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F R Leach

Publications and source records attributed to F R Leach.

At least 19 recordsLinked to original sources
PubMed1997

Cloning and sequencing of a cDNA for firefly luciferase from Photuris pennsylvanica.

The first cDNA from the Photurinae subfamily of the Lampyridae encoding a firefly luciferase from lantern mRNA of Photuris pennsylvanica has been cloned, sequenced, the amino-acid sequence predicted and the sequence reported to GenBank. The cDNA was about 1.8 kb in length with the largest open reading frame coding for a 545-residue protein. The 5' noncoding region is 61 bp long and the 3' noncoding region is 135 bp in length. There is a 24-nucleotide poly(A) tail. When the amino-acid residues are aligned, P. pennsylvanica contains 154 (about 28% of the total residues) that are conserved in all 16 of the deduced luciferase sequences that are presently available. In this P. pennsylvanica luciferase, the amino acids at 276 of the positions are the same at corresponding positions of at least one of the other enzymes. There are two amino-acid differences between this luciferase and the unpublished sequence obtained by Dr. Keith Wood for a putative larval Photuris firefly luciferase cloned from a Maryland firefly. Signature amino-acid sequences and domains found in the deduced sequence are for adenylate kinase, the putative AMP-binding domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.

Amino Acid Sequence↗
PubMed1995

Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity?

Light production by firefly luciferase is limited by product release resulting in flash kinetics. Several compounds (CoA, PPi, and nucleotides) transform the flash-form of light production into continuous light production. The sulfhydryl group of CoA is required; however, since nucleotides are also active, at least two mechanisms (sites) must exist.

Adenine Nucleotides↗
PubMed1994

Effect of periodate-oxidized ATP and other nucleotides on firefly luciferase.

Addition of periodate-oxidized ATP (oATP) to firefly luciferase-containing reaction mixtures enhanced light production when the reaction mixture contained > approximately 8 microM ATP. The time course of light production was changed from a flash pattern to a constant light output during incubations of < approximately 10 min. During longer incubation, firefly luciferase was inactivated in a concentration-dependent fashion by oATP. Firefly luciferase has two different time courses of light production that depend on ATP concentration (DeLuca and McElroy, Biochem. Biophys. Res. Commun., 123, 764, 1984). The enhancement of light production occurred only when higher ATP concentrations (> 8 microM) were used. There is little effect of oATP on firefly luciferase activity at low ATP concentrations (< 2 microM) which gave steady production of light. ATP did not antagonize the inactivation of firefly luciferase by oATP. When the oATP was chemically reduced with sodium borohydride (giving or ATP), there was no inactivation of firefly luciferase on incubation. When or ATP was used in a short incubation the enhancement of light production and time course change were the same as those observed with oATP. The corresponding AMP and adenosine compounds (o and or) were slightly inhibitory to firefly luciferase activity. ADP was without effect but both oADP and or ADP enhanced light production. Of these periodate-oxidized ADP, AMP, and adenosine derivatives only oADP inactivated firefly luciferase. The activating effect can be explained by a change in the conformation of the enzyme-product complex so that the product is released faster. In addition there is an inactivation of the enzyme by certain periodate-oxidized nucleotides during longer incubations.

Adenine Nucleotides↗
PubMed1992

Enhancement of firefly luciferase activity by cytidine nucleotides.

The temporal pattern of light production by firefly luciferase depends on the ATP concentration. With low concentrations of ATP a constant production of light occurred while at high concentrations of ATP (greater than 10 microM) there was a flash of light followed by a decline in light production. This time course of light production with high ATP concentrations was changed from the flash pattern to a pattern with a constant production of light by several cytidine nucleotides. CTP, CDP, dCTP, dCDP, dideoxyCTP, periodate-oxidized CTP and CDP, and the etheno derivatives of CTP and CDP produced that change. CMP, cytidine, CDP-glycerol, CDP-glucose, CDP-ethanolamine, and benzoylbenzoylCTP either were inhibitory to firefly luciferase or were not effective in changing the flash time course. Coenzyme A and related compounds also changed the time course of light production. The changes in time course produced by either cytidine nucleotides or CoA were inhibited by desulfoCoA. These compounds apparently enhanced light production by promoting the dissociation of the inhibitory product, oxidized luciferin, from the enzyme. When the activating compounds were used with high concentrations of ATP, the sensitivity of assay for firefly luciferase was increased. This increased sensitivity is important when using the firefly luciferase gene as a reporter.

Adenine Nucleotides↗
PubMed1992

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases.

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differences are probably attributable to differences in conformation.

Adenosine Triphosphate↗
PubMed1980

Stimulation of Bacillus subtilis transformation by spermidine.

Addition of spermidine in millimolar concentrations to Bacillus subtilis cells curing competence development increases transformability. The spermidine must be added at least 30 min before DNA for maximum stimulation. An incubation period of about 30 minutes is also required for the maximum uptake of labeled spermidine. The amount of DNA initially attached and the rate of DNA uptake are increased to the same extent as transformation. The rate of protein synthesis is also equivalently increased. These observations are consistent with an increase in the number of competent cells in the cell population; this increase is mediated by a spermidine-stimulated protein synthesis.

Bacillus subtilis↗
PubMed1978

The initial attachment of transforming DNA to competent Bacillus subtilis.

The initial attachment of transforming DNA to competent Bacillus subtilis is temperature independent between 25 degrees and 45 degrees. However, below 15 degrees there is a significant reduction in the amount of DNA attached to compentent cells. The DNA that is attached at 4 degrees can lead to transformation or interfere effectively with the subsequent attachment of a distinctive DNA when the cells are shifted to a permissive temperature (37 degrees). These data suggest that the attachment of DNA at 4 degrees is to sites normally involved in the transformation process. The amount of DNA that is initially attached to the bacteria at 4 degrees or 37 degrees after perturbation of the cells by ionic strength changes, repetitive washings, or periodate oxidation varies with the temperature at which the treatment occurs. These results are consistent with a reorientation of the DNA attachment sites upon lowering the temperature to 4 degrees, such that their affinity for DNA and susceptibility inhibitory treatments are reduced.

Bacillus subtilis↗