Subject(s)
Proteins/chemistry , Proteins/physiology , Spectrometry, Fluorescence/methods , Animals , Binding Sites , Biophysical Phenomena , Biophysics , Biotechnology , Energy Transfer , Enzymes/chemistry , Enzymes/metabolism , Fluorescence Polarization/methods , Fluorescent Dyes , Humans , In Vitro Techniques , Kinetics , Ligands , Molecular Structure , Protein Folding , Spectrometry, Fluorescence/instrumentation , Technology, Pharmaceutical , ThermodynamicsABSTRACT
Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.
Subject(s)
Fluoresceins/metabolism , Galactosides/metabolism , Lac Operon , Staining and Labeling/methods , beta-Galactosidase/metabolism , Animals , Cell Death , Cells, Cultured , Fluoresceins/chemical synthesis , Galactosides/chemical synthesis , Gene Expression , Mammals/genetics , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Saccharomyces cerevisiae/genetics , Transformation, GeneticABSTRACT
Methyl beta-D-xylopyranoside in a mixture of N,N-dimethylformamide and 2-methoxypropene containing a little hydrogen chloride gave preponderantly the 2,3-O-isopropylidene derivative, which was readily converted into its 4-trifluoromethanesulfonate. The facile displacement of the triflate group gave a 4-azido-4-deoxy-alpha-L-arabinopyranoside derivative, and this, on mild acid treatment, was hydrolyzed to the 2,3-diol, or under more vigorous conditions to 4-azido-4-deoxy-L-arabinose. Methyl 2,3-di-O-acetyl-4-azido-4-deoxy-alpha-L-arabinopyranoside, from the diol, appears (1H-n.m.r. data) to exist as an equilibrating mixture of the 4C1 and 1C4 conformers in chloroform solution. The reduction of the azido sugar by hydrogen over Pd/C in .6M HCl yielded 4-amino-4-deoxy-L-arabinopyranose as its hydrochloride; in 0.1M HCl, further reactions occurred to give 1,4-dideoxy-1,4-imino-L-arabinitol as the final product. The aminodeoxypentose from lipid A precursor IIA, isolated from a Salmonella mutant by Raetz et al. in 1985, was shown to be identical with the synthetic aminoarabinose by t.l.c., 1H-n.m.r. spectroscopy, and g.l.c. of the acetylated reduction products.
