Limits...
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.

Show MeSH
Typical walking-PCR product patterns. Different Walking-PCR products, which were successfully used for direct sequencing with nested specific primers, are shown. Patterns show big variety based on different templates and different primers. 5 µl aliquots were loaded on a 1% agarose gel and subjected to electrophoresis. 1 kb DNA Ladder (Invitrogen, USA) is shown in first and in last lane. Numbers stand for corresponding fragment lengths of respective bands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175320&req=5

Figure 2: Typical walking-PCR product patterns. Different Walking-PCR products, which were successfully used for direct sequencing with nested specific primers, are shown. Patterns show big variety based on different templates and different primers. 5 µl aliquots were loaded on a 1% agarose gel and subjected to electrophoresis. 1 kb DNA Ladder (Invitrogen, USA) is shown in first and in last lane. Numbers stand for corresponding fragment lengths of respective bands.

Mentions: The Walking-PCR products analyzed on agarose gels showed in most cases a pattern representing fragments of different sizes (Figure 2). This can be explained by different drop off sites of the polymerase and due to different binding sites of the primer during the unspecific-annealing cycle. It results in a mixture of specific PCR products, which are heterogeneous in length. Also, a fraction of unspecific fragments is expected, because the primer could also bind unspecifically in other regions of the genome during the low stringency cycle. To estimate the portion of unspecific fragments produced during the Walking-PCR, cloning experiments were performed (data not shown). Some Walking-PCR products, with which gene walking as described above was successful, were selected. The proportion of clones with specific inserts did not exceed the proportion with unspecific inserts. This suggests that there is a significant fraction of unspecific amplification during the Walking-PCR, nevertheless the unspecific products do not interfere with the following step of direct sequencing. Therefore, direct sequencing with a specific nested primer has to be favored over a cloning and sequencing approach.Figure 2.


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)

Typical walking-PCR product patterns. Different Walking-PCR products, which were successfully used for direct sequencing with nested specific primers, are shown. Patterns show big variety based on different templates and different primers. 5 µl aliquots were loaded on a 1% agarose gel and subjected to electrophoresis. 1 kb DNA Ladder (Invitrogen, USA) is shown in first and in last lane. Numbers stand for corresponding fragment lengths of respective bands.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Typical walking-PCR product patterns. Different Walking-PCR products, which were successfully used for direct sequencing with nested specific primers, are shown. Patterns show big variety based on different templates and different primers. 5 µl aliquots were loaded on a 1% agarose gel and subjected to electrophoresis. 1 kb DNA Ladder (Invitrogen, USA) is shown in first and in last lane. Numbers stand for corresponding fragment lengths of respective bands.
Mentions: The Walking-PCR products analyzed on agarose gels showed in most cases a pattern representing fragments of different sizes (Figure 2). This can be explained by different drop off sites of the polymerase and due to different binding sites of the primer during the unspecific-annealing cycle. It results in a mixture of specific PCR products, which are heterogeneous in length. Also, a fraction of unspecific fragments is expected, because the primer could also bind unspecifically in other regions of the genome during the low stringency cycle. To estimate the portion of unspecific fragments produced during the Walking-PCR, cloning experiments were performed (data not shown). Some Walking-PCR products, with which gene walking as described above was successful, were selected. The proportion of clones with specific inserts did not exceed the proportion with unspecific inserts. This suggests that there is a significant fraction of unspecific amplification during the Walking-PCR, nevertheless the unspecific products do not interfere with the following step of direct sequencing. Therefore, direct sequencing with a specific nested primer has to be favored over a cloning and sequencing approach.Figure 2.

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