The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both Si nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell Unes stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30-to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.The luciferase isolated from the common North American firefly Photinus pyralis is one of the most extensively studied of the enzymes that catalyze light production in bioluminescent organisms (for reviews, see references 8, 35, and 36). P. pyralis luciferase has an apparent molecular weight of 62,000 (59) and requires luciferin, ATP, and 02 as substrates. The structure of firefly luciferin has been determined, and the chemical synthesis of this heterocyclic carboxylic acid has been reported (4, 57). The reactions catalyzed by firefly luciferase are: Mg2+ luciferase + luciferin + ATP * -luciferase luciferyl-AMP cDNA library (10). The cDNA hybridized to a single firefly lantern poly(A)+ RNA species estimated to be 1.95 kilobases (kb) in length. The largest cDNA clone isolated, XLuc23, contained -1.8 kb of luciferase cDNA consisting of two EcoRI fragments (the terminal EcoRI sites were added as synthetic linkers during the construction of the cDNA library). This cDNA was inserted into an Escherichia coli expression plasmid and expressed a fusion protein in E. coli that exhibited the ATP-and luciferin-dependent lightemitting activity of firefly luciferase. luciferase luciferyl-AMP + 02 -+ luciferase + oxyluciferin + AMP + C02 + hv The first reaction is the formation of an enzyme-bound luciferyl-adenylate. During the second reaction, the luciferyl-adenylate undergoes an oxidative decarboxylation which results in the production of CO2, oxyluciferin, AMP, and light. When excess substrates are added to firefly luciferase the reaction produces a flash of light that is proportional to...

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColEl. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2O67) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColEl plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.Bacterial plasmids of the P-I incompatibility group (Inc P-1) are capable of replication in a wide range of Gram-negative bacteria (1-4). We have been examining the replication properties of RK2, an Inc P-1 antibiotic-resistance plasmid (5), in an effort to determine the molecular basis of its broad host range. RK2 [56.4 kilobases (kb)] is self-transmissible and codes for resistance to kanamycin (Km), ampicillin (Ap), and tetracycline (Tc) (5, 6). In Escherichia coli the molecule replicates unidirectionally from a fixed origin located approximately 12 kb from the single EcoRI cleavage site (7). From studies of RK2 molecules manipulated in tntro with restriction endonucleases, it appears that the genes essential for replication and maintenance in E. coli are distributed over a 24-kb portion of the plasmid that includes the origin of replication (ref. 8; unpublished data). This finding is in contrast to the considerably smaller replication regions of plasmids of other incompatibility groups that appear to have a more limited host range than does RK2.To locate and characterize individual genes required for replication, the RK2 essential region has been further subdivided by the construction of hybrid plasmids carrying RK2 genes on unrelated replicons. In this paper, we report that at least one RK2 function can act in trans to permit the replication and maintenance of a recircularized fragment of RK2 that contains the RK2 origin of replication. This origin-containing region is a defective replicon but can be maintained in the cell as a separate covalently closed circular (CCC) molecule in the presence of the cloned gene(s...

A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2 RK2 is a bacterial plasmid of incompatibility group P-1 that is very similar, if not identical, to plasmids having the designation RP1, RP4, and R68 (1). It confers resistance to the antibiotics ampicillin, tetracycline, and kanamycin and exists at approximately five to eight copies per chromosomal equivalent in Escherichia coli (2). A general feature of P-I plasmids is their extensive host range. Such plasmids are capable of conjugal self-transfer to a wide variety of Gram-negative bacteria (3,4). This unique property has been used as the basis for development of a plasmid cloning system in E. coli with widespread applicability. Although native RK2 DNA can be used directly as a recombinant DNA cloning vector, its large size [56 kilobase pairs (kb)] is a serious drawback to routine use. In order to reduce the size and still retain overall broad host range transfer capability, a cloning system has been devised that separates RK2 transfer and replication functions onto separate plasmids. The tetracycline-resistant plasmid component of this system, pRK290, contains a functional RK2 replicon and can be mobilized at high frequency by using a helper plasmid, but is non-self-transmissible. pRK290 contains single EcoRI and Bgl II sites where DNA can be inserted without loss of essential functions. The kanamycin-resistant helper plasmid, pRK2013, consists of the RK2 transfer genes cloned onto a ColEl replicon (5). Its sole function in this system is to trans-complement the vector for mobilization. This paper describes the construction of pRK290, its properties as a cloning vector, and its use in constructing a gene bank of the agriculturally important bacterium Rhizobium meliloti. As an initial test of the gene bank, DNA containing the nitrogenase structural gene region of Klebsiella pneumoniae (6, 7) was used as a hybridization probe to identify clones carrying the nitrogenase region of R. meliloti. One of the members of the bank was found to contain a 26-kb insert with homology to this K. pneumoniae probe. Enzymes. Restriction endonuclease EcoRI was purified in our laboratory; Bgl II was a gift from C. Yanofsky; all other restriction enzymes were obtained from New England BioLabs. T4 DNA ligase was obtained from Bethesda Research Laboratories (Rockville, MD), and was used at a concentration of 1 unit/ml for ligations. Bacterial alkaline phosphatase was obtained from Miles and was dialyzed into 10 mM glycine, pH 9.5/0.1 mM ZnCl2 for storage. DNA was treated with this enzyme at 65°C for 90 min in 10 mM Tris-HCl (pH 9.5). The reaction was terminated by phenol extraction.Bacterial Matings. Matings were performed by mixing 109 cells each of the donor and recipient and filtering the suspension onto 0.45-,um Millipore filters. The filters were incubated at 30°C on nonselective agar plates for 3-6 hr before the cells were resuspended a...

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