw data of sequence at the end of one specific shorter Walking-PCR fragment and continuing with the sequence of one specific longer Walking-PCR fragment. This feature only occurs in <3% of all sequences. Boxed sequence highlights the binding region of the Walking-PCR primer. Upper sequence displays reverse complement sequence of the Walking-PCR primer; identical to higher peaks. Lower sequence displays real genomic sequence obtained through sequencing of reverse strand of same PCR product; identical to lower peaks. One or two additional double peaks at 3-prime end emerge due to terminal transferase activity (of adenosines) of used polymerase (indicated with asterisk).
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Figure 3: Features of sequence raw data. Panels show sequence raw data of three different Walking-PCR products, sequenced directly with specific nested primers. (A) displays high-quality sequence in region 800–830 bp; numbers indicate sequenced base pairs. (B) shows the sequence raw data at the end of a Walking-PCR fragment. Boxed sequence highlights Walking-PCR primer-binding region. Upper sequence displays reverse complement sequence of PCR primer, which is mainly recovered by peaks. Lower sequence displays real genomic sequence obtained through another walking step. (C) shows raw data of sequence at the end of one specific shorter Walking-PCR fragment and continuing with the sequence of one specific longer Walking-PCR fragment. This feature only occurs in <3% of all sequences. Boxed sequence highlights the binding region of the Walking-PCR primer. Upper sequence displays reverse complement sequence of the Walking-PCR primer; identical to higher peaks. Lower sequence displays real genomic sequence obtained through sequencing of reverse strand of same PCR product; identical to lower peaks. One or two additional double peaks at 3-prime end emerge due to terminal transferase activity (of adenosines) of used polymerase (indicated with asterisk).

Mentions: The chromatograms of successful sequencing attempts showed different typical features. In most cases, the sequence quality was high up to 600–900 bp (Figure 3A). Partially sequenced PCR products could be completely characterized using a newly designed nested primer, which was used for sequencing of the same PCR product (primer walking). In most cases, the Walking-PCR products were long enough to use them in multiple successive sequencing reactions. Thus, there is no need for the performance of a new Walking-PCR before the sequence of a Walking-PCR product is completely characterized through primer walking. This is crucial for the high efficiency of the Two-Step Gene Walking method.Figure 3.


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)

Features of sequence raw data. Panels show sequence raw data of three different Walking-PCR products, sequenced directly with specific nested primers. (A) displays high-quality sequence in region 800–830 bp; numbers indicate sequenced base pairs. (B) shows the sequence raw data at the end of a Walking-PCR fragment. Boxed sequence highlights Walking-PCR primer-binding region. Upper sequence displays reverse complement sequence of PCR primer, which is mainly recovered by peaks. Lower sequence displays real genomic sequence obtained through another walking step. (C) shows raw data of sequence at the end of one specific shorter Walking-PCR fragment and continuing with the sequence of one specific longer Walking-PCR fragment. This feature only occurs in <3% of all sequences. Boxed sequence highlights the binding region of the Walking-PCR primer. Upper sequence displays reverse complement sequence of the Walking-PCR primer; identical to higher peaks. Lower sequence displays real genomic sequence obtained through sequencing of reverse strand of same PCR product; identical to lower peaks. One or two additional double peaks at 3-prime end emerge due to terminal transferase activity (of adenosines) of used polymerase (indicated with asterisk).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Features of sequence raw data. Panels show sequence raw data of three different Walking-PCR products, sequenced directly with specific nested primers. (A) displays high-quality sequence in region 800–830 bp; numbers indicate sequenced base pairs. (B) shows the sequence raw data at the end of a Walking-PCR fragment. Boxed sequence highlights Walking-PCR primer-binding region. Upper sequence displays reverse complement sequence of PCR primer, which is mainly recovered by peaks. Lower sequence displays real genomic sequence obtained through another walking step. (C) shows raw data of sequence at the end of one specific shorter Walking-PCR fragment and continuing with the sequence of one specific longer Walking-PCR fragment. This feature only occurs in <3% of all sequences. Boxed sequence highlights the binding region of the Walking-PCR primer. Upper sequence displays reverse complement sequence of the Walking-PCR primer; identical to higher peaks. Lower sequence displays real genomic sequence obtained through sequencing of reverse strand of same PCR product; identical to lower peaks. One or two additional double peaks at 3-prime end emerge due to terminal transferase activity (of adenosines) of used polymerase (indicated with asterisk).
Mentions: The chromatograms of successful sequencing attempts showed different typical features. In most cases, the sequence quality was high up to 600–900 bp (Figure 3A). Partially sequenced PCR products could be completely characterized using a newly designed nested primer, which was used for sequencing of the same PCR product (primer walking). In most cases, the Walking-PCR products were long enough to use them in multiple successive sequencing reactions. Thus, there is no need for the performance of a new Walking-PCR before the sequence of a Walking-PCR product is completely characterized through primer walking. This is crucial for the high efficiency of the Two-Step Gene Walking method.Figure 3.

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