The luciferase cDNA from the 'Genji' firefly, Luciola cruciata, was mutated with hydroxylamine to isolate mutant luciferases. Some of the isolated mutant enzymes produced different colors of light, ranging from green to red. Five such mutants, producing green (lambda max = 558 nm), yellow-orange (lambda max = 595 nm), orange (lambda max = 607 nm) and red light (lambda max = 609 and 612 nm), were analyzed. The mutations were found to be single amino acid changes, from Val239 to Ile, Pro452 to Ser, Ser286 to Asn, Gly326 to Ser and His433 to Tyr respectively.
Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 101 and 108 DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.
The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW XL . This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of D-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)؉ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5-RACE (rapid amplification of cDNA ends) and 3-RACE. The primary structure of LRE from P. pyralis deduced from the nucleotide sequence was shown to consist of 308 amino acids with a molecular weight of 33,619. The cDNA was successfully expressed under the control of the tac promoter in Escherichia coli.Firefly luciferase (Luc, EC 1.13.12.7) catalyzes the oxidative decarboxylation of luciferin (LH 2 ) in the presence of ATP, O 2 , and Mg 2ϩ , producing yellow-green light ( max ϭ 560 nm) as described in the following reaction sequence:
Random mutagenesis of the luciferase cDNA from "Genji" firefly, Luciola cruciata, was induced by hydroxylamine in an attempt to isolate thermostable mutants. Three mutants were isolated, and the cDNAs encoding these proteins were sequenced. All mutant cDNAs carried the same C to T transition mutation that conferred an amino acid substitution of Thr by Ile at position 217. The wild-type luciferase and the thermostable variant (Thr217Ile) were purified to homogeneity, and their enzymatic properties were determined. Thr217Ile was superior to wild-type in thermal and pH stability. Furthermore, the specific activity of the Thr217Ile mutant was increased to 130% of that of the wild-type. In order to examine the effect of amino acid residue substitution at position 217 on the thermostability of luciferase, we replaced the Thr residue at position 217 with all of the rest of the possible amino acid residues by site-directed mutagenesis. The thermostability of these substitution mutants seemed to correlate with the hydrophobicity of the substituted amino acid residue.
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