TETRACYCLINE ASSAY METHOD

Abstract: The invention relates to a method for the determination of a tetracycline in a sample. The method is characterized in that the sample is brought into contact with prokaryotic cells encompassing a DNA vector including a nucleotide sequence encoding a light producing enzyme under transcriptional control of a tetracycline repressor and a tetracycline promoter, detecting the luminescence emitted from the cells, and comparing the emitted luminescence to the luminescence emitted from cells in a control containing no tetracycline. The invention also concerns recombinant prokaryotic cells capable of emitting light in response to the existence of a tetracycline in a sample. Furthermore, the invention relates to novel DNA vectors useful for the construction of said prokaryotic cells.

Description: Tetracycline assay method. HELD OF THE INVENTION This invention relates to a method for the determination of a tetracycline in a sample. The invention also concerns recombinant prokaryotic cells capable of emitting light in response to the existence of a tetracycline in a sample. Furthermore, the invention relates to novel DNA vectors useful for the construction of said prokaryotic cells. BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference. Whole cells can be used in methods based on the use of living cells or organisms as sensor tools of detection. Many of these methods utilize bacterial or yeast cells. Prokaryotic organisms and especially Escherichia coli bacterium are very well characterized and maps of genes and their sequences at nucleotide level are known. Therefore the behavior of the whole cell sensor can be better understood. Because of this fact it is also possible to develop analyte or group specific sensors utilizing different regulatory regions of genomes and also various microbial strains. Whole cells can be utilized in biosensors which are devices consisting of 1) a sensor, 2) a recording unit and 3) a possible connector such as fiber optic guide between 1 and 2. The recording unit has several choices of what is the physical background of the measurement. It can be change in heat, conductance, color reaction, changes in fluorescent properties, emission of endogenous light from the sensor cells etc. Antibiotics used as medicines against microbial invasion are detected from body fluids in order to study the dosage and penetration of the medicine. Often the effective therapeutic range of the antibiotic is rather narrow and the risks of overdosage might be too big. It is also important to measure the presence or concentration of antibiotics from meat and milk due to syndrome of allergic people. In the course of cheese production milk used as starting material should not contain antibiotics due to the fact that cheesemaking bacteria are not able to work on contaminated milk. Conventional tests for the measurement of toxic substances such as antimicrobial agents (antibiotics) are based on the inhibition of growth. Growth inhibition can be followed by monitoring the zone where the growth of microbes is inhibited on a nutrient agar plate around a disk onto which an antibiotic dilution was pipetted. Typical examples of agar diffusion tests are cylindrical, hole or disk methods. The difference in these tests is only restricted in the way the sample is applied on the agar and also the way the bacteria in the test is used. Another means is to follow the metabolism of the test organisms by estimating the intensity of a color reaction which is affected by the inhibitory antibiotic present and comparing it to the uninhibited control (e.g. the commercial products: Delvo Test™, Brilliant black- reduction test, Charm Farm Test, Charm AIM-96 and Valio TlOl-test). Since microbiological methods utilize bacteria or their spores it is the sensitivity of the test bacteria which is of utmost importance. Thus far one had to make compromises in the choice of a suitable test strain since great sensitivity against antimicrobial agents and other characteristics needed for the test strain have not been common features for the same strain of bacteria. A major drawback when using microbes in antibiotic residue tests is slow and unsensitive performance. Since in these methods one always controls in a way or other the growth of the tester strain one cannot imagine the test to be performed in an hour. This is due to the fact that the growth of the microbe is a slow phenomenon even at its fastest mode. Also in many cases microbes are in spores or freeze-dried, the regeneration of which makes the tests even more slow to perform. OBJECT AND SUMMARY OF THE INVENTION The object of the invention is to provide a novel method of determining a tertracycline in a sample where said method is rapid and selective for tetracyclines, i.e. the method is able to distinguish tetracyclines from other antimicrobial agents. According to one aspect of the invention a method for the determination of a tetracycline in a sample is provided, wherein the method is characterized in that the sample is brought into contact with prokaryotic cells encompassing a DNA vector including a nucleotide sequence encoding a light producing enzyme under transcriptional control of a tetracycline repressor and a tetracycline promoter, detecting the luminescense emitted from the cells, and comparing the emitted luminescence to the luminescence emitted from cells in a control containing no tetracycline wherein a detectable luminescence higher than a luminescence of the control indicates the presence of tetracycline in the sample. According to another aspect, the invention concerns a recombinant prokaryotic cell which encompasses a DNA vector including a nucleotide sequence encoding a light producing enzyme, tetracycline repressor and tetracycline promoter. According to yet another aspect, the invention concerns a plasmid which comprises either - the luxCDABE genes (SEQ ID NO: 3), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from T lO, or the insect lucif erase gene (SEQ ID NO: 1), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from ThlO. BRIEF DESCRIPTION OF THE DRAWINGS Figure la shows schematically the method according to this invention, where cells cloned with the plasmid pTetLuxl (SEQ ID NO: 3) are used. Figure lb shows schematically the method according to this invention, where cells cloned with the plasmid pTetLucl (SEQ ID NO: 1) are used. Figure lc shows schematically the production of the lucif erase enzyme, Figure 2 shows the plasmid pTetLuxl (SEQ ID NO: 3). Figure 3 shows the plasmid pTetLucl (SEQ ID NO: 1). Figure 4a shows the production of light (induction factor) versus concentration of tetracycline in samples for three different tetracyclines, Figure 4b shows the production of light (induction factor) versus concentration of tetracycline in samples for further four different tetracyclines. Figure 5 shows the effect of magnesium ions on the sensitivity of the method according to the invention. Figure 6 illustrates possibilities of changing the assay window for the method of the invention by adjusting magnesium ion concentration and pH. Figure 7 shows the induction factor versus tetracycline concentration when using freeze-dried E. coli in the determination of tetracycline. Figure 8 shows a comparison of the assays based on using cells with the plasmid pTetLucl (SΕQ ID NO: 1) and with the plasmid pTetLuxl (SΕQ ID NO: 3). Figure 9 shows induction factors versus antibiotic concentrations of a pig serum sample (cells E. coli K12, pTetLuxl). Figure 10 shows the effect of ΕDTA in a milk sample assay, and Figure 11 shows the light emission versus time for an assay according to the invention. DETAILED DESCRIPTION OF THE INVENTION The term "tetracycline" shall be understood to include any compound covered by the general structure formula and particularly the specific commercially available compounds listed in the table below. Furthermore, the term "tetracycline" shall be understood to cover the metabolic and other reformulation/decomposition products thereof. The cells useful in the method of the invention are preferably Escherichia coli, which are stored in dried form, e.g. in lyophilized form before their use in the method according to the invention. Also freshly cultivated cells can be used. According to a preferred embodiment, the DNA vector including a nucleotide sequence encoding a light producing enzyme is a plasmid containing the luxCDABE genes (SEQ LD NO: 3), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from transposon TnlO. Particularly preferable is the plasmid pTetLuxl (SEQ ID NO: 3). According to another preferred embodiment, the DNA vector including a nucleotide sequence encoding a light producing enzyme is a plasmid containing the insect luciferase gene, tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from ThlO. In this case the substrate for insect luciferase reaction, D-luciferin, is added to the mixture of the sample and the cells in order to initiate the luminescence of the cells. The plasmid is preferably pTetLucl (SEQ ID NO: 1). The method according to this invention is useful for the determination of tetracycline in various kinds of samples. As examples can be mentioned milk, fish, meat, infant formula, eggs, honey, vegetables, serum, plasma, whole blood or the like. The luminescence of the cells is preferably measured using an X-ray or polaroid film, a CCD-camera (Charge Coupled Device), a liquid scintillation counter or, most preferably, a luminometer. The sensitivity of this analysis method with respect to the tetracycline can be controlled by increasing or decreasing the concentration of divalent metal ions, e.g. magnesium ions, in the mixture of the sample and the cells, by adjusting the pH or by combined adjusting of the divalent metal ion concentration and the pH. Increasing concentration of magnesium ions decreases the sensitivity and vice versa. Increasing pH will also cause a decreasing sensitivity. The sensitivity of the analysis with respect to the tetracycline can be increased by the use of cells which are especially antibiotic sensitive mutant strains. Chelating agents such as EDTA can be added to further sensitize the sensor system for tetracyclines. Figures 1 show a schematic representation of a method based on specific detection of the presence of tetracyclines using microbial cells cloned with either the plasmid pTetLuxl (SEQ ID NO: 3) (Figure la) or with the plasmid pTetLucl (SEQ ID NO: 1) (Figure lb). The figures show that cells containing either of the plasmids can be triggered to produce light by adding a chemical agent (a tetracycline). Light production is a consequence of tetracycline responsive promoter activation due to removal of the tet-repressor protein (SEQ ID NO: 11) leading to the production of luciferase specific mRNA and luciferase protein (SEQ ID NO: 2, 4-8) itself. The principle is demonstrated in Figure lc. In case of the usage of full length bacterial luciferase operon (SEQ ID NO: 3) containing luxC, luxD, luxA, luxB and luxE genes (SEQ ID NO: 3) (Figure la), one is able to get light emission without addition of any substance. In case of insect (e.g. firefly) luciferase (SEQ ID NO: 2) (Figure lb), light is emitted only after the addition of D-luciferin. It should be noticed that the triggering of luciferase synthesis and hght production commences immediately when the cells are introduced to the inducer molecules (tetracyclines). Therefore there is no need to use dividing cells and hence there is no need to use long cultivation of microbial cells such as the case is with conventional methods. Therefore, if needed, one can get results in minutes rather than in hours or days which is the case when conventional methods are used. Figure 2a shows the plasmid pTetLuxl (SEQ ID NO: 3), in which the production of bacterial luciferase (SEQ ID NO: 4-8) of Photorhabdus luminescens (formerly Xenorhabdus luminescens; the lux-operon structure and the full-length nucleotide sequence of P. luminescens was published in Szittner, R. and Meighen, E. (1990) J. Biol. Chem. 265, 16581-16587) can be switched on by the addition of a chemical agent belonging to the tetracycline family of antimicrobial agents in a cloned E. coli bacterium. SEQ ID NO: 3 shows the nucleotide sequence of the plasmid pTetLuxl. This plasmid construct is devised to contain the five genes from P. luminescens luciferase operon necessary for the light production without any additions of substrates, i.e. cells cloned with such a construct produce substrates endogenously. By incubating E. coli cells containing this plasmid (or any other microbial strain whereto similar regulation/reporter gene system is incorporated containing the necessary secondary regulatory sequences in the constructs such as correct ribosome binding region, transcriptional termination etc.) in the presence of very small amounts of tetracyclines one is able to obtain light production the intensity of which is proportional to the concentration of tetracycline used. Any E. coli mutant strain and especially those strains having a mutation in the export/import machinery of the membranes or otherwise leaky character making it possible for large molecules to easily penetrate inside the cell would be beneficial to use in the method described in this invention. Also other gram-negative bacteria such as strains belonging to genus Salmonella, Shigella, Enterobacter, Citrobacter, Klebsiella, Erwinia, Pseudomonas, Serratia as well as gram-positive organisms such as those belonging to genus Bacillus (especially B. subtϊlis, B. licheniformis, B. pumilus, B. globigii, B. natto, B. amyloliquefaciens as well as B. niger, B. brevis, B. megaterium ), Streptomyces, Lactobacillus (especially L. lactis, L. casei ) and Streptococcus (especially S. thermophilus, S. cremoris, S. agalactiae ) come into question. Especially asporogenic strains of Bacilli or Lactobacilli are suitable. Figure 3 shows the plasmid pTetLucl (SEQ ID NO: 1), in which the production of firefly luciferase (SEQ ID NO: 2) of Photinus pyralis (The gene encoding firefly luciferase was originally cloned and sequenced in the middle of the 1980's by DeWet, J. et al. (1987) Mol. Cell. Biol. 7, 725-737) can be switched on by the addition of a chemical agent belonging to the tetracycline family of antimicrobial agents in a cloned E. coli bacterium. SEQ ID NO: 1 shows the nucleotide sequence of this plasmid. By incubating E. coli cells containing this plasmid (or any other microbial strain whereto similar regulation/reporter gene system is incorporated containing the necessary secondary regulatory sequences in the constructs such as correct ribosome binding region, transcriptional termination etc.) in the presence of very small amounts of tetracyclines one is able to obtain light production by the addition of D-luciferin, which is the substrate of firefly luciferase. The intensity of light emission is proportional to the concentration of tetracycline used. Figures 4a and 4b shows the effect of altogether seven different tetracyclines on the production of light as a function of concentration of each tetracycline. As controls different non-tetracycline antibiotics were included in this study to show that the sensor strain is specific for the tetracyclines. The luminescense was emitted from E. coli containing the plasmid pTetLuxl (SEQ ID NO: 3). The detection was made after an incubation of 90 min. All tetracyclines tested behaved in a very similar manner and induction efficiencies were at the same antibiotic concentration area. This makes this sensor even more attractive for analytical use for the determination of the tetracycline group of antibiotics. It should be noted that the accumulation of various tetracyclines into microbial cells is very strongly affected by the extracellular concentration of Mg 2+ ions. Figure 5 shows the effect of increasing concentrations of Mg ions on the behavior of E. coli cells containing the plasmid pTetLuxl (SEQ ID NO: 3). As can be seen the tetracycline response curve is shifted to the right as a function of increasing concentrations of added Mg 2+ ions. Thus by increasing the Mg 2+ ion concentration one is able to decrease the sensitivity of the tetracycline sensor described in this invention. This fact is of great importance in cases where one does not need a high sensitivity of the measurement and where the approximate concentration of the ion is roughly constant and known such as in milk, serum and plasma. The sensitivity can be increased by removing magnesium ions from the assay mixture e.g. by adding a chelating agent forming a complex with magnesium. Figure 6 shows the possibilities to change the assay window for tetracyclines by adjusting the magnesium ion concentration and by combined adjustment of the magnesium ion concentration and pH. The sensitivity of the assay can be increased by the use of cells which are especially antibiotic sensitive mutant strains. Hundreds of specific mutations for bacteria are known with which it is possible to study the activity of specific reactions. For instance trace amounts of antibiotics cause visible changes in the metabolism or in the cell membranes of antibiotic sensitive bacterial mutants. Mutations in cell wall structural components or biosynthetic enzymes as well as in transport and efflux proteins such as porins might have an effect on the behavior of each sensor. Using these kinds of mutations one is able to develop tests measuring residual antibiotics from biological material very sensitively. It is also rather simple to transfer new characteristics into bacterial cells by genetic engineering techniques. This phenomenon broadens the applicability of these organisms in tests utilizing whole cell sensor. Measurement of light emission can be done by using X-ray or polaroid film, using a liquid scintillation counter, a CCD-camera or a luminometer. The CCD-camera is an instrument which is capable of detecting very low levels of light. In the applications of this invention such kind of a device could be used for the detection of tetracycline residues in food material such as vegetables or meat. The detection of light emission could be directly monitored from the surface of the food material sprayed with engineered luminescent bacteria. Either chemiluminescent (such as peroxidase - luminol) or bioluminescent (such as luciferase - luciferin) reactions can be utilized. The luminometric method is performed with the aid of genes encoding either bacterial or beetle luciferases such as those described in the Figures 2 and 4. Several luminescent bacterial species such as V. harveyi, V. fischeri, P. leiognathi, P. phosphoreum, Xenorhabdus luminescens etc. exist. Luminescent beetles are for example Luciola mingrelica, Photinus pyralis, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pholas dactylus, etc. Also several eukaryotic species in the sea which luminesce, such as marine ostracod Vargula hilgendo i, jellyfish Aequorea victoria, batrachoidid fish Porichtys notatus, pempherid fish Parapriacanthus ransonneti etc. exist. Fluorescent reporter proteins such as green fluorescent protein (GFP) or any of its variants could be used in the methods described in this invention (Li, X. et al. (1997) J. Biol. Chem. 272, 28545-28549). In this invention high detection sensitivity of the luminescent enzyme labels inside a living cell associated with tetracycline-specific induction of label synthesis is based on the use of optimal concentration of all the reactants inside the cell including the necessary cof actors and accessory enzymes. All luciferase genes from these organisms would presumably work in a similar manner as the two examples shown in this invention. These systems together with enhancers and modulators (wavelength, emission kinetics etc.) of light emission has been described in more detail in Campbell, A. "Chemiluminescence; principles and applications in biology and medicine", Weinheim; Deerfield Beach, FL; VCH; Chichester: Horwood, 1988. Peroxidases or oxidases can be used together with compounds such as luminol or acridines (for instance lucigenin) to yield luminescent signals suitable for a detection system described here. Enzymatically generated chemiluminescence offers great sensitivity and rapid detection, too, in assays described in this invention. Thermally stable dioxetanes (such as AMPPD and Lumigen PPD) can be enzymatically (such as alkaline phosphatase or β-galactosidase) triggered to produce chemiluminescence (Schaap, A.P. et al. (1989) Clin. Chem. 35, 1863-1864). The only difference to the luciferase enzymes would be that these enzymes are capable of cleaving a man-made substrate which gives light emission (chemiluminescence) and the lucif erases cleave natural substrates to produce light (bioluminescence). Tetracycline-controlled expression systems are developed to express heterologous proteins in procaryotic and eucaryotic cells for the purpose of production under a tight control of tet-regulatory system ( Skerra, A. (1994) Gene 151, 131-135; Gossen, M. and Bujard, H. (1995) US Patent 5,464,758 ; Lutz, R. and Bujard, H. (1997) Nucleic Acids Res. 25, 1203-1210). A method to study various tetracyclines and their mode of action was developed by Chopra et al. (Chopra, I. et al. (1990) Antimicrob. Agents Chemother. 34, 111-116) The assay system developed in this study was based on expression of β-galactosidase gene inserted under the control of tetA-gene. The method resulted in less sensitive detection of tetracyclines compared to the invention described here. However in order to obtain maximum sensitivities Chopra et al. showed that it was necessary to add cyclic AMP (cAMP) to the medium which is an extremely expensive molecule to be used in routine applications. Furthermore, the method described by Chopra et al. contains a cell disruption stage by sonication in order to assay for the reporter gene activity, β-galactosidase, which step is not practical. Instead, the method described in this invention does not contain any cell disruption. The activity of luciferase can be measured directly from living cells in real-time and in the case of pTetLuxl (SEQ ID NO: 3) there is no need of addition of any substrates. Therefore, promoter activation due to the presense/absense of tetracycline can be monitored continuously. EXPERIMENTS As cloning hosts and in antibiotic residue measurements various E. coli MCI 061 (cl+, αr D139, _ {ara-leu)l '696, lacX14, gafU, galK, hsr, hsm, strA) (Casadaban, M.J. and Cohen, S.N. (1980) J. Mol. Biol. 138, 179-207), BW322 (CGSC, r/α210::TnlO, thi-1, relAl, spoTl, pyrE) and K-12 (M72 Sm R / cZm-Δbt ' σwvrB, tr ?EA2, Nam7Nam53cI857 HI) (Remaut, E. et al. (1981) Gene 15, 81-93) can be used. Especially the strain LH530 (Hirvas, L. et al. (1997) Microbiology 143, 73-81) which has a decreased rate of lipid A biosynthesis. It has proven to be hypersusceptible to many different antibiotics. Cells were grown on appropriate minimal agar-plates and were kept maximally one month at +4 °C after which new plates were stroked. The strains were kept also in 15% glycerol at -70 °C, where from growth was started through minimal plates. The cells were first cultivated in 5 ml of 2xTY medium (16 g Bacto tryptone, 8 g Yeast extract, 8 g ΝaCl, H 2 0 ad 1 1, pH 7.4, with appropriate antibiotic) 10 h at 30 °C in a shaker after which the cultivation was transferred to a bigger volume for 10 h with same medium. Construction of tetracycline-responsive sensor plasmids: To construct a recombinant DΝA vector carrying luciferase genes under the control of a tetracycline responsive elements two new vectors were created. In the first one modified firefly luciferase gene (SEQ ID NO: 1) from vector pBLuc* (Bonin, A.L. et al. (1994) Gene 141, 75-77) was excised by using restriction enzymes Xbal and H DIII and the 1.7 kb fragment was isolated from LGT-agarose gel and purified using Qiagen gel extraction kit. This DNA-fragment containing the entire Photinus pyralis luciferase gene (SEQ ID NO: 1) was ligated using T4-DNA-ligase enzyme to vector pASK75 (Skerra, A. (1994) Gene 151, 131-135) which was previously restricted with the same restriction enzymes Xbal and HmDIII and calf intestinal phosphatase treated to remove the protruding phosphate groups in order to prevent self-ligation. The resulting ligation mixture was incubated 3 hours at room temperature after which one μl of the mixture was electroporated according to Dower et al. (Dower, W.J. et al. (1988) Nucleic Acids Res. 16, 6126-6144) into electrocompetent E. coli MCI 061 cells. A plasmid was extracted from one of the colonies obtained and checked for the estimated structure by appropriate restriction enzyme digestions and agarose gel electrophoretic techniques. The plasmid obtained was named as pTetLucl (SEQ ID NO: 1). The plasmid containing the luxCDABE genes (SEQ ID NO: 3) of Photorhabdus luminescens under the control of tetracycline responsive element was created as follows: Plasmid pASK75 was cut with restriction enzyme EcoRl and CIP-treated. The linearized plasmid was separated on a LGT-agarose gel electrophoresis and the agarose was removed by using the Qiagen kit. The lux operon was excised with EcoRI from plasmid pCGLS-11 (Frackman, S. et al. (1990) J. Bacteriol. 172, 5767- 5773), gel purified as above and ligated to pASK75 by using T4-DNA-ligase at 16 °C overnight. The ligation mixture was electroporated into E. coli MCI 061 cells as described above and correct transformants were screened for their ability to produce light (as measured with a BioOrbit 1250 manual luminometer) which production was increased in the presence of 1 μg/ml of tetracycline-HCl. The plasmid was further verified by restriction enzyme digestions and the correct structure was named as pTetLuxl (SEQ ID NO: 3). All the DNA-manipulations were performed according to Sambrook et al, "Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. The vector pASK75 was utilized in the construction of tet-sensor plasmids shown in this invention. The vector pASK75 was originally developed for protein production and purification purposes. It contains a signal sequence for secretion of the recombinant protein into the periplasmic space of E. coli. Also a C-terminal fusion between a purification tail, the Strept-tag, was incorporated into the vector to facilitate purification of recombinant protein using streptavidin affinity agarose gel chromatography. The element controlling recombinant gene expression in the vector is tetA promoter/operator system that allows efficient regulation of the expression, which in Skerra's paper was described for the production and one-step purification of a murine single-chain antibody fragment. The tetA promoter/operator (SEQ ID NO: 9) is controlled by tetR-repressor (SEQ ID NO: 9) which is produced by the corresponding gene (SEQ ID NO: 9). Some of the above mentioned elements were eliminated from the present plasmids due to unnecessary features with respect to this invention. Transfer of the tetracycline sensor vectors to the antibiotic sensitive E. coli strain: Either pTetLuxl (SEQ LD NO: 3) or pTetLucl (SEQ ID NO: 1) was transformed into E. coli LH530 cells by electroporation as described above. The transformed cells were restreaked on agar plates and kept maximally for 2 weeks at +4 °C after which a new plate was streaked. Use of the manipulated E. coli in tetracycline determination methods: Example 1 Freeze-dried E. coli K-12/pTetLuxl were reconstituted with 1.0 ml of L-broth and bacteria were diluted 1:10 with 25 mM MES buffer in M9 minimal medium, pH 6.0. 190 μl bacterial suspension was added to microtiter plate wells containing 10 μl of tetracycline dilutions. The plate was incubated 90 minutes at 37 °C after which the plate was measured with Labsystems Luminoskan luminometer. As seen from Figure 7 the sensitivity of the assay of tetracycline is very high and comparable to that of fresh cells. Example 2 Two different types of sensor DNA vector construct were compared. Strains E. coli K-12/pTetLuxl and E. coli K-12/pTetLucl were cultivated in L-broth media until optical density measured at 600 nm (OD600) was 1.5. The cells were diluted 1 to 50 with 25 mM MES-buffer in M9 minimal medium, pH 6.0 (Sambrook et al, 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor) and 190 μl was added to micro titration plate wells and 10 μl of sample dilution of tetracycline was added. After a 60 min incubation at 37 °C the light emission was measured using a Labsystems Luminoskan luminometer. Figure 8 shows the bioluminescence dose response curve as a function of tetracycline added. As seen from the figure both systems (bacterial and insect luciferase) give roughly equal sensitivity of tetracycline detection. One is able to use different luciferases instead of bacterial luciferase (SEQ ID NO: 4-8) from P. luminescens without losing sensitivity or other performance of the test. Figure 8 shows an analogous measurement to the one in Figure 4b. In the plasmid used in this test (pTetLucl) the bacterial luciferase was compensated with firefly luciferase (SEQ ID NO: 2) as described in Figure 3. The test was done essentially as with bacterial luciferase except that after the cells had been incubated with or without tetracycline 10 minutes at 37 °C the cells were measured for light production after 15 minutes incubation time at 37 °C by adding 100 μl of solution containing 1 mM D-luciferin, in 0.1 M Na-citrate buffer, pH 5.0. Thereafter light production was measured using a manual luminometer 1250 (LKB-Wallac, Turku, Finland). As can be seen from Figure 8 sensitivity of the method to detect tetracycline hydrochloride is extremely high and comparable to the detection made with bacterial luciferase. Example 3 A lipemic pig serum was spiked at different concentrations of tetracycline, chlorotetracycline and oxy tetracycline. Fresh E. coli K-12/pTetLuxl were diluted 1:50 with 25 mM MES buffer in M9 minimal medium, pH 6.0. 100 μl bacterial suspension was added to microtiter plate wells containing 100 μl of pig serum spiked with different tetracyclines. The plate was incubated 90 minutes at 37 °C after which the plate was measured with Labsystems Luminoskan luminometer. As seen from Figure 9 the sensitivity of the assay of different tetracyclines in pig serum matrix is very high. Example 4 Tetracyclines will form chelate complexes with Ca 2+ and Mg 2+ in samples (e.g. milk), and loose their antimicrobial and induction activity in our assay system. Tetracyclines can be displaced from cation chelates by using strong chelating agents such as EDTA. Figure 10 shows the determination of tetracycline from a milk sample, which is spiked with different concentrations of tetracycline. Different amounts of EDTA were added to milk samples and this kind of displacement of cation-tetracycline complex clearly improved the sensitivity of the assay. In the assay we used freeze-dried E. coli K12/pTetLuxl that were reconstituted with L-broth 10 minutes in room temperature before the assay. Example 5 Figure 11 shows the kinetics of bacterial bioluminescence after exposure of E. coli K-12/pTetLuxl to different dilutions of tetracycline antibiotics. The specific induction of tetracycline is very fast and specific light emission is seen already at the 10 minutes measuring point in the assay. It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: KORPELA, Matti (B) STREET: Maijamaentie 13 (C) CITY: Naantali (E) COUNTRY: Finland (F) POSTAL CODE (ZIP) : FIN-21100 (A) NAME: KARP, Matti (B) STREET: Kampa atu 1 (C) CITY: Kaarina (E) COUNTRY: Finland (F) POSTAL CODE (ZIP) : FIN-20660 (A) NAME: KURITTU, Jussi (B) STREET: Puutarhakatu 16 A 20 (C) CITY: Turku (E) COUNTRY: Finland (F) POSTAL CODE (ZIP) : FIN-20100 (ii) TITLE OF INVENTION: A NEW ASSAY METHOD (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: FI 974235 (B) FILING DATE: 14-NOV-1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4846 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Photinus pyralis (vii) IMMEDIATE SOURCE: (B) CLONE: pTetLucl (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: Plasmid ( ix) FEATURE : (A) NAME/KEY: misc_feature (B) LOCATION: 1..3098 (D) OTHER INFORMATION: /standard_name= "Vector pASK75" /note= "Part of plasmid originating from vector pASK75; feature description below, SEQ ID 9-11." /citation= ([2]) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3119..4768 (D) OTHER INFORMATION: /product= "Photinus pyralis luciferase" /citation= ([1]) (x) PUBLICATION INFORMATION: (A) AUTHORS: Bonin, (B) TITLE: Photinus pyralis luciferase: vectors that contain a modified luc coding sequence allowing convenient transfer into other systems (C) JOURNAL: Gene (D) VOLUME: 141 (F) PAGES: 75-77 (G) DATE: 1994 (K) RELEVANT RESIDUES IN SEQ ID NO: 1: FROM 3099 TO 4772 (X) PUBLICATION INFORMATION: (A) AUTHORS: Skerra, A (B) TITLE: Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli (C) JOURNAL: Gene (D) VOLUME: 151 (E) ISSUE: 1-2 (F) PAGES: 131-135 (G) DATE: 30-DEC-1994 (K) RELEVANT RESIDUES IN SEQ ID NO: 1: FROM 1 TO 3098 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGCTTGACCT GTGAAGTGAA AAATGGCGCA CATTGTGCGA CATTTTTTTT GTCTGCCGTT 60 TACCGCTACT GCGTCACGGA TCTCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT 120 GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC 180 GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG 240 GGGCTCCCTT TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT 300 TAGGGTGATG GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG 360 TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT 420 ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA 480 AATGAGCTGA TTTAACAAAA ATTTAACGCG AATTTTAACA AAATATTAAC GCTTACAATT 540 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA 600 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA 660 AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA 720 TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT 780 CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG 840 AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC 900 GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT 960 CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA 1020 GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT 1080 CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT 1140 GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT 1200 GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA 1260 CTTACTCTAG CTTCCCGGCA ACAATTGATA GACTGGATGG AGGCGGATAA AGTTGCAGGA 1320 CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT 1380 GAGCGTGGCT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC 1440 GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT 1500 GAGATAGGTG CCTCACTGAT TAAGCATTGG TAGGAATTAA TGATGTCTCG TTTAGATAAA 1560 AGTAAAGTGA TTAACAGCGC ATTAGAGCTG CTTAATGAGG TCGGAATCGA AGGTTTAACA 1620 ACCCGTAAAC TCGCCCAGAA GCTAGGTGTA GAGCAGCCTA CATTGTATTG GCATGTAAAA 1680 AATAAGCGGG CTTTGCTCGA CGCCTTAGCC ATTGAGATGT TAGATAGGCA CCATACTCAC 1740 TTTTGCCCTT TAGAAGGGGA AAGCTGGCAA GATTTTTTAC GTAATAACGC TAAAAGTTTT 1800 AGATGTGCTT TACTAAGTCA TCGCGATGGA GCAAAAGTAC ATTTAGGTAC ACGGCCTACA 1860 GAAAAACAGT ATGAAACTCT CGAAAATCAA TTAGCCTTTT TATGCCAACA AGGTTTTTCA 1920 CTAGAGAATG CATTATATGC ACTCAGCGCA GTGGGGCATT TTACTTTAGG TTGCGTATTG 1980 GAAGATCAAG AGCATCAAGT CGCTAAAGAA GAAAGGGAAA CACCTACTAC TGATAGTATG 2040 CCGCCATTAT TACGACAAGC TATCGAATTA TTTGATCACC AAGGTGCAGA GCCAGCCTTC 2100 TTATTCGGCC TTGAATTGAT CATATGCGGA TTAGAAAAAC AACTTAAATG TGAAAGTGGG 2160 TCTTAAAAGC AGCATAACCT TTTTCCGTGA TGGTAACTTC ACTAGTTTAA AAGGATCTAG 2220 GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC 2280 TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC 2340 GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT 2400 CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT 2460 ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT 2520 ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT 2580 CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG 2640 GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA 2700 CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG 2760 GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG 2820 TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC 2880 TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG 2940 GCCTTTTGCT GGCCTTTTGC TCACATGACC CGACACCATC GAATGGCCAG ATGATTAATT 3000 CCTAATTTTT GTTGACACTC TATCATTGAT AGAGTTATTT TACCACTCCC TATCAGTGAT 3060 AGAGAAAAGT GAAATGAATA GTTCGACAAA AATCTAGAAC TAGTGGATCC CCCGTACC 3118 ATG GAA GAC GCC AAA AAC ATA AAG AAA GGC CCG GCG CCA TTC TAT CCG 3166 Met Glu Asp Ala Lys Asn lie Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 CTA GAG GAT GGA ACC GCT GGA GAG CAA CTG CAT AAG GCT ATG AAG AGA 3214 Leu Glu Asp Gly Thr Ala Gly Glu Gin Leu His Lys Ala Met Lys Arg 20 25 30 TAC GCC CTG GTT CCT GGA ACA ATT GCT TTT ACA GAT GCA CAT ATC GAG 3262 Tyr Ala Leu Val Pro Gly Thr lie Ala Phe Thr Asp Ala His lie Glu 35 40 45 GTG AAC ATC ACG TAC GCG GAA TAC TTC GAA ATG TCC GTT CGG TTG GCA 3310 Val Asn lie Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 GAA GCT ATG AAA CGA TAT GGG CTG AAT ACA AAT CAC AGA ATC GTC GTA 3358 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg lie Val Val 65 70 75 80 TGC AGT GAA AAC TCT CTT CAA TTC TTT ATG CCG GTG TTG GGC GCG TTA 3406 Cys Ser Glu Asn Ser Leu Gin Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 TTT ATC GGA GTT GCA GTT GCG CCC GCG AAC GAC ATT TAT AAT GAA CGT 3454 Phe lie Gly Val Ala Val Ala Pro Ala Asn Asp lie Tyr Asn Glu Arg 100 105 110 GAA TTG CTC AAC AGT ATG AAC ATT TCG CAG CCT ACC GTA GTG TTT GTT 3502 Glu Leu Leu Asn Ser Met Asn lie Ser Gin Pro Thr Val Val Phe Val 115 120 125 TCC AAA AAG GGG TTG CAA AAA ATT TTG AAC GTG CAA AAA AAA TTA CCA 3550 Ser Lys Lys Gly Leu Gin Lys lie Leu Asn Val Gin Lys Lys Leu Pro 130 135 140 ATA ATC CAG AAA ATT ATT ATC ATG GAT TCT AAA ACG GAT TAC CAG GGA 3598 lie lie Gin Lys He He He Met Asp Ser Lys Thr Asp Tyr Gin Gly 145 150 155 160 TTT CAG TCG ATG TAC ACG TTC GTC ACA TCT CAT CTA CCT CCC GGT TTT 3646 Phe Gin Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 AAT GAA TAC GAT TTT GTA CCA GAG TCC TTT GAT CGT GAC AAA ACA ATT 3694 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr He 180 185 190 GCA CTG ATA ATG AAC TCC TCT GGA TCT ACT GGG TTA CCT AAG GGT GTG 3742 Ala Leu He Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 GCC CTT CCG CAT AGA ACT GCC TGC GTC AGA TTC TCG CAT GCC AGA GAT 3790 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 CCT ATT TTT GGC AAT CAA ATC ATT CCG GAT ACT GCG ATT TTA AGT GTT 3838 Pro He Phe Gly Asn Gin He He Pro Asp Thr Ala He Leu Ser Val 225 230 235 240 GTT CCA TTC CAT CAC GGT TTT GGA ATG TTT ACT ACA CTC GGA TAT TTG 3886 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 ATA TGT GGA TTT CGA GTC GTC TTA ATG TAT AGA TTT GAA GAA GAG CTG 3934 He Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 TTT TTA CGA TCC CTT CAG GAT TAC AAA ATT CAA AGT GCG TTG CTA GTA 3982 Phe Leu Arg Ser Leu Gin Asp Tyr Lys He Gin Ser Ala Leu Leu Val 275 280 285 CCA ACC CTA TTT TCA TTC TTC GCC AAA AGC ACT CTG ATT GAC AAA TAC 4030 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu He Asp Lys Tyr 290 295 300 GAT TTA TCT AAT TTA CAC GAA ATT GCT TCT GGG GGC GCA CCT CTT TCG 4078 Asp Leu Ser Asn Leu His Glu He Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 AAA GAA GTC GGG GAA GCG GTT GCA AAA CGC TTC CAT CTT CCA GGG ATA 4126 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly He 325 330 335 CGA CAA GGA TAT GGG CTC ACT GAG ACT ACA TCA GCT ATT CTG ATT ACA 4174 Arg Gin Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala He Leu He Thr 340 345 350 CCC GAG GGG GAT GAT AAA CCG GGC GCG GTC GGT AAA GTT GTT CCA TTT 4222 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 TTT GAA GCG AAG GTT GTG GAT CTG GAT ACC GGG AAA ACG CTG GGC GTT 4270 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 AAT CAG AGA GGC GAA TTA TGT GTC AGA GGA CCT ATG ATT ATG TCC GGT 4318 Asn Gin Arg Gly Glu Leu Cys Val Arg Gly Pro Met He Met Ser Gly 385 390 395 400 TAT GTA AAC AAT CCG GAA GCG ACC AAC GCC TTG ATT GAC AAG GAT GGA 4366 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu He Asp Lys Asp Gly 405 410 415 TGG CTA CAT TCT GGA GAC ATA GCT TAC TGG GAC GAA GAC GAA CAC TTC 4414 Trp Leu His Ser Gly Asp He Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 TTC ATA GTT GAC CGC TTG AAG TCT TTA ATT AAA TAC AAA GGA TAC CAG 4462 Phe He Val Asp Arg Leu Lys Ser Leu He Lys Tyr Lys Gly Tyr Gin 435 440 445 GTG GCC CCC GCT GAA TTG GAG TCG ATA TTG TTA CAA CAC CCC AAC ATC 4510 Val Ala Pro Ala Glu Leu Glu Ser He Leu Leu Gin His Pro Asn He 450 455 460 TTC GAC GCG GGC GTG GCA GGT CTT CCC GAC GAT GAC GCC GGT GAA CTT 4558 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 CCC GCC GCC GTT GTT GTT TTG GAG CAC GGA AAG ACG ATG ACG GAA AAA 4606 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 GAG ATC GTG GAT TAC GTC GCC AGT CAA GTA ACA ACC GCC AAA AAG TTG 4654 Glu He Val Asp Tyr Val Ala Ser Gin Val Thr Thr Ala Lys Lys Leu 500 505 510 CGC GGA GGA GTT GTG TTT GTG GAC GAA GTA CCG AAA GGT CTT ACC GGA 4702 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 AAA CTC GAC GCA AGA AAA ATC AGA GAG ATC CTC ATA AAG GCC AAG AAG 4750 Lys Leu Asp Ala Arg Lys He Arg Glu He Leu He Lys Ala Lys Lys 530 535 540 GGC GGA AAG TCC AAA TTG TAAAATGTAA CTGTATTCAG CGATGACGAA 4798 Gly Gly Lys Ser Lys Leu 545 550 ATTCTTAGCT ATTGTAATAC TCTAGCGGGC TGCAGGAATT CGATATCA 4846 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 550 a ino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Glu Asp Ala Lys Asn He Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gin Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr He Ala Phe Thr Asp Ala His He Glu 35 40 45 Val Asn He Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg He Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gin Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe He Gly Val Ala Val Ala Pro Ala Asn Asp He Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Asn He Ser Gin Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gin Lys He Leu Asn Val Gin Lys Lys Leu Pro 130 135 140 He He Gin Lys He He He Met Asp Ser Lys Thr Asp Tyr Gin Gly 145 150 155 160 Phe Gin Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr He 180 185 190 Ala Leu He Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro He Phe Gly Asn Gin He He Pro Asp Thr Ala He Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 He Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gin Asp Tyr Lys He Gin Ser Ala Leu Leu Val 275 280 285 ~ Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu He Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu He Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly He 325 330 335 Arg Gin Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala He Leu He Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gin Arg Gly Glu Leu Cys Val Arg Gly Pro Met He Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu He Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp He Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe He Val Asp Arg Leu Lys Ser Leu He Lys Tyr Lys Gly Tyr Gin 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser He Leu Leu Gin His Pro Asn He 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu He Val Asp Tyr Val Ala Ser Gin Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys He Arg Glu He Leu He Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ser Lys Leu 545 550 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10220 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Photorhabdus luminescens (vii) IMMEDIATE SOURCE: (B) CLONE: pTetLuxl (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: join(l..3190, 10140..10220) (D) OTHER INFORMATION: /standard_name= "vector pASK75" /note= "Parts of plasmid originating from vector pASK75; feature description below, SEQ ID NO: 9-11." /citation= ([2]) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3634..5082 (D) OTHER INFORMATION: /product= "Lux C" /citation= ( [1] ) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 5097..6017 (D) OTHER INFORMATION: /product= "Lux D" /citation= ( [1] ) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 6069..7148 (D) OTHER INFORMATION: /product= "Lux A" /citation= ([1]) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 7166..8146 (D) OTHER INFORMATION: /product= "Lux B" /citation= ([1]) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 8256..9437 (D) OTHER INFORMATION: /product= "Lux E" /citation= ( [1] ) (x) PUBLICATION INFORMATION: (A) AUTHORS: Frackman, (B) TITLE: Cloning, organization and expression of the bioluminescence genes of Xenorhabdus lumiminescenss (C) JOURNAL: J. Bacteriol. (D) VOLUME: 172 (F) PAGES: 5767-5773 (G) DATE: 1990 (K) RELEVANT RESIDUES IN SEQ ID NO: 3: FROM 3191 TO 10139 (x) PUBLICATION INFORMATION: (A) AUTHORS: Skerra, A (B) TITLE: Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli (C) JOURNAL: Gene (D) VOLUME: 151 (E) ISSUE: 1-2 (F) PAGES: 131-135 (G) DATE: 30-DEC-1994 (K) RELEVANT RESIDUES IN SEQ ID NO: 3: FROM 1 TO 3190 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AGCTTGACCT GTGAAGTGAA AAATGGCGCA CATTGTGCGA CATTTTTTTT GTCTGCCGTT 60 TACCGCTACT GCGTCACGGA TCTCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT 120 GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC 180 GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG 240 GGGCTCCCTT TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT 300 TAGGGTGATG GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG 360 TTGGAGTCCA CGTTC TTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT 420 ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA 480 AATGAGCTGA TTTAACAAAA ATTTAACGCG AATTTTAACA AAATATTAAC GCTTACAATT 540 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA 600 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA 660 AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT TTTTGCGGCA 720 TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA TGCTGAAGAT 780 CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA GATCCTTGAG 840 AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT GCTATGTGGC ' 900 GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT ACACTATTCT 960 CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA TGGCATGACA 1020 GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT 1080 CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT 1140 GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT 1200 GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA 1260 CTTACTCTAG CTTCCCGGCA ACAATTGATA GACTGGATGG AGGCGGATAA AGTTGCAGGA 1320 CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT 1380 GAGCGTGGCT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC 1440 GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG ACAGATCGCT 1500 GAGATAGGTG CCTCACTGAT TAAGCATTGG TAGGAATTAA TGATGTCTCG TTTAGATAAA 1560 AGTAAAGTGA TTAACAGCGC ATTAGAGCTG CTTAATGAGG TCGGAATCGA AGGTTTAACA 1620 ACCCGTAAAC TCGCCCAGAA GCTAGGTGTA GAGCAGCCTA CATTGTATTG GCATGTAAAA 1680 AATAAGCGGG CTTTGCTCGA CGCCTTAGCC ATTGAGATGT TAGATAGGCA CCATACTCAC 1740 TTTTGCCCTT TAGAAGGGGA AAGCTGGCAA GATTTTTTAC GTAATAACGC TAAAAGTTTT 1800 AGATGTGCTT TACTAAGTCA TCGCGATGGA GCAAAAGTAC ATTTAGGTAC ACGGCCTACA 1860 GAAAAACAGT ATGAAACTCT CGAAAATCAA TTAGCCTTTT TATGCCAACA AGGTTTTTCA 1920 CTAGAGAATG CATTATATGC ACTCAGCGCA GTGGGGCATT TTACTTTAGG TTGCGTATTG 1980 GAAGATCAAG AGCATCAAGT CGCTAAAGAA GAAAGGGAAA CACCTACTAC TGATAGTATG 2040 CCGCCATTAT TACGACAAGC TATCGAATTA TTTGATCACC AAGGTGCAGA GCCAGCCTTC 2100 TTATTCGGCC TTGAATTGAT CATATGCGGA TTAGAAAAAC AACTTAAATG TGAAAGTGGG 2160 TCTTAAAAGC AGCATAACCT TTTTCCGTGA TGGTAACTTC ACTAGTTTAA AAGGATCTAG 2220 GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC 2280 TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC 2340 GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT 2400 CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT 2460 ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT 2520 ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT 2580 CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG 2640 GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA 2700 CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG 2760 GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG 2820 TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC 2880 TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG 2940 GCCTTTTGCT GGCCTTTTGC TCACATGACC CGACACCATC GAATGGCCAG ATGATTAATT 3000 CCTAATTTTT GTTGACACTC TATCATTGAT AGAGTTATTT TACCACTCCC TATCAGTGAT 3060 AGAGAAAAGT GAAATGAATA GTTCGACAAA AATCTAGATA ACGAGGGCAA AAAATGAAAA 3120 AGACAGCTAT CGCGATTGCA GTGGCACTGG CTGGTTTCGC TACCGTAGCG CAGGCCTGAG 3180 ACCAGAATTC TTCTTTAGAA ATCTGCCGGT AAAAATTAGA TTGCTATTCA ATCTATTTCT 3240 ATCGGTATTT GTGAAATAAT ACTCAGGATA ATAATTTACA TAAATATTAT CACGCATTAG 3300 AGAAGAGCAT GACTTTTTTA ATTTAAACTT TTCATTAACA AATCTTGTTG ATATGAAAAT 3360 TTTCCTTTGC TATTTTAACA GATATTAAAA CGGGAATAGG CGTTATATTG ACGATCCATT 3420 CAGTTAGATT AAAAACCTTG AGCAGAAAAT TTATATTATT ATCATAATTA TGACGAAAGT 3480 TACAGGCCAG GAACCACGTA GTCAGAATCT GATTTTCTAT ATATTTGTTA TTTACATCGT 3540 CATAACACAA AAATATAAGA AGCAAGTGTT GGTACGACCA GTTCGCAAGA TAGTTAAACA 3600 GCAACTTAAG TTGAAATTAC CCCCATTAAA TGG ATG GCA AAT ATG ACT AAA AAA 3654 Met Ala Asn Met Thr Lys Lys 555 ATT TCA TTC ATT ATT AAC GGC CAG GTT GAA ATC TTT CCC GAA AGT GAT 3702 He Ser Phe He He Asn Gly Gin Val Glu He Phe Pro Glu Ser Asp 560 565 570 GAT TTA GTG CAA TCC ATT AAT TTT GGT GAT AAT AGT GTT TAC CTG CCA 3750 Asp Leu Val Gin Ser He Asn Phe Gly Asp Asn Ser Val Tyr Leu Pro 575 580 585 ATA TTG AAT GAC TCT CAT GTA AAA AAC ATT ATT GAT TGT AAT GGA AAT 3798 He Leu Asn Asp Ser His Val Lys Asn He He Asp Cys Asn Gly Asn 590 595 600 605 AAC GAA TTA CGG TTG CAT AAC ATT GTC AAT TTT CTC TAT ACG GTA GGG 3846 Asn Glu Leu Arg Leu His Asn He Val Asn Phe Leu Tyr Thr Val Gly 610 615 620 CAA AGA TGG AAA AAT GAA GAA TAC TCA AGA CGC AGG ACA TAC ATT CGT 3894 Gin Arg Trp Lys Asn Glu Glu Tyr Ser Arg Arg Arg Thr Tyr He Arg 625 630 635 GAC TTA AAA AAA TAT ATG GGA TAT TCA GAA GAA ATG GCT AAG CTA GAG 3942 Asp Leu Lys Lys Tyr Met Gly Tyr Ser Glu Glu Met Ala Lys Leu Glu 640 645 650 GCC AAT TGG ATA TCT ATG ATT TTA TGT TCT AAA GGC GGC CTT TAT GAT 3990 Ala Asn Trp He Ser Met He Leu Cys Ser Lys Gly Gly Leu Tyr Asp 655 660 665 GTT GTA GAA AAT GAA CTT GGT TCT CGC CAT ATC ATG GAT GAA TGG CTA 4038 Val Val Glu Asn Glu Leu Gly Ser Arg His He Met Asp Glu Trp Leu 670 675 680 685 CCT CAG GAT GAA AGT TAT GTT CGG GCT TTT CCG AAA GGT AAA TCT GTA 4086 Pro Gin Asp Glu Ser Tyr Val Arg Ala Phe Pro Lys Gly Lys Ser Val 690 695 700 CAT CTG TTG GCA GGT AAT GTT CCA TTA TCT GGG ATC ATG TCT ATA TTA 4134 His Leu Leu Ala Gly Asn Val Pro Leu Ser Gly He Met Ser He Leu 705 710 715 CGC GCA ATT TTA ACT AAG AAT CAG TGT ATT ATA AAA ACA TCG TCA ACC 4182 Arg Ala He Leu Thr Lys Asn Gin Cys He He Lys Thr Ser Ser Thr 720 725 730 GAT CCT TTT ACC GCT AAT GCA TTA GCG TTA AGT TTT ATT GAT GTA GAC 4230 Asp Pro Phe Thr Ala Asn Ala Leu Ala Leu Ser Phe He Asp Val Asp 735 740 745 CCT AAT CAT CCG ATA ACG CGC TCT TTA TCT GTT ATA TAT TGG CCC CAC 4278 Pro Asn His Pro He Thr Arg Ser Leu Ser Val He Tyr Trp Pro His 750 755 760 765 CAA GGT GAT ACA TCA CTC GCA AAA GAA ATT ATG CGA CAT GCG GAT GTT 4326 Gin Gly Asp Thr Ser Leu Ala Lys Glu He Met Arg His Ala Asp Val 770 775 780 ATT GTC GCT TGG GGA GGG CCA GAT GCG ATT AAT TGG GCG GTA GAG CAT 4374 He Val Ala Trp Gly Gly Pro Asp Ala He Asn Trp Ala Val Glu His 785 790 795 GCG CCA TCT TAT GCT GAT GTG ATT AAA TTT GGT TCT AAA AAG AGT CTT 4422 Ala Pro Ser Tyr Ala Asp Val He Lys Phe Gly Ser Lys Lys Ser Leu 800 805 810 TGC ATT ATC GAT AAT CCT GTT GAT TTG ACG TCC GCA GCG ACA GGT GCG 4470 Cys He He Asp Asn Pro Val Asp Leu Thr Ser Ala Ala Thr Gly Ala 815 820 825 GCT CAT GAT GTT TGT TTT TAC GAT CAG CGA GCT TGT TTT TCT GCC CAA 4518 Ala His Asp Val Cys Phe Tyr Asp Gin Arg Ala Cys Phe Ser Ala Gin 830 835 840 845 AAC ATA TAT TAC ATG GGA AAT CAT TAT GAG GAA TTT AAG TTA GCG TTG 4566 Asn He Tyr Tyr Met Gly Asn His Tyr Glu Glu Phe Lys Leu Ala Leu 850 855 860 ATA GAA AAA CTT AAT CTA TAT GCG CAT ATA TTA CCG AAT GCC AAA AAA 4614 He Glu Lys Leu Asn Leu Tyr Ala His He Leu Pro Asn Ala Lys Lys 865 870 875 GAT TTT GAT GAA AAG GCG GCC TAT TCT TTA GTT CAA AAA GAA AGC TTG 4662 Asp Phe Asp Glu Lys Ala Ala Tyr Ser Leu Val Gin Lys Glu Ser Leu 880 885 890 TTT GCT GGA TTA AAA GTA GAG GTG GAT ATT CAT CAA CGT TGG ATG ATT 4710 Phe Ala Gly Leu Lys Val Glu Val Asp He His Gin Arg Trp Met He 895 900 905 ATT GAG TCA AAT GCA GGT GTG GAA TTT AAT CAA CCA CTT GGC AGA TGT 4758 He Glu Ser Asn Ala Gly Val Glu Phe Asn Gin Pro Leu Gly Arg Cys 910 915 920 925 GTG TAC CTT CAT CAC GTC GAT AAT ATT GAG CAA ATA TTG CCT TAT GTT 4806 Val Tyr Leu His His Val Asp Asn He Glu Gin He Leu Pro Tyr Val 930 935 940 CAA AAA AAT AAG ACG CAA ACC ATA TCT ATT TTT CCT TGG GAG TCA TCA 4854 Gin Lys Asn Lys Thr Gin Thr He Ser He Phe Pro Trp Glu Ser Ser 945 950 955 TTT AAA TAT CGA GAT GCG TTA GCA TTA AAA GGT GCG GAA AGG ATT GTA 4902 Phe Lys Tyr Arg Asp Ala Leu Ala Leu Lys Gly Ala Glu Arg He Val 960 965 970 GAA GCA GGA ATG AAT AAC ATA TTT CGA GTT GGT GGA TCT CAT GAC GGA 4950 Glu Ala Gly Met Asn Asn He Phe Arg Val Gly Gly Ser His Asp Gly 975 980 985 ATG AGA CCG TTG CAA CGA TTA GTG ACA TAT ATT TCT CAT GAA AGG CCA 4998 Met Arg Pro Leu Gin Arg Leu Val Thr Tyr He Ser His Glu Arg Pro 990 995 1000 1005 TCT AAC TAT ACG GCT AAG GAT GTT GCG GTT GAA ATA GAA CAG ACT CGA 5046 Ser Asn Tyr Thr Ala Lys Asp Val Ala Val Glu He Glu Gin Thr Arg 1010 1015 1020 TTC CTG GAA GAA GAT AAG TTC CTT GTA TTT GTC CCA TAATAGGTAA 5092 Phe Leu Glu Glu Asp Lys Phe Leu Val Phe Val Pro 1025 1030 AAGT ATG GAA AAT GAA TCA AAA TAT AAA ACC ATC GAC CAC GTT ATT TGT 5141 Met Glu Asn Glu Ser Lys Tyr Lys Thr He Asp His Val He Cys 1 5 10 15 GTT GAA GGA AAT AAA AAA ATT CAT GTT TGG GAA ACG CTG CCA GAA GAA 5189 Val Glu Gly Asn Lys Lys He His Val Trp Glu Thr Leu Pro Glu Glu 20 25 30 AAC AGC CCA AAG AGA AAG AAT GCC ATT ATT ATT GCG TCT GGT TTT GCC 5237 Asn Ser Pro Lys Arg Lys Asn Ala He He He Ala Ser Gly Phe Ala 35 40 45 CGC AGG ATG GAT CAT TTT GCT GGT CTG GCG GAA TAT TTA TCG CGG AAT 5285 Arg Arg Met Asp His Phe Ala Gly Leu Ala Glu Tyr Leu Ser Arg Asn 50 55 60 GGA TTT CAT GTG ATC CGC TAT GAT TCG CTT CAC CAC GTT GGA TTG AGT 5333 Gly Phe His Val He Arg Tyr Asp Ser Leu His His Val Gly Leu Ser 65 70 75 TCA GGG ACA ATT GAT GAA TTT ACA ATG TCT ATA GGA AAG CAG AGC TTG 5381 Ser Gly Thr He Asp Glu Phe Thr Met Ser He Gly Lys Gin Ser Leu 80 85 90 95 TTA GCA GTG GTT GAT TGG TTA ACT ACA CGA AAA ATA AAT AAC TTC GGT 5429 Leu Ala Val Val Asp Trp Leu Thr Thr Arg Lys He Asn Asn Phe Gly 100 105 110 ATG TTG GCT TCA AGC TTA TCT GCG CGG ATA GCT TAT GCA AGC CTA TCT 5477 Met Leu Ala Ser Ser Leu Ser Ala Arg He Ala Tyr Ala Ser Leu Ser 115 120 125 GAA ATC AAT GCT TCG TTT TTA ATC ACC GCA GTC GGT GTT GTT AAC TTA 5525 Glu He Asn Ala Ser Phe Leu He Thr Ala Val Gly Val Val Asn Leu 130 135 140 AGA TAT TCT CTT GAA AGA GCT TTA GGG TTT GAT TAT CTC AGT CTA CCC 5573 Arg Tyr Ser Leu Glu Arg Ala Leu Gly Phe Asp Tyr Leu Ser Leu Pro 145 150 155 ATT AAT GAA TTG CCG GAT AAT CTA GAT TTT GAA GGC CAT AAA TTG GGT 5621 He Asn Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly 160 165 170 175 GCT GAA GTC TTT GCG AGA GAT TGT CTT GAT TTT GGT TGG GAA GAT TTA 5669 Ala Glu Val Phe Ala Arg Asp Cys Leu Asp Phe Gly Trp Glu Asp Leu 180 185 190 GCT TCT ACA ATT AAT AAC ATG ATG TAT CTT GAT ATA CCG TTT ATT GCT 5717 Ala Ser Thr He Asn Asn Met Met Tyr Leu Asp He Pro Phe He Ala 195 200 205 TTT ACT GCA AAT AAC GAT AAT TGG GTC AAG CAA GAT GAA GTT ATC ACA 5765 Phe Thr Ala Asn Asn Asp Asn Trp Val Lys Gin Asp Glu Val He Thr 210 215 220 TTG TTA TCA AAT ATT CGT AGT AAT CGA TGC AAG ATA TAT TCT TTG TTA 5813 Leu Leu Ser Asn He Arg Ser Asn Arg Cys Lys He Tyr Ser Leu Leu 225 230 235 GGA AGT TCG CAT GAC TTG AGT GAA AAT TTA GTG GTC CTG CGC AAT TTT 5861 Gly Ser Ser His Asp Leu Ser Glu Asn Leu Val Val Leu Arg Asn Phe 240 245 250 255 TAT CAA TCG GTT ACG AAA GCC GCT ATC GCG ATG GAT AAT GAT CAT CTG 5909 Tyr Gin Ser Val Thr Lys Ala Ala He Ala Met Asp Asn Asp His Leu 260 265 270 GAT ATT GAT GTT GAT ATT ACT GAA CCG TCA TTT GAA CAT TTA ACT ATT 5957 Asp He Asp Val Asp He Thr Glu Pro Ser Phe Glu His Leu Thr He 275 280 285 GCG ACA GTC AAT GAA CGC CGA ATG AGA ATT GAG ATT GAA AAT CAA GCA 6005 Ala Thr Val Asn Glu Arg Arg Met Arg He Glu He Glu Asn Gin Ala 290 295 300 ATT TCT CTG TCT TAAAATCTAT TGAGATATTC TATCACTCAA ATAGCAATAT 6057 He Ser Leu Ser 305 AAGGACTCTC T ATG AAA TTT GGA AAC TTT TTG CTT ACA TAC CAA CCT CCC 6107 Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gin Pro Pro 1 5 10 CAA TTT TCT CAA ACA GAG GTA ATG AAA CGT TTG GTT AAA TTA GGT CGC 6155 Gin Phe Ser Gin Thr Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg 15 20 25 ATC TCT GAG GAG TGT GGT TTT GAT ACC GTA TGG TTA CTG GAG CAT CAT 6203 He Ser Glu Glu Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His His 30 35 40 45 TTC ACG GAG TTT GGT TTG CTT GGT AAC CCT TAT GTC GCT GCT GCA TAT 6251 Phe Thr Glu Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr 50 55 60 TTA CTT GGC GCG ACT AAA AAA TTG AAT GTA GGA ACT GCC GCT ATT GTT 6299 Leu Leu Gly Ala Thr Lys Lys Leu Asn Val Gly Thr Ala Ala He Val 65 70 75 CTT CCC ACA GCC CAT CCA GTA CGC CAA CTT GAA GAT GTG AAT TTA TTG 6347 Leu Pro Thr Ala His Pro Val Arg Gin Leu Glu Asp Val Asn Leu Leu 80 85 90 GAT CAA ATG TCA AAA GGA CGA TTT CGG TTT GGT ATT TGC CGA GGG CTT 6395 Asp Gin Met Ser Lys Gly Arg Phe Arg Phe Gly He Cys Arg Gly Leu 95 100 105 TAC AAC AAG GAC TTT CGC GTA TTC GGC ACA GAT ATG AAT AAC AGT CGC 6443 Tyr Asn Lys Asp Phe Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg 110 115 120 125 GCC TTA GCG GAA TGC TGG TAC GGG CTG ATA AAG AAT GGC ATG ACA GAG 6491 Ala Leu Ala Glu Cys Trp Tyr Gly Leu He Lys Asn Gly Met Thr Glu 130 135 140 GGA TAT ATG GAA GCT GAT AAT GAA CAT ATC AAG TTC CAT AAG GTA AAA 6539 Gly Tyr Met Glu Ala Asp Asn Glu His He Lys Phe His Lys Val Lys 145 150 155 GTA AAC CCC GCG GCG TAT AGC AGA GGT GGC GCA CCG GTT TAT GTG GTG 6587 Val Asn Pro Ala Ala Tyr Ser Arg Gly Gly Ala Pro Val Tyr Val Val 160 165 170 GCT GAA TCA GCT TCG ACG ACT GAG TGG GCT GCT CAA TTT GGC CTA CCG 6635 Ala Glu Ser Ala Ser Thr Thr Glu Trp Ala Ala Gin Phe Gly Leu Pro 175 180 185 ATG ATA TTA AGT TGG ATT ATA AAT ACT AAC GAA AAG AAA GCA CAA CTT 6683 Met He Leu Ser Trp He He Asn Thr Asn Glu Lys Lys Ala Gin Leu 190 195 200 205 GAG CTT TAT AAT GAA GTG GCT CAA GAA TAT GGG CAC GAT ATT CAT AAT 6731 Glu Leu Tyr Asn Glu Val Ala Gin Glu Tyr Gly His Asp He His Asn 210 215 220 ATC GAC CAT TGC TTA TCA TAT ATA ACA TCT GTA GAT CAT GAC TCA ATT 6779 He Asp His Cys Leu Ser Tyr He Thr Ser Val Asp His Asp Ser He 225 230 235 AAA GCG AAA GAG ATT TGC CGG AAA TTT CTG GGG CAT TGG TAT GAT TCT 6827 Lys Ala Lys Glu He Cys Arg Lys Phe Leu Gly His Trp Tyr Asp Ser 240 245 250 TAT GTG AAT GCT ACG ACT ATT TTT GAT GAT TCA GAC CAA ACA AGA GGT 6875 Tyr Val Asn Ala Thr Thr He Phe Asp Asp Ser Asp Gin Thr Arg Gly 255 260 265 TAT GAT TTC AAT AAA GGG CAG TGG CGT GAC TTT GTA TTA AAA GGA CAT 6923 Tyr Asp Phe Asn Lys Gly Gin Trp Arg Asp Phe Val Leu Lys Gly His 270 275 280 285 AAA GAT ACT AAT CGC CGT ATT GAT TAC AGT TAC GAA ATC AAT CCC GTG 6971 Lys Asp Thr Asn Arg Arg He Asp Tyr Ser Tyr Glu He Asn Pro Val 290 295 300 GGA ACG CCG CAG GAA TGT ATT GAC ATA ATT CAA AAA GAC ATT GAT GCT 7019 Gly Thr Pro Gin Glu Cys He Asp He He Gin Lys Asp He Asp Ala 305 310 " 315 ACA GGA ATA TCA AAT ATT TGT TGT GGA TTT GAA GCT AAT GGA ACA GTA 7067 Thr Gly He Ser Asn He Cys Cys Gly Phe Glu Ala Asn Gly Thr Val 320 325 330 GAC GAA ATT ATT GCT TCC ATG AAG CTC TTC CAG TCT GAT GTC ATG CCA 7115 Asp Glu He He Ala Ser Met Lys Leu Phe Gin Ser Asp Val Met Pro 335 340 345 TTT CTT AAA GAA AAA CAA CGT TCG CTA TTA TAT TAGCTAAGGA GAAAGAA 7165 Phe Leu Lys Glu Lys Gin Arg Ser Leu Leu Tyr 350 355 360 ATG AAA TTT GGA TTG TTC TTC CTT AAC TTC ATC AAT TCA ACA ACT GTT 7213 Met Lys Phe Gly Leu Phe Phe Leu Asn Phe He Asn Ser Thr Thr Val 1 5 10 15 CAA GAA CAA AGT ATA GTT CGC ATG CAG GAA ATA ACG GAG TAT GTT GAT 7261 Gin Glu Gin Ser He Val Arg Met Gin Glu He Thr Glu Tyr Val Asp 20 25 30 AAG TTG AAT TTT GAA CAG ATT TTA GTG TAT GAA AAT CAT TTT TCA GAT 7309 Lys Leu Asn Phe Glu Gin He Leu Val Tyr Glu Asn His Phe Ser Asp 35 40 45 AAT GGT GTT GTC GGC GCT CCT CTG ACT GTT TCT GGT TTT CTG CTC GGT 7357 Asn Gly Val Val Gly Ala Pro Leu Thr Val Ser Gly Phe Leu Leu Gly 50 55 60 TTA ACA GAG AAA ATT AAA ATT GGT TCA TTA AAT CAC ATC ATT ACA ACT 7405 Leu Thr Glu Lys He Lys He Gly Ser Leu Asn His He He Thr Thr 65 70 75 80 CAT CAT CCT GTC GCC ATA GCG GAG GAA GCT TGC TTA TTG GAT CAG TTA 7453 His His Pro Val Ala He Ala Glu Glu Ala Cys Leu Leu Asp Gin Leu 85 90 95 AGT GAA GGG AGA TTT ATT TTA GGG TTT AGT GAT TGC GAA AAA AAA GAT 7501 Ser Glu Gly Arg Phe He Leu Gly Phe Ser Asp Cys Glu Lys Lys Asp 100 105 110 GAA ATG CAT TTT TTT AAT CGC CCG GTT GAA TAT CAA CAG CAA CTA TTT 7549 Glu Met His Phe Phe Asn Arg Pro Val Glu Tyr Gin Gin Gin Leu Phe 115 120 125 GAA GAG TGT TAT GAA ATC ATT AAC GAT GCT TTA ACA ACA GGC TAT TGT 7597 Glu Glu Cys Tyr Glu He He Asn Asp Ala Leu Thr Thr Gly Tyr Cys 130 135 140 AAT CCA GAT AAC GAT TTT TAT AGC TTC CCT AAA ATA TCT GTA AAT CCC 7645 Asn Pro Asp Asn Asp Phe Tyr Ser Phe Pro Lys He Ser Val Asn Pro 145 150 155 160 CAT GCT TAT ACG CCA GGC GGA CCT CGG AAA TAT GTA ACA GCA ACC AGT 7693 His Ala Tyr Thr Pro Gly Gly Pro Arg Lys Tyr Val Thr Ala Thr Ser 165 170 175 CAT CAT ATT GTT GAG TGG GCG GCC AAA AAA GGT ATT CCT CTC ATC TTT 7741 His His He Val Glu Trp Ala Ala Lys Lys Gly He Pro Leu He Phe 180 185 190 AAG TGG GAT GAT TCT AAT GAT GTT AGA TAT GAA TAT GCT GAA AGA TAT 7789 Lys Trp Asp Asp Ser Asn Asp Val Arg Tyr Glu Tyr Ala Glu Arg Tyr 195 200 205 AAA GCC GTT GCG GAT AAA TAT GAC GTT GAC CTA TCA GAG ATA GAC CAT 7837 Lys Ala Val Ala Asp Lys Tyr Asp Val Asp Leu Ser Glu He Asp His 210 215 220 CAG TTA ATG ATA TTA GTT AAC TAT AAC GAA GAT AGT AAT AAA GCT AAA 7885 Gin Leu Met He Leu Val Asn Tyr Asn Glu Asp Ser Asn Lys Ala Lys 225 230 235 240 CAA GAG ACG CGT GCA TTT ATT AGT GAT TAT GTT CTT GAA ATG CAC CCT 7933 Gin Glu Thr Arg Ala Phe He Ser Asp Tyr Val Leu Glu Met His Pro 245 250 255 AAT GAA AAT TTC GAA AAT AAA CTT GAA GAA ATA ATT GCA GAA AAC GCT 7981 Asn Glu Asn Phe Glu Asn Lys Leu Glu Glu He He Ala Glu Asn Ala 260 265 270 GTC GGA AAT TAT ACG GAG TGT ATA ACT GCG GCT AAG TTG GCA ATT GAA 8029 Val Gly Asn Tyr Thr Glu Cys He Thr Ala Ala Lys Leu Ala He Glu 275 280 285 AAG TGT GGT GCG AAA AGT GTA TTG CTG TCC TTT GAA CCA ATG AAT GAT 8077 Lys Cys Gly Ala Lys Ser Val Leu Leu Ser Phe Glu Pro Met Asn Asp 290 295 300 TTG ATG AGC CAA AAA AAT GTA ATC AAT ATT GTT GAT GAT AAT ATT AAG 8125 Leu Met Ser Gin Lys Asn Val He Asn He Val Asp Asp Asn He Lys 305 310 315 320 AAG TAC CAC ATG GAA TAT ACC TAATAGATTT CGAGTTGCAG CGAGGCGGCA 8176 Lys Tyr His Met Glu Tyr Thr 325 AGTGAACGAA TCCCCAGGAG CATAGATAAC TATGTGACTG GGGTGAGTGA AAGCAGCCAA 8236 CAAAGCAGCA GCTTGAAAG ATG AAG GGT ATA AAA GAG TAT GAC AGC AGT GCT 8288 Met Lys Gly He Lys Glu Tyr Asp Ser Ser Ala 1 5 10 GCC ATA CTT TCT AAT ATT ATC TTG AGG AGT AAA ACA GGT ATG ACT TCA 8336 Ala He Leu Ser Asn He He Leu Arg Ser Lys Thr Gly Met Thr Ser 15 20 25 TAT GTT GAT AAA CAA GAA ATT ACA GCA AGC TCA GAA ATT GAT GAT TTG 8384 Tyr Val Asp Lys Gin Glu He Thr Ala Ser Ser Glu He Asp Asp Leu 30 35 40 ATT TTT TCG AGC GAT CCA TTA GTG TGG TCT TAC GAC GAG CAG GAA AAA 8432 He Phe Ser Ser Asp Pro Leu Val Trp Ser Tyr Asp Glu Gin Glu Lys 45 50 55 ATC AGA AAG AAA CTT GTG CTT GAT GCA TTT CGT AAT CAT TAT AAA CAT 8480 He Arg Lys Lys Leu Val Leu Asp Ala Phe Arg Asn His Tyr Lys His 60 65 70 75 TGT CGA GAA TAT CGT CAC TAC TGT CAG GCA CAC AAA GTA GAT GAC AAT 8528 Cys Arg Glu Tyr Arg His Tyr Cys Gin Ala His Lys Val Asp Asp Asn 80 85 90 ATT ACG GAA ATT GAT GAC ATA CCT GTA TTC CCA ACA TCG GTT TTT AAG 8576 He Thr Glu He Asp Asp He Pro Val Phe Pro Thr Ser Val Phe Lys 95 100 105 TTT ACT CGC TTA TTA ACT TCT CAG GAA AAC GAG ATT GAA AGT TGG TTT 8624 Phe Thr Arg Leu Leu Thr Ser Gin Glu Asn Glu He Glu Ser Trp Phe 110 115 120 ACC AGT AGC GGC ACG AAT GGT TTA AAA AGT CAG GTG GCG CGT GAC AGA 8672 Thr Ser Ser Gly Thr Asn Gly Leu Lys Ser Gin Val Ala Arg Asp Arg 125 130 135 TTA AGT ATT GAG AGA CTC TTA GGC TCT GTG AGT TAT GGC ATG AAA TAT 8720 Leu Ser He Glu Arg Leu Leu Gly Ser Val Ser Tyr Gly Met Lys Tyr 140 145 150 155 GTT GGT AGT TGG TTT GAT CAT CAA ATA GAA TTA GTC AAT TTG GGA CCA 8768 Val Gly Ser Trp Phe Asp His Gin He Glu Leu Val Asn Leu Gly Pro 160 165 170 GAT AGA TTT AAT GCT CAT AAT ATT TGG TTT AAA TAT GTT ATG AGT TTG 8816 Asp Arg Phe Asn Ala His Asn He Trp Phe Lys Tyr Val Met Ser Leu 175 180 185 GTG GAA TTG TTA TAT CCT ACG ACA TTT ACC GTA ACA GAA GAA CGA ATA 8864 Val Glu Leu Leu Tyr Pro Thr Thr Phe Thr Val Thr Glu Glu Arg He 190 195 200 GAT TTT GTT AAA ACA TTG AAT AGT CTT GAA CGA ATA AAA AAT CAA GGG 8912 Asp Phe Val Lys Thr Leu Asn Ser Leu Glu Arg He Lys Asn Gin Gly 205 210 215 AAA GAT CTT TGT CTT ATT GGT TCG CCA TAC TTT ATT TAT TTA CTC TGC 8960 Lys Asp Leu Cys Leu He Gly Ser Pro Tyr Phe He Tyr Leu Leu Cys 220 225 230 235 CAT TAT ATG AAA GAT AAA AAA ATC TCA TTT TCT GGA GAT AAA AGC CTT 9008 His Tyr Met Lys Asp Lys Lys He Ser Phe Ser Gly Asp Lys Ser Leu 240 245 250 TAT ATC ATA ACC GGA GGC GGC TGG AAA AGT TAC GAA AAA GAA TCT CTG 9056 Tyr He He Thr Gly Gly Gly Trp Lys Ser Tyr Glu Lys Glu Ser Leu 255 260 265 AAA CGT GAT GAT TTC AAT CAT CTT TTA TTT GAT ACT TTC AAT CTC AGT 9104 Lys Arg Asp Asp Phe Asn His Leu Leu Phe Asp Thr Phe Asn Leu Ser 270 275 280 GAT ATT AGT CAG ATC CGA GAT ATA TTT AAT CAA GTT GAA CTC AAC ACT 9152 Asp He Ser Gin He Arg Asp He Phe Asn Gin Val Glu Leu Asn Thr 285 290 295 TGT TTC TTT GAG GAT GAA ATG CAG CGT AAA CAT GTT CCG CCG TGG GTA 9200 Cys Phe Phe Glu Asp Glu Met Gin Arg Lys His Val Pro Pro Trp Val 300 305 310 315 TAT GCG CGA GCG CTT GAT CCT GAA ACG TTG AAA CCT GTA CCT GAT GGA 9248 Tyr Ala Arg Ala Leu Asp Pro Glu Thr Leu Lys Pro Val Pro Asp Gly 320 325 330 ACG CCG GGG TTG ATG AGT TAT ATG GAT GCG TCA GCA ACC AGT TAT CCA 9296 Thr Pro Gly Leu Met Ser Tyr Met Asp Ala Ser Ala Thr Ser Tyr Pro 335 340 345 GCA TTT ATT GTT ACC GAT GAT GTC GGG ATA ATT AGC AGA GAA TAT GGT 9344 Ala Phe He Val Thr Asp Asp Val Gly He He Ser Arg Glu Tyr Gly 350 355 360 AAG TAT CCC GGC GTG CTC GTT GAA ATT TTA CGT CGC GTC AAT ACG AGG 9392 Lys Tyr Pro Gly Val Leu Val Glu He Leu Arg Arg Val Asn Thr Arg 365 370 375 ACG CAG AAA GGG TGT GCT TTA AGC TTA ACC GAA GCG TTT GAT AGT 9437 Thr Gin Lys Gly Cys Ala Leu Ser Leu Thr Glu Ala Phe Asp Ser 380 385 390 TGATATCCTT TGCCTAATTG TAAGTGGAAT GCTTGCGTTA TATAAATCTG AATGACATCT 9497 ACACTTTACA AAATTCTCCA AAACATCCAC ATTTGGGTAC TTGATAGAGG TTTATGGGGT 9557 TGGCTTAACA TTGTTCTCAT TGTTATTATT GGCTCAAAGC AAAAGGAGAT AACATGAAAA 9617 AATTGGCAGT TATGCTTGCA TTGGGAATGA TTAGCTTTGG TGCAATGGCA GTTGATGGGT 9677 ATAAAGATGC AAAGTTTGGC ATGACAGAAG AAGAGTTTCT TTCGAAGAGG TTATGTGATT 9737 TTGAAAAATT TGAGGGAGAT TCTCGAATAG AAGAAGTATC ACTTTATTCA TGTTCTGACT 9797 TTTCGTTTGC TAACAAAAAG CGTGAAGCAA TGGCATTTTT TTTAAATGGG AAATTTAAAA 9857 GATTAGAGAT TAATATTGGC AGACTTGTGA AGCCAGTAAG CAAATCGTTA ACGAAAAAGT 9917 ACGGAGATGG ATCATCGTAT CCATCAAAAG AAGAATTTGA GAACGCGCTA AAATACAATG 9977 GAACTATGTC TATAGGTTAT GATAATAATA CGGTATTAGT TGATATACAT ATAATATGTG 10037 GCAAAGAAGG CATAGAAACC AGTCAACTGA TTTATACGAG TCCAGATGTT TATACGCTCC 10097 CAGATTTCGG AGAAAAAATC CAGGAATTAA AGGGATTAAA GGAATTCGAG CTCGGTACCC 10157 GGGGATCCCT CGAGGTCGAC CTGCAGGCAG CGCTTGGCGT CACCCGCAGT TCGGTGGTTA 10217 ATA 10220 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 483 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ala Asn Met Thr Lys Lys He Ser Phe He He Asn Gly Gin Val 1 5 10 15 Glu He Phe Pro Glu Ser Asp Asp Leu Val Gin Ser He Asn Phe Gly 20 25 30 Asp Asn Ser Val Tyr Leu Pro He Leu Asn Asp Ser His Val Lys Asn 35 40 45 He He Asp Cys Asn Gly Asn Asn Glu Leu Arg Leu His Asn He Val 50 55 60 Asn Phe Leu Tyr Thr Val Gly Gin Arg Trp Lys Asn Glu Glu Tyr Ser 65 70 75 80 Arg Arg Arg Thr Tyr He Arg Asp Leu Lys Lys Tyr Met Gly Tyr Ser 85 90 95 Glu Glu Met Ala Lys Leu Glu Ala Asn Trp He Ser Met He Leu Cys 100 105 110 Ser Lys Gly Gly Leu Tyr Asp Val Val Glu Asn Glu Leu Gly Ser Arg 115 120 125 His He Met Asp Glu Trp Leu Pro Gin Asp Glu Ser Tyr Val Arg Ala 130 135 140 Phe Pro Lys Gly Lys Ser Val His Leu Leu Ala Gly Asn Val Pro Leu 145 150 155 160 Ser Gly He Met Ser He Leu Arg Ala He Leu Thr Lys Asn Gin Cys 165 170 175 He He Lys Thr Ser Ser Thr Asp Pro Phe Thr Ala Asn Ala Leu Ala 180 185 190 Leu Ser Phe He Asp Val Asp Pro Asn His Pro He Thr Arg Ser Leu 195 200 205 Ser Val He Tyr Trp Pro His Gin Gly Asp Thr Ser Leu Ala Lys Glu 210 215 220 He Met Arg His Ala Asp Val He Val Ala Trp Gly Gly Pro Asp Ala 225 230 235 240 He Asn Trp Ala Val Glu His Ala Pro Ser Tyr Ala Asp Val He Lys 245 250 255 Phe Gly Ser Lys Lys Ser Leu Cys He He Asp Asn Pro Val Asp Leu 260 265 270 Thr Ser Ala Ala Thr Gly Ala Ala His Asp Val Cys Phe Tyr Asp Gin 275 280 285 Arg Ala Cys Phe Ser Ala Gin Asn He Tyr Tyr Met Gly Asn His Tyr 290 295 300 Glu Glu Phe Lys Leu Ala Leu He Glu Lys Leu Asn Leu Tyr Ala His 305 310 315 320 He Leu Pro Asn Ala Lys Lys Asp Phe Asp Glu Lys Ala Ala Tyr Ser 325 330 335 Leu Val Gin Lys Glu Ser Leu Phe Ala Gly Leu Lys Val Glu Val Asp 340 345 350 He His Gin Arg Trp Met He He Glu Ser Asn Ala Gly Val Glu Phe 355 360 365 Asn Gin Pro Leu Gly Arg Cys Val Tyr Leu His His Val Asp Asn He 370 375 380 Glu Gin He Leu Pro Tyr Val Gin Lys Asn Lys Thr Gin Thr He Ser 385 390 395 400 He Phe Pro Trp Glu Ser Ser Phe Lys Tyr Arg Asp Ala Leu Ala Leu 405 410 415 Lys Gly Ala Glu Arg He Val Glu Ala Gly Met Asn Asn He Phe Arg 420 425 430 Val Gly Gly Ser His Asp Gly Met Arg Pro Leu Gin Arg Leu Val Thr 435 440 445 Tyr He Ser His Glu Arg Pro Ser Asn Tyr Thr Ala Lys Asp Val Ala 450 455 460 Val Glu He Glu Gin Thr Arg Phe Leu Glu Glu Asp Lys Phe Leu Val 465 470 475 480 Phe Val Pro (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 307 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Glu Asn Glu Ser Lys Tyr Lys Thr He Asp His Val He Cys Val 1 5 10 15 Glu Gly Asn Lys Lys He His Val Trp Glu Thr Leu Pro Glu Glu Asn 20 25 30 Ser Pro Lys Arg Lys Asn Ala He He He Ala Ser Gly Phe Ala Arg 35 40 45 Arg Met Asp His Phe Ala Gly Leu Ala Glu Tyr Leu Ser Arg Asn Gly 50 55 60 Phe His Val He Arg Tyr Asp Ser Leu His His Val Gly Leu Ser Ser 65 70 75 80 Gly Thr He Asp Glu Phe Thr Met Ser He Gly Lys Gin Ser Leu Leu 85 90 95 Ala Val Val Asp Trp Leu Thr Thr Arg Lys He Asn Asn Phe Gly Met 100 105 110 Leu Ala Ser Ser Leu Ser Ala Arg He Ala Tyr Ala Ser Leu Ser Glu 115 120 125 He Asn Ala Ser Phe Leu He Thr Ala Val Gly Val Val Asn Leu Arg 130 135 140 Tyr Ser Leu Glu Arg Ala Leu Gly Phe Asp Tyr Leu Ser Leu Pro He 145 150 155 160 Asn Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly Ala 165 170 175 Glu Val Phe Ala Arg Asp Cys Leu Asp Phe Gly Trp Glu Asp Leu Ala 180 185 190 Ser Thr He Asn Asn Met Met Tyr Leu Asp He Pro Phe He Ala Phe 195 200 205 Thr Ala Asn Asn Asp Asn Trp Val Lys Gin Asp Glu Val He Thr Leu 210 215 220 Leu Ser Asn He Arg Ser Asn Arg Cys Lys He Tyr Ser Leu Leu Gly 225 230 235 240 Ser Ser His Asp Leu Ser Glu Asn Leu Val Val Leu Arg Asn Phe Tyr 245 250 255 Gin Ser Val Thr Lys Ala Ala He Ala Met Asp Asn Asp His Leu Asp 260 265 270 He Asp Val Asp He Thr Glu Pro Ser Phe Glu His Leu Thr He Ala 275 280 285 Thr Val Asn Glu Arg Arg Met Arg He Glu He Glu Asn Gin Ala He 290 295 300 Ser Leu Ser 305 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 360 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gin Pro Pro Gin Phe Ser 1 5 10 15 Gin Thr Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg He Ser Glu 20 25 30 Glu Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His His Phe Thr Glu 35 40 45 Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr Leu Leu Gly 50 55 60 Ala Thr Lys Lys Leu Asn Val Gly Thr Ala Ala He Val Leu Pro Thr 65 70 75 80 Ala His Pro Val Arg Gin Leu Glu Asp Val Asn Leu Leu Asp Gin Met 85 90 95 Ser Lys Gly Arg Phe Arg Phe Gly He Cys Arg Gly Leu Tyr Asn Lys 100 105 110 Asp Phe Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg Ala Leu Ala 115 120 125 Glu Cys Trp Tyr Gly Leu He Lys Asn Gly Met Thr Glu Gly Tyr Met 130 135 140 Glu Ala Asp Asn Glu His He Lys Phe His Lys Val Lys Val Asn Pro 145 150 155 160 Ala Ala Tyr Ser Arg Gly Gly Ala Pro Val Tyr Val Val Ala Glu Ser 165 170 175 Ala Ser Thr Thr Glu Trp Ala Ala Gin Phe Gly Leu Pro Met He Leu 180 185 190 Ser Trp He He Asn Thr Asn Glu Lys Lys Ala Gin Leu Glu Leu Tyr 195 200 205 Asn Glu Val Ala Gin Glu Tyr Gly His Asp He His Asn He Asp His 210 215 220 Cys Leu Ser Tyr He Thr Ser Val Asp His Asp Ser He Lys Ala Lys 225 230 235 240 Glu He Cys Arg Lys Phe Leu Gly His Trp Tyr Asp Ser Tyr Val Asn 245 250 255 Ala Thr Thr He Phe Asp Asp Ser Asp Gin Thr Arg Gly Tyr Asp Phe 260 265 270 sn Lys Gly Gin Trp Arg Asp Phe Val Leu Lys Gly His Lys Asp Thr 275 280 285 Asn Arg Arg He Asp Tyr Ser Tyr Glu He Asn Pro Val Gly Thr Pro 290 295 300 Gin Glu Cys He Asp He He Gin Lys Asp He Asp Ala Thr Gly He 305 310 315 320 Ser Asn He Cys Cys Gly Phe Glu Ala Asn Gly Thr Val Asp Glu He 325 330 335 He Ala Ser Met Lys Leu Phe Gin Ser Asp Val Met Pro Phe Leu Lys 340 345 350 Glu Lys Gin Arg Ser Leu Leu Tyr 355 360 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 327 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Lys Phe Gly Leu Phe Phe Leu Asn Phe He Asn Ser Thr Thr Val 1 5 10 15 Gin Glu Gin Ser He Val Arg Met Gin Glu He Thr Glu Tyr Val Asp 20 25 30 Lys Leu Asn Phe Glu Gin He Leu Val Tyr Glu Asn His Phe Ser Asp 35 40 45 Asn Gly Val Val Gly Ala Pro Leu Thr Val Ser Gly Phe Leu Leu Gly 50 55 60 Leu Thr Glu Lys He Lys He Gly Ser Leu Asn His He He Thr Thr 65 70 75 80 His His Pro Val Ala He Ala Glu Glu Ala Cys Leu Leu Asp Gin Leu 85 90 95 Ser Glu Gly Arg Phe He Leu Gly Phe Ser Asp Cys Glu Lys Lys Asp 100 105 110 Glu Met His Phe Phe Asn Arg Pro Val Glu Tyr Gin Gin Gin Leu Phe 115 120 125 Glu Glu Cys Tyr Glu He He Asn Asp Ala Leu Thr Thr Gly Tyr Cys 130 135 140 Asn Pro Asp Asn Asp Phe Tyr Ser Phe Pro Lys He Ser Val Asn Pro 145 150 155 160 His Ala Tyr Thr Pro Gly Gly Pro Arg Lys Tyr Val Thr Ala Thr Ser 165 170 175 His His He Val Glu Trp Ala Ala Lys Lys Gly He Pro Leu He Phe 180 185 190 Lys Trp Asp Asp Ser Asn Asp Val Arg Tyr Glu Tyr Ala Glu Arg Tyr 195 200 205 Lys Ala Val Ala Asp Lys Tyr Asp Val Asp Leu Ser Glu He Asp His 210 215 220 Gin Leu Met He Leu Val Asn Tyr Asn Glu Asp Ser Asn Lys Ala Lys 225 230 235 240 Gin Glu Thr Arg Ala Phe He Ser Asp Tyr Val Leu Glu Met His Pro 245 250 255 Asn Glu Asn Phe Glu Asn Lys Leu Glu Glu He He Ala Glu Asn Ala 260 265 270 Val Gly Asn Tyr Thr Glu Cys He Thr Ala Ala Lys Leu Ala He Glu 275 280 285 Lys Cys Gly Ala Lys Ser Val Leu Leu Ser Phe Glu Pro Met Asn Asp 290 295 300 Leu Met Ser Gin Lys Asn Val He Asn He Val Asp Asp Asn He Lys 305 310 315 320 Lys Tyr His Met Glu Tyr Thr 325 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 394 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Lys Gly He Lys Glu Tyr Asp Ser Ser Ala Ala He Leu Ser Asn 1 5 10 15 He He Leu Arg Ser Lys Thr Gly Met Thr Ser Tyr Val Asp Lys Gin 20 25 30 Glu He Thr Ala Ser Ser Glu He Asp Asp Leu He Phe Ser Ser Asp 35 40 45 Pro Leu Val Trp Ser Tyr Asp Glu Gin Glu Lys He Arg Lys Lys Leu 50 55 60 Val Leu Asp Ala Phe Arg Asn His Tyr Lys His Cys Arg Glu Tyr Arg 65 70 75 80 His Tyr Cys Gin Ala His Lys Val Asp Asp Asn He Thr Glu He Asp 85 90 95 Asp He Pro Val Phe Pro Thr Ser Val Phe Lys Phe Thr Arg Leu Leu 100 105 110 Thr Ser Gin Glu Asn Glu He Glu Ser Trp Phe Thr Ser Ser Gly Thr 115 120 125 Asn Gly Leu Lys Ser Gin Val Ala Arg Asp Arg Leu Ser He Glu Arg 130 135 140 Leu Leu Gly Ser Val Ser Tyr Gly Met Lys Tyr Val Gly Ser Trp Phe 145 150 155 160 Asp His Gin He Glu Leu Val Asn Leu Gly Pro Asp Arg Phe Asn Ala 165 170 175 His Asn He Trp Phe Lys Tyr Val Met Ser Leu Val Glu Leu Leu Tyr 180 185 190 Pro Thr Thr Phe Thr Val Thr Glu Glu Arg He Asp Phe Val Lys Thr 195 200 205 Leu Asn Ser Leu Glu Arg He Lys Asn Gin Gly Lys Asp Leu Cys Leu 210 215 220 He Gly Ser Pro Tyr Phe He Tyr Leu Leu Cys His Tyr Met Lys Asp 225 230 235 240 Lys Lys He Ser Phe Ser Gly Asp Lys Ser Leu Tyr He He Thr Gly 245 250 255 Gly Gly Trp Lys Ser Tyr Glu Lys Glu Ser Leu Lys Arg Asp Asp Phe 260 265 270 Asn His Leu Leu Phe Asp Thr Phe Asn Leu Ser Asp He Ser Gin He 275 280 285 Arg Asp He Phe Asn Gin Val Glu Leu Asn Thr Cys Phe Phe Glu Asp 290 295 300 Glu Met Gin Arg Lys His Val Pro Pro Trp Val Tyr Ala Arg Ala Leu 305 310 315 320 Asp Pro Glu Thr Leu Lys Pro Val Pro Asp Gly Thr Pro Gly Leu Met 325 330 335 Ser Tyr Met Asp Ala Ser Ala Thr Ser Tyr Pro Ala Phe He Val Thr 340 345 350 Asp Asp Val Gly He He Ser Arg Glu Tyr Gly Lys Tyr Pro Gly Val 355 360 365 Leu Val Glu He Leu Arg Arg Val Asn Thr Arg Thr Gin Lys Gly Cys 370 375 380 Ala Leu Ser Leu Thr Glu Ala Phe Asp Ser 385 390 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3098 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vii) IMMEDIATE SOURCE: (B) CLONE: pASK75 (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: vector (ix) FEATURE: (A) NAME/KEY: promoter (B) LOCATION: 542..672 (D) OTHER INFORMATION :/function= "beta-la promoter" /label= beta-la /citation= ([1]) ( ix) FEATURE : (A) NAME/KEY: CDS (B) LOCATION: 673..1530 (D) OTHER INFORMATION: /product= "beta-la" /citation^ ([1]) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1543..2163 (D) OTHER INFORMATION: /product= "tetR" /citations ([1]) (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 2713..2950 (D) OTHER INFORMATION: /functions "ORI" /label= ORI /citations ([1]) (ix) FEATURE: (A) NAME/KEY: promoter (B) LOCATION: 2976..3073 (D) OTHER INFORMATION: /functions "p tetA promoter" /citations ( [i] ) (X) PUBLICATION INFORMATION: (A) AUTHORS: Skerra, A (B) TITLE: Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli (C) JOURNAL: Gene (D) VOLUME: 151 (E) ISSUE: 1-2 (F) PAGES: 131-135 (G) DATE: 30-DEC-1994 (K) RELEVANT RESIDUES IN SEQ ID NO: 9: FROM 1 TO 3098 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGCTTGACCT GTGAAGTGAA AAATGGCGCA CATTGTGCGA CATTTTTTTT GTCTGCCGTT 60 TACCGCTACT GCGTCACGGA TCTCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT 120 GTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC 180 GCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG 240 GGGCTCCCTT TAGGGTTCCG ATTTAGTGCT TTACGGCACC TCGACCCCAA AAAACTTGAT 300 TAGGGTGATG GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG 360 TTGGAGTCCA CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT 420 ATCTCGGTCT ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA 480 AATGAGCTGA TTTAACAAAA ATTTAACGCG AATTTTAACA AAATATTAAC GCTTACAATT 540 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA 600 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA 660 AAAAGGAAGA GT ATG AGT ATT CAA CAT TTC CGT GTC GCC CTT ATT CCC 708 Met Ser He Gin His Phe Arg Val Ala Leu He Pro 395 400 405 TTT TTT GCG GCA TTT TGC CTT CCT GTT TTT GCT CAC CCA GAA ACG CTG 756 Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu 410 415 420 GTG AAA GTA AAA GAT GCT GAA GAT CAG TTG GGT GCA CGA GTG GGT TAC 804 Val Lys Val Lys Asp Ala Glu Asp Gin Leu Gly Ala Arg Val Gly Tyr 425 430 435 ATC GAA CTG GAT CTC AAC AGC GGT AAG ATC CTT GAG AGT TTT CGC CCC 852 He Glu Leu Asp Leu Asn Ser Gly Lys He Leu Glu Ser Phe Arg Pro 440 445 450 GAA GAA CGT TTT CCA ATG ATG AGC ACT TTT AAA GTT CTG CTA TGT GGC 900 Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly 455 460 465 470 GCG GTA TTA TCC CGT ATT GAC GCC GGG CAA GAG CAA CTC GGT CGC CGC 948 Ala Val Leu Ser Arg He Asp Ala Gly Gin Glu Gin Leu Gly Arg Arg 475 480 485 ATA CAC TAT TCT CAG AAT GAC TTG GTT GAG TAC TCA CCA GTC ACA GAA 996 He His Tyr Ser Gin Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu 490 495 500 AAG CAT CTT ACG GAT GGC ATG ACA GTA AGA GAA TTA TGC AGT GCT GCC 1044 Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala 505 510 515 ATA ACC ATG AGT GAT AAC ACT GCG GCC AAC TTA CTT CTG ACA ACG ATC 1092 He Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr He 520 525 530 GGA GGA CCG AAG GAG CTA ACC GCT TTT TTG CAC AAC ATG GGG GAT CAT 1140 Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His 535 540 545 550 GTA ACT CGC CTT GAT CGT TGG GAA CCG GAG CTG AAT GAA GCC ATA CCA 1188 Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala He Pro 555 560 565 AAC GAC GAG CGT GAC ACC ACG ATG CCT GTA GCA ATG GCA ACA ACG TTG 1236 Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu 570 575 580 CGC AAA CTA TTA ACT GGC GAA CTA CTT ACT CTA GCT TCC CGG CAA CAA 1284 Arg Lys Leu Leu Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gin Gin 585 590 595 TTG ATA GAC TGG ATG GAG GCG GAT AAA GTT GCA GGA CCA CTT CTG CGC 1332 Leu He Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg 600 605 610 TCG GCC CTT CCG GCT GGC TGG TTT ATT GCT GAT AAA TCT GGA GCC GGT 1380 Ser Ala Leu Pro Ala Gly Trp Phe He Ala Asp Lys Ser Gly Ala Gly 615 620 625 630 GAG CGT GGC TCT CGC GGT ATC ATT GCA GCA CTG GGG CCA GAT GGT AAG 1428 Glu Arg Gly Ser Arg Gly He He Ala Ala Leu Gly Pro Asp Gly Lys 635 640 645 CCC TCC CGT ATC GTA GTT ATC TAC ACG ACG GGG AGT CAG GCA ACT ATG 1476 Pro Ser Arg He Val Val He Tyr Thr Thr Gly Ser Gin Ala Thr Met 650 655 660 GAT GAA CGA AAT AGA CAG ATC GCT GAG ATA GGT GCC TCA CTG ATT AAG 1524 Asp Glu Arg Asn Arg Gin He Ala Glu He Gly Ala Ser Leu He Lys 665 670 675 CAT TGG TAGGAATTAA TG ATG TCT CGT TTA GAT AAA AGT AAA GTG ATT 1572 His Trp Met Ser Arg Leu Asp Lys Ser Lys Val He 680 1 5 10 AAC AGC GCA TTA GAG CTG CTT AAT GAG GTC GGA ATC GAA GGT TTA ACA 1620 Asn Ser Ala Leu Glu Leu Leu Asn Glu Val Gly He Glu Gly Leu Thr 15 20 25 ACC CGT AAA CTC GCC CAG AAG CTA GGT GTA GAG CAG CCT ACA TTG TAT 1668 Thr Arg Lys Leu Ala Gin Lys Leu Gly Val Glu Gin Pro Thr Leu Tyr 30 35 40 TGG CAT GTA AAA AAT AAG CGG GCT TTG CTC GAC GCC TTA GCC ATT GAG 1716 Trp His Val Lys Asn Lys Arg Ala Leu Leu Asp Ala Leu Ala He Glu 45 50 55 ATG TTA GAT AGG CAC CAT ACT CAC TTT TGC CCT TTA GAA GGG GAA AGC 1764 Met Leu Asp Arg His His Thr His Phe Cys Pro Leu Glu Gly Glu Ser 60 65 70 TGG CAA GAT TTT TTA CGT AAT AAC GCT AAA AGT TTT AGA TGT GCT TTA 1812 Trp Gin Asp Phe Leu Arg Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu 75 80 85 90 CTA AGT CAT CGC GAT GGA GCA AAA GTA CAT TTA GGT ACA CGG CCT ACA 1860 Leu Ser His Arg Asp Gly Ala Lys Val His Leu Gly Thr Arg Pro Thr 95 100 105 GAA AAA CAG TAT GAA ACT CTC GAA AAT CAA TTA GCC TTT TTA TGC CAA 1908 Glu Lys Gin Tyr Glu Thr Leu Glu Asn Gin Leu Ala Phe Leu Cys Gin 110 115 120 CAA GGT TTT TCA CTA GAG AAT GCA TTA TAT GCA CTC AGC GCA GTG GGG 1956 Gin Gly Phe Ser Leu Glu Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly 125 130 135 CAT TTT ACT TTA GGT TGC GTA TTG GAA GAT CAA GAG CAT CAA GTC GCT 2004 His Phe Thr Leu Gly Cys Val Leu Glu Asp Gin Glu His Gin Val Ala 140 145 150 AAA GAA GAA AGG GAA ACA CCT ACT ACT GAT AGT ATG CCG CCA TTA TTA 2052 Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp Ser Met Pro Pro Leu Leu 155 160 165 170 CGA CAA GCT ATC GAA TTA TTT GAT CAC CAA GGT GCA GAG CCA GCC TTC 2100 Arg Gin Ala He Glu Leu Phe Asp His Gin Gly Ala Glu Pro Ala Phe 175 180 185 TTA TTC GGC CTT GAA TTG ATC ATA TGC GGA TTA GAA AAA CAA CTT AAA 2148 Leu Phe Gly Leu Glu Leu He He Cys Gly Leu Glu Lys Gin Leu Lys 190 195 200 TGT GAA AGT GGG TCT TAAAAGCAGC ATAACCTTTT TCCGTGATGG TAACTTCACT 2203 Cys Glu Ser Gly Ser 205 AGTTTAAAAG GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC 2263 GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG 2323 ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG 2383 TGGTTTGTTT GCCGGACAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA 2443 GAGCGCAGAT ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA 2503 ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA 2563 GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC 2623 AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA 2683 CCGAACTGAG ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA 2743 AGGCGGACAG GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC 2803 CAGGGGGAAA CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC 2863 GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG 2923 CCTTTTTACG GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGACCCGA CACCATCGAA 2983 TGGCCAGATG ATTAATTCCT AATTTTTGTT GACACTCTAT CATTGATAGA GTTATTTTAC 3043 CACTCCCTAT CAGTGATAGA GAAAAGTGAA ATGAATAGTT CGACAAAAAT CTAGA 3098 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 286 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Ser He Gin His Phe Arg Val Ala Leu He Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gin Leu Gly Ala Arg Val Gly Tyr He Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys He Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg He Asp Ala Gly Gin Glu Gin Leu Gly Arg Arg He His Tyr Ser 85 90 95 Gin Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr 100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala He Thr Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr He Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala He Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly Glu Leu Leu Thr Leu Ala Ser Arg Gin Gin Leu He Asp Trp 195 200 205 Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215 220 Ala Gly Trp Phe He Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser 225 230 235 240 Arg Gly He He Ala Ala Leu Gly Pro Asp Gly Lys Pro Ser Arg He 245 250 255 Val Val He Tyr Thr Thr Gly Ser Gin Ala Thr Met Asp Glu Arg Asn 260 265 270 Arg Gin He Ala Glu He Gly Ala Ser Leu He Lys His Trp 275 280 285 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 207 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Ser Arg Leu Asp Lys Ser Lys Val He Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly He Glu Gly Leu Thr Thr Arg Lys Leu Ala Gin 20 25 30 Lys Leu Gly Val Glu Gin Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala He Glu Met Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gin Asp Phe Leu Arg 65 70 75 80 Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gin Tyr Glu Thr 100 105 110 Leu Glu Asn Gin Leu Ala Phe Leu Cys Gin Gin Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gin Glu His Gin Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gin Ala He Glu Leu 165 170 175 Phe Asp His Gin Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 He He Cys Gly Leu Glu Lys Gin Leu Lys Cys Glu Ser Gly Ser 195 200 205

Claims: CLAIMS 1. A method for the determination of a tetracycline in a sample characterized in that the sample is brought into contact with prokaryotic cells encompassing a DNA vector including a nucleotide sequence encoding a light producing enzyme under transcriptional control of a tetracycline repressor and a tetracycline promoter, detecting the luminescense emitted from the cells, and comparing the emitted luminescence to the luminescence emitted from cells in a control containing no tetracycline - wherein a detectable luminescence higher than a luminescence of the control indicates the presence of tetracycline in the sample. 2. The method according to claim 1 characterized in that the cells are Escherichia coli. 3. The method according to claim 1 or 2 characterized in that the DNA vector is a plasmid containing the luxCDABE genes (SEQ ID NO: 3), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from 7 0. 4. The method according to claim 3 characterized in that the DNA vector is the plasmid pTetLuxl (SEQ ID NO: 3). 5. The method according to claim 1 or 2 characterized in that - the DNA vector is a plasmid containing the insect luciferase gene (SEQ ID NO: 1), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from TnlQ, and that D-luciferin is added to the mixture of the sample and the cells in order to initiate the luminescence of the cells. 6. The method according to claim 5 characterized in that the DNA vector is the plasmid pTetLucl (SEQ ID NO: 1). 7. The method according to any of the claims 1 - 6 characterized in that the sensitivity of the analysis with respect to the tetracycline is controlled by increasing or decreasing the concentration of divalent metal ions, e.g. magnesium ions, or adjusting the pH, or combined adjusting of the divalent metal ion concentration and the pH. 8. The method according to any of the claims 1 - 6 characterized in that the sensitivity of the analysis with respect to the tetracycline derivative is increased by the use of cells which are especially antibiotic sensitive mutant strains. 9. The method according to any of the claims 1 - 8 characterized in that the luminescence is measured using an X-ray or polaroid film, a CCD-camera, a Uquid scintillation counter or a luminometer. 10. The method according to any of the claims 1 - 9 characterized in that the sample to be analyzed is milk, fish, meat, infant formula, eggs, honey, vegetables, serum, plasma, whole blood or the like. 11. A recombinant prokaryotic cell characterized in that it encompasses a DNA vector including a nucleotide sequence encoding a light producing enzyme, tetracycline repressor and tetracycline promoter. 12. The cell according to claim 11 characterized in that the DNA vector is a plasmid containing either - the luxCDABE genes (SEQ ID NO: 3), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from 7nl0 , or the insect luciferase gene (SEQ ID NO: 1), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from 7nl0. 13. The cell according to claim 11 or 12 characterized in that it is Escherichia coli. 14. The cell according to claim 12, 13 or 14, characterized in that it is in dried form, e.g. in lyophilized form. 15. A plasmid characterized in that it comprises either - the luxCDABE genes (SEQ ID NO: 3), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from TnlO , or - the insect luciferase gene (SEQ ID NO: 1), tetracycline repressor (TetR) (SEQ ID NO: 11) and tetracycline promotor (TetA) (SEQ ID NO: 9) from ThlO. 16. A plasmid according to claim 15 characterized in that it is pTetLuxl (SEQ ID NO: 3) or pTetLucl (SEQ ID NO: 1).

Inventors: KARP MATTI, KORPELA MATTI, KURITTU JUSSI, KARP, MATTI, KORPELA, MATTI, KURITTU, JUSSI
Applicants: KARP MATTI, KORPELA MATTI, KURITTU JUSSI, KARP, MATTI, KORPELA, MATTI, KURITTU, JUSSI
Application year: 1998
Main classification: C12Q1
IPC codes: C12N9, C12N15