Gene, 96 (1990) 205-211 Elsevier
205
GENE 003744
Prophage lambda libraries for isolating c D N A clones by functional screening (Recombinant DNA; phage 2g~i1; lysogeny; expression vector; ligand screening; slime mold; Dictyostelium discoideum; cAMP-binding proteins; calmoddlin-binding proteins)
Rupert Mutzel, Annette Baeuefle, Stephan Jung and Heike Dammann FakultiitJ~r Biologie, Universiti~t Konstanz, D 7750 Konstanz (F.R.G.) Received by J.K.C. Knowles: 1 February 1990 Revised: 14 June 1990 Accepted: 16 July 1990
SUMMARY
Isolation of cDNA clones from Agtl I phage libraries by functional screening is limited by the low amount of lacZ-cDNAencoded fusion protein synthesized in an isolated phage plaque. The mnount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia co!i was lysogenized with a Agtl I cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the Agtl 1 prophage by elimination of nonlysogenic parental cells with a ~cl phage. The usefulness of the lysogea library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands, cDNA clones encoding a well-characterized D. discoideum protein, the: regulatory subunit of the cAMP-dependent p~otein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [ 3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-bi~ding protein(s) from D. discoideum by functional screening with [ 125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.
INTRODUCTION
Since the pioneering work of Young and Davis (1983a,b) isolation of cDNAs encoding eukaryotic proteins from phage A expression libraries has become a standard tool in Correspondence to: Dr. R. Mutzel, Fakult~t filr Biologie, Universitat Kostanz, Postfach 5560, D 7750 Konstanz (F.R.G.) Tel. (07531)882479; Fax (07531)882966. Abbreviations: Ap, amp~cillin;/~Gal,/?-galactosidase;BSA, bovine serum albumin; CaM, calmodulin; D., Dictyostelium;DMSO, dimethyl sulfoxicle; EGTA, ethylene glycol bis L&ami~oethylether)-N,N,N',N'-tetraacetic acid; IPTG, isopropyl-/?-D-thiogalactopyranoside;moi, multiplicity of infection; nt, nucleotide(s); oligo, oligodeoxyribonucleotide;pf,u, plaqueforming units; L broth, 0.1% bactotryptone/0.05% yeast extract/0.1% NaCI/pH 7.0; R subunR, regulatory subunit of cAMP-dependent pro:ein kinase; SDS, sodium dodecyl sulfate; TBS, 0.01 M Tris. HCI pH 7.2/0.15 M NaCI; TBSII, TBS/0.1 mM CaCIz/0.05% Tween-20. 0~q78-1! 19/90/$03.50 © 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
molecular biology. In the Agtl 1 system, cDNAs axe cloned into a unique EcoRl site located near the 3' end of the E. coil lacZ gene, and lacZ-cDNA-encoded fusion proteins are synthesized upon induction of the iac promoter with IPTG, prnvided the cDNA coding strand is properly oriented and in-frame with the coding phase of the lacZ gene. Two different approaches for the identification of cDNA clones by expression of their encoded proteins with antisera were originally proposed: first, lysogenization of ggtl 1 phages in an E. coli hflA host and screening fox' the proteins synthesized by bacteria h~boring the ~.gtl1 ?rophage (Young and Davis, 1983a) and, second, scceening of the proteins synthesized in phage plaques (Young and Davis, 1983b). The former technique has since been abandoned, probably because the amount of specific protein synthesized in a phage plaque is sufficient for detection with most antisera and phage plaques can be plated at higher
206 densities than bacterial colonies; moreover, failure of some recombinant 2gtl 1 phages to form stable lysogens has been cited (H u y n h et al., 1985). Expression screening of phage Z libraries has become even more attractive by the observation that at least certain eukaryotic proteins can retain functionality when expressed in a fusion form with/~Gal ( K a u f m a n et al., 1986; Bernier et al., 1087; Mutzel et al., 1988) and a n u m b e r ofeukaryotic c D N A s have been cloned by ligaud binding to lacZ-cDNAencoded fusion proteins in 2gtl 1 phage plaques (Lacombe et al., 1987; Sikela and Hahn, 1987; Singh et al., 1988). Functional cloning appears particularly useful whenever specific antibodies or oligo probes are not available, or heterologous antibodies or D N A probes fail to specifically recognize their counterparts from evolutionary distant organisms. However, the method is limited by the low quantities of fusion protein expressed by a tingle phage plaque ( < 1 fmol) and hence the need for a high-affinity ligand with high specific radioactivity ( > 1 0 0 0 C i / m m o l ; Lacomt,e et al., 1987). Sikela and H a h n (1987) reported the isolation of a c D N A clone encoding a protein homologous to C a M kinase I~
from a rat brain 2gtl 1 c D N A expression library by binding of [ ~25I]CaM te phage plaques. In a similar a p p r o a c h to analyze CaM-binding proteins in the cellular slime mold, D. discoideum, functional screening o f a ;tgtl 1 phage library failed. To allow increased expression of c D N A - e n c o d e d proteins in individual clones, we have re-introduced the lysogen screenhlg technique for functional isolation of ~,DNA clones.
RESULTS AND DISCUSSION (a) Preparation of the 2gtll lysogen library E. coil was lysogenized with the 2gtl 1 e D N A library constructed by L a c o m b e et al. (1986) from D. discoideum A X 2 cells: strain Y1089 (AlacU169, Alon, araD139, strA, hflA [ c h r : : T n l 0 ] , p M C 9 ; Young and Davis, 1983b) was grown to saturation in L broth containing 100 #g Ap/ml and 0.2% maltose. Bacteria were diluted to an A6oo of 0.2 in 10 m M MgSO4 a n d 1 ml o f t h e suspension w a s incubated with 5 x l0 s pfu of the 2gtl I library for 25 min at room temperature. U n d e r the conditions described by H u y n h
TABLE I Characterization of cDNA clones encoding the R subunit Clone a
Size of antigenic compound
IPTG inducible c
cAMP-binding activity in crude extract (pmol/mg protein) d
Size of eDNA insert (kb) e
Hybridization with R subunit eDNA r
+ + + + +
6.6 n.d. 2.2 14.9 14.5
1.25 1.25 0.9 1.15 1.1
+ + + + +
(kDa) b
R6 R8 R! ! R22 R30
160 160 140 160 150
a Clones were isolated by colony screening with anti R subunit antiserum. The lysogen library was diluted to 5 x 104 cells/ml in L broth and 0.1 ml aliquots were plated on L agdr plates (90 mm diameter) containing 100 #g Ap/ml. Colonies were grown for 16 h at 28°C. Nitrocellulose filter circles (BA 85, Schleicher & Schuell) were placed on the plates, transferred to petri dishes containing a Wh,~.tman3 MM filter paper circle saturated with 2.5 mi L broth, and regrown for 1-2 h at 28 ° C. The filters were then transferred to fresh petri dishes with filter paper soaked in L broth containing 2 mM IPTG, incubated 15 min at 42°C and incubated at 37°C for 1 h. To lyse filter-bound bacteria, the filters were frozen twice on a steel replica block immersed in a dry ice/ethanol bath. Debris were removed by rinsing the filters with TBS buffer. Filters were blocked with 3 % BSA in TBS buffer for I h at room temperatut~ and reacted with preadsorbed antiserum against the R subunit ofD. discoideumcAMP-dependent protein kinase as previously described (Mutzel et al., 1987); bound antibody was labeled with peroxidase-conjugated goat anti-rabbit IgG (Dianova, Hamburg, F.R.G.) and the complex was stained with 2-chloronaphthol/H202 (Towbin et al,, 1979). b Crude extracts from liquid cultures of the lysogens were prepared and chromatographed on 0.1% SDS-7.5~ polyacrylamide gels (Laemmli, 1970) as described (Mutzel et al., 1988). Proteins were electrophoretically transferred to nitrocellulose filters (BA 85, Schleicher & Schuell) according to Kyhse-Andcrsen (1984) and antigenic material was detected with anti-R subunit antiserum. Prestained Mr marker proteins (Sigma) were used as a standard. In extracts from clones R6, R22, and R30, proteo!ytic degradation products of the fusion proteins were observed. The Mr of the largest band is given. c Extracts from both IPTG-induced (! mM) and non-induced cultures were compared by Western blotting. d Binding of 10- ~ M[3H]cAMP(35-40 Ci/mmol, Amersham) to R subunit in crude lysogen lysates was measured as previously described (Mutzel et al., 1988). Extracts were prepared after induction with I mM IPTG; cAMP-binding in each non-induced extract was 3000 Ci/mmol had to be enzymatically synthesized and purified (Lacombe et al., 1987) commercially available tritiated c AM P sufficed to show cAMP binding directly on bacterial colony blot~.
(2) The preparation of a lysogen library from an existing 2gtl 1 phage library can easily and conveniently be achieved in one day. The risk of a particular 2 g t l l phage falling to form a stable lysogen (Huynh et al., 1985) may thus he balanced by the possibility to isolate, by functional screening, c D N A clone~ which proved otherwise inaccessible.
ACKNOWLEDGEMENTS We thank M.L. Lacombe, H.W. Hofer, D. Malchow and B. Wurster for helpful discussions. We are grateful to E. Bremer for most valuable suggestions, comments on the manuscript, and a gift of 2h80cI. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 156.
REFERENCES Bernier, L., Alvarez, F., Norgard, E.M., Raible, D.W., Mentaberry, A., Schembri,J.G., Sabatini, D.D. and Colman, D.R.: Molecular cloning of a 2',T-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues. J. Neurosci. 7 (1987) 2703-2710. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Anal. Bioehem. 72 (1976) 248-254. Chirala, S.S.: The nucleotide sequence of the lac operon and phage junction in lambda gtl 1. Nucleic Acids Res. 14 (1986) 5935. Cohen, P. and Klee, C.B.: Criieria required to demonstrate calmodulin. dependent effects in vivo. In Cohen, P. and Klee, C.B. (Eds.), Calmodulin, Elsevier, Amsterdam, 1988, pp. 357-364. Huynh, T.V., Young, R.A. and Davis, R.W.: Constructing and screening eDNA libraries in 2gtl0 and 2gtll. In Glover, D.M. (Ed.), DNA Cloning; A Practical Approach, Vol. I. IRL Press, Oxford, 1985, pp. 49-78. Kanfman, D.L., McGinnis, J.F., Krieg~r, N.R. and Tobin, A.J.: Brain glutamate decarboxylase cloned in 2gt-I 1: fusion protein produces y-aminobutyricacid. Science 232 (1986) 1138-1140. Kyhse-Andersen,J.: Electroblotting of multiple gels: a simple apparatus without buffertank for rapid transfer of proteins from polyacrylamide to nitrocellulose,j. Biochem. Biophys. Methods 10 (1984) 203-209. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Lacombe, M.L., Ladant, D., Mutzel, R. and V6ron, M.: Gene isolation by direct in situ cAMP binding. Gene 58 (1987) 29-36. Mutzel, R., Lacombe, M.L., Simon, M.N., de Gunzburg, J. and V6ron, M.: Cloningand eDNA sequence of the regulatory subunit ofcAMPdependent protein kinase from Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 84 (1987) 6-10. Mutzel, R., Simon, M.N., Lacombe, M.L. and V~ron,M.: Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by 2gtl 1 eDNA clones. Biochemistry27 (1988) 481-486. Rangel-Aldao, R., Schwartz, D. and Rubin, C.S.: Rapid a~say for cyclic AMP and cyclicGMP phosphodiesterases. Anal. Biochem. 87 (1978) 367-375. Sikela, J.M. and Hahn, W.E.: Screening an expression library with a ligund probe: isolation and sequence of a eDNA corresponding to a
2!1 brain calmodulin-binding protein. Proc. Natl. Acad. Sci. USA 84 (1987) 3038-3042. Sin'.on, M.N., Mutzel, R., Mutzel, H. and V~ron, M.: Vectors for expres~ sion of truncated coding sequences in Escherichia coll. Plasmid 19' (1988) 94-102. Simpson, P.J., Chou, W.-G., Whitbeck, A.A., Young, F.E. and Zain, S.: Evidence for prokaryotic transcription and translation control regions in the human factor IX gene. Gene 61 (1987) 373-383. Singh, H., LeBowitz, J.H., Baldwin Jr., A.S. and Sharp, P.A.: Molecular cloning of an enhancer binding protein: isolation by screening of an
expressioa library with a recognition site DNA. Cell 52 (1988) 415-423. Towbin, H., Staehelin, T. and Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proe. Natl. Acad. Sci. USA 76 (1979) 4350-4354. Young, R.A. and Davis, R.W.: Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80 (1983a) 1194-1198. Young, R.A. and Davis, R.W.: Yeast RNA polymerase II genes: isolation with antibody probes. Scienc~ 222 (1983b) 778-782.
205
GENE 003744
Prophage lambda libraries for isolating c D N A clones by functional screening (Recombinant DNA; phage 2g~i1; lysogeny; expression vector; ligand screening; slime mold; Dictyostelium discoideum; cAMP-binding proteins; calmoddlin-binding proteins)
Rupert Mutzel, Annette Baeuefle, Stephan Jung and Heike Dammann FakultiitJ~r Biologie, Universiti~t Konstanz, D 7750 Konstanz (F.R.G.) Received by J.K.C. Knowles: 1 February 1990 Revised: 14 June 1990 Accepted: 16 July 1990
SUMMARY
Isolation of cDNA clones from Agtl I phage libraries by functional screening is limited by the low amount of lacZ-cDNAencoded fusion protein synthesized in an isolated phage plaque. The mnount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia co!i was lysogenized with a Agtl I cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the Agtl 1 prophage by elimination of nonlysogenic parental cells with a ~cl phage. The usefulness of the lysogea library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands, cDNA clones encoding a well-characterized D. discoideum protein, the: regulatory subunit of the cAMP-dependent p~otein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [ 3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-bi~ding protein(s) from D. discoideum by functional screening with [ 125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.
INTRODUCTION
Since the pioneering work of Young and Davis (1983a,b) isolation of cDNAs encoding eukaryotic proteins from phage A expression libraries has become a standard tool in Correspondence to: Dr. R. Mutzel, Fakult~t filr Biologie, Universitat Kostanz, Postfach 5560, D 7750 Konstanz (F.R.G.) Tel. (07531)882479; Fax (07531)882966. Abbreviations: Ap, amp~cillin;/~Gal,/?-galactosidase;BSA, bovine serum albumin; CaM, calmodulin; D., Dictyostelium;DMSO, dimethyl sulfoxicle; EGTA, ethylene glycol bis L&ami~oethylether)-N,N,N',N'-tetraacetic acid; IPTG, isopropyl-/?-D-thiogalactopyranoside;moi, multiplicity of infection; nt, nucleotide(s); oligo, oligodeoxyribonucleotide;pf,u, plaqueforming units; L broth, 0.1% bactotryptone/0.05% yeast extract/0.1% NaCI/pH 7.0; R subunR, regulatory subunit of cAMP-dependent pro:ein kinase; SDS, sodium dodecyl sulfate; TBS, 0.01 M Tris. HCI pH 7.2/0.15 M NaCI; TBSII, TBS/0.1 mM CaCIz/0.05% Tween-20. 0~q78-1! 19/90/$03.50 © 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)
molecular biology. In the Agtl 1 system, cDNAs axe cloned into a unique EcoRl site located near the 3' end of the E. coil lacZ gene, and lacZ-cDNA-encoded fusion proteins are synthesized upon induction of the iac promoter with IPTG, prnvided the cDNA coding strand is properly oriented and in-frame with the coding phase of the lacZ gene. Two different approaches for the identification of cDNA clones by expression of their encoded proteins with antisera were originally proposed: first, lysogenization of ggtl 1 phages in an E. coli hflA host and screening fox' the proteins synthesized by bacteria h~boring the ~.gtl1 ?rophage (Young and Davis, 1983a) and, second, scceening of the proteins synthesized in phage plaques (Young and Davis, 1983b). The former technique has since been abandoned, probably because the amount of specific protein synthesized in a phage plaque is sufficient for detection with most antisera and phage plaques can be plated at higher
206 densities than bacterial colonies; moreover, failure of some recombinant 2gtl 1 phages to form stable lysogens has been cited (H u y n h et al., 1985). Expression screening of phage Z libraries has become even more attractive by the observation that at least certain eukaryotic proteins can retain functionality when expressed in a fusion form with/~Gal ( K a u f m a n et al., 1986; Bernier et al., 1087; Mutzel et al., 1988) and a n u m b e r ofeukaryotic c D N A s have been cloned by ligaud binding to lacZ-cDNAencoded fusion proteins in 2gtl 1 phage plaques (Lacombe et al., 1987; Sikela and Hahn, 1987; Singh et al., 1988). Functional cloning appears particularly useful whenever specific antibodies or oligo probes are not available, or heterologous antibodies or D N A probes fail to specifically recognize their counterparts from evolutionary distant organisms. However, the method is limited by the low quantities of fusion protein expressed by a tingle phage plaque ( < 1 fmol) and hence the need for a high-affinity ligand with high specific radioactivity ( > 1 0 0 0 C i / m m o l ; Lacomt,e et al., 1987). Sikela and H a h n (1987) reported the isolation of a c D N A clone encoding a protein homologous to C a M kinase I~
from a rat brain 2gtl 1 c D N A expression library by binding of [ ~25I]CaM te phage plaques. In a similar a p p r o a c h to analyze CaM-binding proteins in the cellular slime mold, D. discoideum, functional screening o f a ;tgtl 1 phage library failed. To allow increased expression of c D N A - e n c o d e d proteins in individual clones, we have re-introduced the lysogen screenhlg technique for functional isolation of ~,DNA clones.
RESULTS AND DISCUSSION (a) Preparation of the 2gtll lysogen library E. coil was lysogenized with the 2gtl 1 e D N A library constructed by L a c o m b e et al. (1986) from D. discoideum A X 2 cells: strain Y1089 (AlacU169, Alon, araD139, strA, hflA [ c h r : : T n l 0 ] , p M C 9 ; Young and Davis, 1983b) was grown to saturation in L broth containing 100 #g Ap/ml and 0.2% maltose. Bacteria were diluted to an A6oo of 0.2 in 10 m M MgSO4 a n d 1 ml o f t h e suspension w a s incubated with 5 x l0 s pfu of the 2gtl I library for 25 min at room temperature. U n d e r the conditions described by H u y n h
TABLE I Characterization of cDNA clones encoding the R subunit Clone a
Size of antigenic compound
IPTG inducible c
cAMP-binding activity in crude extract (pmol/mg protein) d
Size of eDNA insert (kb) e
Hybridization with R subunit eDNA r
+ + + + +
6.6 n.d. 2.2 14.9 14.5
1.25 1.25 0.9 1.15 1.1
+ + + + +
(kDa) b
R6 R8 R! ! R22 R30
160 160 140 160 150
a Clones were isolated by colony screening with anti R subunit antiserum. The lysogen library was diluted to 5 x 104 cells/ml in L broth and 0.1 ml aliquots were plated on L agdr plates (90 mm diameter) containing 100 #g Ap/ml. Colonies were grown for 16 h at 28°C. Nitrocellulose filter circles (BA 85, Schleicher & Schuell) were placed on the plates, transferred to petri dishes containing a Wh,~.tman3 MM filter paper circle saturated with 2.5 mi L broth, and regrown for 1-2 h at 28 ° C. The filters were then transferred to fresh petri dishes with filter paper soaked in L broth containing 2 mM IPTG, incubated 15 min at 42°C and incubated at 37°C for 1 h. To lyse filter-bound bacteria, the filters were frozen twice on a steel replica block immersed in a dry ice/ethanol bath. Debris were removed by rinsing the filters with TBS buffer. Filters were blocked with 3 % BSA in TBS buffer for I h at room temperatut~ and reacted with preadsorbed antiserum against the R subunit ofD. discoideumcAMP-dependent protein kinase as previously described (Mutzel et al., 1987); bound antibody was labeled with peroxidase-conjugated goat anti-rabbit IgG (Dianova, Hamburg, F.R.G.) and the complex was stained with 2-chloronaphthol/H202 (Towbin et al,, 1979). b Crude extracts from liquid cultures of the lysogens were prepared and chromatographed on 0.1% SDS-7.5~ polyacrylamide gels (Laemmli, 1970) as described (Mutzel et al., 1988). Proteins were electrophoretically transferred to nitrocellulose filters (BA 85, Schleicher & Schuell) according to Kyhse-Andcrsen (1984) and antigenic material was detected with anti-R subunit antiserum. Prestained Mr marker proteins (Sigma) were used as a standard. In extracts from clones R6, R22, and R30, proteo!ytic degradation products of the fusion proteins were observed. The Mr of the largest band is given. c Extracts from both IPTG-induced (! mM) and non-induced cultures were compared by Western blotting. d Binding of 10- ~ M[3H]cAMP(35-40 Ci/mmol, Amersham) to R subunit in crude lysogen lysates was measured as previously described (Mutzel et al., 1988). Extracts were prepared after induction with I mM IPTG; cAMP-binding in each non-induced extract was 3000 Ci/mmol had to be enzymatically synthesized and purified (Lacombe et al., 1987) commercially available tritiated c AM P sufficed to show cAMP binding directly on bacterial colony blot~.
(2) The preparation of a lysogen library from an existing 2gtl 1 phage library can easily and conveniently be achieved in one day. The risk of a particular 2 g t l l phage falling to form a stable lysogen (Huynh et al., 1985) may thus he balanced by the possibility to isolate, by functional screening, c D N A clone~ which proved otherwise inaccessible.
ACKNOWLEDGEMENTS We thank M.L. Lacombe, H.W. Hofer, D. Malchow and B. Wurster for helpful discussions. We are grateful to E. Bremer for most valuable suggestions, comments on the manuscript, and a gift of 2h80cI. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 156.
REFERENCES Bernier, L., Alvarez, F., Norgard, E.M., Raible, D.W., Mentaberry, A., Schembri,J.G., Sabatini, D.D. and Colman, D.R.: Molecular cloning of a 2',T-cyclic nucleotide 3'-phosphodiesterase: mRNAs with different 5' ends encode the same set of proteins in nervous and lymphoid tissues. J. Neurosci. 7 (1987) 2703-2710. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Anal. Bioehem. 72 (1976) 248-254. Chirala, S.S.: The nucleotide sequence of the lac operon and phage junction in lambda gtl 1. Nucleic Acids Res. 14 (1986) 5935. Cohen, P. and Klee, C.B.: Criieria required to demonstrate calmodulin. dependent effects in vivo. In Cohen, P. and Klee, C.B. (Eds.), Calmodulin, Elsevier, Amsterdam, 1988, pp. 357-364. Huynh, T.V., Young, R.A. and Davis, R.W.: Constructing and screening eDNA libraries in 2gtl0 and 2gtll. In Glover, D.M. (Ed.), DNA Cloning; A Practical Approach, Vol. I. IRL Press, Oxford, 1985, pp. 49-78. Kanfman, D.L., McGinnis, J.F., Krieg~r, N.R. and Tobin, A.J.: Brain glutamate decarboxylase cloned in 2gt-I 1: fusion protein produces y-aminobutyricacid. Science 232 (1986) 1138-1140. Kyhse-Andersen,J.: Electroblotting of multiple gels: a simple apparatus without buffertank for rapid transfer of proteins from polyacrylamide to nitrocellulose,j. Biochem. Biophys. Methods 10 (1984) 203-209. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Lacombe, M.L., Ladant, D., Mutzel, R. and V6ron, M.: Gene isolation by direct in situ cAMP binding. Gene 58 (1987) 29-36. Mutzel, R., Lacombe, M.L., Simon, M.N., de Gunzburg, J. and V6ron, M.: Cloningand eDNA sequence of the regulatory subunit ofcAMPdependent protein kinase from Dictyostelium discoideum. Proc. Natl. Acad. Sci. USA 84 (1987) 6-10. Mutzel, R., Simon, M.N., Lacombe, M.L. and V~ron,M.: Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by 2gtl 1 eDNA clones. Biochemistry27 (1988) 481-486. Rangel-Aldao, R., Schwartz, D. and Rubin, C.S.: Rapid a~say for cyclic AMP and cyclicGMP phosphodiesterases. Anal. Biochem. 87 (1978) 367-375. Sikela, J.M. and Hahn, W.E.: Screening an expression library with a ligund probe: isolation and sequence of a eDNA corresponding to a
2!1 brain calmodulin-binding protein. Proc. Natl. Acad. Sci. USA 84 (1987) 3038-3042. Sin'.on, M.N., Mutzel, R., Mutzel, H. and V~ron, M.: Vectors for expres~ sion of truncated coding sequences in Escherichia coll. Plasmid 19' (1988) 94-102. Simpson, P.J., Chou, W.-G., Whitbeck, A.A., Young, F.E. and Zain, S.: Evidence for prokaryotic transcription and translation control regions in the human factor IX gene. Gene 61 (1987) 373-383. Singh, H., LeBowitz, J.H., Baldwin Jr., A.S. and Sharp, P.A.: Molecular cloning of an enhancer binding protein: isolation by screening of an
expressioa library with a recognition site DNA. Cell 52 (1988) 415-423. Towbin, H., Staehelin, T. and Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proe. Natl. Acad. Sci. USA 76 (1979) 4350-4354. Young, R.A. and Davis, R.W.: Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80 (1983a) 1194-1198. Young, R.A. and Davis, R.W.: Yeast RNA polymerase II genes: isolation with antibody probes. Scienc~ 222 (1983b) 778-782.
