Detection of amylase

Functional analysis of a barley high-pI alpha-amylase gene promoter has identified a gibberellin (GA) response complex in the region between -174 and -108. The sequence of the central element, TAACAAA, is very similar to the c-Myb and v-Myb consensus binding site. We investigated the possibility that a GA-regulated Myb transactivates alpha-amylase gene expression in barley aleurone cells. A cDNA clone, GAmyb, which encodes a novel Myb, was isolated from a barley aleurone cDNA library. RNA blot analysis revealed that GAmyb expression in isolated barley aleurone layers is up-regulated by GA. The kinetics of GAmyb expression indicates that it is an early event in GA-regulated gene expression and precedes alpha-amylase gene expression. Cycloheximide blocked alpha-amylase gene expression but failed to block GAmyb gene expression, indicating that protein synthesis is not required for GAmyb gene expression. Gel mobility shift experiments with recombinant GAMyb showed that GAMyb binds specifically to the TAACAAA box in vitro. We demonstrated in transient expression experiments that GAMyb activates transcription of a high-pI alpha-amylase promoter fused to a beta-glucuronidase reporter gene in the absence of GA. Our results indicate that the GAMyb is the sole GA-regulated transcription factor required for transcriptional activation of the high-pI alpha-amylase promoter. We therefore postulate that GAMyb is a part of the GA-response pathway leading to alpha-amylase gene expression in aleurone cells.

laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with direct on-site demonstration of a bioallergen exposure hazard. OBJECTIVE: In a field study, we evaluated a recently developed LFIA for fungal alpha-amylase, an important bakery allergen. METHODS: Airborne and surface dust (wipe) samples and samples from flours and baking additives used at the workplace were collected in 5 industrial bakeries and tested in the LFIA for fungal amylase. For comparison, amylase was measured in sample eluates with the reference EIA method. RESULTS: Sensitivity of the LFIA was 1 to 10 ng/mL, and of EIA, approximately 25 pg/mL. In LFIA, most flour samples, 84% of wipe samples, 26% of personal airborne dust, and none of the 26 ambient air dust samples produced a visible reaction. Wipe samples from dough-making areas and flour samples gave the strongest reactions. All extracts with >5 ng allergen per milliliter showed a positive LFIA reaction. CONCLUSION: The LFIA for fungal amylase is an easy and rapid method to demonstrate the allergen directly at the worksite in less than 10 to 20 minutes. Similar LFIA methods may be used for other occupational allergens in other work environments. CLINICAL IMPLICATIONS: Lateral flow immunoassays for occupational allergens may be of great value in occupational hygiene surveys to demonstrate directly to workers and supervisors the hazards of work-related bioallergen exposure. Publication Types: Comparative Study, Research Support, Non-U.S. Gov't

The following examples are provided as a means of illustrating the present invention and are not to be construed as a limitation thereon.

EXAMPLE 1

Detection of an αamylase gene in Bacillus caldolyticus

(a) Preparation of Probes Amy-1 and Amy-2

The oligonucleotide probes Amy-1 and Amy-2 were prepared using solid-phase synthetic methods (Alkinson et al, 1984). The scheme for synthesis of the oligomers was as outlined by Matteucci et al, supra (1981) utilizing proton activated, protected 2'-deoxy-ribonucleotide phosphoramidites (Beaucage et al, supra 1981). All sequential steps were performed in an automated manner on an Applied Biosystems Model 380 DNA Synthesizer using protected nucleotides, solvents, chemicals and reagents, all of which were obtained from Applied Biosystems, Foster City, Calif. U.S.A. The solid-phase support (also from Applied Biosystems) was controlled pore glass to which the starting 3'-nucleotide was already attached. Certain modifications were introduced into the automated reaction cycle in accordance with the manufacturer's recommendations. Upon completion of the synthesis, the oligomers were deblocked and cleaved from the solid support within the DNA synthesizer according to the manufacturer's recommendations.

Removal of the blocking groups was completed by heating the aqueous solution containing the oligomer with concentrated ammonium hydroxide at 55° centigrade (C) for from 4 to 24 hours in a sealed vial. The resulting solution was evaporated, the residue dissolved in 0.01 molar (M) triethylammonium bicarbonate buffer, pH 7.0 (TEAB buffer). This solution was chromatographed over Sephadex-G50® Gel Filtration Resin. This column was prepared in, and eluted with, the same TEAB buffer. Material eluting with the void volume was pooled and the solution evaporated. A portion of the residue (10 to 40% of the absorbance units at 260 nanometers), dissolved in loading buffer (composition: 0.1% Bromophenol Blue, 0.1% Xylene Cyanol, 10 millimolar disodium EDTA, in formamide) was further purified by electrophoresis on polyacrylamide gels. The gel size was 18×32 centimeters (cm) with a thickness of 1.5 millimeters (mm). The well size for each oligomer purified in this manner was 2 to 5 cm in width and up to five oligomers were purified using a single gel. The concentration of acrylamide in the gel varied from 14 to 20%, depending on the chain length of the desired product. For longer oligomers, the 14% gel is preferred, while shorter oligomers were purified on up to a 20% acrylamide gel. The gel also contained 7M urea and Tris-borate-EDTA buffer (0.1 M Tris, 0.1M borate, 2 millimolar EDTA, pH 8.3). The running buffer was the same Tris-Borate-EDTA mixture. Electrophoresis was carried out at 20 to 60 watts, constant power, for from 6 to 18 hours.

Following completion of the electrophoresis, the gel was encased in plastic wrap and the oligomers were visualized by shadowing with ultraviolet light. This shadowing was accomplished by placing the wrapped gel on a fluorescent thin layer chromatography plate and viewing the gel with a short wave length ultraviolet light source. The desired product appeared as the slowest migrating, major blue band by this shadowing technique. The desired band was excised from the gel. The DNA oligomer was eluted from the gel slice onto powdered diethylaminoethyl (DEAE) cellulose using an EpiGene (Baltimore, Md., U.S.A.) D-Gel® electrophoresis apparatus. The oligomer was recovered from the cellulose by elution with 1M TEAB buffer. The buffer solution containing the oligomer was evaporated, the residue dissolved in 0.01M TEAB buffer, and then desalted by passage over a column of Sephadex-G50® as described previously. The material eluting in the void volume was pooled and lyophilized to give the final product. Using these procedures, about 0.5 to 5.0 A 260 units of each of the purified oligomers was obtained.

(b) Radiolabeling of Probes Amy-1 and Amy-2

Each oligonucleotide probe (prepared as described in Example 1a) was radiolabeled in a reaction composed of the following: 0.02 A 260 units of DNA oligomer, 4.0 units of T 4 polynucleotide kinase (Boehringer-Mannheim), 2.5 microliter (μl ) of a 10X Tris buffer (as recommended by the manufacturer), 200 microcurie (μCi) γ -32 -P adenosine triphosphate (ATP) supplied by Amersham in a total volume of 25 μl. The reaction was incubated at 37° C. for 30 minutes. Then 75 μl of 0.1X saline sodium citrate (SSC) buffer (1X SSC is 0.15M NaCl, 0.015M sodium citrate, pH 8.0) was added to stop the reaction. The mixture was passed through a 1.0 milliliter (ml) Sephadex G-50 column, which retained the unincorporated ATP. The eluant consisted of 100 μl of labeled oligonucleotide with a total activity of approximately 2.5×10 8 counts per minute (cpm), as determined by Cerenkov counting in a liquid scintillation counter.

(c) Detection of an α-amylase gene in Bacillus caldolyticus

A 250 ml nutrient broth culture of Bacillus caldolyticus was grown for 18 hours at 60° C. The cells were then washed in 0.1M sodium phosphate (pH 6. and then suspended in a buffer containing 20 millimolar (mM) sodium phosphate (pH 6., 1 mM MgCl 2 and 25 percent sucrose and then treated with 500 units mutanolysin (Sigma Chemical Co., St. Louis, Mo. U.S.A.) for 90 minutes at 50° C. The cells were then lysed by the addition of a detergent containing 1% Brij 58, 0.4% sodium deoxycholate, 0.062M ethylene-diaminetetraacetic acid (EDTA) and 0.05M Tris-HCl (pH 8.0). The lysed solution was then extracted once with an equal volume of water-saturated phenol and once with an equal volume of chloroform. The resultant aqueous phase was then made 0.3M in sodium acetate. Two volumes of 95% ethanol were added, resulting in the precipitation of the DNA. The DNA was resuspended in 0.1X SSC buffer and treated sequentially with RNAse (pancreatic, from Boehringer-Mannheim, 100 microgram (μg) per ml for 60 minutes at 37° C.) then protease K (Boehringer-Mannheim, 100 μg/ml for 60 minutes at 37° C). The resulting DNA was again extracted once with 1 volume of phenol and once with 1 volume of chloroform. The resulting DNA was then dialyzed against 10 mM Tris-HCl (pH 8.0), 1.0 mM EDTA (pH 8.0) and 0.1M NaCl and then against 10 mM Tris HCl (pH 8.0) and 1.0 mM EDTA.

1.0 μg aliquots of the DNA prepared as above were digested separately with the restriction endonucleases EcoRI, SalI, BamHI, BclI, PstI and AccI under the conditions recommended by the manufacturer (International Biotechnologies, Inc.). Each reaction was subjected to electrophoresis on two identical 0.8% agarose gels under conditions sufficient to separate the fragments, usually 5 volts/cm for 8 hours. The gels were then dried for one hour at 60° C. onto Whatman 3MM filter paper in a vacuum gel dryer. The dried gels were then soaked in a solution of 0.5 normal (N) NaOH, 0.15M NaCl for 30 minutes and then soaked again in 0.5M Tris-HCl (pH 8.0) for 1 hour at 0° C. Following this, the gels were then submerged in 0.25% non-fat dry milk in 2X SSC buffer for 1 hour at 65° C.

5×10 7 cpm of each of the labeled probe Amy-1 and Amy-2 (prepared as in step b, above) were added separately to each of 10 ml aliquots of the non-fat dry milk solution described above. Each probe solution was then placed with a gel in a sealed plastic bag, using a separate bag for each probe and gel. The bag was incubated for 2 hours at 40° C. The gels were then removed and washed three times with 500 ml of 2X saline sodium citrate and 0.1% sodium dodecylsulfate at 45° C. The gels were then wrapped in plastic wrap and exposed to X-ray film (Kodak AR) for 48 hours at -70° C. The resulting autoradiograms revealed that both probes hybridized to specific bands in each restriction endonuclease digest of Bacillus caldolyticus DNA. (In some restriction digests, the size of the hybridizing bands was the same for probe Amy-1 and probe Amy-2, indicating that the particular enzyme did not cut the DNA at a position between the locations where these probes bind). Both probes also hybridized to an α-amylase gene cloned from Bacillus licheniformis (as described in Example 2) but not to bacteriophage lambda DNA, evidencing the identification of an α-amylase gene from Bacillus caldolyticus.

EXAMPLE 2

Detection of an α-amylase gene in Bacillus licheniformis

Bacillus licheniformis produces an amylase which has been characterized as a true α-amylase (Chiang et al, Starch, 31:3, 86-92, 1979). The α-amylase gene from this organism has been previously cloned, said clone consisting of a 3.5 kilobase EcoR1 fragment on the vector plasmid pUB110 which directs the synthesis of α-amylase when introduced into a strain of Bacillus subtilis which does not normally produce this protein. Probes Amy-1 and Amy-2 were prepared as described in Examples la and lb. The hybridization was conducted as described in Example 1c. Probes Amy-1 and Amy-2 were found to hybridize to the cloned α-amylase gene (at 45° C.) but not to the DNA from the vector plasmid pUB110, and also to hybridize specifically to Bacillus licheniformis genomic DNA (at 45° C.). This result indicates that probes Amy-1 and Amy-2 are hybridizing to DNA encoding the α-amylase gene of Bacillus licheniformis itself and not coincidentally to unrelated DNA of this strain.



EXAMPLE 3

Detection of an amylase gene in Bacillus alkalophilus

Using the radiolabeled probes Amy-1 and Amy-2 (prepared as described in Examples 1a and 1b), an amylase gene was detected in Bacillus alkalophilus subspecies halodurans. The DNA was released from the cells by treatment with lysozyme and the hybridization was otherwise as conducted in Example 1c (except for incubation of the probes and gels at 35° C. and subsequent washing of the gels at 40° C.). The resulting autoradiogram revealed hybridized probe to the amylase gene. The amylase produced by this organism is believed to be an endo-acting amylase producing substrate degradation products in the β-configuration. See Boyer et al, Starch, 31, 166-171 (1979). This suggests that probes Amy-1 and Amy-2 may also have utility in identifying genes encoding α-amylase-like enzymes such as that produced by Bacillus alkalophilus subspecies halodurans which cannot be classified as true α- or true β-amylases.

EXAMPLE 4

Detection of an α-amylase gene in E. coli

An α-amylase produced by E. coli has been reported by Freundlieb and Boos (J. Biol. Chem., 261, No. 6, 2946-2953, 1986). Using the radiolabeled probe Amy-1 (prepared as described in Examples 1a and 1b), an α-amylase gene was detected in E. coli. The methodologies of Example 1c were used except that lysozyme was used to release the DNA from the cells.

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