[5]

CLONING OF HORMONE GENES

[5] C l o n i n g

75

of Hormone Genes from a Mixture of cDNA Molecules 1

B y HOWARD M. GOODMAN AND RAYMOND J. MACDONALD

The molecular cloning of complementary D N A (cDNA) synthesized from a purified m R N A is a well-established method for obtaining purified D N A for sequence analysis and use as a hybridization probe. H o w e v e r , only a limited number of purified m R N A s are currently available, namely, those from specialized cells producing predominantly a single protein such as globin, immunoglubin, or ovalbumin. We have recently described the isolation and amplification in bacteria of the D N A sequences complementary to m R N A for several polypeptide hormones: rat insulin, z rat growth hormone, 3 human chorionic somatomammotropin, 4 and human growth hormone. 5 All these clones were isolated after transformation of bacteria with an enriched but impure population of c D N A molecules ligated to a plasmid vector. This chapter describes the procedures we have used to isolate and clone specific hormone c D N A s from an impure m R N A population. Principle of the M e t h o d A novel purification procedure for obtaining c D N A clones containing nucleotide sequences complementary to an individual m R N A species has been developed. 6 The method employs restriction endonuclease cleavage o f double-stranded c D N A transcribed from a complex mixture of m R N A . The method does not require any extensive purification o f R N A but instead makes use of transcription of R N A into c D N A , sequence-specific fragmentation of this c D N A with one or two restriction endonucleases, and fractionation of the c D N A restriction fragments on the basis of their 1 Support for writing this chapter came from grants from the United States Public Health Service: Grant Nos. CA 14026 and AM 19997. H.M.G. is an investigator at the Howard Hughes Medical Institute. R. J. M. was supported by Grant AM21344to WiliamJ. Rutter. A. Ullrich, J. Shine, J. Chirgwin, R. Pictet, E. Tischer, W. J. Rutter, and H. M. Goodman, Science 196, 1313 0977). a p. H. Seeburg, J. Shine, J. A. Martial, J. D. Baxter, and H. M. Goodman, Nature (London) 270, 486 0977). 4 j. Shine, P. H. Seeburg, J. A. Martial, J. D. Baxter, and H. M. Goodman, Nature (London) 270, 494 (1977). 5 H. M. Goodman, P. H. Seeburg, J. Shine, J..A. Martial, and J. D. Baxter. Specific Eukaryotic Genes: Struct. Organ. Funct., Proc. Alfred Benzon Syrup., 13th, 1978, p. 179 0979). 8 p. H. Seeburg, J. Shine, J. A. Martial, A. Ullrich, J. D. Baxter, and H. M. Goodman, Cell 12, 157 (1977). METHODS IN ENZYMOLOGY, VOL. 68

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181968-X

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[5]

lengths. The use of restriction endonucleases eliminates size heterogeneity and produces homogeneous-length DNA fragments from any cDNA species that contains at least two restriction sites. From the initially heterogeneous population of cDNA transcripts, uniform-sized fragments of desired sequence are produced. The fragments may be several hundred nucleotides in length and may in some instances include part or all of the structural gene for a particular protein. The length of the fragments depends on the number of nucleotides separating the restriction sites and is usually different for different regions of DNA. Fractionation by length allows purification of an enriched population of fragments containing the desired sequence. Current Separation and analysis methods permit the isolation of such fragments from a RNA preparation in which a specific mRNA species represents at least 2% of the mass of the RNA transcribed. The use of standard RNA fractionation methods to prepurify the mRNA before transcription will result in lowering the actual lower limit of detection to less than 2% of the total mRNA isolated from the organism. Specific sequences purified by the procedure outlined above may be further purified by a second specific cleavage with a restriction endonuclease capable of cleaving the desired sequence at an internal site. This cleavage results in formation of two subfragments of the desired sequence, separable on the basis of their lengths. The subfragments are separated from uncleaved and specifically cleaved contaminating sequences which had substantially the same original size prior to the second cleavage. The method is founded upon the rarity and randomness of placement of restriction endonuclease recognition sites, which results in an extremely low probability that a contaminant having the same original length as the desired molecule will be cleaved by the same second enzyme to yield fragments having the same length as those yielded by the desired sequence. After separation from the contaminants, the subfragments of the desired sequence may be rejoined using DNA ligase to reconstitute the original sequence. 4 The two subfragments must be prevented from joining together in the reverse order relative to the original sequence. This is accomplished by treatment with alkaline phosphatase prior to the second restriction endonuclease digestion and separation and rejoining of the subfragments. The purified fragments can then be recombined with a cloning vector and transformed into a suitable host strain. Materials and Reagents

Reagents 1. Oligo(dT) (dT12-18), P-L Biochemicals 2. AMV reverse transcriptase, Office of Program Resources and Logistics, Life Sciences, Inc.

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CLONING OF HORMONE GENES

77

3. Restriction site oligonucleotide linkers, e.g., d(CCAAGCTTGG), Collaborative Research, Inc. 4. T4 DNA ligase, Bethesda Research Laboratories, Inc. (5224) or PL Biochemicals (0870) 5. Restriction endonucleases (e.g., HindlII), Biolabs, Inc. 6. Radioactive nucleoside triphosphates, New England Nuclear or Amersham/Searle 7. S1 nuclease, Miles Laboratories 8. Nonradioactive nucleoside triphosphates, P-L Biochemicals 9. T4 polynucleotide kinase, Boehringer Mannheim Biochemicals (174 645) 10. Escherichia coli DNA polymerase I, Boehringer Mannheim Biochemicals (104 485) 11. Calf intestinal alkaline phosphatase (CLAP), Sigma Type VII (P-4502) Bacterial Strains and Plasmids r-9

1. X1776, EK2 host 7 constructed by Roy Curtiss, III, Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35294 2. Plasmids pMB9, pBR313, and pBR322 s'9 obtained from Herbert W. Boyer, University of California, San Francisco, San Francisco, California 94143 Materials

1. TEN 9 buffer: 100 mM NaC1, 20 mM Tris-HC1, pH 9.0, 1 mM NazEDTA 2. S1 buffer: 30 mM NaOAc, pH 4.5, 0.3 M NaC1, 3 mM ZnC12, 10 p.g/ml denatured calf thymus DNA 3. S1 stock buffer: 30 mM NaOAc, pH 4.5, 0.3 M NaCI, 3 mM ZnClz, 50% glycerol 4. TBE buffer: 10.8 g Tris base, 5.5 g boric acid, 0.93 g Na2EDTA per liter of H20 5. TBEss (sample solution): 0.2 ml 10× TBE buffer, 0.3 ml 0.2M Na2EDTA, pH 8, 0.5 ml 4% sarkosyl, 1.0 ml glycerol, 0.5 ml 0.5% bromphenol blue 6. 10× Poll buffer: 0.6 M Tris-HC1, pH 7.5, 80 mM MgCI2 7. 10 × HindlII buffer: 60 mM Tris-HCl, pH 7.5, 60 mM MgCI~, 0.6 M NaCI 7 R. Curtiss, III, D. A. Pereira, J. C. Hsu, S. C. Hull, J. E. Clark, L. F. Maturin, Sr., R. Goldschmidt, R. Moody, M. Inoue, and L. Alexander, Proc. Miles Int. Syrup., lOth, p. 45 (1977). F. Bolivar, R. L. Rodriguez, M. C. Betlach, and H. W. Boyer, Gene 2, 75 (1977). 9 F. Bolivar, R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow, Gene 2, 95 (1977).

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OF D N A

[5]

8. 10x Kinase buffer: 0.7 M Tris-HC1, pH 7.6, 0.1 M MgCI2, 50 mM DTT 9. 10 × Ligase buffer: 0.66M Tris-HCl, pH 7.6, 66 mM MgC12, 0.1 M DTT 10. X medium: 10 g Bactotryptone, 5 g yeast extract, 10 g NaCI, 100 mg diaminopimelic acid, 40 mg thymine, 1 liter tap-distilled H20, 0.33 ml 6 N NaOH. Autoclave 20 min. Add 10 ml sterile 50% glucose and 10 ml filter-sterilized nalidixic acid (7.5 mg/ml) in 50 mM NaOH. 11. M X wash buffer: 0.1 M NaC1, 5 mM MgC12, 5 mM Tris-HCl, pH 7.6 12. M× Ca 2+ buffer: 75 mM CaC12, 250 mM KCI, 5 mM MgC12, 5 mM Tris-HCl, pH 7.6 13. X plates: X medium containing 15 g Bactoagar per liter 14. Triton lysis mix: 1 ml 10% Triton, 75 ml 0.25 M EDTA, pH 8, 15 ml 1 M Tris, pH 8, 9 ml H20 Methods The complete scheme for cDNA cloning is illustrated in Fig. 1. Step 1. m R N A Purification. 6 The source of the RNA is obviously of critical importance in isolating the particular hormone cDNA clone of interest. As examples, we have used human term placentas to isolate human chorionic somatomammotropin clones,4 rat islets of Langerhans to isolate rat insulin clones, 2 a rat pituitary tumor cell line (GC cells) to isolate rate growth hormone, a and growth hormone-producing tumors of pituitary origin from patients with acromegaly as a source for human growth hormone sequences. 5 In all cases the tissue is quick-frozen in liquid nitrogen and stored at - 70°. For extraction of total RNA, 40 g of the frozen tissue was broken into small pieces and dissolved with the aid of a blender in 140 ml of a freshly prepared solution of 7 M guanidinium-HCl,1° 20 mM Tris-HCI, pH 7.5, 1 mM EDTA, and 1% sarcosyl at 0°. After adding 0.5 g CsCl to each milliliter, the dark-brown solution was heated at 65° for 5 min, quick-cooled in ice, layered on top of a 5-ml cushion of 5.7 M CsCl, 10 mM Tris-HC1, pH 7.5, 1 mM EDTA in 1 × 3½ in. nitrocellulose tubes and centrifuged u in an SW27 rotor at 27,000 rpm for 16 hr at 15°. After centrifugation, the tube contents were decanted, the tubes were drained, and the bottom ½cm containing the clear RNA pellet was cut off with a razor blade. Pellets were transferred to a sterile Erlenmeyer flask and dissolved in 20 ml 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, 5% sarcosyl, and 5% phenol. The solution was then made 0.1 M in NaCl and 10 R. A. Cox, this series, Vol. 12, p. 120. u V. Glisin, R. Crkvenjakov, and C. B y u s ,

Biochemistry 13, 2633 (1974).

[5]

CLONING

(1)

OF

HORMONE

79

GENES

mRNAmriflcation

(2) cDNA ~ ~ t h e s i s !Phenol, G-75 (3)

RNA hydrolysis

(4)

dscDNA synthesis

l

Phenoi, G-75

(5)

Restriction endonuclease digestion

~ (5)

Restriction endonuclease digestion

(6) SIdig]stionofd s c D N A / (7) 3~p HindIII linker preparation

(8)

HindIIIlinkerligation withPol I treatment

(9)

HindIII digestion of 32p linker dscDNA

l

Phenol

(I0) Preparativegel electrophoresis (11)

(12)

Preparation of pBR322, HindIII, ClAP

~- (13)

Eleetroelution

Ligation with pBR322, HindlTl, ClAP

(14)

Transformation of ;(1776

(15)

Selection of Amp R colonies

(16)

Test f ! r Tet s

(17)

Screening for recombinant clones

!

FIG. 1. S c h e m e for c D N A c l o n i n g .

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SYNTHESIS AND PURIFICATION OF D N A

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vigorously shaken with 40 ml of a 50% phenol-50% chloroform mixture. RNA was precipitated from the aqueous phase with ethanol in the presence of 0.2 M sodium acetate, pH 5.5. RNA pellets were washed with 95% ethanol, dried, and dissolved in sterile H20. For example, 40 g of placental tissue usually yielded about 30 mg of RNA from which approximately 300 /xg of polyadenylated RNA was obtained after being chromatographed twice on oligo(dT) cellulose. TM Step 2. cDNA Synthesis. Dry 1 mCi of 3H-dCTP ( - 3 5 Ci/mmol) in an appropriate tube under a stream of nitrogen. Add 250/.d 0.2 M Tris-HCl, pH 8.3, 10 ~ 1 M MgC12, 20/.d 1 M dithioerythritol (DTT), 10/xl of 2.5 mg/ml oligo(dT12_ls), 60/xl of 5 mM dCTP, 200/xl of 5 mM each dATP, dGTP, dTTP, - 5 0 /~g poly(A ÷) RNA, and H20 to 1 ml. If a smaller amount of poly(A ÷) RNA is used, scale down the reaction volumes proportionally while keeping the enzyme/RNA ratio constant. The final concentrations of dNTPs are dCTP, 0.3 mM, and dATP, dTTP, and dGTP, 1.0 mM each. The reaction is started with the addition of 40/zl of AMV reverse transcriptase (or the equivalent of 250 units). After incubation for 15 min at 45 ° the reaction is stopped by the addition of~o volume (104)/xl of 0.2 M EDTA, pH 8.5. The time course of the reaction is assayed by taking 2-/zl samples at 0 time and 15 min and adding them to 0.5 ml 10 mM EDTA, pH 7.0, containing 200/zg tRNA carrier. After adding 1 ml of 10% trichloroacetic acid (TCA) and leaving on ice for 30 min, the precipitates are filtered through GFC filters, washed with 5% TCA, and counted in a liquid scintillation counter. The reaction should yield about 1-10/xg of cDNA with a specific activity of - 6 × 106 dpm//zg. The entire reaction mixture is mixed with ½ volume (0.5 ml) of phenol and then with an equal volume (0.5 ml) of CHC1a-isoamyl alcohol (24:1). After centrifugation to separate the phases [an Eppendorf microcentrifuge (Brinkman) is very convenient for all centrifugation steps] the aqueous layer is removed and the organic phase and interface reextracted with a small volume o f T E N 9 buffer (100 mM NaCI, 20 mM Tris-HC1, pH 9.0, 1 mM Na2EDTA). The combined aqueous extracts are mixed with glycerol (10% final concentration) and chromatographed on a Sephadex G-75 column in TEN 9 buffer. A 5-ml plastic disposable pipette is suitable as a column for small loading volumes (< 200/xl) and a 10-ml pipette for larger volumes. Collect 0.2-ml fractions for small columns and 0.4-ml fractions for large columns. After counting appropriate size aliquots, pool the excluded peak fractions containing the cDNA. Step 3. RNA Hydrolysis 12 H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972).

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STEP a. Ethanol precipitation of pooled G-75 fractions: Add ~ volume of 2 M NaC1 and 2.5 volumes 95% ethanol (EtOH). After 30 min in a dry i c e - E t O H bath, or overnight at - 2 0 °, centrifuge for 20 min in the Brinkman microfuge or 60 min at 10,000 rpm in the HB4 rotor o f a Sorvall centrifuge. Check the supernatant for counts before discarding and dry the pellets under vacuum briefly. STEP b. RNA hydrolysisXa: Dissolve the dried pellet in fresh 0.1 M NaOH (0.5-5 times the original reaction volume) and incubate at 70° for 20 min. Neutralize with ~o volume 1 M Tris-HCl, pH 7.8, and enough 1 M HCI to bring to pH 8 (check with pH paper). STEP C. Second ethanol precipitation: Precipitate hydrolyzed sample as in step a. After removing the supernatant, add 70% EtOH at - 2 0 ° to the pellet, mix, and place at - 7 0 ° for 30 min; centrifuge 10 min in the microfuge, remove the supernatant, and dry the pellet under vacuum. (Check the supernatant for counts before discarding.) Step 4. Double-Stranded cDNA Synthesis. To 57/~l of single-stranded [all] cDNA (1 /zg) from step 3 (add H20 to 57 #l) add 25/A 0.2 M TrisHC1, pH 8.3, 2 /A 1 M DTT, 1 /A 1 M MgCI2, and 10/A of 5 mM each dNTP (0.5 mM each final). Heat the mixture for 3 min at 68° and quickcool on ice. Add 5/~l (15-30 units) AMV reverse transcriptase and incubate 1.5 hr at 420.14"15Take 2-/~1 time points at 0, 45, and 90 min and assay TCA-precipitable counts. This test measures the stability of the tritiated cDNA first strand. The double-stranded cDNA (dscDNA) is isolated by Sephadex G-75 chromatography as described for single-stranded cDNA in step 1. The formation of a double-stranded hairpin structure can be assayed using S1 nuclease as follows. STEP a. Add about 5000 cpm of dscDNA directly to 1 ml of S 1 buffer (30 mM NaOAc, pH 4.5, 0.3 M NaC1, 3 mM ZnC12, l0/zg/ml denatured calf thymus DNA) at 0°. Divide into two 0.5-ml aliquots and add 5/A S1 nuclease to one of the two tubes. 16 Stock S1 nuclease (Miles 37-637-1) is stored concentrated in S1 stock buffer (S1 buffer without DNA and containing 50% glycerol) and should be diluted before use about 10-fold in S1 13 Hydrolysis of RNA with 0.3 N NaOH at room temperature overnight results in less single-strand scissions in the cDNA. 14 An alternate protocol can be found in W. Salser, in "Genetic Engineering" (A. M. Chakrabarty, ed.), p. 53. CRC Press, Cleveland, Ohio, 1979. 15 We usually obtain yields of 3-20% [micrograms of cDNA per microgram of poly(A +) RNA] for the first-strand synthesis and 30-100% for the second strand. In preliminary experiments using a simpler method for synthesizing dscDNA as described by M. P. Wickens, G. N. Buell, and R. T. Schimke [J. Biol. Chem. 253, 2483 (1978)], we obtained better yields than using the method described above. le Use Siliclad-treated glass tubes.

82

SYNTHESIS AND PURIFICATION OF D N A

[5]

stock buffer so that 5/zl contains 300 units. After incubation of both tubes at 37 ° for 60 min, TCA-precipitate and count. 17 About 70% of the counts should be Sl-resistant. STEP b. Add about 5000 cpm of d s c D N A to 1 ml of S1 buffer, heat at 100° for 3 min, and quick-cool on ice. Divide in half and add 5/zl o f $1 nuclease to one tube. Process as in step a. If a large fraction of the dscDNA is the form of a hairpin, about 5 0 - 6 0 % of the counts should still be S1resistant. STEP c. After following the procedure in step a, heat both tubes at 100 ° for 3 min, quick-cool on ice, and add 5/zl S 1 nuclease to the same tube that had S1 nuclease in step a. Incubate 60 min at 37 ° and TCAprecipitate. If the first S1 nuclease treatment digested the hairpin, only about 10% of the counts will be TCA-precipitable after the second S1 reaction. Step 5. Restriction Endonuclease Digestion. When c D N A is transcribed from a heterogeneous m R N A preparation, it shows pronounced size heterogeneity not only because of the sequence complexity of the R N A template but most probably also as a result of " s l i p p a g e " of the oligo(dT) primer on the much longer poly(A) stretch of varying length and the difficulty of obtaining full-length c D N A transcripts from long RNA molecules. To determine whether c D N A sequences from a particular m R N A are present in such a heterogeneous c D N A preparation, the c D N A is fragmented with restriction endonucleases. 6 By this approach the effect o f size heterogeneity is reduced and it is possible to detect discrete restriction fragments generated from any predominant c D N A species. To facilitate radioautography in subsequent steps of this analysis [a-32p]dCTP ( - 2 5 Ci/mmol) is substituted for [aH]dCTP in the synthesis of the first a n d / o r second c D N A strand (steps 1-4). For restriction endonuclease digestions of single-stranded c D N A 5-p.l analytical reactions are terminated after incubation by the addition of 20 #1 of ice-cold water. T h e y are then boiled for 2 min, quick-cooled on ice, and made 7 mM in MgC12. Small aliquots ( ~ 2 × l05 cpm) are digested with an excess of one of the restriction endonucleases (HaeIII, H h a I , or HinfI) capable of cleaving single-stranded D N A ts,la or with any appropriate restriction e n z y m e when d s c D N A is used as the substrate. ~° The amount of each e n z y m e used is empirically determined to be in excess of the amount needed to digest completely an equivalent amount of

1~Add 2 ml of 10% TCA to each tube and retain o n ice for 30 rain. Collect the precipitates o n GFC filters, dry them under a heat lamp, and c o u n t in a t o l u e n e - b a s e d fluor in a scintillation c o u n t e r .

is K. Horiuchi and N. D. Zinder, Proc. Natl. Acad. Sci. U.S.A. 72, 2555 (1975). 1~R. W. Blakesley and R. D. Wells, Nature (London) 257, 421 (1975).

[5]

CLONING OF HORMONE GENES

83

restriction-sensitive DNA under identical reaction conditions. (Restriction endonuclease fragments of single-stranded bacteriophage DNAs such as ~bX174 and M13 are convenient markers, as they can also be isolated as double-stranded replicative forms.) Reactions are stopped with 5/xl of 20 mM EDTA, 20% sucrose, and 0.05% bromphenol blue, heated to 100° for 1 min, and then analyzed by polyacrylamide gel electrophoresis. For example, a convenient system is a composite 4.5-10% polyacrylamide slab gel n run for 2.5 hr at 150 V in Tris-borate-EDTA buffer (10.8 g Tris base, 5.5 g boric acid, 0.93 g Na2EDTA per liter) and visualized by autoradiography of the dried gel. Discrete bands on the polyacrylamide gel should be readily detectable on top of the heterodisperse cDNA "background" provided that the corresponding mRNA species represents at least 2% of the mass of the RNA template and that the cDNA species contains at least two cleavage sites for the enzyme(s) used. These conditions can usually be met, if necessary, by prior purification of the poly(A ÷) RNA (e.g., on a sucrose gradient) and/or careful selection of the restriction enzyme(s) employed. These bands can be identified as resulting from a specific hormone mRNA by (1) direct sequence analysis of the cDNA band after excision from gel and comparison with the known amino acid sequence of the hormone, 3-6 (2) a similar analysis after cloning of the band (see below), 2-4 or (3) correlation of the intensity of a particular band with the translational activity of the mRNA after sucrose gradient fractionation. Step 6. S I Nuclease Digestion of Double-Stranded cDNA. To - 1/xg of full-length dscDNA in 170/xl of H20 add 20/zl of 10× S1 buffer (0.3 M NaOAc, pH 4.5, 3 M NaCI, 45 mM ZnC12) and 10/xl of S1 nuclease (240 units of enzyme, final concentration 1200 units/ml). Incubate the reaction at 22° for 30 min and then at 3 - 4 ° for 5 min. Terminate the reaction by addition of~o volume 1 M Tris-HC1, pH 9, and ~ volume 0.2 M Na~EDTA, pH 8. After addition of tRNA carrier to 50/xg/ml extract once with an equal volume of phenol and then with an equal volume of CHCla-isoamyl alcohol (24: 1). Separate and remove the aqueous layer and reextract the organic phase and interface with 50/zl TEN 9 buffer. After combining the aqueous phases and adding glycerol (10% final) chromatograph the dscDNA on a 4-ml column of Sephadex G-75 using TEN 9 buffer. Seven-drop fractions are collected and to the excluded peak containing the dscDNA are added NaC1 to 0.2 M and 2.5 volumes of 95% EtOH. Pellet the cDNA in the microfuge and dissolve at about 10 n g / # / i n 6 mM Tris-HC1, pH 7.5, and 0.8 mM MgCI2. This mild S1 nuclease treatment digests the hairpin structure of the dscDNA and yields a final product of duplex dscDNA. ~o j. L. Roberts, P. H. Seeburg, J, Shine, E. Herbert, J. D. Baxter, and H. M. Goodman, Proc. Natl. Acad. Sci. U.S.A. 76, 2153 (1979).

84

SYNTHESIS AND PURIFICATION OF D N A

[5]

Step 7. 32P-Restriction Site Oligonucleotide Linker Preparation STEP a. END-LABELING OF THE LINKER. Dry 50 /xCi of [y-32p]ATP (usually -1000 Ci/mmol) under a stream of nitrogen in a microfuge tube. Add 7/xl of HindlII (dCCAAGCTTGG) or other suitable restriction site oligonucleotide linker (e.g., BamH1, SalI) at 75 ng//xl in HsO, 1 /zl 10× kinase buffer (0.7 M Tris-HC1, pH 7.6, 0.1 M MgCIz, 50 mM DTT), and 2/zl T4 polynucleotide kinase (5 units, Boehringer 174 645) and incubate 30 min at 37°. Then add 1/xl 10x kinase buffer, 1/zl 10 mM ATP, 2/xl T4 polynucleotide kinase, and 6/xl H20. Incubate for an additional 30 min at 37°. Terminate the reaction by extracting once with 20 /xl phenolCHCla-isoamyl alcohol (25:24: 1) and twice with 20/zl CHCI3. Remove residual CHCI3 in the aqueous layer by gently blowing a stream of nitogen on it for a few minutes. STEP b. TESTING THE asp LINKERS. Mix the following components: 1/xl asp linkers, 1/zl 10× T4 DNA ligase buffer (0.66 M Tris-HCl, pH 7.6, 66 mM MgCl~, 0.1 M DDT), 1/~l l0 mM ATP, 6/zl H20, and 1/zl T4 DNA ligase (1 unit/~l, Bethesda Research Laboratories 5224). Incubate for 2 hr at 14°C. Remove 5/zl of the reaction mixture and combine with 5/~l TBEss (sample solution: 0.2 ml 10× TBE buffer, 0.3 ml 0.2 M NasEDTA, pH 8, 0.5 ml 4% sarkosyl, 1.0 ml glycerol, 0.5 ml 0.5% bromphenol blue). Analyze on a prerun 7% polyacrylamide gel (20:1 acrylamide/bis) and run at - 150 V for about 1 hr or until the bromphenol blue is about halfway down the gel. Wrap the gel in Saran Wrap and autoradiograph with Kodak No-Screen film for 2 hr. about/>50% of the label should be in oligomers rather than monomers, sl Step 8. asP-Linker Addition to Double-Stranded cDNA. Treatment of S1 nuclease-digested dscDNA with E. coli DNA polymerase I increases the efficiency of blunt-end linker addition about 10-fold. Treat the dscDNA from step 6 with E. coli DNA polymerase I as follows. To 26.5/xl containing - 2 0 0 ng of 3H- and/or asP-labeled S1 nuclease-treated dscDNA (step 6), or a restriction fragment isolated by preparative gel electrophoresis, 4 add 3.9/zl of 10× PolI buffer (0.6 M Tris-HC1, pH 7.5, 80 mM MgC12), 2/zl 0.2 M fl-mercaptoethanol, 4/xl l0 mM ATP, 1.6/xl of a mixture of the four dNTPs at 5 mM each, and 2 /xl (4 units) of E. coli DNA polymerase I z~ (Boehringer, 104485). Incubate for l0 min at l0 °. This step ensures that the dscDNA is a complete duplex structure without protruding single-strand tails, a,4 Now add 1.5 /xl 10× PolI buffer, 0.8/zl 2x Poor polymerization can be caused by a low concentration of a2P-labeled 5'-ends, bad [y-a2P]ATP, bad kinase, or a ligase preparation which works poorly for blunt-end ligation. See A. Sugino, H. M. Goodman, H. L. Heyneker, J. Shine, H. W. Boyer, and N. R. Cozzarelli [J. Biol. Chem. 252, 3987 (1977)] for a more detailed discussion. 22 The E. coli Poll should be free of contaminating DNase activity.

[5]

C L O N I N G OF H O R M O N E GENES

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0.2 M/3-mercaptoethanol, 1.5/A 10 mM ATP, 9.2/A zzp linkers, and 2/A T4 DNA ligase (1 unit//.d). Incubate at 14° for 3-4 hr. Remove 2/zl of the reaction into 5/xl TBEss and freeze the remainder of the reaction. Analyze the 2-/xl sample on a 7% polyacrylamide gel as in step 7. Autoradiograph for 4 hr. The linker polymerization should be nearly as efficient as the linker test ligation in step 7.

Step 9. Restriction Endonuclease Digestion of the zzP-LinkerDouble-Stranded cDNA S T E P a. R E S T R I C T I O N E N D O N U C L E A S E DIGESTION. When HindlII linkers have been used, dilute the ligation reaction from step 8 (-53/xl remaining) with 250/zl 7 mM MgC12 and 60 mM NaCI. Heat at 65° for 5 min. Add 7/zl ofHindlII endonuclease (70 units, Biolabs, Inc.) and additional 7-/zl aliquots at 1.5 hr and 3 hr of incubation at 37°. Incubate for a total time of 4-5 hr at 37°. Remove 15 /zl from the reaction (freeze the remainder at - 2 0 °) and mix with 10/zl TBEs~. Analyze on a 7% polyacrylamide gel. Radioautography should show that no linker polymers remain and that a band (or smear) is present at the length expected for the input dscDNA, z-4 If the gel is satisfactory, thaw the reaction mix and extract once with phenol-CHC13-isoamyl alcohol (25:24:1). Remove the aqueous layer and add ~0 volume 2 M NaC1 and 2.5 volumes 95% EtOH. Leave overnight at - 2 0 °. STEP b. PREPARATIVEELECTROPHORESIS.Centrifuge the sample for 15 min at 4° in the Brinkman microfuge. Dissolve the pellet in 30/xl of 0.25 × TBE and add 5/zl TBEss and 5/zl glycerol. Separate on a 5% polyacrylamide gel as described in step 10.23 Step 10. Preparative Polyacrylamide Gel Electrophoresis. 24,~5The following stock solutions are required: (a) 10× TBE buffer (108 g Tris base, 55 g boric acid, 9.3 g Na2EDTA, H20 to 1 liter); (b) 20:1 AcBis (20 g acrylamide, 1 g bisacrylamide, H20 to 100 ml); (c) 2% ammonium persulfate (store for less than 1 week at 4°); and (d) TBEss (see step 7). For a gel 15 × 13 cm and 1.5 mm thick use the amounts shown in Table I (for a longer gel, 15 cm × 1.5 mm, double the quantities). Prerun the short gel (13 cm) at 150 V for 30 min and run at 150 V. Prerun the long gel (30 cm) at 250 V for 30 min and run at 250 V. Continue the electrophoresis for an appropriate time (depending on An alternate method for separating the asp linkers from the linkers attached to the cDNA is to chromatograph the mixture on a 10-ml column of BioGel A-5m (200-400 mesh, Bio-Rad Laboratories) in l0 mMM Tris-HCl pH 7.5, 100 mM NaCl, and 1 mM EDTA as described by A. E. Sippel, H. Land, W. Lindenmaier, M. C. Nguyen-Huu, T. Wurtz, K. N. Timmis, K. Giesecke, and G. Schutz [Nucleic Acids Res. 5, 3275 (1978)]. This procedure eliminates steps l0 and 1 l; i.e., after step 9 and chromatography proceed to step 12. 24 C. W. Dingman and A. L. Peacock, Biochemistry 7, 659 (1968). 25 T. Maniatis, A. Jeffrey, and H. van de Sande, Biochemistry 14, 3787 (1975). 23

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TABLE I POLYACRYLAMIDE GEL COMPOSITION Component

5% gel

7% gel

AcBis 10X TBE buffer 2% Ammonium persulfate TEMED H~O

10 ml 4 ml 2.5 ml 13 p.I 23.5 ml 40 ml

14 ml 4 ml 2.5 ml 13/zl 19.5 ml 40 ml

the-fragment size) and radioautograph the wet gel after covering it with Saran Wrap. Step 11. Electroelution. Using the autoradiograph of the gel as a guide excise the a2p-labeled "high"-molecular weight dscDNA with a scapel. Place the gel slice and 3.5 ml of 0.25 × TBE buffer together with l0/zg/ml tRNA in medium-sized (~-in-diameter) dialysis tubing. Place in the electroelution apparatus described by McDonell et al. 26 Orient the bag parallel to the electrodes and fill the apparatus with about 1.5 liters of 0.5 × TBE buffer. Electroelute at 100 mA COlastant current ( - 3 5 V) for at least 8 hr. Check the efficiency of elution by Cerenkov counting of the eluate and the gel piece plus bag. Precipitate the eluted DNA by the addition of A volume of 2 M NaC1 and 2.5 volumes of 95% EtOH. Store overnight at - 2 0 ° and centrifuge. Dissolve the pellet in 0.3 ml l0 mM Tris-HCl, pH 8, containing 1 mM EDTA. Centrifuge out the debris for 1 min in the microfuge. Ethanol-precipitate the DNA again in a 1.5-ml microfuge tube. Step 12. Preparation of Phosphatase-Treated Linear pBR322. Prepare supercoiled pBR322 plasmid DNA as described in references 8 and 9 and digest with the appropriate restriction endonuclease (i.e., the same enzyme as used for the linkers, e.g., HindIII endonuclease for HindIII linker, BamH1 endonuclease for BamH1 linkers). In the case of HindIII add 5/zl of 10× HindIII buffer (60 mM Tris-HCl, pH 7.5, 60 mM MgCl2, 0.6 M NaC1) to 15/~g of pBR322 DNA and bring to a final volume of 46/zl with H20. Add 2/zl (20 units) ofHindIII endonuclease at 0, 40, and 80 min of incubation at 37°. Incubate a total of 2 hr. Check the digest by analysis on a 1% agarose gel and save a 5-p.l sample of the reaction for a control in the transformation step 14. Dilute the reaction with 450/zl H20 and heat at 65 ° for 10 min to inactivate the enzyme. Add 5/zl 1 M Tris-HCl, pH 9.0, and 1.5 /zl (3 units) of CIAP. Incubate 30 min at 65 °, add another 1.5/xl CIAP, and incubate an additional 30 min at 65 °. Extract the reaction three times with an equal volume of phenol-CHCla-isoamyl alcohol (25:24: l) 2e M. W. McDonell, M. N. Simon, and F. W. Studier, J. Mol. Biol. U0, 119 (1977).

[5]

CLONING OF HORMONE GENES

87

and then three times with CHCla-isoamyl alcohol (24: 1). Add NaCI to 0.2 M to the aqueous phase and precipitate with 2.5 volumes of EtOH. Centrifuge, resuspend, and reprecipitate with EtOH. Dissolve the DNA in 0.2 ml 10 mM Tris-HC1, pH 7.5, and 1 mM EDTA and determine the concentration spectrophotometrically (1 mg/ml DNA = 20 A260). Step 13. Ligation of "a2P-Linker-Double-Stranded cDNA to Phosphatase-Treated HindlII-Digested pBR322. Dissolve the isolated 32p-linker-dscDNA after electroelution or chromatography in sufficient 0.2× ligase buffer 27 to give 50-100 ng in 8 ~1 (based on count recovery). To the 8 /xl of a2p-linker-dscDNA ( - 5 0 ng) add 2 /A HindlII-digested, phosphatase-treated pBR322 from step 12 (150 ng), 1.5 /zl 10× ligase buffer, and 1.5 /zl 10 mM ATP. Heat 2 min at 37°, add 2/zl (2 units) T4 DNA ligase, and incubate at 14° for i>4 hr. It is possible to assay for ligation by removing 1-/xl samples at 0 time and at 4 hr and analyzing them on a 5% polyacrylamide gel. Dry the gel and expose it at - 7 0 ° using a Lightning Plus intensifying screen with Kodak XR-2 film. Successful ligation is indicated by azp incorporation into the region of the gel where linear pBR322 migrates. To the remainder of the reaction add NaCI to 0.2 M, 2.5 volumes of 95% EtOH, and precipitate overnight at - 20°. Centrifuge for 15 min in the microfuge and dry the pellet briefly under vacuum. Dissolve the pellet in 50/xl 10 mM Tris-HCl, pH 7.6. Step 14. Transformation of X1776.2s STEP a. Grow a 10-ml culture ofE. coli strain X1776 at 37° overnight in X medium (19 g Bactotryptone, 5 g yeast extract, 10 g NaCI, 100 mg diaminopimelic acid, 40 mg thymine, 1 liter tap-distilled H20, 0.33 ml 6 N NaOH. Autoclave 20 min and then add 10 ml sterile 50% glucose and 10 ml of 7.5 mg/ml filter-sterilized nalidixate in 50 mM NaOH). STEP b. Dilute the overnight culture to 0.05 A600 in 100 ml X medium. Grow at 37 ° with shaking to 0.4 A600. STEP C. Centrifuge the culture in the Sorvall SS34 rotor (6000 rpm, 5 min, 5°) in screw-cap polycarbonate tubes. Resuspend the cell pellets in 60 ml cold (4°) M x wash buffer (0.1 M NaCI, 5 mM MgC12, 5 mM TrisHC1, pH 7.6). Centrifuge again. Resuspend the cell pellets in 30 ml of M x wash buffer, combine into one tube, and centrifuge (6000 rpm, 5 min, 5°). STEP d. Resuspend the cell pellet in 30 ml cold M x Ca 2÷ buffer (75 mM CaC12, 250 mM KCI, 5 mM MgCI2, 5 mM Tris-HC1, pH 7.6). Allow to stand on ice for 20 rain. STEP e. Centrifuge again (6000 rpm, 5 rain, 5°). Resuspend in 0.5 ml 2r Ligase buffer (10x): 0.66 M Tris-HCl, pH 7.6, 66 mM MgC12, 0.1 M DTT. 2 s See also M. V. Nargard, K. Keem, and J. J. Monahan [Gene 3, 279 (1978)] for another high-efficiency X1776 transformation procedure.

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M x Ca 2+ buffer. Do all further manipulations in the appropriate physical containment facility as specified by the NIH guidelines on recombinant DNA research. STEP f. Add 100 pJ of the cell suspension (from step e) to 50/zl of the ligated DNA solution (in 10 mM Tris, pH 7.6) in a 0.5-ml polypropylene microfuge tube. Mix well. Hold on ice for 60 min. STEP g. Heat-shock the mixture for 90 sec at 42° and cool briefly to room temperature. Transfer the solution to a sterile 10-ml culture tube and add 1.35 ml X medium at room temperature. STEP h. Incubate the culture for 1 hr at 37° with shaking. Step 15. Selection of Ampicillin Resistant (Amp R) Colonies. Plate all of the 1.5 ml culture in 0.2-ml aliquots on x-ampicillin plates. (Add 15 g Bactoagar before autoclaving and 5 ml of 10 mg/ml filter-sterilized ampicillin after autoclaving to the X medium recipe. Glucose and nalidixate are also added after autoclaving, as in the × medium recipe.) Incubate plates at 37° overnight or longer. We obtain several million transformants per microgram of supercoiled pBR322 DNA. The following tests are useful in accessing the quality of the plasmid vector used in the transformation: TEST a. EFFICIENCY OF HindIII DIGESTION. The starting plasmid, pBR322, should migrate as a supercoiled DNA molecule on a 1% agarose gel and give a high transformation frequency ( - 4 x 10n Amp R transformants per microgram of DNA). After HindIII digestion the supercoils and nicked circles should disappear from the gel profile, and a low background of Amp R transformants should be obtained after transformation (

Cloning of hormone genes from a mixture of cDNA molecules.

[5] CLONING OF HORMONE GENES [5] C l o n i n g 75 of Hormone Genes from a Mixture of cDNA Molecules 1 B y HOWARD M. GOODMAN AND RAYMOND J. MACDON...
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