- Email: [email protected]
[78] Preparation of cDNA, ds cDNA, and tailed ds cDNA from interferon mRNA templates
[78]
PREPARATIONOF INTERFERONcDNA
601
the corresponding native and recombinant species show identical activities. Accordingly, it appears that differences in their primary sequences lead to differences in their interaction with a variety of cells. In addition to the large number of natural interferons that can be isolated, it is possible to prepare hybrid and modified interferons by recombination, reconstruction, and alteration of the natural recombinants. Thus, it may be possible to tailor interferons for specific biological activities. Several cloned human interferons have been expressed 1'7-a and we have purified several of them. Recombinant leukocyte interferon A (IFLrA) was purified to homogeneity. 2s,2a On January 15, 1981, clinical trials with IFLrA were begun by Hoffmann-La Roche. Crystals of IFLrA were obtained with the intention of beginning X-ray crystallography to determine its tertiary structure. 3°,31 It is evident that interferon research has taken several giant steps forward. The availability of large amounts of pure interferon for both basic and clinical research will stimulate the advancement of knowledge about interferon much further. The availability of the cDNA and genomic recombinants will lead to understanding how interferon synthesis is regulated by viruses and double-stranded RNA. Recombinants containing the IFLrA sequence have been expressed in an E, coli DNA-dependent cellfree extract. ~2 Such studies with the genomic clones in a eukaryotic cellfree system would be most rewarding. As with most productive areas of research, there is now very much more to do than there was before. M. Evinger, S. Maeda, and S. Pestka, J. Biol. Chem. 256, 2113 (1981). zr R. B. Herberman, J. R. Ortaldo, A. Mantovani, D. S. Hobbs, H.-F. Kung, and S. Pestka, Nature (London) (in press). ~8 T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, J. Biol. Chem. (in press). T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, this series, Vol. 78 [72]. 30 D. L. Miller, H.-F. Kung, T. Staehelin, and S. Pestka, this volume [1]. 31 D. L. Miller, H.-F. Kung, T. Staehelin, and S. Pestka, in preparation. 32 H. Weissbach, D. V. Goeddel, R. McCandliss, S. Maeda, P. C. FamiUetti, B. Redfield, T. Staehelin, and S. Pestka, Arch. Biochem. Biophys. (in press).
[78] P r e p a r a t i o n o f c D N A , ds c D N A , a n d T a i l e d ds c D N A from Interferon mRNA Templates B y RUSSELL M C C A N D L I S S , A L A N SLOMA,
and
H I D E O TAKESHIMA
In order to isolate recombinants containing specific sequences corresponding to messenger RNA (mRNA), it is necessary to make complementary DNA (cDNA) copies of the mRNA, to make the cDNA copy
METHODS IN ENZYMOLOGY.VOL. 79
CopyrightO 1981by AcademicPress. Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181979-5
602
CLONING OF INTERFERON GENES
[78]
d o u b l e - s t r a n d e d , a n d to m o d i f y t h e d o u b l e - s t r a n d e d e D N A ( d s e D N A ) so t h a t it c a n b e i n c o r p o r a t e d into a c l o n i n g v e h i c l e , M a n y c D N A s , i n c l u d i n g t h o s e f o r insulin, m g r o w t h h o r m o n e , 8 g l o b i n , ~ o v a l b u m i n , s d i h y d r o f o l a t e r e d u c t a s e , ° a n d i n t e r f e r o n s , r-~s h a v e b e e n c l o n e d b y t h i s a p p r o a c h , G e n e r a l t e c h n i q u e s f o r t h e s e r e a c t i o n s h a v e b e e n p r e s e n t e d in Vol. 68 o f t h i s s e r i e s . I n t h i s c h a p t e r , specific d e t a i l s o f t h e p r e p a r a t i o n o f i n t e r f e r o n eDNA for cloning r are described.
Synthesis o f
cDNA
M e s s e n g e r R N A c a n be c o p i e d into a e D N A m o l e c u l e w i t h r e v e r s e t r a n s c d p t a s e . TM I n t e r f e r o n m R N A h a s b e e n s h o w n to be p o l y a d e n y l a t e d , 14-~8 w h i c h p e r m i t s o l i g o ( d T ) to b e u s e d as a p r i m e r for e D N A s y n t h e s i s . C o m p l e m e n t a r y D N A is s y n t h e s i z e d b y t h e f o l l o w i n g m e t h o d . Stock Solutions and Materials
Tris • H C I , 0.5 M , p H 8.3 K C I , 1.4 M MgCI~, 0.25 M 1 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter, and H. M. Goodman, Science 196, 1313 (1977). 2 L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 3 p. H. Seeburg, J. Shine, J. A. Martial, J. D. Baxter, and H. M. Goodman, Nature (London) 270, 486 (1977). 4 F. Rougeon, P. Kourilsky, and B. Mach, Nucleic Acids Res. 2, 2365 (1975). P. Humphries, M. Cochet, A. Krust, P. Gerlinger, P. Kourilsky, and P. Chambon, Nucleic Acids Res. 4, 2389 (1977). A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Sehimke, and S. N. Cohen, Nature (London) 275, 617 (1978). S. Maeda, R. MeCandliss, M. Gross, A. Sloma, P. C. Familletti, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980). 8 S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. EesGdi, W. Boll, K. Cantell, and C. Weissmann, Nature (London) 284, 316 (1980). g T. Taniguchi, M. Sakai, Y. Fujii-Kuriyama, M. Muramatsu, S. Kobayashi, and T. Sudo, Proc. Jpn. Acad. Set. B 55, 464 (1979). 1oR. Derynck, J. Content, E. De Clercq, G. Volekaert, J. Tavernier, R. Devos, and W. Fiers, Nature (London) 285, 542 (1980). 11 M. Houghton, A. G. Stewart, S. M. Doel, J. S. Emtage, M. A. W. Eaton, J. C. Smith, T. P. Patel, H. M. Lewis, A. G. Porter, J. R. Birch, T. Cartwright, and N. H. Carey, Nucleic Acids Res. 8, 1913 (1980). 12 D. V. Goeddel, H. M. Shepard, E. Yelverton, D. Leung, R. Crea, A. SIGma, and S. Pestka, Nucleic Acids Res. 8, 4057 (1980). 13D. L. Kacian, and J. C. Myers, Proc. Natl. Acad. Sci. U.S.A. 73, 2191 (1976). 14F. H. Reynolds, Jr., and P. M. Pitha, Biochem. Biophys. Res. Commun. 59, 1023 (1974). 1~ S. Pestka, J. Mclnnes, E. A. Havell, and J. Vil~ek, Proc. Natl. Acad. Sci. U.S.A. 72, 3898 (1975). ~3R. McCandliss, A. Sloma, and S. Pestka, this volume [8].
[78]
PREPARATION OF INTERFERON e D N A
603
dATE 0.05 M, pH 7.0 dGTE 0.05 M, pH 7.0 dCTE 0.05 M, pH 7.0 TTE 0.05 M, pH 7.0 [a-a2P]dCTE 400 Ci/mmol, 1 mCi/ml (Amersham) Dithiothreitol (DTT), 0.01 M Oligo(dT)n_la, 250/xg/ml (Collaborative Research) Actinomycin D, 500/xg/ml (Calbiochem) Disodium ethylenediaminetetraacetate (EDTA), 0.2 M, pH 8.0 Avian myeloblastosis virus (AMV) reverse transcriptase, approximately 10,000 units/ml (obtained from Research Resources Branch, Viral Oncology Program, National Cancer Institute) All buffers and salt solutions are autoclaved. The other solutions are prepared with sterile glass-distilled water and stored in sterile containers. All stock solutions are stored frozen. Procedure. As a template for cDNA synthesis, 12 S interferon mRNA isolated as previously described TM is used. In order to follow synthesis, either [3H]dCTP or [a-3zP]dCTP may be used. For preparation of recombinants, the eDNA must be located on polyacrylamide gels; therefore, it is more convenient to use 32p as the radioactive label. The radioactive compound is dried by lyophilization, by blowing off the solvent with a stream of nitrogen, or by drying in a Savant Speed Vac Concentrator. For each microgram of mRNA, 5/~Ci of [a-a~P]dCTP at a specific activity of 400 Ci/mmol are used. The dried material is dissolved in a 2 × reaction mixture consisting of0.1M Tris • HCI, pH 8.3, 140 mM KC1, 20 mM MgCl2, I mM dATE 1 mM dCTE 1 mM dGTE 1 mM TTE and 0.4 mM DTT. This solution is kept on ice. To this solution are added mRNA (50/~g/rnl, final concentration), oligo(dT)n_ls (25 ftg/ml), actinomycin D (40/zg/ml), AMV reverse transcriptase (800 units/ml), and enough water to dilute the 2× mix to 1×. After 5 rain on ice, the reaction mixture is incubated at 46° for l0 min. After the incubation, EDTA is added to a final concentration of 25 mM. The solution is extracted one time with an equal volume of phenol : chloroform (1/1; v/v), and the aqueous phase is chromatographed on a column of Sephadex G-100 (0.7 x 20 cm) equilibrated with 10 mM Tris • HC1, pH 8.0, I mM EDTA, 0.1M NaC1. The cDNA in the excluded volume is precipitated by addition of 0.1 volume of 2.4 M sodium acetate, pH 5, and 2.5 volumes of ethanol. To remove the mRNA template, the cDNA is sedimented by centrifugation, dissolved in 0.3 ml of 0.1 M NaOH, and incubated at 70° for 20 min. The solution is neutralized with 1.0 M HCI and precipitated with ethanol as described above. The yield of eDNA is 10-20% of the mRNA used. The conditions described are optimized for full-length eDNA synthesis, but not for mammal incorporation, which would be important in the synthesis of a cDNA probe.
604
CLONING OF INTERFERON GENES
[78]
Synthesis of ds cDNA
Synthesis of ds cDNA from cDNA is performed with the use of DNA polymerase I (Klenow fragment), which lacks the 5' ---> 3' exonuclease activity. 1~ No additional primer is needed because of the 3' loop on most cDNA molecules made with AMV reverse transcriptase. TM In order to make ds cDNA, the 3' loop must be cleaved by Aspergillus oryzae S1 nuclease. TM To select for full-length copies of the mRNA, the ds cDNA is sized on a polyacrylamide gel, and long fragments are located, excised, and electroeluted from the gel. Stock Solutions and Materials Potassium phosphate, 0.5 M, pH 7.4 MgCI~, 0.25 M DTT, 0.1 M dATE 0.05 M, pH 7.0 dGTP, 0.05 M, pH 7.0 dCTP, 0.05 M, pH 7.0 TTP, 0.05 M, pH 7.0 [8-aH]dCTP, 22 Ci/mmol, 1 mCi/ml (Amersham) Escherichia coli DNA polymerase I (Klenow fragment), approximately 1000 units/ml (Boehringer-Mannheim) 5× S1 nuclease buffer: 0.167 M sodium acetate, pH 4.5; 5 mM ZnCI2 Procedure. For cloning, it is not necessary to use a radioactive label in the second strand if the first strand is labeled. However, to follow second-strand synthesis, it is convenient to use [aH]dCTE The labeled compound is dried as described above. Ten microcuries of [3H]dCTP (specific activity, 22 Ci/mmol) are used for each microgram of mRNA used for cDNA synthesis. The dried [aH]dCTP is dissolved in a 2× reaction mixture consisting of 0.2 M potassium phosphate, pH 7.4, 20 mM MgCl~, 2 mM DTT, 0.4 mM each of dATP, dGTP, dCTP, and TTE This mixture is kept on ice, cDNA in water is added, E. coli DNA polymerase I (Klenow fragment) is added to 100 units/ml, and water is added to dilute the reaction mixture to 1 ×. The solution is incubated a minimum of 2 hr at 15° . It is convenient to allow this reaction to incubate overnight. After the incubation, EDTA is added to 25 mM, the solution is extracted once with an equal volume of phenol : chloroform (1/1; v/v), and the aqueous phase is chromatographed on a 0.7 × 20 cm column of Sephadex G-100 equilibrated with 10 mM Tris • HC1, pH 8.0, 1 mM EDTA, and 0.1 M NaCl. The DNA in the excluded fractions is precipitated with ethanol as described lr H. Klenow, K. Overgaard-Hansen, and S. A. Patkar, Eur. J. Biochem. 22, 371 (1971). is A. Efstratiadis, F. C. Kafatos, A. M. Maxam, and T. Maniatis, Cell 7, 279 (1976). 19 V. M. Vogt, Eur. J. Biochem. 33, 192 (1973).
[78]
PREPARATIONOF INTERFERONcDNA
605
above. The yield of dsDNA is 50-100% of the amount of cDNA used as template. At this point, the ds cDNA contains a hairpin loop. The singlestranded loop is removed by digestion withAspergillus oryzae S 1 nuclease, prepared by the method of Vogt. TMThe ds cDNA is dissolved in water, and 0.25 volume of 5× S1 buffer is added. An appropriate amount of S1 nuclease is added, and the solution is incubated 20 min at 37°. The amount of enzyme to be added must be determined empirically for each enzyme preparation, since the activity varies from one preparation to another. This is done by measuring the decrease in trichloroacetic acid-precipitable counts from the ds cDNA. Usually, 50-75% of the ds cDNA is resistant to S1 nuclease. However, care must be exercised in order to avoid overdigestion due to low levels of contaminating nucleases in the S I nuclease preparation. The Sl-digested ds cDNA is extracted once with phenol : chloroform, and the aqueous phase is precipitated with ethanol as described above. It is desirable to have full-length copies of the gene. Since interferon mRNA is about 12 S on sucrose gradients, 16 ds cDNA having about 900 base pairs should contain the entire sequence. Molecules of this size can be isolated by electrophoresis on an 8% polyacrylamide gel run in Trisborate-EDTA (TBE)(0.09 M Tris, 0.09 M borate, 0.025 M EDTA, pH 8.3) as described by Maniatis et al. 2° Plasmid pBR322 digested with TaqI restriction endonuclease provides molecular weight markers of convenient size. 21 After electrophoresis, the gel is stained with 0.5 /zg of ethidium bromide per milliliter to visualize the markers. The wet gel is then exposed to Kodak X-Omat R film for a short period of time (dependent upon the number of counts applied to the gel) to locate the labeled ds cDNA. The region of the gel containing DNA molecules between 500 and 1300 base pairs long is cut out, and the DNA is eluted electrophoretically. The gel slice is placed in a dialysis bag containing 0.3-0.6 ml of 0.1 × TBE, and the bag is placed in an electrophoresis chamber similar to that described by Smith. 22 Electrophoresis is performed for 1.5 hr at 100 V. The solution from inside the dialysis bag is extracted once with phenol:chloroform (1/1; v/v), and the ds cDNA is precipitated with ethanol. Recovery can be estimated by monitoring Cerenkov radiation of the gel slice and the solutions. Homopolyrner Tailing of ds cDNA A convenient means of inserting ds cDNA into a cloning vector is to add single-stranded homopolymer tails to the 3'-OH termini of the ds z0 T. Maniatis, A. Jeffrey, and H. van deSande, Biochemistry 14, 3787 (1975). 21 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978). 2~ H. O. Smith, this series, Vol. 65, p. 371.
606
CLONING OF INTERFERON GENES
[78]
YIELDS OF PRODUCTS DURING SYNTHESIS OF
dC-TAILED ds cDNA SYNTHESIS
Step
Amount (gg)
Percent yield
mRNA cDNA Sized ds cDNA dC-ds cDNA
13 1.5 0.24 0.23
11.5 1.18 (15) a 1.8 (96)
Numbers in parentheses represent the stepwise yields.
cDNA and complementary homopolymer tails to the 3'-OH termini of the vector, and to anneal the two tailed DNA molecules. Methods of homopolymer addition have been described in detail by Nelson and Brutlag. z3 Because cloning into the plasmid pBR322 at thePstI site by G-C tailing regenerates the PstI recognition sequence at each end of the inserted DNA sequence, z4 it is convenient to use this procedure. Stock Solutions and Materials
Potassium cacodylate, 1.4 M; 0.3 M "Iris; pH 7.6 (pH becomes 7.2 when diluted 1 : 10) CoCI~, 15 mM DTT, 10 mM dCTP, 4 mM, pH 7.0 Procedure. Sizing of the ds cDNA allows an accurate estimation of the concentration of 3' termini. Addition of dCMP residues to the 3' ends may be followed by incorporation of [SH]dCTP into acid-precipitable material. Twenty-five microcuries of [aH]dCTP are used for each microgram of mRNA used for the original cDNA synthesis. The radioactive compound is dried and redissolved in the reaction mixture. Double-stranded cDNA is dissolved in appropriate amounts of stock solutions to give final concentrations as follows: 0.14 M potassium cacodylate, 0.03 M Tris, pH 7.2; 1 mM DTT; 0.1 mM [SH]dCTP (1 Ci/mmol); 1.5 mM COC12; and 2 x 10-SM 3' termini. The solution without COC12 is warmed to 37°; the CoCI~ is then added. Terminal deoxynucleotidyltransferase purified by the method of Chang and Bollum 25 is added to a final concentration of 100 units/ml. The T. Nelson and D. Brutlag, this series, Vol. 68, p. 41. F. Bolivar, R. L. Rodriguez, P. J. Greene, M. C. Baflach, H. L. Heyneker, H. W. Buyer, J. H. Crosa, and S. Falkow, Gene 2, 95 (1977). 25 L. M. S. Chang and F. J. Bollum, J. Biol. Chem. 246, 909 (1971).
[79]
OLIGO(dG)-TAILING OF
PLASMIDDNA
607
reaction is allowed to proceed at 37° for 5 min. At this time, a sample is taken to measure incorporation of [3H]dCMP into acid-precipitable material. If tails of suffacient length have not been added, the reaction can be continued by placing the solution at 37° again for the desired length of time. We have obtained the best transformation eltieiencies with homopolymer tails of about 10-20 dCMP residues. ~ When tails of the desired length have been generated, EDTA is added (10 mM, final concentration). The solution is extracted once with phenol : chloroform (1/I; v/v), and the aqueous phase is precipitated with ethanol. The dC-tailed ds cDNA at this point is ready for insertion into a dG-tailed pBR322 vector as described by Maeda et al. ~ Results of a representative preparation are given in the table. 56 S. Maeda, this volume [80].
[79] P r e p a r a t i o n o f P l a s m i d s a n d T a i l i n g w i t h Oligodeoxyguanylic Acid B y SHUICHIRO I~xAEDA
The plasmid pBR3221 was used for the cloning of complementary DNA (eDNA) copies of mRNA from interferon-producing human cells. Deoxyguanylic acid (dG) residues are added to the P s t I restriction termini with terminal deoxynueleotidyltransferase. 2 The tailed plasmid DNA is annealed with cDNA which has been tailed with oligodeoxycytidylic acid (dC) residues, s Because the homopolymer-joining method reconstructs the P s t I sites at each end of the inserted eDNA, 1 the inserted eDNA and flanking oligo(dG) • oligo(dC) sequences can be excised from the reeombinants with PstI. Since the P s t I site lies in the ~-lactamase gene of the plasmid, the eDNA may be expressed as a fusion protein 4 or, in some cases, as an independent protein. 5 1 F. Bolivar, R. L. Rodriguez, P. J. Green, M. C. Betlach, H. L. Heyneker, H. W. Boyer, and S. Falkow, Gene 2, 95 (1977). 2 R. Roychoudhury and R. Wu, this series, Vol. 65, p. 43. 3 R. McCandliss, A. Sloma, and H. Takeshima, this volume [78]. 4 L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 5 A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen, Nature (London) 275, 617 (1978).
METHODSIN ENZYMOLOGY.VOL. 79
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181979-5
PREPARATIONOF INTERFERONcDNA
601
the corresponding native and recombinant species show identical activities. Accordingly, it appears that differences in their primary sequences lead to differences in their interaction with a variety of cells. In addition to the large number of natural interferons that can be isolated, it is possible to prepare hybrid and modified interferons by recombination, reconstruction, and alteration of the natural recombinants. Thus, it may be possible to tailor interferons for specific biological activities. Several cloned human interferons have been expressed 1'7-a and we have purified several of them. Recombinant leukocyte interferon A (IFLrA) was purified to homogeneity. 2s,2a On January 15, 1981, clinical trials with IFLrA were begun by Hoffmann-La Roche. Crystals of IFLrA were obtained with the intention of beginning X-ray crystallography to determine its tertiary structure. 3°,31 It is evident that interferon research has taken several giant steps forward. The availability of large amounts of pure interferon for both basic and clinical research will stimulate the advancement of knowledge about interferon much further. The availability of the cDNA and genomic recombinants will lead to understanding how interferon synthesis is regulated by viruses and double-stranded RNA. Recombinants containing the IFLrA sequence have been expressed in an E, coli DNA-dependent cellfree extract. ~2 Such studies with the genomic clones in a eukaryotic cellfree system would be most rewarding. As with most productive areas of research, there is now very much more to do than there was before. M. Evinger, S. Maeda, and S. Pestka, J. Biol. Chem. 256, 2113 (1981). zr R. B. Herberman, J. R. Ortaldo, A. Mantovani, D. S. Hobbs, H.-F. Kung, and S. Pestka, Nature (London) (in press). ~8 T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, J. Biol. Chem. (in press). T. Staehelin, D. S. Hobbs, H.-F. Kung, and S. Pestka, this series, Vol. 78 [72]. 30 D. L. Miller, H.-F. Kung, T. Staehelin, and S. Pestka, this volume [1]. 31 D. L. Miller, H.-F. Kung, T. Staehelin, and S. Pestka, in preparation. 32 H. Weissbach, D. V. Goeddel, R. McCandliss, S. Maeda, P. C. FamiUetti, B. Redfield, T. Staehelin, and S. Pestka, Arch. Biochem. Biophys. (in press).
[78] P r e p a r a t i o n o f c D N A , ds c D N A , a n d T a i l e d ds c D N A from Interferon mRNA Templates B y RUSSELL M C C A N D L I S S , A L A N SLOMA,
and
H I D E O TAKESHIMA
In order to isolate recombinants containing specific sequences corresponding to messenger RNA (mRNA), it is necessary to make complementary DNA (cDNA) copies of the mRNA, to make the cDNA copy
METHODS IN ENZYMOLOGY.VOL. 79
CopyrightO 1981by AcademicPress. Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181979-5
602
CLONING OF INTERFERON GENES
[78]
d o u b l e - s t r a n d e d , a n d to m o d i f y t h e d o u b l e - s t r a n d e d e D N A ( d s e D N A ) so t h a t it c a n b e i n c o r p o r a t e d into a c l o n i n g v e h i c l e , M a n y c D N A s , i n c l u d i n g t h o s e f o r insulin, m g r o w t h h o r m o n e , 8 g l o b i n , ~ o v a l b u m i n , s d i h y d r o f o l a t e r e d u c t a s e , ° a n d i n t e r f e r o n s , r-~s h a v e b e e n c l o n e d b y t h i s a p p r o a c h , G e n e r a l t e c h n i q u e s f o r t h e s e r e a c t i o n s h a v e b e e n p r e s e n t e d in Vol. 68 o f t h i s s e r i e s . I n t h i s c h a p t e r , specific d e t a i l s o f t h e p r e p a r a t i o n o f i n t e r f e r o n eDNA for cloning r are described.
Synthesis o f
cDNA
M e s s e n g e r R N A c a n be c o p i e d into a e D N A m o l e c u l e w i t h r e v e r s e t r a n s c d p t a s e . TM I n t e r f e r o n m R N A h a s b e e n s h o w n to be p o l y a d e n y l a t e d , 14-~8 w h i c h p e r m i t s o l i g o ( d T ) to b e u s e d as a p r i m e r for e D N A s y n t h e s i s . C o m p l e m e n t a r y D N A is s y n t h e s i z e d b y t h e f o l l o w i n g m e t h o d . Stock Solutions and Materials
Tris • H C I , 0.5 M , p H 8.3 K C I , 1.4 M MgCI~, 0.25 M 1 A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter, and H. M. Goodman, Science 196, 1313 (1977). 2 L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 3 p. H. Seeburg, J. Shine, J. A. Martial, J. D. Baxter, and H. M. Goodman, Nature (London) 270, 486 (1977). 4 F. Rougeon, P. Kourilsky, and B. Mach, Nucleic Acids Res. 2, 2365 (1975). P. Humphries, M. Cochet, A. Krust, P. Gerlinger, P. Kourilsky, and P. Chambon, Nucleic Acids Res. 4, 2389 (1977). A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Sehimke, and S. N. Cohen, Nature (London) 275, 617 (1978). S. Maeda, R. MeCandliss, M. Gross, A. Sloma, P. C. Familletti, J. M. Tabor, M. Evinger, W. P. Levy, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 77, 7010 (1980). 8 S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. EesGdi, W. Boll, K. Cantell, and C. Weissmann, Nature (London) 284, 316 (1980). g T. Taniguchi, M. Sakai, Y. Fujii-Kuriyama, M. Muramatsu, S. Kobayashi, and T. Sudo, Proc. Jpn. Acad. Set. B 55, 464 (1979). 1oR. Derynck, J. Content, E. De Clercq, G. Volekaert, J. Tavernier, R. Devos, and W. Fiers, Nature (London) 285, 542 (1980). 11 M. Houghton, A. G. Stewart, S. M. Doel, J. S. Emtage, M. A. W. Eaton, J. C. Smith, T. P. Patel, H. M. Lewis, A. G. Porter, J. R. Birch, T. Cartwright, and N. H. Carey, Nucleic Acids Res. 8, 1913 (1980). 12 D. V. Goeddel, H. M. Shepard, E. Yelverton, D. Leung, R. Crea, A. SIGma, and S. Pestka, Nucleic Acids Res. 8, 4057 (1980). 13D. L. Kacian, and J. C. Myers, Proc. Natl. Acad. Sci. U.S.A. 73, 2191 (1976). 14F. H. Reynolds, Jr., and P. M. Pitha, Biochem. Biophys. Res. Commun. 59, 1023 (1974). 1~ S. Pestka, J. Mclnnes, E. A. Havell, and J. Vil~ek, Proc. Natl. Acad. Sci. U.S.A. 72, 3898 (1975). ~3R. McCandliss, A. Sloma, and S. Pestka, this volume [8].
[78]
PREPARATION OF INTERFERON e D N A
603
dATE 0.05 M, pH 7.0 dGTE 0.05 M, pH 7.0 dCTE 0.05 M, pH 7.0 TTE 0.05 M, pH 7.0 [a-a2P]dCTE 400 Ci/mmol, 1 mCi/ml (Amersham) Dithiothreitol (DTT), 0.01 M Oligo(dT)n_la, 250/xg/ml (Collaborative Research) Actinomycin D, 500/xg/ml (Calbiochem) Disodium ethylenediaminetetraacetate (EDTA), 0.2 M, pH 8.0 Avian myeloblastosis virus (AMV) reverse transcriptase, approximately 10,000 units/ml (obtained from Research Resources Branch, Viral Oncology Program, National Cancer Institute) All buffers and salt solutions are autoclaved. The other solutions are prepared with sterile glass-distilled water and stored in sterile containers. All stock solutions are stored frozen. Procedure. As a template for cDNA synthesis, 12 S interferon mRNA isolated as previously described TM is used. In order to follow synthesis, either [3H]dCTP or [a-3zP]dCTP may be used. For preparation of recombinants, the eDNA must be located on polyacrylamide gels; therefore, it is more convenient to use 32p as the radioactive label. The radioactive compound is dried by lyophilization, by blowing off the solvent with a stream of nitrogen, or by drying in a Savant Speed Vac Concentrator. For each microgram of mRNA, 5/~Ci of [a-a~P]dCTP at a specific activity of 400 Ci/mmol are used. The dried material is dissolved in a 2 × reaction mixture consisting of0.1M Tris • HCI, pH 8.3, 140 mM KC1, 20 mM MgCl2, I mM dATE 1 mM dCTE 1 mM dGTE 1 mM TTE and 0.4 mM DTT. This solution is kept on ice. To this solution are added mRNA (50/~g/rnl, final concentration), oligo(dT)n_ls (25 ftg/ml), actinomycin D (40/zg/ml), AMV reverse transcriptase (800 units/ml), and enough water to dilute the 2× mix to 1×. After 5 rain on ice, the reaction mixture is incubated at 46° for l0 min. After the incubation, EDTA is added to a final concentration of 25 mM. The solution is extracted one time with an equal volume of phenol : chloroform (1/1; v/v), and the aqueous phase is chromatographed on a column of Sephadex G-100 (0.7 x 20 cm) equilibrated with 10 mM Tris • HC1, pH 8.0, I mM EDTA, 0.1M NaC1. The cDNA in the excluded volume is precipitated by addition of 0.1 volume of 2.4 M sodium acetate, pH 5, and 2.5 volumes of ethanol. To remove the mRNA template, the cDNA is sedimented by centrifugation, dissolved in 0.3 ml of 0.1 M NaOH, and incubated at 70° for 20 min. The solution is neutralized with 1.0 M HCI and precipitated with ethanol as described above. The yield of eDNA is 10-20% of the mRNA used. The conditions described are optimized for full-length eDNA synthesis, but not for mammal incorporation, which would be important in the synthesis of a cDNA probe.
604
CLONING OF INTERFERON GENES
[78]
Synthesis of ds cDNA
Synthesis of ds cDNA from cDNA is performed with the use of DNA polymerase I (Klenow fragment), which lacks the 5' ---> 3' exonuclease activity. 1~ No additional primer is needed because of the 3' loop on most cDNA molecules made with AMV reverse transcriptase. TM In order to make ds cDNA, the 3' loop must be cleaved by Aspergillus oryzae S1 nuclease. TM To select for full-length copies of the mRNA, the ds cDNA is sized on a polyacrylamide gel, and long fragments are located, excised, and electroeluted from the gel. Stock Solutions and Materials Potassium phosphate, 0.5 M, pH 7.4 MgCI~, 0.25 M DTT, 0.1 M dATE 0.05 M, pH 7.0 dGTP, 0.05 M, pH 7.0 dCTP, 0.05 M, pH 7.0 TTP, 0.05 M, pH 7.0 [8-aH]dCTP, 22 Ci/mmol, 1 mCi/ml (Amersham) Escherichia coli DNA polymerase I (Klenow fragment), approximately 1000 units/ml (Boehringer-Mannheim) 5× S1 nuclease buffer: 0.167 M sodium acetate, pH 4.5; 5 mM ZnCI2 Procedure. For cloning, it is not necessary to use a radioactive label in the second strand if the first strand is labeled. However, to follow second-strand synthesis, it is convenient to use [aH]dCTE The labeled compound is dried as described above. Ten microcuries of [3H]dCTP (specific activity, 22 Ci/mmol) are used for each microgram of mRNA used for cDNA synthesis. The dried [aH]dCTP is dissolved in a 2× reaction mixture consisting of 0.2 M potassium phosphate, pH 7.4, 20 mM MgCl~, 2 mM DTT, 0.4 mM each of dATP, dGTP, dCTP, and TTE This mixture is kept on ice, cDNA in water is added, E. coli DNA polymerase I (Klenow fragment) is added to 100 units/ml, and water is added to dilute the reaction mixture to 1 ×. The solution is incubated a minimum of 2 hr at 15° . It is convenient to allow this reaction to incubate overnight. After the incubation, EDTA is added to 25 mM, the solution is extracted once with an equal volume of phenol : chloroform (1/1; v/v), and the aqueous phase is chromatographed on a 0.7 × 20 cm column of Sephadex G-100 equilibrated with 10 mM Tris • HC1, pH 8.0, 1 mM EDTA, and 0.1 M NaCl. The DNA in the excluded fractions is precipitated with ethanol as described lr H. Klenow, K. Overgaard-Hansen, and S. A. Patkar, Eur. J. Biochem. 22, 371 (1971). is A. Efstratiadis, F. C. Kafatos, A. M. Maxam, and T. Maniatis, Cell 7, 279 (1976). 19 V. M. Vogt, Eur. J. Biochem. 33, 192 (1973).
[78]
PREPARATIONOF INTERFERONcDNA
605
above. The yield of dsDNA is 50-100% of the amount of cDNA used as template. At this point, the ds cDNA contains a hairpin loop. The singlestranded loop is removed by digestion withAspergillus oryzae S 1 nuclease, prepared by the method of Vogt. TMThe ds cDNA is dissolved in water, and 0.25 volume of 5× S1 buffer is added. An appropriate amount of S1 nuclease is added, and the solution is incubated 20 min at 37°. The amount of enzyme to be added must be determined empirically for each enzyme preparation, since the activity varies from one preparation to another. This is done by measuring the decrease in trichloroacetic acid-precipitable counts from the ds cDNA. Usually, 50-75% of the ds cDNA is resistant to S1 nuclease. However, care must be exercised in order to avoid overdigestion due to low levels of contaminating nucleases in the S I nuclease preparation. The Sl-digested ds cDNA is extracted once with phenol : chloroform, and the aqueous phase is precipitated with ethanol as described above. It is desirable to have full-length copies of the gene. Since interferon mRNA is about 12 S on sucrose gradients, 16 ds cDNA having about 900 base pairs should contain the entire sequence. Molecules of this size can be isolated by electrophoresis on an 8% polyacrylamide gel run in Trisborate-EDTA (TBE)(0.09 M Tris, 0.09 M borate, 0.025 M EDTA, pH 8.3) as described by Maniatis et al. 2° Plasmid pBR322 digested with TaqI restriction endonuclease provides molecular weight markers of convenient size. 21 After electrophoresis, the gel is stained with 0.5 /zg of ethidium bromide per milliliter to visualize the markers. The wet gel is then exposed to Kodak X-Omat R film for a short period of time (dependent upon the number of counts applied to the gel) to locate the labeled ds cDNA. The region of the gel containing DNA molecules between 500 and 1300 base pairs long is cut out, and the DNA is eluted electrophoretically. The gel slice is placed in a dialysis bag containing 0.3-0.6 ml of 0.1 × TBE, and the bag is placed in an electrophoresis chamber similar to that described by Smith. 22 Electrophoresis is performed for 1.5 hr at 100 V. The solution from inside the dialysis bag is extracted once with phenol:chloroform (1/1; v/v), and the ds cDNA is precipitated with ethanol. Recovery can be estimated by monitoring Cerenkov radiation of the gel slice and the solutions. Homopolyrner Tailing of ds cDNA A convenient means of inserting ds cDNA into a cloning vector is to add single-stranded homopolymer tails to the 3'-OH termini of the ds z0 T. Maniatis, A. Jeffrey, and H. van deSande, Biochemistry 14, 3787 (1975). 21 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978). 2~ H. O. Smith, this series, Vol. 65, p. 371.
606
CLONING OF INTERFERON GENES
[78]
YIELDS OF PRODUCTS DURING SYNTHESIS OF
dC-TAILED ds cDNA SYNTHESIS
Step
Amount (gg)
Percent yield
mRNA cDNA Sized ds cDNA dC-ds cDNA
13 1.5 0.24 0.23
11.5 1.18 (15) a 1.8 (96)
Numbers in parentheses represent the stepwise yields.
cDNA and complementary homopolymer tails to the 3'-OH termini of the vector, and to anneal the two tailed DNA molecules. Methods of homopolymer addition have been described in detail by Nelson and Brutlag. z3 Because cloning into the plasmid pBR322 at thePstI site by G-C tailing regenerates the PstI recognition sequence at each end of the inserted DNA sequence, z4 it is convenient to use this procedure. Stock Solutions and Materials
Potassium cacodylate, 1.4 M; 0.3 M "Iris; pH 7.6 (pH becomes 7.2 when diluted 1 : 10) CoCI~, 15 mM DTT, 10 mM dCTP, 4 mM, pH 7.0 Procedure. Sizing of the ds cDNA allows an accurate estimation of the concentration of 3' termini. Addition of dCMP residues to the 3' ends may be followed by incorporation of [SH]dCTP into acid-precipitable material. Twenty-five microcuries of [aH]dCTP are used for each microgram of mRNA used for the original cDNA synthesis. The radioactive compound is dried and redissolved in the reaction mixture. Double-stranded cDNA is dissolved in appropriate amounts of stock solutions to give final concentrations as follows: 0.14 M potassium cacodylate, 0.03 M Tris, pH 7.2; 1 mM DTT; 0.1 mM [SH]dCTP (1 Ci/mmol); 1.5 mM COC12; and 2 x 10-SM 3' termini. The solution without COC12 is warmed to 37°; the CoCI~ is then added. Terminal deoxynucleotidyltransferase purified by the method of Chang and Bollum 25 is added to a final concentration of 100 units/ml. The T. Nelson and D. Brutlag, this series, Vol. 68, p. 41. F. Bolivar, R. L. Rodriguez, P. J. Greene, M. C. Baflach, H. L. Heyneker, H. W. Buyer, J. H. Crosa, and S. Falkow, Gene 2, 95 (1977). 25 L. M. S. Chang and F. J. Bollum, J. Biol. Chem. 246, 909 (1971).
[79]
OLIGO(dG)-TAILING OF
PLASMIDDNA
607
reaction is allowed to proceed at 37° for 5 min. At this time, a sample is taken to measure incorporation of [3H]dCMP into acid-precipitable material. If tails of suffacient length have not been added, the reaction can be continued by placing the solution at 37° again for the desired length of time. We have obtained the best transformation eltieiencies with homopolymer tails of about 10-20 dCMP residues. ~ When tails of the desired length have been generated, EDTA is added (10 mM, final concentration). The solution is extracted once with phenol : chloroform (1/I; v/v), and the aqueous phase is precipitated with ethanol. The dC-tailed ds cDNA at this point is ready for insertion into a dG-tailed pBR322 vector as described by Maeda et al. ~ Results of a representative preparation are given in the table. 56 S. Maeda, this volume [80].
[79] P r e p a r a t i o n o f P l a s m i d s a n d T a i l i n g w i t h Oligodeoxyguanylic Acid B y SHUICHIRO I~xAEDA
The plasmid pBR3221 was used for the cloning of complementary DNA (eDNA) copies of mRNA from interferon-producing human cells. Deoxyguanylic acid (dG) residues are added to the P s t I restriction termini with terminal deoxynueleotidyltransferase. 2 The tailed plasmid DNA is annealed with cDNA which has been tailed with oligodeoxycytidylic acid (dC) residues, s Because the homopolymer-joining method reconstructs the P s t I sites at each end of the inserted eDNA, 1 the inserted eDNA and flanking oligo(dG) • oligo(dC) sequences can be excised from the reeombinants with PstI. Since the P s t I site lies in the ~-lactamase gene of the plasmid, the eDNA may be expressed as a fusion protein 4 or, in some cases, as an independent protein. 5 1 F. Bolivar, R. L. Rodriguez, P. J. Green, M. C. Betlach, H. L. Heyneker, H. W. Boyer, and S. Falkow, Gene 2, 95 (1977). 2 R. Roychoudhury and R. Wu, this series, Vol. 65, p. 43. 3 R. McCandliss, A. Sloma, and H. Takeshima, this volume [78]. 4 L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). 5 A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen, Nature (London) 275, 617 (1978).
METHODSIN ENZYMOLOGY.VOL. 79
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181979-5