alexa Cloning by metabolic interference in yeast and enzymatic characterization of Arabidopsis thaliana sterol delta 7-reductase.
Agri and Aquaculture

Agri and Aquaculture

Rice Research: Open Access

Author(s): Lecain E, Chenivesse X, Spagnoli R, Pompon D, Lecain E, Chenivesse X, Spagnoli R, Pompon D, Lecain E, Chenivesse X, Spagnoli R, Pompon D, Lecain E, Chenivesse X, Spagnoli R, Pompon D

Abstract Share this page

Abstract Reduction of the delta 7 double bond of sterols, a key biosynthetic step in higher eukaryotes, is lacking in lower eukaryotes like the yeast Saccharomyces cerevisiae, leading to terminal sterols with a delta 5,7-conjugated diene structure. Genes encoding two sterol reductases involved, respectively, in the reduction of sterol delta 14 and delta 24(28) double bonds have been cloned to date, but no sequence information was available on the enzyme responsible for delta 7-bond reduction. This study presents the cloning of the NADPH-sterol delta 7-reductase (delta 7-red) from Arabidopsis thaliana, based on a metabolic interference approach in yeast. The principle is the functional expression of a plant cDNA library in the yeast strain FY1679-28C tolerant to sterol modifications and the selection of clones resistant to the polyene fungicide nystatin. The toxicity of this compound is dependent on the presence of delta 5,7-unsaturated sterols in the yeast plasma membrane. One clone out of 10(5) transformants exhibits a cDNA-dependent alteration of cell sterol composition. The 1290-base pair cDNA open reading frame was isolated and sequenced. The corresponding protein presents a significant sequence similarity with yeast delta 14- and delta 24(28)-reductases and with human lamin B receptor. The coding sequence was extracted by polymerase chain reaction and inserted into a galactose-inducible yeast expression vector to optimize expression. Analysis using transformed wild type yeast or sterol altered mutants, indicated that delta 5,7-ergosta- and cholesta-sterols are efficiently reduced in vivo, regardless of the structural variations on the side chain. No reductase activity was observed toward the delta 14 or the delta 5 positions of sterols. In vivo extensive delta 7-reduction of the free and esterified pools of sterols was observed upon induction of the enzyme. Ergosterol present before induction was reduced into ergosta-5,22-dieneol, whereas ergosta-5-eneol is the new end product of sterol neosynthesis, indicating that the yeast delta 22 desaturase may be no longer active on C-7-saturated sterols. In vitro tests indicated that delta 7-reductase activity is preferentially associated with the endoplasmic reticulum membrane and confirmed the previous finding that NADPH is the reducing agent.
This article was published in J Biol Chem and referenced in Rice Research: Open Access

Relevant Expert PPTs

Relevant Speaker PPTs

Recommended Conferences

Relevant Topics

OMICS International Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

agrifoodaquavet@omicsonline.com

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

clinical_biochem@omicsonline.com

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

business@omicsonline.com

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

chemicaleng_chemistry@omicsonline.com

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

environmentalsci@omicsonline.com

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

engineering@omicsonline.com

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

generalsci_healthcare@omicsonline.com

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

genetics_molbio@omicsonline.com

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

immuno_microbio@omicsonline.com

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

omics@omicsonline.com

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

materialsci@omicsonline.com

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

mathematics_physics@omicsonline.com

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

medical@omicsonline.com

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

neuro_psychology@omicsonline.com

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

pharma@omicsonline.com

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

social_politicalsci@omicsonline.com

1-702-714-7001 Extn: 9042

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version