ARVO Annual Meeting Abstract | May 2003
Use of the Yeast Saccharomyces cerevisiae as a Model Organism to Study Human Retinal Disease Gene Mutations
Abstract
Abstract: : Purpose: To describe the development of an in vitro yeast expressionsystem for the study of the biochemical and cellular effects of specific mutations in the ELOVL4 macular disease gene encoding a putative fatty acid elongation enzyme.Methods: Library screening was performed to isolate the human wild-type ELOVL4 cDNA which served as a template for site-directed mutagenesis to introducesequence variations associated with human retinal degenerative phenotypes. Standard molecular cloning techniques enabled the construction of plasmids for expression in the yeast Saccharomyces cerevisiae. Western blotting of extracts from yeast cells expressing the engineered plamids will confirm expression of protein originating from the exogenous cDNAs. Preparation of fatty acid extracts from transformed yeast cells grown on media supplemented with various fatty acid substrates followed by gas chromatography and mass spectroscopy enables characterization of the enzymatic activities of the exogenous wild-type and mutant ELOVL4 cDNA constructs. Results: Wild-type strain SC334 yeast cells grown on rich media in the absence of exogenous fatty acids were found to contain saturated and monounsaturated fatty acid species up to 18 carbon atoms in length but not polyunsaturated fatty acids (PUFAs) or longer chain species, suggesting that the yeast cells lack the biochemical components required to synthesize long-chain and polyunsaturated fatty acids, enabling the characterization of exogenous enzyme activities on the elongation of long-chain PUFA substrates. ELOVL4 cDNA constructs containing either the 797-801delAACTT or 790delT+794delT macular disease-associated mutations were synthesized by site-directed mutagenesis of a human brain-derived wild-type ELOVL4 cDNA clone. The wild-type and mutant cDNAs have been inserted into the yeast expression plasmids pYES2 and pYPGE15 for inducible and constitutive expression respectively. Assessment of substrate specificity of the wild-type ELOVL4 cDNA and defects in enzymatic function of the mutant cDNAs in the yeast functional assay system is currently underway. Conclusions: The yeast Saccharomyces cerevisiae is a valuable model system that can be used to study the effects of exogenously expressed fatty acid biosynthetic enzymes on lipid metabolism. Through its use we aim to gain insight into the biochemical function of the ELOVL4 gene in the normal and degenerating retina.
- دوازده سال تحقیق و مطالعه برای یافتن جواب سوالم ...
- Amplification, Sequencing and Cloning of Iranian N...
- Expression of the Bacillus subtilis levanase gene ...
- Codon usage patterns in Escherichia coli, Bacillus...
- شناسایی سویه های بومی باسیلوس مولد آلفا آمیلاز و ک...
- Itaconic acid/ اسید ایتاکونیک/ در رابطه با عملیات ...
- ازمایشات بر روی انسان ها توسط ارتش امریکا (عملیات...
- ترک اعتیاد به مواد مخدر مستلزم دوباره سازی شخصیت ب...
- Isolation of β-glucan from the cell wall of Saccha...
- β-Glucan exacerbates allergic airway responses to ...
- β-Glucan supplementation, allergy symptoms, and qu...
- B-Glucan-بتاگلوکان
- Fibronectin-فیبرونکتین
- Hemoglobin Differentially Induces Binding of Candi...
- Intracellular expression of Vitreoscilla hemoglobi...
- Hemoglobin differentially induces binding of Candi...
- Detection of plasma (1 → 3)-β-d-glucan in patients...
- Saccharomyces cerevisiae in the respiratory system...
- Active transport of basic amino acids driven by a ...
- مکمل غذایی حاوی اس آدنوزیل متیونین ممکن است به درم...
- رابطه بین تغذیه و افزایش ناهنجاریهای اجتماعی با پو...
- چرا درد دل کردن باعث آرامش انسان می شود /اعتیاد به...
- tetraphenyl porphyrin sulfateعلت علمی ناهنجاریهای ...
- Scientists name bacterium for Frank Zappa
- How grapevines got acne bacteria
- Propionibacterium acnes type zappae
- Chronic Endophthalmitis Caused by Propionibacteriu...
- Propionibacterium acnes endophthalmitis /Case Pres...
- Propionibacterium acnes endogenous endophthalmitis...
هیچ نظری موجود نیست:
ارسال یک نظر