ABSTRACT
The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN.PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 microg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days' batch cultivation.
Subject(s)
Alkyl and Aryl Transferases/metabolism , Artemisia annua/enzymology , Artemisinins/metabolism , Genetic Engineering/methods , Saccharomyces cerevisiae/genetics , Sesquiterpenes/metabolism , Alkyl and Aryl Transferases/genetics , Artemisia annua/genetics , Artemisinins/chemistry , Blotting, Western , Gas Chromatography-Mass Spectrometry , Genome, Fungal/genetics , Molecular Structure , Plasmids/genetics , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/metabolism , Recombination, Genetic , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Sesquiterpenes/chemistry , Transformation, GeneticABSTRACT
OBJECTIVE: To validate self-reported smoking habits in smoking pregnant women and estimate the prevalence of substantial exposure to passive smoking in non-smoking pregnant women. DESIGN: Retrospective, quantitative study, consecutively collected samples. SETTING: One antenatal clinic in Blekinge County, Sweden. SUBJECTS: 509 pregnant women. METHOD: Information about smoking habits were taken from the antenatal records of 496 women (97%). Serum samples from these women were tested for cotinine, a nicotine metabolite. MAIN OUTCOME MEASURES: Self-reported smoking habits and cotinine levels in ng/mL. RESULTS: Of 407 women, reporting to be non-smokers, 6% were most likely smokers, and 3% had cotinine levels suggesting exposure to substantial passive smoking. Of 60 women, reporting smoking 1-10 cigarettes per day, 32% were likely to smoke more. CONCLUSION: If the true facts about exposure to tobacco smoke are not revealed, a number of women who might benefit from information and support at the antenatal clinic will miss the opportunity of such assistance, resulting in increased risks for both the woman and her fetus.
