Firefly luciferase as a reporter enzyme for measuring gene expression in vegetative and symbiotic Rhizobium meliloti and other gram-negative bacteria.

A DNA segment carrying a cDNA copy of the luciferase gene (luc) of the North American firefly Photinus pyralis, fused to the lambda PR promoter and expressed in Escherichia coli [de Wet et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7870-7873], was inserted into a broad-host-range plasmid vector and established in a variety of Gram-negative bacteria. Luciferase activity, expressed from the lambda PR promoter, was detected in both intact cells and extracts prepared from cells of strains of Rhizobium meliloti, R. phaseoli, R. fredii, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Acinetobacter calcoaceticus and Azotobacter vinelandii. The highest levels of activity, determined by measurements of both intact cells and extracts, were observed for P. aeruginosa and the three species of Rhizobium examined. Expression of luciferase activity also was relatively high in R. meliloti bacteroids of mature alfalfa nodules. This activity was readily detectable in intact nodules using x-ray film or in extracts prepared from purified bacteroids.

Symmetrical erosive peripheral polyarthritis in the Late Archaic Period of Alabama.

Rheumatoid arthritis was first described unambiguously in 1800, but its etiology and historical origins are still obscure. Definite rheumatoid arthritis has not been demonstrated in pre-19th century Old World skeletal remains. Six individuals who lived 3000 to 5000 years ago in northwestern Alabama and present erosive polyarthritis characteristic of rheumatoid arthritis are described. The diagnosis raises the possibility that rheumatoid arthritis can be associated with a New World pathogen or allergen.

Firefly and bacterial luminescence: basic science and applications.

The basic chemistry of the reactions leading to light emission in the firefly and in bacteria are briefly reviewed. With excess firefly reagents, the light intensity is proportional to the ATP concentration. For this reason, the reagents have been used for ATP determination in a number of important biological systems. A number of such applications are reviewed. With excess bacterial reagents, the light intensity is directly proportional to the reduced pyridine nucleotide concentration (NADH or NADPH). The applications of this system for studying reactions involving dehydrogenases using NAD or NADP as electron acceptors are presented. Many assays have now been developed using enzymes immobilized on Sepharose. The advantages of using the immobilized enzymes are greater stability of the immobilized enzymes over the soluble forms; increased sensitivity of detection relative to the soluble forms, and reusability of the immobilized enzymes. A comparison of the immobilized bioluminescent assay for 7 alpha-hydroxysteroid with gas-liquid chromatography and radioimmunoassay is presented. Coimmobilized enzymes can be packed in a flow cell and used in an automated instrument with good reproducibility. It is likely that future developments of bioluminescent assays for ATP or NAD(P)H will be with immobilized enzymes using an automated instrument.

Identification of a lysine residue at a nucleotide binding site in the firefly luciferase with p-fluorosulfonyl[14C]benzoyl-5'-adenosine.

Firefly luciferase is 80-90% inactivated within 3 h upon incubation with the adenine nucleotide analogue p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). Although 4 mol of 14C-FSBA/mol of enzyme is irreversibly bound during inactivation, only 1 mol of 14C-FSBA appears to be specifically directed to an adenine nucleotide binding site on the enzyme. The other 3 mol of 14C-FSBA is bound to the protein nonspecifically. The major radioactive peptide in a tryptic digest os labeled luciferase was isolated and shown to have the following amino acid sequence: *Lys-Gly-Glx-Asx-Ser-Lys, where *Lys is the radioactive derivative of the lysine residue that was sulfonylated during the inactivation.

Metabolism of adenine nucleotides in the cultured fetal mouse heart.

Intact beating fetal mouse hearts in organ culture were deprived of oxygen and glucose for up to 4 h, resulting in loss of beating, an 80% fall in ATP, reduction of energy charge from 0.85 to 0.48, and doubling of total nucleoside concentration. Radiolabeled adenine nucleotides were degraded to hypoxanthine and inosine, which were lost from the hearts into the medium during the deprivation period. Adenosine and adenine also appeared in the medium when adenosine deaminase was inhibited. After 24 h of O2 and glucose resupply, ATP returned to 60% of control, and energy charge rose to 0.76. Labeled nucleosides and bases remaining in the heart or exogenous labeled adenine were utilized to resynthesize ATP. [14C]glycine was rapidly taken up by recovering hearts but was not used for de novo adenine nucleotide synthesis. Ability to recover ATP and spontaneous contraction appear related to residual nucleotide and nucleoside content rather than to energy charge.