Aug 29, 1989 - chose bacterial rather than firefly luciferase to report tRNA- mediated nonsense suppression. The active enzyme is an AB heterodimer, and we ...
BACTERIOLOGY, Feb. 1990,
Vol. 172, No. 2
0021-9193/90/020595-08$02.00/0 Copyright C 1990, American Society for Microbiology
A Simple and Sensitive In Vivo Luciferase Assay for tRNAMediated Nonsense Suppression DENNIS W. SCHULTZ AND MICHAEL YARUS* Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 Received 29 August 1989/Accepted 6 November 1989
We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing ,-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of .40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous I8-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The 1-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.
sense suppression has also been used to investigate the effect of ribosomal mutations such as rpsL on the accuracy of translation (28) as well as the effects of context or polarity (7a, 18). In short, virtually any process that modifies the translational activity of a tRNA can be examined by measuring nonsense suppression. The in vivo activity of many naturally occurring tRNAs can be quantified by appropriately altering their anticodons to nonsense complements (20). Thus nonsense suppression is applicable not only to numerous processes but also to many tRNA sequences. Nonsense suppression by cognate tRNAs, by its very nature, lends itself to in vivo measurements in cells undergoing steady-state growth. Therefore, one advantage of suppression assays is that they can reflect conditions within cells. The nondisruptive, rapid measurement of light emission from intact cells complements and extends this quality of suppression experiments. A translational reporter gene should also have other qualities. The gene's product should normally be absent in cells to favor low backgrounds and the highest sensitivity. Furthermore, the activity of the reporter should be relatively insensitive to the chemical character of the amino acid inserted at the site of the nonsense codon. Ideally, the reporter gene should be insensitive to ancillary phenomena, such as polarity, that may complicate interpretation. Although an extremely polar nonsense mutation within the reporter gene may not affect the discrimination among strong suppressors, it will exaggerate the discrimination among weak suppressors and depress the measurements of the weakest suppressors into the background. In addition, it would be convenient if the cellular concentration of the reporter (and any endogenous substrate; in this case, FMNH2) were insensitive to growth phase and if the product of their reaction were easily, rapidly, and accurately assayed. Remarkably, the luciferase assay for tRNA suppressor action meets all of these criteria.
As a result of the simplicity, rapidity, and sensitivity of the measurement of light, luciferase seems likely to become the reporter gene of choice for the assay of both procaryotic and
eucaryotic gene activity. Bacterial luciferase has been used analogously to P-galactosidase to report gene expression in procaryotes (2, 7). Bacterial luciferase is also the mutated gene in a mutagenicity assay that is 100 times more sensitive than the Ames test (30). In mammalian cells, detection of firefly luciferase activity is 1,000 times more sensitive than the detection of chloramphenicol acetyltransferase activity (6). In this paper, we describe a new assay for suppressor tRNA action that exploits the advantages of photon counting.
Firefly luciferase requires, and can be used to assay, ATP. The firefly enzyme catalyzes the ATP-dependent oxidation of luciferin, a high-molecular-weight substrate that crosses cell membranes slowly (19). Bacterial luciferase requires, and can be used to assay, NAD(P)H or FMNH2 (19). Bacterial luciferase catalyzes the FMNH2-dependent oxidation of n-decyl aldehyde, a small lipophilic substrate that crosses cell membranes rapidly. Because of this rapid uptake, it seemed easier to supply an excess of the substrate n-decyl aldehyde to the intracellular enzyme. Therefore, we chose bacterial rather than firefly luciferase to report tRNAmediated nonsense suppression. The active enzyme is an AB heterodimer, and we have utilized nonsense mutations in its essential B subunit. Suppressor efficiency is taken to be the fraction of the light from cells containing a suppressed nonsense mutant luxB to that from a wild-type lux strain. Nonsense suppression by tRNAs has found frequent application in the study of translation, tRNA biosynthesis, and tRNA function. Measurement of suppression reproducibly quantifies tRNA synthesis and stability (26), processing (13), modification (21), aminoacylation (11, 15), tRNA efficiency (5, 32), and tRNA interactions on the ribosome (25). Non*
Corresponding author. 595
SCHULTZ AND YARUS
B pLUX I
TTC TTC CTC
AAT TTT ATG AAT TCA AAG CGT TCT.
FIG. 1. (A) Principal components of pLUX1 and their sources. The H3 (HindlIl) fragment containing luxAB was derived from the HindIII-PvuII fragment of pLAV1 by adding a HindIlI linker (triple arc). The EcoRI-B* fragment, containing the HindlIl site into which luxAB was inserted (double arc), was obtained from pSWC101. The remainder of the plasmid (quadruple arc) was derived from pJC27. B* is an inactivated BamHI site at the junction of the pJC27 and pSWC101 fragments used to construct pCAM203. (B) DNA sequence corresponding to the first 16 codons of the luxB gene (12) and the site-directed changes in plasmids pLUX2, pLUX3, and pLUX4.
MATERIALS AND METHODS Bacterial strains. All strains are derivatives of Escherichia coli K-12. MY347, which is S90C [ara thi A(lac-pro) rpsL(F' laclq Alac-14 proB+)], and MY349, which is S90CA24 [ara thi l(lac-pro) rpsLIF' lacJPA24(Am) A(lac-14) proB+], from J. Miller (18) were used for ,-galactosidase and Lux assay comparisons, and MY349 was used to test Lux response to cell density. A(lac-14) fuses lacI to lacZ. XAC-1 [ara A(lacproB)XIII nalA rif argE(Am)/F' lacI373 lacZU118(Am) proB+] (20), in combination with various suppressor tRNAs, was used for testing the response of luciferase to amino acid substitution. MY649 [A(lac-pro) ara thi recA56 F-] (5) was used for all other luciferase experiments. BW313 (ung dut spoT thi-J relAIF' lysA) (14) was the host for the preparation of single-stranded template for site-directed mutagenesis. Plasmids. Synthetic amber-suppressing tRNAs carried on pGFIB were obtained from J. H. Miller (20). These insert
alanine, cysteine, glutamic acid, glycine, histidine, phenylalanine, and proline at UAG. pLAV1, which was constructed from pUC9 (16) and pTB7 (1) by Lawrence Chlumsky, was a gift from T. 0. Baldwin. pLUX1 incorporates the HindIII-PvuII fragment of pLAV1, which contains the luxA and luxB genes from Vibrio harveyi. This fragment, after the addition of a HindlIl linker to its PvuII end, was cloned into the HindIl site of pCAM203 to form pLUX1 (Fig. 1A). This HindlIl cloning site in pCAM203 lies within the normal promoter P2 (29) for the gene for tetracycline resistance (tet). Insertion of this fragment at this HindIll site disrupts P2. However, the Lux fragment can be transcribed in a direction opposite to tet transcription from promoter P1 (29),
located in the intact 5' end of the tet gene. The EcoRIBamHI fragment of pLUX1, containing the 5' end of the tet gene with the HindIII cloning site and the P1 promoter, was derived from pSWC101 (22). The remainder of pLUX1 was derived from pJC27 (5), which supplied the gene for chloramphenicol acetyltransferase, the P15A double-stranded replication origin, and the fl origin. The combination of these latter two fragments has been used in this laboratory as pCAM203 (Fig. 1A), constructed by James F. Curran. All tRNAs, which are derivatives of Su+7 (3), are contained in pOP203.3 (8). Reagents. N-Decyl aldehyde and o-nitrophenyl-P3-D-galactopyranoside were purchased from Sigma Chemical Co. Site-directed mutagenesis. Nonsense mutations at amino acid position 13 in luxB were created by site-directed mutagenesis as previously described (5). Single-stranded template was isolated from BW313 transformed with pLUX1. In a darkroom, colonies containing pLUXi with prospective luxB nonsense mutations [designated luxB13(Am), luxB13(0c), and luxB13(Op), residing on plasmids pLUX2, pLUX3, and pLUX4, respectively] were identified by their failure to luminesce on closed agar plates under air saturated with n-decyl aldehyde (the aldehyde was spread on the inside of the top). The presence of the site-directed nonsense mutations was confirmed by DNA sequencing by the method of Sanger et al. (24). When amber (UAG), ochre (UAA), and opal (UGA) tRNA suppressors were introduced into the above strains, each gave its characteristic pattern of suppression. Strains and media for Lux assays. Strains for luciferase assays were constructed by transforming MY649, MY347, and MY349 with pLUX1 or pLUX2 and, in most cases, with pOP203.3 containing suppressor tRNAs derived from Su+7. Transformants were purified twice on selective plates before use. Plasmids encoding Lux were selected and maintained in media containing 31 ptg of chloramphenicol per ml, whereas suppressor tRNA plasmids were selected and maintained in media containing 15 ,ug of tetracycline per ml. Overnight cultures were grown in the minimal E medium of Vogel and Bonner (31) supplemented with 0.4% glucose, 10 ,ug of thiamine per ml, and the appropriate antibiotics. MY649 derivatives were also supplemented with 30 ,ug of proline per ml and 0.1% Casamino Acids. Lux assays. Saturated overnight cultures were diluted 1:100 into the same medium and grown at 37°C to an A6. between 0.2 and 0.7. Cultures of cells with either the luxB wild type or luxB13(Am) with strong tRNA suppressors were diluted 10- or 100-fold into their growth media for assay, whereas cultures containing cells with luxB13(Am) and weak or no suppressors were used directly. N-Decyl aldehyde was diluted 1:1,000 in water just before each experiment and then sonicated to disperse the organic phase. To assay, 20 ,ul of the n-decyl aldehyde in water was added to 200 ,ul of medium in a small glass tube (Kimble; 6by 50-mm disposable borosilicate), and the mixture was quickly vortexed. Light emission at room temperature was immediately determined in a Turner Designs TD20e luminometer (Turner Designs, Mountain View, Calif.), integrating for 20 s. These operations could normally be carried out under conventional room illumination. The optical density of cultures was determined immediately after initiation of light measurements. The entire analysis was complete in less than a minute. Cells incubated in growth media with or without 0.01% decanal for 60 min yielded indistinguishable colonyforming titers. This simple protocol was also the most reproducible of the
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VOL. 172, 1990
assay methods we tried. Cells subjected to any changes in the suspending medium during dilution, or cells kept on ice, yielded fluctuations in light production that decreased reproducibility. The initial rate of light production by a freshly sampled culture was convenient to measure and faithfully reflected the cellular content of luciferase, whereas longer measurements did not. P-Galactosidase assays. For comparison of Lux and 13galactosidase assays, cultures of MY347 and MY349 containing both Lux and tRNA plasmids were grown as described above. The same cultures used for the Lux assay were chilled for 10 to 20 min and used for 1-galactosidase assays, which were performed as described previously (22). j3-Galactosidase activity was linear for the entirety of 24-h incubations. Nevertheless, to match assay times, wild-type lacIZ strains containing weak suppressors were diluted 100-fold for ,B-galactosidase assays so that their incubation times were roughly equivalent to that of their sister lacIZ (Am) strains containing the same weak suppressor. Suppressor efficiency. Nonsense suppressor efficiency is a percentage or fraction, approximately equal to the fraction of ribosomal transits in which the suppressor tRNA translates its codon, excluding the relevant peptide release factor. Experimentally, it is the ratio of activity from two strains, one containing a reporter gene with the nonsense mutation and a suppressor tRNA and the second containing the wild-type reporter gene and the same source of suppressor tRNA. Electroporation and rapid Lux assay. MY649(pLUX2) was grown in superbroth (32 g of tryptone, 20 g of yeast extract, 5 g of NaCl, and 5 ml of 1 N NaOH per liter) to an A600 of 1.0 and chilled for 15 to 30 min. Cells were centrifuged and suspended sequentially in 1.0 volume of 1 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.0), 0.5 volume of 1 mM HEPES, 0.02 volume of 10% glycerol, and finally 0.002 volume of 10% glycerol. DNA was added to 40 ,ul of concentrated cells and electroporated in a Bio-Rad Gene Pulser set at 25 ,uF, 1.8 KV, and 400 Ql. Time constants were 85 to 90% of theoretical maxima. Cells were immediately diluted into 10 ml of superbroth and shaken at 37°C. Lux assays and spectrophotometer readings were performed as described above on 1:3 dilutions of this expression culture. Stable transformants, measured by plating at 60 min after electroporation, were 2 x 108 to 6 x 108 per ,ug of
plasmid. Curve fitting. Straight lines were fitted to the data in Fig. 2 through 4 by using the Marquardt algorithm. The curves shown are best fits, equivalent to least-squares lines. RESULTS Response of Lux assay to cell number. To show that light production varies as expected with the mass of Lux protein in the sample, the response of light emission to dilution was determined. Strain MY649 transformed with pLUX1 was grown to the midlog phase (A650, 0.36). Serial dilutions were made, and samples were assayed for light emission in a Beckman LS-8000 liquid scintillation counter with coincidence correction turned off, permitting photon counting (Fig. 2A). Samples from the same dilutions were assayed pari passu for light emission in the luminometer (Fig. 2B). Both plots showed a linear relationship between cell number and light emission over the entire range of each instrument, and both instruments exhibited roughly the same sensitivity of detection. However, the greater maximum, and thus greater range of light measurement, in the luminometer
A 6 (2
B 3 2 F-
0 -J -
LOG DILUTION FIG. 2. Response of Lux assay to cell number and amount of lux gene product in the assay volume. (A) Log1o (light intensity, as scintillations per minute in a counter) versus log1o (cell number, expressed as a fraction of 1 ml of culture). (B) Log1o (light intensity in luminometer units) versus log1o (cell number, expressed as a fraction of 1 ml of culture).
makes assays more convenient than in the liquid scintillation counter. Light detected by the luminometer accurately followed the amount of bacterial luciferase in the sample over a 40,000-fold range (Fig. 2B). This result also suggests that cell concentration and the operations of dilution have no detectable effect on light emission. Response of Lux assay to growth phase. A convenient assay requires that specific light output be constant throughout a reasonable period of culture. To test the effect of cell growth, an overnight culture of MY349(pLUX1) was diluted 100-fold and shaken at 37°C. Light emission from 0.2-ml samples was measured periodically between A550s of 0.012 and 0.746. Light from MY349(pLUX1) accurately followed the growth of the cells over nearly this entire range (Fig. 3). Luminometer readings may become erratic above an A550 of 0.6 or 0.7 (Fig. 3 and data not shown). Similar results were obtained for MY349(pLUX2) with no suppressor (data not
SCHULTZ AND YARUS 0. 0-
-3. 0 -4. 5
EFF- f LUX) FIG. 4. Relation between the log1o efficiencies of amber suppres-
FIG. 3. Light intensity and cell mass during culture growth. Strain MY349(pLUX1) was diluted 1:100 into minimal medium and grown for at least 2 h before readings were taken. Light intensity (by luminometer) is plotted versus turbidity (A5s0)-
sion as determined in concurrent Lux and 3-galactosidase assays. Samples for Lux assays were taken directly from growing cultures or from 10' or 10 2 dilutions. Turbidities were measured simultaneously with Lux assays. ,B-Galactosidase assays were initiated on the same cultures after 10 to 20 min on ice.
shown). Therefore the amount of bacterial luciferase per cell and the cellular supply of FMNH2 appear to be effectively constant throughout the log phase. Cultures need not be assayed at the same turbidity as long as the assays are performed during logarithmic growth. Comparison of tRNA suppression efficiencies from Lux and ,B-galactosidase assays. The efficiencies of tRNA suppression derived from the Lux assay were compared with those derived from a concurrent P-galactosidase assay on the same cells. A set of Su+7 tRNA derivatives with known efficiencies of suppression (32) was used for this comparison. Two sets of strains were constructed. Both carried a plasmid expressing the Su+7 tRNA derivative. One also carried pLUX1, containing wild-type luxAB, as well as an F' that contained a wild-type lacIZ fusion gene. The other also carried pLUX2, containing luxBJ3(Am), as well as an F' that contained lacIZ with amber A24. The efficiency of suppression of each Su+7 derivative is simply the ratio of the Lux or the ratio of ,3-galactosidase activity in the second strain to that in the first. Each determination of suppressor activity (Fig. 4) represents the mean of six to eight determinations from independent transformants of each strain (see below). Because both the Lux and ,-galactosidase measurements refer to the same cells, all physiological parameters that influence tRNA suppression are intrinsically controlled. However, in comparisons of ,B-galactosidase and Lux assays, different efficiencies of suppression in the two assays are expected because of differences between the two messages and episomes. The net influence of all these unshared factors must be relatively small, because there was an excellent correlation between the Lux and ,3-galactosidase assays for tRNAs with either high or intermediate levels of suppression (Fig. 4). The slope of this line is not exactly 1, however, and suppressor efficiencies derived from the Lux assay were consistently slightly higher than those from the ,-galactosidase assay. This may indicate a slightly better context or less polarity in the Lux message or reflect some other difference between the two episomes. Nevertheless, both genes responded in a very similar way to increasing tRNA activity (Fig. 4). Therefore, measurement of luciferase produces an index of
tRNA activity that is commensurate with that of the widely used 3-galactosidase assay. Sensitivity of Lux assay compared with that of the Bgalactosidase assay. Figure 4 indicates an increasing discrepancy between the values of efficiency of suppression derived from the two assays for very weak tRNA suppressors. This discrepancy results because the ,B-galactosidase assay cannot distinguish weak tRNA suppressors. In the strains constructed for this comparison, the level of suppression was reduced from published values (32) because of hyperaccurate (rpsL) ribosomes that constrain suppressor efficiency (28). However, the activity of all but the very weakest of tRNA suppressors could be distinguished from background with the Lux assay. In both the Lux and 1-galactosidase assays, the background was determined by the level of readthrough of the nonsense mutation in their respective messages. Therefore, the 80-fold greater sensitivity of the Lux assay was, in part, a result of the lower background readthrough of the Lux message (see Discussion). Reproducibility of Lux and 0-galactosidase assays. To test the reproducibility of these assays, six to eight independent transformants of MY347 and MY349 containing the Lux and tRNA plasmids were purified twice by restreaking and assayed for Lux and 3-galactosidase activity. The suppression efficiencies plotted in Fig. 4, derived from either assay, were calculated from the means of those assays. The standard error of the mean for sets of independent transformants was ca. 7% for the 3-galactosidase assay and 10% for the Lux assay. These standard errors are not significantly different. Therefore, we conclude that the assays are similarly reproducible. Response to amino acid substitution at the amber codon in LuxB. A generally applicable assay for tRNA suppression should be relatively insensitive to the side chain of the amino acid at the nonsense site in the protein. The amino acid at position 13 in LuxB is not phylogenetically conserved (T. 0. Baldwin, personal communication). Therefore, it seemed likely that a variety of amino acids would support Lux activity. To determine whether the Lux assay can utilize a variety
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of amber suppressing tRNAs, seven strains containing suppressor tRNAs that insert seven different amino acids (alanine, cysteine, glutamine, glycine, histidine, phenylalanine, and proline) were transformed with pLUX1 and pLUX2. When patched on selective media, all seven suppressor strains containing pLUX2 emitted light (Fig. 5A), demonstrating that all seven can suppress luxB13(Am). Figure 5A also shows that the light emitted from the luxBJ3(Am) strains is due to suppression by these tRNAs and not to readthrough of this allele, since a strain with pLUX2 and no tRNA suppressor was dark. Because the plasmids containing the suppressor tRNAs affected growth of this strain, determination of suppression required comparison to an isogenic suppressor strain carrying wild-type lux (Fig. 5A). The levels of suppression discernible in Fig. 5A correlate well with those determined quantitatively (data not shown). Because both large and small side chains (phenylalanine and glycine) as well as acidic (glutamic acid), basic (histidine), and hydrophobic (alanine, proline, and phenylalanine) amino acids were included in this set, we conclude that a variety of amino acids substituted at the 13th codon of luxB will yield a functional luciferase. Therefore, the Lux assay should generalize to most, if not all, tRNA amber suppressors. However, these data do not establish that all amino acids are quantitatively identical in function at position 13 of luxB. Homotopic set of nonsense codons. To extend the utility of the Lux assay, a homotopic set of nonsense codons at amino acid position 13 in luxB was constructed. Therefore it should be possible to use the Lux assay in conjunction with any tRNA suppressor for amber (UAG, pLUX2), ochre (UAA, pLUX3), or opal (UGA, pLUX4) mutations (Fig. 1B). Rapid assay for tRNA suppression. We wished to combine the rapid Lux assay with electroporation, which allows the reproducible introduction of plasmid-borne tRNA genes into bacterial cells. Measuring light produced shortly after electroporation uncouples the determination of translational activity from the formation of stable transformants, if desired. This could be useful in screening constructions that are difficult to express stably (23), or it could provide quick assays to test the progress of a plasmid construction. Such measurements can be performed with little additional effort after any transformation. Figure 6 shows the kinetics of light production after cells containing pLUX2 were electroporated in the presence of 10 ng of tRNA expression plasmids carrying an efficient (Su+7; efficiency, 0.78 ) amber suppressor, an inefficient (Su+7 A32; efficiency, 0.09 ) amber suppressor, and a control parental tRNATIp gene. The light produced did reflect the relative efficiencies of these suppressors as measured independently in stable transformants. Such relative values may be measured quickly, starting at 90 min after electroporation, depending on the sensitivity required (Fig. 6). However, light production from suppression was much closer to the control light level (due to readthrough of the pLUX2 amber; Fig. 6) than with stable transformants. This occurred because light from readthrough comes from all cells, whereas suppression occurs in only the minority of newly transformed cells. Therefore the useful range of this electroporation assay is reduced compared with those of assays on purified transformants. In this experiment ca. 10-3 of cells were transformed; thus the background was elevated by orders of magnitude. However, the useful range may be increased, and the time for detection of incremental light production may be decreased, by increasing the plasmid
Lux ASSAY FOR tRNA SUPPRESSION
DNA concentration or by any other measure that increases the fraction of cells transformed. DISCUSSION We have devised a new assay to measure tRNA suppression at nonsense codons that is (i) rapid, because the entire Lux assay can be performed in less than a minute, compared with hours required for the P-galactosidase assay; (ii) easy to perform, because it involves only one addition to an otherwise unperturbed culture; and (iii) sensitive, because the background is 80-fold less than in a simultaneous P-galactosidase assay. The Lux assay is therefore especially apt for weak tRNA suppressors. The assay is linear with cell number and luciferase content in both the liquid scintillation counter and the luminometer. Light emission per cell is also constant throughout logarithmic growth, so that numerous unsynchronized cultures can easily be assayed and subsequently accurately normalized to turbidometric measurements of culture density. The Lux assay also responds as expected to the translational activities of tRNAs and yields suppressor efficiencies that vary similarly to those from simultaneous determinations using P-galactosidase activity. The homotopic set of nonsense codons at amino acid position 13 in luxB and the relative insensitivity of the protein to amino acid replacement there make the Lux assay a general one, likely to be usable for most tRNA nonsense suppressors. The nucleotide context of these codons is not extreme (7a, 18) but should give moderately efficient suppression. A codon near the N terminus was chosen to facilitate amino acid sequencing when required. With our current instrumentation, we reliably detect light from approximately 102 cells. With a few times this many cells, reliable measurements of suppressor activity may be made. Therefore, reasonably accurate quantitative screening of individual products of a recombinant DNA construction is possible by suspending a small cell sample from individual transformant colonies. In fact, the dark-adapted human eye often supplies useful screening information on individual colonies in situ (see Materials and Methods and pLUX1 and pLUX2 in Fig. 5). Save for better quantitation, the Lux method is comparable to color production by P-galactosidase in this respect. However, with more sensitive instrumentation, suppressor assays (or other expression assays) appear possible on single E. coli cells, after which a viable cell could be recovered. P-Galactosidase has been the most widely used reporter in procaryotes. The lacZ operon is particularly well understood, which is helpful in designing specialized reporters. Also, lacIZ fusions allow promiscuous replacement of amino acids in the Lacd portion of the protein without changes in specific activity (18). However, the large size of the ,galactosidase gene results in polarity for promoter-proximal nonsense mutations (27, 33) and poor codon contexts (7a). Although P-galactosidase assays for nonsense suppression work well with strong tRNA suppressors, they can be tedious and inaccurate for weak tRNA suppressors. For accurate colorimetry, one must often wait overnight for sufficient reaction. Protracted reactions may require adjustments for enzyme half-life (particularly for fusions) and may introduce relatively large backgrounds due to the spontaneous breakdown of o-nitrophenyl-13-D-galactopyranoside (or breakdown due to other enzymes in the cell). Such backgrounds hinder the accurate measurement of weak activities. The Lux assay is superior in these respects. The measurement of light from cells with luxB13(Am) and weak suppres-
-The I~ rore
FIG. 5. Response of Lux to amino acid substitution. (A) Photograph of light emitted from Lux-containing bacteria. The leftmost column of patches contains XAC-1 harboring various suppressor tRNA plasmids and pLUX1 (luxB wild type). The next column of patches is XAC-1 harboring suppressor tRNA plasmids and pLUX2 [luxBl3(Am)]. These amber suppressors insert (from top) alanine, cysteine, glutamic acid, glycine, histidine, phenylalanine, and proline, as indicated at the left. The rightmost luminescent patch is XAC-1(pLUX1). Invisible and to its right is XAC-1(pLUX2). Exposure was for 15 s at f5.6 with ASA 3200 film. (B) The same plates, photographed with incident light. 600
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Lux ASSAY FOR tRNA SUPPRESSION
3. 5 -
-J1.5 C)1. 5
90 1 20 150 1 80 2 10
TIME FIG. 6. Kinetics of light production per cell mass (log1o [light after introduction of plasmid-borne suppressor tRNA genes by electroporation. MY649(pLUX2) was subjected to electroporation in the presence of 10 ng of pMY228 Su+7, 10 ng of pSWC232 Su+7 A32, or 10 ng of pMY231 Su-7. These are homologous plasmids containing an efficient amber suppressor, an inefficient suppressor, and the wild-type tRNATrP gene, respectively. At intervals after the addition of broth, samples were taken for measurement of turbidity and light production. Symbols: A, Su+7; 0, Su+7 A32; 0, Su-7.
sors can be performed in the same short time as for strong suppressors (20 s). In fact, the overall assay time for weak suppressors is shorter because dilution is not required. Such short assay intervals are also advantageous in principle because they have a clear meaning; they determine the instantaneous steady state in the cell rather than an average state integrated over periods of growth or other change. ,B-Galactosidase constitutes about 5% of the total protein in induced cells (4), corresponding to -1,000 Miller units (17). If 0.1 Miller unit is taken as the absolute minimum required for the P-galactosidase assay, then the activity of 109 molecules of ,-galactosidase is required. In contrast, it is possible to measure as little as 0.01 light units on the luminometer or about 3,000 cpm on the liquid scintillation counter (counter background is -3,000 cpm with no coincidence correction). From the measurements of Hastings and Weber (10), this corresponds to 3 x 104 quanta per s or about i05 molecules of luciferase (9). Therefore the Lux assay is potentially 104 times more sensitive than the 3-galactosidase assay. In practice, our Lux assay was 80-fold more sensitive than a simultaneous 3-galactosidase assay. Therefore the full difference in sensitivity was not realized. Although there are other differences between the Lux and 3-galactosidase measurements, this is primarily because the background in both assays was determined by the readthrough of the amber mutations in their respective mRNAs and not by the physical limits of detection of the product of the enzymes. The readthrough of nonsense mutations is strongly affected by the genetic constitution of the translational apparatus. The rpsL allele present in the strains used to compare the ,-galactosidase and Lux assays was responsible for a 30-fold decrease in the readthrough of the Lux message (data not shown). In view of this, it is not the absolute sensitivity of the Lux assay that is likely to be indispensible, but the availability of extreme sensitivity from a rapid measurement within other experiments. Nevertheless, there are other
biological systems (e.g., an assay for mRNA splicing) in which the background would presumably be much lower, so that the full and unique range of the Lux assay would become accessible. The availability of short assay times in minimally perturbed cells suggests the use of Lux to measure the kinetics of any slow process that determines the translational activity of an aminoacylated tRNA. This is illustrated in Fig. 6, where the light output is monitored during the establishment of transformants after electroporation. The light/A6. ratio increased continuously through the period shown. For up to 60 min, these kinetics reflected the recovery of the cells from electroporation. This is consistent with the fact that, at 60 min (Fig. 6), the total light output was about 1 order of magnitude below that expected for the number of stable transformants measured at the same time. Even more revealing, at 60 min the light output of the control with no suppressor was similar to those of the suppressed strains. Finally, shortly after 60 min, A600 began to show slow net cell multiplication (data not shown). From 90 min on, the expression and maturation of the suppressor tRNA became evident in the greater specific light output from suppressor strains. In other experiments, the light output varied with the efficiency of the transformation. Therefore one simple application of this technique might be to rapidly determine the success of critical transformations, electroporations, or ligations. ACKNOWLEDGMENTS We thank T. 0. Baldwin for pLAV1 and discussion of unpublished sequences for several luxB genes. We are grateful to Ann Olin for the skillful execution of the experiment shown in Fig. 6. This work was supported by Public Health Service grant GM30881 to M.Y. from the National Institutes of Health. LITERATURE CITED 1. Baldwin, T. O., T. Berends, T. A. Bunch, T. F. Holzman, S. K. Rausch, L. Shamansky, M. L. Treat, and M. M. Ziegler. 1984. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry 23:3663-3667. 2. Carmi, 0. A., G. S. A. B. Stewart, S. Ulitzur, and J. Kuhn. 1987. Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp. J. Bacteriol. 169:2163-2170. 3. Cline, S. W., M. Yarus, and P. Wier. 1986. Construction of a systematic set of tRNA mutants by ligation of synthetic oligonucleotides into defined single-stranded gaps. DNA 5:37-51. 4. Cohn, M. 1957. Contributions of studies on the beta-galactosidase of Escherichia coli to our understanding of enzyme synthesis. Bacteriol. Rev. 21:140-168. 5. Curran, J. F., and M. Yarus. 1986. Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance. Proc. Natl. Acad. Sci. USA 83:6538-6542. 6. DeWet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725-735. 7. Engebrecht, J., M. Simon, and M. Silverman. 1985. Measuring gene expression with light. Science 227:1345-1347. 7a.Folley, L. S., and M. Yarus. 1989. Codon contexts observed in weakly expressed genes reduce gene output in vivo. J. Mol. Biol. 209:359-378. 8. Fuller, F. 1982. A family of cloning vectors containing the lacUV5 promoter. Gene 19:43-54. 9. Hastings, J. W., W. H. Riley, and J. Massa. 1965. The purification, properties, and chemiluminescent quantum yield of bacterial luciferase. J. Biol. Chem. 240:1473-1481. 10. Hastings, J. W., and G. Weber. 1963. Total quantum flux of isotropic sources. J. Opt. Soc. Am. 53:1410-1415.
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