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Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa
The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an arra...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
F1000Research
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670013/ https://www.ncbi.nlm.nih.gov/pubmed/26674490 http://dx.doi.org/10.12688/f1000research.2-99.v3 |
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author | Fonseca, Érica L. Vicente, Ana Carolina Paulo |
author_facet | Fonseca, Érica L. Vicente, Ana Carolina Paulo |
author_sort | Fonseca, Érica L. |
collection | PubMed |
description | The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-bla (GES-1) /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla (GES-1) /aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of bla (GES-1) /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla (GES-1) and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla (GES-1) /aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla (GES-1)/ aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream bla (GES-1)/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla (GES-1)/ aacA4 free circular forms, but not to bla (GES-1) and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription. |
format | Online Article Text |
id | pubmed-4670013 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | F1000Research |
record_format | MEDLINE/PubMed |
spelling | pubmed-46700132015-12-14 Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa Fonseca, Érica L. Vicente, Ana Carolina Paulo F1000Res Research Article The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site ( attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-bla (GES-1) /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla (GES-1) /aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of bla (GES-1) /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla (GES-1) and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla (GES-1) /aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than bla (GES-1)/ aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream bla (GES-1)/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla (GES-1)/ aacA4 free circular forms, but not to bla (GES-1) and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription. F1000Research 2015-11-27 /pmc/articles/PMC4670013/ /pubmed/26674490 http://dx.doi.org/10.12688/f1000research.2-99.v3 Text en Copyright: © 2015 Fonseca ÉL and Vicente ACP http://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Fonseca, Érica L. Vicente, Ana Carolina Paulo Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa |
title | Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
Pseudomonas aeruginosa
|
title_full | Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
Pseudomonas aeruginosa
|
title_fullStr | Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
Pseudomonas aeruginosa
|
title_full_unstemmed | Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
Pseudomonas aeruginosa
|
title_short | Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
Pseudomonas aeruginosa
|
title_sort | polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from
pseudomonas aeruginosa |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670013/ https://www.ncbi.nlm.nih.gov/pubmed/26674490 http://dx.doi.org/10.12688/f1000research.2-99.v3 |
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