Fluorescence resonance energy transfer dye nucleotide terminators: a new synthetic approach for high-throughout DNA sequencing.

Fluorescence resonance energy transfer (FRET) based dye-nucleotide terminators (10-13) were designed, synthesized, and formulated with Thermo Sequenase II DNA polymerase into a robust kit for high throughput DNA sequencing. The key energy transfer (ET) rigid and linear linker (2), required for the syntheses of energy transfer cassettes (6-9) was synthesized via Heck coupling reaction on t-Boc-L-4-iodo-phenylalanine (1) with N-TFA-propargylamine.

Early effect of myo-inositol deficiency on phosphatidylinositol metabolism in rat liver.

Young rats (100 g) were fed either a myo-inositol-deficient or supplemented (control) diet for up to 14 days following a 12 h fast. At various times during this period animals were killed, livers were removed, and a microsomal fraction was prepared and assayed for CDPdiacylglycerol inositol transferase activity and for phosphatidylinositol-inositol exchange activity. Within 2 days after beginning the regimen, rats consuming the deficient diet had a 40% lower activity of the transferase than rats consuming the control diet. This difference was maintained throughout the feeding period and developed simultaneously with the accumulation of triacylglycerol in the deficient livers. In contrast, the specific activity of the exchange enzyme was unchanged by feeding the deficient diet.

Early effect of myo-inositol deficiency on fatty acid synthetic enzymes of rat liver.

Young rats (100 g) were fed either a purified myo-inositol-deficient balanced diet or a control diet containing 0.5% by weight myo-inositol, ad libitum, for up to 2 weeks following a 48 h fast. Weight gain was the same for animals in both groups. Liver triacylglycerol levels in the deficient animals were 1.8-, 3.5- and 3.0-fold higher than the corresponding levels in the control animals after 4, 8 and 14 days of feeding, respectively. In the myo-inositol-deficient group the specific activities of liver fatty acid synthetase and acetyl-CoA carboxylase were elevated 1.5-2.0-fold over controls, reaching a maximum after 3-4 days of feeding. Subsequently, activities declined to control levels. Rates of fatty acid synthetase synthesis in the deficient group, as measured by [3H]leucine incorporation into immunoprecipitable fatty acid synthetase polypeptide, were significantly higher (1.5-2.0-fold) than controls after 12-18 h of feeding and then declined to control levels by 1 day. No difference was noted between groups in either the rate of total, soluble liver protein synthesis or the half-life of fatty acid synthetase over this time period. These results suggest that the liver lipodystrophy observed during myo-inositol deficiency in rats may be due in part to elevated levels of lipogenic enzymes in this tissue in the early stage of the deficiency.

Translation of rat liver fatty acid synthetase mRNA in a cell-free system derived from wheat germ.

Total liver polysomes were isolated from rats that had fasted for 48 hr and that then had been re-fed a high-carbohydrate, fat-free diet for 20-24 hr. Indirect immunoprecipitation of the polysomes with purified antibody to rat liver fatty acid synthetase and deproteination on sodium dodecyl sulfate-containing sucrose gradients gave an RNA fraction which, when translated in a cell-free system derived from wheat germ, yielded a major polypeptide of apparent molecular weight 225,000 when the translation products were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The polypeptide was specifically precipitated with antibody against rat liver fatty acid synthetase and competed with unlabeled fatty acid synthetase for binding to the antibody. It was somewhat smaller than native fatty acid synthetase subunits (molecular weight 240,000). The peptide accounted for approximately 65% of the radioactive, antibody-precipitable product, the remainder being peptides in the molecular weight range 100,000-150,000. Synthesis of the polypeptide was optimized with respect to K(+), Mg(2+), and spermine concentrations. The quantity of fatty acid synthetase mRNA obtained by the above procedure and measured by translation was a function of the nutritional state of the animal. The relative activity in fasting rats compared to rats that were re-fed for 12 hr was 1:12. The data suggest that rat liver fatty acid synthetase is synthesized as intact subunits from a large mRNA molecule or molecules.

Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver.

The induction of fatty acid synthetase activity in rat liver is markedly reduced by feeding a diet containing a source of linoleate. Within 48 h after changing from a fat-free, high carbohydrate diet to one containing 15% by weight of safflower oil, the specific activity of rat liver fatty acid synthetase is approximately 2-fold lower than that from rats fed a fat-free diet througout. In contrast, feeding a diet containing 15% hydrogenated coconut oil, a source of saturated fatty acids, or 5% methyl oleate for the same length of time has little effect on the activity of the enzyme. The rate of synthesis of the enzyme is reduced by safflower oil feeding from the fat-free (control) level and after 48 h is approximately one-half that of the control. The rate of degradation of fatty acid synthetase is markedly increased in the safflower oil-fed animals over the control; the half-lives are 1.8 days and 3.8 days, respectively. It is suggested that polyunsaturated fatty acids, or a product derived from them, may directly or indirectly regulate the transcription or translation (or both) of fatty acid synthetase messenger RNA.

Reversible inhibition of the fatty acid synthetase complex from Mycobacterium smegmatis by palmitoyl-coenzyme A.

Palmitoyl-CoA dissociates the fatty acid synthetase complex from Mycobacterium smegmatis into inactive subunits of molecular weight 250,000 as determined by sucrose density gradient centrifugation. Palmitoyl-CoA binds to the subunits but the binding can be prevented and reversed by the mycobacterial 3-O-methylmannose-containing polysaccharide. When the palmitoyl-CoA containing inactive subunits were isolated by gel filtration on Sepharose 6B, and then concentrated and dialyzed against 0.5 M phosphate buffer, pH 7.0, containing 3 mM of the complexing agent heptakis-(2,6-di-O-methyl)-beta-cyclodextrin, activity was regenerated to the level of 40 percent of a control sample. The reversibility of the dissociation and inactivation of the synthetase by palmitoyl-CoA suggests that this end product might play a regulatory role by acting as a feedback inhibitor.