A yeast-based model system for cloning secreted and membrane proteins
Autor(a) principal: | |
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Data de Publicação: | 2002 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Anais da Academia Brasileira de Ciências (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652002000400005 |
Resumo: | The targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5´-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5´-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139) with a higher expression level in green buds and stem cells, and the other one (YE290) with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control. |
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Anais da Academia Brasileira de Ciências (Online) |
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A yeast-based model system for cloning secreted and membrane proteinssignal sequencespotassium channelyeast acid phosphataselibrary screeningThe targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5´-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5´-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139) with a higher expression level in green buds and stem cells, and the other one (YE290) with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control.Academia Brasileira de Ciências2002-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652002000400005Anais da Academia Brasileira de Ciências v.74 n.4 2002reponame:Anais da Academia Brasileira de Ciências (Online)instname:Academia Brasileira de Ciências (ABC)instacron:ABC10.1590/S0001-37652002000400005info:eu-repo/semantics/openAccessSURPILI,MARCELO J.MÜLLER-RÖBER,BERNDWILLMITZER,LOTHAReng2003-01-24T00:00:00Zoai:scielo:S0001-37652002000400005Revistahttp://www.scielo.br/aabchttps://old.scielo.br/oai/scielo-oai.php||aabc@abc.org.br1678-26900001-3765opendoar:2003-01-24T00:00Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC)false |
dc.title.none.fl_str_mv |
A yeast-based model system for cloning secreted and membrane proteins |
title |
A yeast-based model system for cloning secreted and membrane proteins |
spellingShingle |
A yeast-based model system for cloning secreted and membrane proteins SURPILI,MARCELO J. signal sequences potassium channel yeast acid phosphatase library screening |
title_short |
A yeast-based model system for cloning secreted and membrane proteins |
title_full |
A yeast-based model system for cloning secreted and membrane proteins |
title_fullStr |
A yeast-based model system for cloning secreted and membrane proteins |
title_full_unstemmed |
A yeast-based model system for cloning secreted and membrane proteins |
title_sort |
A yeast-based model system for cloning secreted and membrane proteins |
author |
SURPILI,MARCELO J. |
author_facet |
SURPILI,MARCELO J. MÜLLER-RÖBER,BERND WILLMITZER,LOTHAR |
author_role |
author |
author2 |
MÜLLER-RÖBER,BERND WILLMITZER,LOTHAR |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
SURPILI,MARCELO J. MÜLLER-RÖBER,BERND WILLMITZER,LOTHAR |
dc.subject.por.fl_str_mv |
signal sequences potassium channel yeast acid phosphatase library screening |
topic |
signal sequences potassium channel yeast acid phosphatase library screening |
description |
The targeting of proteins to cell organelles and membranes, or of proteins destined to secretion, is coordinated by signal sequences located at the 5´-end of their respective genes. A signal sequence trap system was envisaged in which a truncated version of the yeast acid phosphatase pho5 gene lacking the start codon and signal sequence could serve as a reporter gene. A fraction enriched in 5´-end fragments obtained by PCR from a potato guard-cell cDNA library was cloned in frame to the acid phosphatase gene and the acid phosphatase activity was assayed directly in yeast colonies grown on selective medium. Putative signal sequences targeting the acid phosphatase to the membrane or to the outside of the cell were used to screen the cDNA bank in order to recover the original full-size sequence which gave rise to the signal sequence. Two unknown sequences displaying marked tissue-specific expression were retrieved, one of them (YE139) with a higher expression level in green buds and stem cells, and the other one (YE290) with a higher expression level in androceum, gyneceum, and roots. The limitations of the system are further analyzed using other sequences as control. |
publishDate |
2002 |
dc.date.none.fl_str_mv |
2002-12-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652002000400005 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652002000400005 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S0001-37652002000400005 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Academia Brasileira de Ciências |
publisher.none.fl_str_mv |
Academia Brasileira de Ciências |
dc.source.none.fl_str_mv |
Anais da Academia Brasileira de Ciências v.74 n.4 2002 reponame:Anais da Academia Brasileira de Ciências (Online) instname:Academia Brasileira de Ciências (ABC) instacron:ABC |
instname_str |
Academia Brasileira de Ciências (ABC) |
instacron_str |
ABC |
institution |
ABC |
reponame_str |
Anais da Academia Brasileira de Ciências (Online) |
collection |
Anais da Academia Brasileira de Ciências (Online) |
repository.name.fl_str_mv |
Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC) |
repository.mail.fl_str_mv |
||aabc@abc.org.br |
_version_ |
1754302855788888065 |