- Email: [email protected]
[29] Cloning of cDNA for the catalytic subunit of cAMP-dependent protein kinase
[29]
CLONINGOF cDNA FOR cAMP-DEPENDENTPROTEINKINASE
311
fected cells at physiological concentrations of cAMP. As expected, the normal stimulation of steroidogenesis in the presence of exogenous cAMP is dramatically inhibited in the transfected cells and they become resistant to the growth inhibitory effects of cAMP. 3° Concluding R e m a r k s In utilizing the approach described above, it is important to fully appreciate the complex regulatory network in which the cAMP-dependent protein kinases function. For example, the overexpression of exogenous C subunit has led to a compensatory increase in regulatory subunits in several cell types. 3~ Expression of wild-type RI or RII subunit genes may compete for the total pool of cellular C subunit and affect the distribution of type I (RI) and type II (RII) kinases in the cell, thus leading to physiological changes that reflect distinct functional roles for the different types of kinase. Chronic expression of the mutant RI subunits described above might also lead to some type of compensation such as an increase in basal levels of cAMP. The utilization of these molecular genetic approaches in studies on cAMP function should prove useful in defining the role of specific PK isoforms in a variety of intracellular regulatory events. 3o Clegg, C. H., Correll, L. A., Cadd, G. G., and McKnight, G. S., J. Biol. Chem. 262, 13111 (1987). 31 Uhler, M. D. and McKnight, G. S., J. Biol. Chem. 262, 15202 (1987).
[29] C l o n i n g o f c D N A for t h e C a t a l y t i c S u b u n i t o f cAMP-Dependent Protein Kinase
By MARK O. SHOWERS and RICHARD A. MAURER Recombinant DNA technology provides a highly useful method for analysis and manipulation of specific gene products. The ability to readily manipulate and express cloned DNA can provide a relatively rapid method to explore structure-function relationships for a particular protein. The recent cloning of one of the regulatory subunits of the cAMPdependent protein kinase ~ will certainly provide new information concerning the enzyme. It seems likely that in the near future recombinant t D. C. Lee, D. F. Carmichael, E. G. Krebs. and G. S. McKnight, Proc. Natl. Acad. Sci. U.S.A. 80, 3608 (1983).
METHODS IN ENZYMOLOGY.VOL. 159
Copyright © 1988by Academic Press, Inc. All rightsof reproductionin any form reserved.
312
CYCLIC NUCLEOTIDE ACTION
[29]
DNA techniques will help provide substantial new information concerning the structure, function, and regulation of protein kinase subunits. The usual starting point for application of recombinant DNA techniques to study of a particular protein involves the isolation of a cloned cDNA containing coding sequences for that protein. We will describe the methods we have utilized to isolate a cDNA for a protein closely related to the catalytic subunit of cAMP-dependent protein kinase. 2 The same general technique could be used to isolate a cloned cDNA for any protein kinase or in fact for any protein for which a portion of the amino acid sequence was known. The general strategy for isolation of a cloned cDNA involves the use of oligonucleotides predicted from the amino acid sequence of the protein. The use of this technique is based on the development of hybridization and washing conditions which could discriminate against a single nucleotide mismatch between an oligonucleotide probe and a target nucleic acid. 3 This allows identification of recombinant clones which contain a portion of the coding sequence of a particular protein. As the genetic code is redundant, it is necessary to synthesize a mixture of oligonucleotides containing all possible coding sequences. It is desirable to select a region of amino acid sequence which predicts a minimum number of coding sequences. Thus, regions containing methionine or tryptophan, which both have only a single codon, are desirable. It is also useful to avoid where possible regions containing amino acids with four or six codons. Usually, a compromise between probe length and coding sequence redundancy must be reached. Probes ranging from 15 to 25 nucleotides have been found to be useful in a number of laboratories. Furthermore, it is highly desirable to prepare oligonucleotide probes representing two different regions of the protein. In our case, we examined the known amino acid sequence of the catalytic subunit of the cAMP-dependent protein kinase 4,5 for amino acid sequences which might predict useful oligonucleotide probes. Two regions, each containing seven amino acids, were selected for use in predicting oligonucleotide probes. One region, which was from the amino-terminal half of the protein, contained two methionine residues while the other sequence was from the carboxy2 M. O. Showers and R. A. Maurer, J. Biol. Chem. 261, 16288 (1986L 3 R. B. Wallace, M. J. Johnson, T. Hirose, T. Miyake, E. H. Kawashima, and K. Itakura, Nucleic Acids Res. 9, 879 (1981). 4 S. Shoji, D. C. Parmalee, R. D. Wade, S. Kumar, L. H. Ericsson, K. A. Walsh, H. Neurath, G. L. Long, J. G. Demaille, E. H. Fischer, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. 78, 848 (1981). s S. Shoji, L. H. Ericsson, K. A. Walsh, E. H. Fischer, and K. Titani, Biochemistry 22, 3702 (1983).
[29]
CLONING OF c D N A FOR ¢AMP-DEPENDENT PROTEIN KINASE
313
terminal half of the protein and contained two tryptophan residues. The amino sequences of these regions and the nucleotide coding sequence are Probe 1
Probe 2
Tyr Met Val Met Glu Tyr Val UAU AUG GUN AUG GAA UAU GU C G C
Lys Ala Val Asp Trp Trp Ala AAA GCN GUN GAU UGG UGG GC G C
Mixed oligonucleotides corresponding to the complement of all possible coding sequences are then synthesized. In our case both probes are 20-mers and probe 1 contains 32 different sequence combinations and probe 2 contains 64 combinations. These probes were synthesized using an automated synthesizer. Alternatively, a number of companies will synthesize oligonucleotides as specified by an individual or manual methods can be utilized for the synthesis of oligonucleotides. The radiolabeled oligonucleotide is then used as a hybridization probe to screen a library of cDNAs cloned in a bacteriophage vector. The cloned c D N A insert can then be recovered from phage which hybridize to the oligonucleotide. Analysis of the nucleotide sequence of the c D N A can then be used to confirm that the cDNA, in fact, contains the coding sequence of the specific protein. Of course, this approach is limited to proteins for which at least a partial amino acid sequence is available. While this is a limitation of the method, it is also in some respects a strength. Comparison of the amino acid sequence derived from the cloned c D N A to the known amino acid sequence of the protein permits unambiguous identification of a particular clone.
Materials
Bacterial Growth and Phage Plating Reagents LB medium: Bacto-tryptone, 10 g; Bacto-yeast extract, 5 g; NaCI, 5 g. Add 1 liter of distilled water and autoclave. For plates add 15 g of Bacto-agar per liter and let the medium cool to about 45 ° before pouring. Plates should be well dried by incubation for 1 to 2 days at room temperature depending on the relative humidity. If the plates are not sufficiently dried, difficulty will be encountered in removing nitrocellulose filters from top agar. A well-dried plate has a slightly wrinkled surface. For top agar add 7 g of agarose/liter and autoclave. Top agar should be held at 55 ° in a water bath for use in plating bacteriophage. The Bacto-tryptone, Bacto-yeast extract, and Bactoagar are obtained from Difco 10 m M MgSO4, autoclaved
314
CYCLIC NUCLEOTIDEACTION
[29]
Phage storage medium (SM): 100 m M NaC1, 10 m M MgSO4, 0.01% gelatin (w/v), 10 m M Tris (pH 7.4), autoclaved The bacterial strains Y1088 and Y1090 for propagation of gtl 1 bacteriophage are available from American Type Culture collection as well as from Promega Biotec and other suppliers who sell 7gtl 1 DNA and bacteriophage DNA packaging reagents.
Plaque Screening Oligonucleotides predicted from the coding sequence of the protein, prepared by an automated synthesizer, manual methods 6,7 or purchased from a commercial laboratory Tris, 0.7 M, pH 7.6, 0.1 M MgCI2, 0.1 M dithiothreitol Spermidine, 20 m M [7-32p]ATP, 1000-3000 Ci/mmol EDTA, 0.2 M TE: 10 m M Tris, pH 7.4, 1 m M EDTA TEN200:10 m M Tris, pH 7.4, 1 m M EDTA, 0.2 M NaC1 TEN1000:I0 m M Tris, pH 7.4, 1 m M EDTA, 1.0 M NaCI Diethylaminoethyl-cellulose (DE-52, Whatman, resuspended in 2 vol of TE and autoclaved) Chromatography column, 0.5 x 10 cm Polynucleotide kinase cDNA library cloned in hgtl 1 or hgtl0. Libraries should be obtained for tissues which have the highest expression of the desired protein. Libraries for a number of human, rat, and mouse tissues can be obtained from Clontech Laboratories, Inc., Palo Alto, California Nitrocellulose filters, 137-mm diameter, from Schleicher and Schuell or Millipore NaOH, 0.5 M, 1.5 M NaC1 Tris-HCl, 0.5 M, pH 7.4, 1.5 M NaCI SSPE: 0.15 M NaC1, 10 m M sodium phosphate, pH 7.4, 1 m M EDTA SET: 0.15 M NaCI, 1 m M EDTA, 20 m M Tris, pH 7.4 Denhardt's solution: 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll Hybridization solution: 6 x SET, 10x Denhardt's solution, 0.1% SDS, 0.1% sodium pyrophosphate, 100/xg/ml salmon sperm DNA (sonicated for several minutes in 10 m M Tris, pH 7.4, 1 m M EDTA, boiled for 10 min immediately before use~ and chilled on ice)
6 M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719 (1980). 7 M. D. Matteucci and M. H. Caruthers, J. A m . Chem. Soc. 103, 3185 (1981).
[29]
CLONING OF c D N A FOR
cAMP-DEPENDENTPROTE1NKINASE
315
A vacuum oven capable of 80° is required for fixing DNA to the nitrocellulose filters 90 x 170 mm crystallization dishes 75 x 150 mm crystallization dishes Tubes, sterile, capped, 17 x 100 mm Conical, snap cap tubes, 1.5 ml X-Ray film, Kodak XAR-5 Intensifying screens, Du Pont Lightning Plus Methods
1. Preparation of Radiolabeled Oligonucleotide The oligonucleotide is radiolabeled by transfer of 3zp from ATP to the 5' terminus of the DNA by polynucleotide kinase and then unincorporated radioactivity is removed by chromatography on DEAE-cellulose. It should be noted that individuals working with 3zp should take adequate precautions to avoid unnecessary exposure to radiation. This should include adequate shielding using low-density materials such as ½-in.-thick Plexiglas. The reaction is assembled in a 1.5-ml conical tube in ice. The reaction includes 0.1/zg of DNA, 2/zl of 0.7 M Tris, pH 7.6, 0.1 M MgC12, 0.1 M dithiothreitol, 1/zl of 20 m M spermidine, 10/zl of [y-32p]ATP, and 4 U of polynucleotide kinase in a total volume of 20/zl. The reaction is incubated 40 min at 37° and then 20/zl of 0.2 M EDTA is added and the mixture incubated 5 min at 68°. After briefly cooling the reaction on ice, 1 ml of TE is added and the sample transferred to a small column containing DE-52. The column has a diameter of 0.5 cm and contains DE-52 to a depth of about 0.5 cm. The column is washed with 4 x l ml of TEN200 in order to elute unincorporated ATP. The radiolabeled oligonucleotide is then eluted with 2 x 1 ml of TENI000. Probes should have specific activities of at least 108 cpm//zg.
2. Hybridization Screening of the Libraries Recombinants containing sequences complementary to the oligonucleotide probes are detected using the in situ plaque hybridization technique of Benton and Davis. 8 The bacteriophage cDNA library is plated on a lawn of bacteria on agar plates. Nitrocellulose filters are placed on the plates and phage particles allowed to absorb to the filters. Phage DNA is 8 W. D. Benton and R. W. Davis, Science 196, 180 (1977).
316
CYCLIC NUCLEOTIDE ACTION
[29]
denatured in situ and fixed to the nitrocellulose. The filters can then be hybridized to the oligonucleotide to identify plaques which contain sequences complementary to the probes. As multiple filters can be placed in succession on a single plate, it is possible to readily prepare duplicate filters for hybridization to more than one probe. Furthermore, as more than 5 x l 0 4 phage can be plated on a single plate, it is possible to screen millions of recombinants using this approach. Bacteria for plating the phage are prepared from an overnight culture of Y1088 or Y1090. A single colony of bacteria is transferred to 20 ml of LB in a 125-ml flask and incubated overnight at 37 ° with vigorous shaking. The culture is transferred to a sterile 50-ml tube, centrifuged at 4000 g for 10 min, and the supernatant discarded. The cells are resuspended in 10 ml of sterile 10 m M MgSO4. Then 0.2 ml of the bacterial cells is combined with 0.1 ml of an appropriate dilution of the bacteriophage library. For nonabundant cDNAs, the library should be diluted with SM to a concentration of approximately 5 x 105 plaque-forming units/ml and a large number of 150-mm plates (perhaps 20) should be screened. For more abundant cDNAs, it might be convenient to start at lower initial densities and/or screen less plates. After gentle mixing of the bacteria and phage, the mixture is incubated for 20 rain at 37 ° in order to allow the phage to absorb to the bacteria. The absorbed phage are then transferred to a sterile 17 x 100 mm tube containing 7.5 ml of top agar held at 55 ° and the tube capped. After mixing by inversion, the contents are immediately poured onto a 150-mm LB plate. After the top agar solidifies, the plates are incubated for 8 to 12 hr at 37 ° and then placed for 1 hr at 4 °. Before placing the nitrocellulose filters on the plates they should be numbered with a permanent marking pen. We have found it convenient to use nonsterile filters although some investigators recommend sterilizing the filters. It is important to wear gloves in order to avoid transferring oil to the filters which prevents uniform wetting and absorption of phage. The filter is held on either side and the middle of the filter touched to the center of the plate. The filter is then smoothly lowered to make contact with the entire agarose surface. Of course, it is important to avoid trapping air bubbles under the filter. To permit subsequent alignment of the filter and the agar plate, a needle, dipped in India ink, is used to pierce the filter. The needle should be covered with enough ink to clearly mark both the filter and agar. If another filter is to be placed on the same plate, sufficient ink should be placed on the agar to adequately mark the second filter. Filters are allowed to remain in contact with the top agar for several minutes. They are then removed with forceps and immersed bottom side up in the following solutions for 1-2 min each: (1) 0.5 M NaOH, 1.5 M NaC1, (2) 0.5 M Tris, pH 7.4, 1.5 M NaCI, (3) 2 x SSPE. It is convenient to
[29]
CLONING OF c D N A FOR cAMP-DEPENDENT PROTEIN KINASE
317
use 90 x 170 mm crystallization dishes for immersion of the filters. The filters are then placed on two sheets of Whatman 3MM paper and allowed to air dry for 15-30 min and then baked for 2 hr at 80° in a vacuum oven. For this step it is convenient to separate the filters from each other using the paper interleaving which comes with the filters. It is also desirable to place a light weight on top of a stack of filters to prevent extreme curling. Prehybridization and hybridization take place in 70 x 150 mm dishes. Alternatively, sealable plastic bags can be used and such bags are desirable for high-temperature incubations. Optimal temperatures for hybridization vary according to both the length of the probe and the nucleotide content. It is possible to predict the melting temperature for an oligonucleotide and hybridization should be performed at 5-10 ° below the apparent melting temperature. 9 For 20-mer probes we have found it sufficient to perform hybridizations at room temperature although this is not the optimal condition. For prehybridization, the filters are mixed with 5 ml of hybridization solution per filter and incubated with gentle agitation on a gyrorotary shaker for several hours at room temperature. The filters are then incubated with 2-3 ml of hybridization solution per filter containing 10 6 cpm/ml of the 32p-labeled oligonucleotide probe. Filters are allowed to hybridize to the oligonucleotide probe for several hours to overnight at room temperature. Although overnight hybridizations may increase sensitivity, high backgrounds may be encountered with some probes. Following hybridization the nitrocellulose filters are washed three times for 20 min each in 6x SET at room temperature. The filters are then blotted dry and mounted on 3MM filter paper sheets. After marking the filter paper with radioactive ink to permit alignment of the filters and X-ray film, the filters are exposed to Kodak XAR-5 film for 1 to several days at -70 ° using Du Pont Cronex Lightning Plus screens. Following development of the X-ray film, the autoradiogram is aligned with the agar plate using the India ink marks. After alignment, regions containing plaques which contain sequences complementary to the oligonucleotide probe can be identified. If more than one probe was used to screen replicate filters, then the autoradiographic signal for the two different probes should align. In our experience it is advisable to use two different probes. Using only a single oligonucleotide probe we have encountered many clones which did not correspond to the desired sequence. Because of the high density of plaques used for the initial screening, it is usually not possible to identify and select a single plaque. Therefore, it is necessary to select a small area of the agar plate for rescreening. The 9 G. Dalbadie-McFarland, L. W. Cohen, A. D. Riggs, C. Morin, K. ltakura, and J. H. Richards, Proc. Natl. Acad. Sci. U.S.A. 79, 6409 (1982).
318
CYCLICNUCLEOTIDEACTION
[30]
appropriate region of the plate can be removed by piercing through the agar layers using the large end of a sterile Pasteur pipet. The resulting agar plug is ejected into 1 ml of SM medium and 50/xl of chloroform are added to prevent bacterial growth. This mixture is incubated for several hours at room temperature to allow the phage to diffuse from the agar plug. The phage eluate can then be rescreened by adsorption to bacteria and plating on agar plates as described above. It is desirable to prepare suitable dilutions of the eluted phage so that approximately 1000 plaque-forming units is plated for rescreening. The plaques are then screened for sequences complementary to the oligonucleotide probe as described above. This process of phage screening and phage isolation are repeated until all of the plaques on a plate hybridize to the oligonucleotide probe. At this point the purified phage are available for analysis of the nucleotide sequence of the cDNA insert using nucleotide sequence analysis by the chemical 1° or chain termination method H and the identity of the cDNA can then be confirmed. ~oA. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. ~1j. Messing, this series, Vol. 101, p. 20.
[30] M o l e c u l a r C l o n i n g o f c D N A for a H o r m o n e - R e g u l a t e d I s o f o r m o f t h e R e g u l a t o r y S u b u n i t o f T y p e II cAMP-Dependent Protein Kinase from Rat Ovaries B y TORE JAHNSEN, LARS HEDIN, VINCE J. KIDD, TANYA SCHULZ, an d JOANNE S. RICHARDS
We have recently purified and characterized the regulatory subunits of cAMP-dependent protein kinases RI and RII from rat ovaries, rat brain, and rat heart. ~,2 Based on immunological data, one-dimensional (1D) and two-dimensional (2D) SDS-PAGE as well as 1D and 2D peptide mapping, three distinct proteins, presumably the products of three separate genes, were identified: RI (Mr 49,000), RII52/51 (Mr 52,000-51,000), and RII54 (Mr 54,000). RII52/51 was present in rat ovary and brain, whereas RII54 was T. Jahnsen, S. M. Lohmann, U. Walter, L. Hedin, and J. S. Richards, J. Biol. Chem. 260, 15980 ( 1 9 8 5 ) .
2 T. Jahnsen, L. Hedin, S. M. Lohmann, U. Walter, and J. S. Richards, J. Biol. Chem. 261, 6637 (1986).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
CLONINGOF cDNA FOR cAMP-DEPENDENTPROTEINKINASE
311
fected cells at physiological concentrations of cAMP. As expected, the normal stimulation of steroidogenesis in the presence of exogenous cAMP is dramatically inhibited in the transfected cells and they become resistant to the growth inhibitory effects of cAMP. 3° Concluding R e m a r k s In utilizing the approach described above, it is important to fully appreciate the complex regulatory network in which the cAMP-dependent protein kinases function. For example, the overexpression of exogenous C subunit has led to a compensatory increase in regulatory subunits in several cell types. 3~ Expression of wild-type RI or RII subunit genes may compete for the total pool of cellular C subunit and affect the distribution of type I (RI) and type II (RII) kinases in the cell, thus leading to physiological changes that reflect distinct functional roles for the different types of kinase. Chronic expression of the mutant RI subunits described above might also lead to some type of compensation such as an increase in basal levels of cAMP. The utilization of these molecular genetic approaches in studies on cAMP function should prove useful in defining the role of specific PK isoforms in a variety of intracellular regulatory events. 3o Clegg, C. H., Correll, L. A., Cadd, G. G., and McKnight, G. S., J. Biol. Chem. 262, 13111 (1987). 31 Uhler, M. D. and McKnight, G. S., J. Biol. Chem. 262, 15202 (1987).
[29] C l o n i n g o f c D N A for t h e C a t a l y t i c S u b u n i t o f cAMP-Dependent Protein Kinase
By MARK O. SHOWERS and RICHARD A. MAURER Recombinant DNA technology provides a highly useful method for analysis and manipulation of specific gene products. The ability to readily manipulate and express cloned DNA can provide a relatively rapid method to explore structure-function relationships for a particular protein. The recent cloning of one of the regulatory subunits of the cAMPdependent protein kinase ~ will certainly provide new information concerning the enzyme. It seems likely that in the near future recombinant t D. C. Lee, D. F. Carmichael, E. G. Krebs. and G. S. McKnight, Proc. Natl. Acad. Sci. U.S.A. 80, 3608 (1983).
METHODS IN ENZYMOLOGY.VOL. 159
Copyright © 1988by Academic Press, Inc. All rightsof reproductionin any form reserved.
312
CYCLIC NUCLEOTIDE ACTION
[29]
DNA techniques will help provide substantial new information concerning the structure, function, and regulation of protein kinase subunits. The usual starting point for application of recombinant DNA techniques to study of a particular protein involves the isolation of a cloned cDNA containing coding sequences for that protein. We will describe the methods we have utilized to isolate a cDNA for a protein closely related to the catalytic subunit of cAMP-dependent protein kinase. 2 The same general technique could be used to isolate a cloned cDNA for any protein kinase or in fact for any protein for which a portion of the amino acid sequence was known. The general strategy for isolation of a cloned cDNA involves the use of oligonucleotides predicted from the amino acid sequence of the protein. The use of this technique is based on the development of hybridization and washing conditions which could discriminate against a single nucleotide mismatch between an oligonucleotide probe and a target nucleic acid. 3 This allows identification of recombinant clones which contain a portion of the coding sequence of a particular protein. As the genetic code is redundant, it is necessary to synthesize a mixture of oligonucleotides containing all possible coding sequences. It is desirable to select a region of amino acid sequence which predicts a minimum number of coding sequences. Thus, regions containing methionine or tryptophan, which both have only a single codon, are desirable. It is also useful to avoid where possible regions containing amino acids with four or six codons. Usually, a compromise between probe length and coding sequence redundancy must be reached. Probes ranging from 15 to 25 nucleotides have been found to be useful in a number of laboratories. Furthermore, it is highly desirable to prepare oligonucleotide probes representing two different regions of the protein. In our case, we examined the known amino acid sequence of the catalytic subunit of the cAMP-dependent protein kinase 4,5 for amino acid sequences which might predict useful oligonucleotide probes. Two regions, each containing seven amino acids, were selected for use in predicting oligonucleotide probes. One region, which was from the amino-terminal half of the protein, contained two methionine residues while the other sequence was from the carboxy2 M. O. Showers and R. A. Maurer, J. Biol. Chem. 261, 16288 (1986L 3 R. B. Wallace, M. J. Johnson, T. Hirose, T. Miyake, E. H. Kawashima, and K. Itakura, Nucleic Acids Res. 9, 879 (1981). 4 S. Shoji, D. C. Parmalee, R. D. Wade, S. Kumar, L. H. Ericsson, K. A. Walsh, H. Neurath, G. L. Long, J. G. Demaille, E. H. Fischer, and K. Titani, Proc. Natl. Acad. Sci. U.S.A. 78, 848 (1981). s S. Shoji, L. H. Ericsson, K. A. Walsh, E. H. Fischer, and K. Titani, Biochemistry 22, 3702 (1983).
[29]
CLONING OF c D N A FOR ¢AMP-DEPENDENT PROTEIN KINASE
313
terminal half of the protein and contained two tryptophan residues. The amino sequences of these regions and the nucleotide coding sequence are Probe 1
Probe 2
Tyr Met Val Met Glu Tyr Val UAU AUG GUN AUG GAA UAU GU C G C
Lys Ala Val Asp Trp Trp Ala AAA GCN GUN GAU UGG UGG GC G C
Mixed oligonucleotides corresponding to the complement of all possible coding sequences are then synthesized. In our case both probes are 20-mers and probe 1 contains 32 different sequence combinations and probe 2 contains 64 combinations. These probes were synthesized using an automated synthesizer. Alternatively, a number of companies will synthesize oligonucleotides as specified by an individual or manual methods can be utilized for the synthesis of oligonucleotides. The radiolabeled oligonucleotide is then used as a hybridization probe to screen a library of cDNAs cloned in a bacteriophage vector. The cloned c D N A insert can then be recovered from phage which hybridize to the oligonucleotide. Analysis of the nucleotide sequence of the c D N A can then be used to confirm that the cDNA, in fact, contains the coding sequence of the specific protein. Of course, this approach is limited to proteins for which at least a partial amino acid sequence is available. While this is a limitation of the method, it is also in some respects a strength. Comparison of the amino acid sequence derived from the cloned c D N A to the known amino acid sequence of the protein permits unambiguous identification of a particular clone.
Materials
Bacterial Growth and Phage Plating Reagents LB medium: Bacto-tryptone, 10 g; Bacto-yeast extract, 5 g; NaCI, 5 g. Add 1 liter of distilled water and autoclave. For plates add 15 g of Bacto-agar per liter and let the medium cool to about 45 ° before pouring. Plates should be well dried by incubation for 1 to 2 days at room temperature depending on the relative humidity. If the plates are not sufficiently dried, difficulty will be encountered in removing nitrocellulose filters from top agar. A well-dried plate has a slightly wrinkled surface. For top agar add 7 g of agarose/liter and autoclave. Top agar should be held at 55 ° in a water bath for use in plating bacteriophage. The Bacto-tryptone, Bacto-yeast extract, and Bactoagar are obtained from Difco 10 m M MgSO4, autoclaved
314
CYCLIC NUCLEOTIDEACTION
[29]
Phage storage medium (SM): 100 m M NaC1, 10 m M MgSO4, 0.01% gelatin (w/v), 10 m M Tris (pH 7.4), autoclaved The bacterial strains Y1088 and Y1090 for propagation of gtl 1 bacteriophage are available from American Type Culture collection as well as from Promega Biotec and other suppliers who sell 7gtl 1 DNA and bacteriophage DNA packaging reagents.
Plaque Screening Oligonucleotides predicted from the coding sequence of the protein, prepared by an automated synthesizer, manual methods 6,7 or purchased from a commercial laboratory Tris, 0.7 M, pH 7.6, 0.1 M MgCI2, 0.1 M dithiothreitol Spermidine, 20 m M [7-32p]ATP, 1000-3000 Ci/mmol EDTA, 0.2 M TE: 10 m M Tris, pH 7.4, 1 m M EDTA TEN200:10 m M Tris, pH 7.4, 1 m M EDTA, 0.2 M NaC1 TEN1000:I0 m M Tris, pH 7.4, 1 m M EDTA, 1.0 M NaCI Diethylaminoethyl-cellulose (DE-52, Whatman, resuspended in 2 vol of TE and autoclaved) Chromatography column, 0.5 x 10 cm Polynucleotide kinase cDNA library cloned in hgtl 1 or hgtl0. Libraries should be obtained for tissues which have the highest expression of the desired protein. Libraries for a number of human, rat, and mouse tissues can be obtained from Clontech Laboratories, Inc., Palo Alto, California Nitrocellulose filters, 137-mm diameter, from Schleicher and Schuell or Millipore NaOH, 0.5 M, 1.5 M NaC1 Tris-HCl, 0.5 M, pH 7.4, 1.5 M NaCI SSPE: 0.15 M NaC1, 10 m M sodium phosphate, pH 7.4, 1 m M EDTA SET: 0.15 M NaCI, 1 m M EDTA, 20 m M Tris, pH 7.4 Denhardt's solution: 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll Hybridization solution: 6 x SET, 10x Denhardt's solution, 0.1% SDS, 0.1% sodium pyrophosphate, 100/xg/ml salmon sperm DNA (sonicated for several minutes in 10 m M Tris, pH 7.4, 1 m M EDTA, boiled for 10 min immediately before use~ and chilled on ice)
6 M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719 (1980). 7 M. D. Matteucci and M. H. Caruthers, J. A m . Chem. Soc. 103, 3185 (1981).
[29]
CLONING OF c D N A FOR
cAMP-DEPENDENTPROTE1NKINASE
315
A vacuum oven capable of 80° is required for fixing DNA to the nitrocellulose filters 90 x 170 mm crystallization dishes 75 x 150 mm crystallization dishes Tubes, sterile, capped, 17 x 100 mm Conical, snap cap tubes, 1.5 ml X-Ray film, Kodak XAR-5 Intensifying screens, Du Pont Lightning Plus Methods
1. Preparation of Radiolabeled Oligonucleotide The oligonucleotide is radiolabeled by transfer of 3zp from ATP to the 5' terminus of the DNA by polynucleotide kinase and then unincorporated radioactivity is removed by chromatography on DEAE-cellulose. It should be noted that individuals working with 3zp should take adequate precautions to avoid unnecessary exposure to radiation. This should include adequate shielding using low-density materials such as ½-in.-thick Plexiglas. The reaction is assembled in a 1.5-ml conical tube in ice. The reaction includes 0.1/zg of DNA, 2/zl of 0.7 M Tris, pH 7.6, 0.1 M MgC12, 0.1 M dithiothreitol, 1/zl of 20 m M spermidine, 10/zl of [y-32p]ATP, and 4 U of polynucleotide kinase in a total volume of 20/zl. The reaction is incubated 40 min at 37° and then 20/zl of 0.2 M EDTA is added and the mixture incubated 5 min at 68°. After briefly cooling the reaction on ice, 1 ml of TE is added and the sample transferred to a small column containing DE-52. The column has a diameter of 0.5 cm and contains DE-52 to a depth of about 0.5 cm. The column is washed with 4 x l ml of TEN200 in order to elute unincorporated ATP. The radiolabeled oligonucleotide is then eluted with 2 x 1 ml of TENI000. Probes should have specific activities of at least 108 cpm//zg.
2. Hybridization Screening of the Libraries Recombinants containing sequences complementary to the oligonucleotide probes are detected using the in situ plaque hybridization technique of Benton and Davis. 8 The bacteriophage cDNA library is plated on a lawn of bacteria on agar plates. Nitrocellulose filters are placed on the plates and phage particles allowed to absorb to the filters. Phage DNA is 8 W. D. Benton and R. W. Davis, Science 196, 180 (1977).
316
CYCLIC NUCLEOTIDE ACTION
[29]
denatured in situ and fixed to the nitrocellulose. The filters can then be hybridized to the oligonucleotide to identify plaques which contain sequences complementary to the probes. As multiple filters can be placed in succession on a single plate, it is possible to readily prepare duplicate filters for hybridization to more than one probe. Furthermore, as more than 5 x l 0 4 phage can be plated on a single plate, it is possible to screen millions of recombinants using this approach. Bacteria for plating the phage are prepared from an overnight culture of Y1088 or Y1090. A single colony of bacteria is transferred to 20 ml of LB in a 125-ml flask and incubated overnight at 37 ° with vigorous shaking. The culture is transferred to a sterile 50-ml tube, centrifuged at 4000 g for 10 min, and the supernatant discarded. The cells are resuspended in 10 ml of sterile 10 m M MgSO4. Then 0.2 ml of the bacterial cells is combined with 0.1 ml of an appropriate dilution of the bacteriophage library. For nonabundant cDNAs, the library should be diluted with SM to a concentration of approximately 5 x 105 plaque-forming units/ml and a large number of 150-mm plates (perhaps 20) should be screened. For more abundant cDNAs, it might be convenient to start at lower initial densities and/or screen less plates. After gentle mixing of the bacteria and phage, the mixture is incubated for 20 rain at 37 ° in order to allow the phage to absorb to the bacteria. The absorbed phage are then transferred to a sterile 17 x 100 mm tube containing 7.5 ml of top agar held at 55 ° and the tube capped. After mixing by inversion, the contents are immediately poured onto a 150-mm LB plate. After the top agar solidifies, the plates are incubated for 8 to 12 hr at 37 ° and then placed for 1 hr at 4 °. Before placing the nitrocellulose filters on the plates they should be numbered with a permanent marking pen. We have found it convenient to use nonsterile filters although some investigators recommend sterilizing the filters. It is important to wear gloves in order to avoid transferring oil to the filters which prevents uniform wetting and absorption of phage. The filter is held on either side and the middle of the filter touched to the center of the plate. The filter is then smoothly lowered to make contact with the entire agarose surface. Of course, it is important to avoid trapping air bubbles under the filter. To permit subsequent alignment of the filter and the agar plate, a needle, dipped in India ink, is used to pierce the filter. The needle should be covered with enough ink to clearly mark both the filter and agar. If another filter is to be placed on the same plate, sufficient ink should be placed on the agar to adequately mark the second filter. Filters are allowed to remain in contact with the top agar for several minutes. They are then removed with forceps and immersed bottom side up in the following solutions for 1-2 min each: (1) 0.5 M NaOH, 1.5 M NaC1, (2) 0.5 M Tris, pH 7.4, 1.5 M NaCI, (3) 2 x SSPE. It is convenient to
[29]
CLONING OF c D N A FOR cAMP-DEPENDENT PROTEIN KINASE
317
use 90 x 170 mm crystallization dishes for immersion of the filters. The filters are then placed on two sheets of Whatman 3MM paper and allowed to air dry for 15-30 min and then baked for 2 hr at 80° in a vacuum oven. For this step it is convenient to separate the filters from each other using the paper interleaving which comes with the filters. It is also desirable to place a light weight on top of a stack of filters to prevent extreme curling. Prehybridization and hybridization take place in 70 x 150 mm dishes. Alternatively, sealable plastic bags can be used and such bags are desirable for high-temperature incubations. Optimal temperatures for hybridization vary according to both the length of the probe and the nucleotide content. It is possible to predict the melting temperature for an oligonucleotide and hybridization should be performed at 5-10 ° below the apparent melting temperature. 9 For 20-mer probes we have found it sufficient to perform hybridizations at room temperature although this is not the optimal condition. For prehybridization, the filters are mixed with 5 ml of hybridization solution per filter and incubated with gentle agitation on a gyrorotary shaker for several hours at room temperature. The filters are then incubated with 2-3 ml of hybridization solution per filter containing 10 6 cpm/ml of the 32p-labeled oligonucleotide probe. Filters are allowed to hybridize to the oligonucleotide probe for several hours to overnight at room temperature. Although overnight hybridizations may increase sensitivity, high backgrounds may be encountered with some probes. Following hybridization the nitrocellulose filters are washed three times for 20 min each in 6x SET at room temperature. The filters are then blotted dry and mounted on 3MM filter paper sheets. After marking the filter paper with radioactive ink to permit alignment of the filters and X-ray film, the filters are exposed to Kodak XAR-5 film for 1 to several days at -70 ° using Du Pont Cronex Lightning Plus screens. Following development of the X-ray film, the autoradiogram is aligned with the agar plate using the India ink marks. After alignment, regions containing plaques which contain sequences complementary to the oligonucleotide probe can be identified. If more than one probe was used to screen replicate filters, then the autoradiographic signal for the two different probes should align. In our experience it is advisable to use two different probes. Using only a single oligonucleotide probe we have encountered many clones which did not correspond to the desired sequence. Because of the high density of plaques used for the initial screening, it is usually not possible to identify and select a single plaque. Therefore, it is necessary to select a small area of the agar plate for rescreening. The 9 G. Dalbadie-McFarland, L. W. Cohen, A. D. Riggs, C. Morin, K. ltakura, and J. H. Richards, Proc. Natl. Acad. Sci. U.S.A. 79, 6409 (1982).
318
CYCLICNUCLEOTIDEACTION
[30]
appropriate region of the plate can be removed by piercing through the agar layers using the large end of a sterile Pasteur pipet. The resulting agar plug is ejected into 1 ml of SM medium and 50/xl of chloroform are added to prevent bacterial growth. This mixture is incubated for several hours at room temperature to allow the phage to diffuse from the agar plug. The phage eluate can then be rescreened by adsorption to bacteria and plating on agar plates as described above. It is desirable to prepare suitable dilutions of the eluted phage so that approximately 1000 plaque-forming units is plated for rescreening. The plaques are then screened for sequences complementary to the oligonucleotide probe as described above. This process of phage screening and phage isolation are repeated until all of the plaques on a plate hybridize to the oligonucleotide probe. At this point the purified phage are available for analysis of the nucleotide sequence of the cDNA insert using nucleotide sequence analysis by the chemical 1° or chain termination method H and the identity of the cDNA can then be confirmed. ~oA. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. ~1j. Messing, this series, Vol. 101, p. 20.
[30] M o l e c u l a r C l o n i n g o f c D N A for a H o r m o n e - R e g u l a t e d I s o f o r m o f t h e R e g u l a t o r y S u b u n i t o f T y p e II cAMP-Dependent Protein Kinase from Rat Ovaries B y TORE JAHNSEN, LARS HEDIN, VINCE J. KIDD, TANYA SCHULZ, an d JOANNE S. RICHARDS
We have recently purified and characterized the regulatory subunits of cAMP-dependent protein kinases RI and RII from rat ovaries, rat brain, and rat heart. ~,2 Based on immunological data, one-dimensional (1D) and two-dimensional (2D) SDS-PAGE as well as 1D and 2D peptide mapping, three distinct proteins, presumably the products of three separate genes, were identified: RI (Mr 49,000), RII52/51 (Mr 52,000-51,000), and RII54 (Mr 54,000). RII52/51 was present in rat ovary and brain, whereas RII54 was T. Jahnsen, S. M. Lohmann, U. Walter, L. Hedin, and J. S. Richards, J. Biol. Chem. 260, 15980 ( 1 9 8 5 ) .
2 T. Jahnsen, L. Hedin, S. M. Lohmann, U. Walter, and J. S. Richards, J. Biol. Chem. 261, 6637 (1986).
METHODS IN ENZYMOLOGY, VOL. 159
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.