Proc. Natl. Acad. Sci. USA Vol. 84, pp. 8893-8897, December 1987 Biochemistry Isolation and sequencing of the gene encoding A5-3-ketosteroid of Pseudomonas testosteroni: Overexpression of the protein ATHAN KULIOPULOS*, DAVID SHORTLE*, AND PAUL TALALAYtt Departments of *Biological Chemistry and tPharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Paul Talalay, August 31, 1987

ABSTRACT We describe the cloning, sequencing, and The steroid isomerase of P. testosteroni has been under overexpression of the steroid isomerase (3-oxosteroid 5-_A4- intensive study in a number of laboratories because of its isomerase, EC 5.3.3.1) gene of Pseudomonas testosteroni. A interesting mechanism and extraordinarily high (diffusion- genomic library of P. testosteroni total DNA constructed from controlled) catalytic efficiency (9). The catalytic process partial EcoRI digests ligated to a XgtWES vector was probed involves a direct intramolecular diaxial transfer of the 4f3 with a 23-base oligonucleotide mixture [ATGAAC(T)ACC- proton to the 6(3 position. Spectroscopic, kinetic isotope, and (A,T)CCG(C,A)GAG(A)CAC(T)ATGAC] corresponding to isotope exchange studies support the view that this proton the NH2-terminal sequence of steroid isomerase. Subclones transfer proceeds via an enolic intermediate and that the derived from a recombinant phage containing a 5400-base-pair cleavage ofthe 4(3 hydrogen to carbon bond is rate-limiting (9, insert were sequenced and found to contain the expected 10). The reaction mechanism has also been probed with 375-nucleotide open reading frame flanked at both ends by -directed (chemically and photochemically reac- in-frame TGA termination codons. The DNA sequence agreed tive) steroid analogues and mechanism-based (suicide sub- with the above 125-amino acid sequence except for codons 22, strate) inhibitors (11-15). Several of these inhibitors react 24, 33, and 38, all of which encoded Asp rather than Asn, and covalently with the carboxyl group of Asp-38, whereas the codon 77, which encoded Glu rather than Gln. A 1370-base- mechanism-based acetylenic secosteroid inhibitors (which pair fragment was inserted into pUC 19 plasmid vector and are enzymatically converted to conjugated allenic ketones) used to construct a strain ofEscherichia coli JM 101 that over- react with Asn-57 to form what is believed to be an enol- expressed the isomerase gene in the presence of isopropyl imidate structure (16, 17). (3-D-thiogalactopyranoside. Cytosolic extracts of this strain Recently, it has been possible to position a spin-labeled contained a major soluble protein that migrated with the substrate analogue by NMR methods into the 2.5-A x-ray steroid A-isomerase subunit on polyacrylamide gel electropho- structure of steroid isomerase. These studies have revealed resis in the presence of sodium dodecyl sulfate. These cytosolic the proximity to the bound steroid of the side chains of extracts had 10-50% of the specific activity of crystalline Tyr-14, Tyr-55, Asp-38, and Asn-57, and the negative end of isomerase, depending on the method of preparation. The a 10-residue a-helix (8). recombinant was crystallized in both monoclinic (flat This paper describes the cloning, sequencing, and overex- plates) and hexagonal (bipyramids) crystal forms, described pression of the isomerase gene.§ Large quantities of the previously for the enzyme isolated from P. testosteroni. The wild-type enzyme and of modified enzyme species in which kinetic properties of the crystalline recombinant enzyme, critical amino acid residues have been altered by site-directed including specific activity, Km for 5-androstene-3,17-dione (340 mutagenesis are now accessible. Hence, further insight can ,LM), and K; for the competitive inhibitor 19-nortestosterone be gained into the molecular structure, steroid-binding do- (11.9 ,iM), agreed closely with the values reported for the main, and detailed mechanism of this unusually active cata- isolated enzyme. lyst. Steroid isomerase (isomerase; 3-oxosteroid A5-A4-isom- MATERIALS AND METHODS erase, EC 5.3.3.1) catalyzes the isomerization of the double Bacterial Strains. P. testosteroni (ATCC 11996) was from bond of 5-androstene-3,17-dione into conjugation with the our own laboratory (18); Escherichia coli JM 101 and E. coli carbonyl group (Fig. 1). This enzymatic reaction was first VCS 257 were obtained from Stratagene Cloning Systems described in Pseudomonas testosteroni (an organism that can (San Diego, CA). grow on steroids as its only source of carbon) and in animal Recombinant DNA Techniques. All restriction tissues by Talalay and Wang (1). were from New England Biolabs. Restriction digestions were The enzyme exists in dilute solution as a noncovalently but done at 370C under conditions described by Maniatis et al. tightly bonded homodimer of 125 amino acids per subunit (2). (19). Small amounts of plasmid DNA were isolated by the The complete amino acid sequence was reported by Benson method ofHolmes and Quigley (20). Large scale preparations et al. (3). Cysteine and tryptophan are both absent. The of plasmid DNA were obtained by the alkaline-lysis proce- isomerase was first crystallized by Kawahara and Talalay (4) dure of Birnboim and Doly (21). Analysis of plasmid DNA in a monoclinic form (5). The structure of a hexagonal and restriction fragments followed by agarose gel electro- crystalline form (6), which is more tractable for high- phoresis and recovery of DNA fragments from low melting resolution x-ray diffraction studies, has been reported at a point agarose were carried out as described with slight 6-A resolution level by Westbrook et al. (7), and a 2.5-A resolution structure is nearing completion [E. M. West- Abbreviation: isomerase, A5-3-ketosteroid isomerase. brook, personal communication; see Kuliopulos et al. (8)]. tTo whom reprint requests should be addressed at: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205. The publication costs of this article were defrayed in part by page charge §This sequence is being deposited in the EMBL/GenBank data base payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, and in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03568). 8893 Downloaded by guest on September 25, 2021 8894 Biochemistry: Kuliopulos et al. Proc. Natl. Acad. Sci. USA 84 (1987) 0 toside). Cells were grown in shaking flasks for 13-16 hr at 370C in Luria-Bertani medium containing 75 mg ofampicillin per liter, and induced by addition of0.67 mM thiogalactoside at the start ofgrowth. Cells obtained from 2.5 liters ofgrowth medium were sedimented by centrifugation and stored at 40C overnight. The pellet was then suspended in 50 ml of cold 50 mM Tris HCI (pH 7.5) and stirred for 1 hr at 40C. Cells were again centrifuged at 11,000 x g at 40C for 10 min and the supernatant fraction was concentrated by ultrafiltration to 6.2 ml with the use of Centricon-10 Microconcentrators (Ami- FIG. 1. Reaction catalyzed by isomerase. The enzyme converts con). Protein was precipitated by adding saturated ammoni- 5-androstene-3,17-dione to 4-androstene-3,17-dione by a direct in- um to tramolecular and stereospecific transfer of the C-4j3 proton to the sulfate, adjusted pH 7.0 with NH40H at 40C, until the C-6f3 position. solution was 60% saturated. The isomerase was then purified by crystallization and recrystallization according to methods previously described (26). The isomerase remaining in the modifications (19). T4 DNA and the Klenow fragment cell pellet after the wash step could be extracted by sonic of E. coli DNA polymerase I were purchased from Boeh- disruption of the pellet resuspended in 15 ml of 50 mM ringer Mannheim and reactions were carried out as described Tris HCl (pH 7.5) and addition of Polymin-P to the sonicated (19). Frozen competent E. coli JM 101 cells were bought from supernatant fraction to a concentration of 0.85% (wt/vol). Stratagene Cloning Systems and used as recommended by DNA and contaminating proteins were then sedimented by the supplier. centrifugation and ammonium sulfate was added to the Isolation of DNA from P. testosteroni. Cultures of P. supernatant fraction to 84% saturation. The isomerase could testosteroni were grown in Erlenmeyer flasks on an orbital be further purified by the crystallization procedure outlined shaker at 30'C in a salt medium containing 10 g of Difco yeast above. Large single crystals ofisomerase were then grown by extract per liter until an absorbance of 0.82 at 650 nm was gradually increasing the ammonium sulfate concentration in reached (22). Cells obtained from 2 liters of culture fluid were hanging drops by the vapor diffusion method. Kinetic studies sedimented by centrifugation and solubilized by incubation at and analysis were performed according to methods described 250C for 20 min with 60 ml of 50 mM Tris HCl, pH 8.0/25% (27). Protein concentrations in crude extracts were deter- (wt/vol) sucrose and 90 mg of lysozyme. The lysate was mined by the method of Bradford (28). When isomerase diluted to 500 ml with 10 mM Tris HCl/1 mM EDTA, pH 8.0 purity exceeded 40%, the protein concentrations were mea- (Tris-EDTA). Sodium dodecyl sulfate [1.5 ml of25% (wt/vol) sured by ultraviolet absorbance, assuming that an isomerase solution] was added and the mixture was shaken overnight at solution containing 1 mg ofprotein per ml had an absorbance 40C. This solution was then incubated successively for 1-hr of 0.336 at 280 nm (29). Crude cell extracts and purified periods at 250C, first with 1.5 mg of pancreatic RNase A and isomerase fractions were analyzed by polyacrylamide gel then with 5 mg of proteinase K. Next, 2 vol of anhydrous electrophoresis in the presence ofsodium dodecyl sulfate and ethanol was added slowly and the resulting gel was spooled stained with Coomassie blue R 250. onto a 10-ml glass pipette and transferred to a 500-ml beaker. The gel was diluted to 125 ml with Tris-EDTA and agitated RESULTS AND DISCUSSION overnight at 250C. The RNase A and proteinase K digestions Twenty positive plaques were obtained by screening 20,000 were then repeated as described above. The solution was recombinant colonies with the synthetic 23-mer oligodeoxy- then adjusted to contain 0.5 M NaCl in a total vol of 200 ml nucleotide probe. Six of these positive clones were purified ofTris-EDTA. Insoluble material was removed by centrifug- and all six shared a 5400-base-pair (bp) EcoRI fragment that ing at 1000 x g for 6 min at 40C. Polyethylene glycol 6000 was hybridized to the 23-mer probe. This fragment was digested then added to 6% (wt/vol) and the solution was shaken at with a series of restriction enzymes, and the fragments were 250C for 1 hr, placed on ice for 1.5 hr in glass centrifuge tubes, subcloned into pUC 19. The restriction map and sequencing and the DNA was sedimented by centrifugation at 1700 x g strategy are shown in Fig. 2. Nucleotide sequences of both for 8 min at 40C. The DNA pellet was dissolved in 8 ml of strands were obtained and are shown in Fig. 3 together with Tris-EDTA, extracted with phenol, and dialyzed overnight the polypeptide translation ofthe 375-bp open-reading frame. against Tris-EDTA at 40C. The gene was found to be flanked at both 5' and 3' ends by Cloning of Genomic DNA and Isolation of the Isomerase in-frame TGA termination codons. Just upstream of the 5' Gene. A partial EcoRI digest of P. testosteroni DNA was termination codon is a 7-bp region (AGAAGGA), which is ligated into the EcoRI site of a XgtWES vector and packaged complementary to the 16S ribosomal RNA (30). into A particles. Plaques were screened using the E. coli strain The DNA sequence of the gene itself agreed with the VCS 257 by hybridizing to a 32P-end-labeled 23-base oligo- previously determined amino acid sequence (3) except for deoxynucleotide mixture [ATGAAC(T)ACC(A,T)CCG- residues 22, 24, 33, and 38, all of which encoded Asp rather (C,A)GAG(A)CAC(T)ATGAC] corresponding to the NH2- than Asn, and residue 77, which encoded Glu rather than Gln. terminal sequence of isomerase (3) by using standard proce- The DNA sequence agrees perfectly with the NH2-terminal dures. The washing was performed at 470C. Positive plaques portion of the isomerase sequenced by automated Edman were purified and restriction fragments of recombinant DNA degradation (31, 32). No independent information on the were subjected to Southern analysis (23). In the subcloning identity of residue 77 is available. experiments, pUC 19 plasmid vector (24) was used for both A 1370-bp fragment containing the isomerase gene was restriction mapping and sequencing. The DNA sequence was inserted 3' to the lac promoter of pUC 19. This recombinant obtained using 32P-end-labeled primers and alkali-denatured plasmid, designated pAK 1370, was then used to construct a supercoiled plasmid as template by the dideoxynucleotide strain of E. coli JM 101 that overexpressed the isomerase method of Sanger et al. (25). gene in the presence of thiogalactoside. The parent bacterial Overexpression of the Isomerase Gene and Purification of strain bearing the pUC 19 vector has no detectable endoge- Enzyme. A Pst I fragment containing the isomerase gene was nous isomerase activity. When the 1370-bp fragment was inserted into pUC 19 vector and used to construct a strain of reversed in orientation within the pUC 19 vector, isomerase E. coli JM 101 that overexpressed the isomerase gene in the activity was only 0.67% of that observed with the insert in the presence of isopropyl 6-D-thiogalactopyranoside (thiogalac- proper orientation and was unaffected by thiogalactoside. Downloaded by guest on September 25, 2021 Biochemistry: Kuliopulos et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8895 0 200 400 600 800 1000 1200 1400 1 1 1 I I

+ *

IF I IVI7Z I p E BS NNA B BSN E B BS E P

4 4-

FIG. 2. Restriction map and sequencing strategy of the cloned DNA plasmid pAK 1370. The 375-bp region encoding the isomerase polypeptide is indicated by the box. The gene is contained within a DNA segment 1370 bp long flanked by Pst I sites and inserted into the Pst I site of the pUC 19 vector. The horizontal arrows above and below the restriction map indicate the start points, the direction, and the extent of the DNA-primed synthesis used for sequence analysis of subclones by the dideoxynucleotide method (25). The arrow originating from the open circle indicates the sequence primed from the synthetic 23-mer oligodeoxynucleotide mixture (see Materials andMethods) that was used to probe for the isomerase gene in the P. testosteroni DNA library. B, BstNI; BS, BssHII; E, Eag I; N, Nar I; NA, Nae I; P, Pst I; S, Sph I.

-330 -320 -310 -300 -290 -280 -270 -260 -250 * * * * * * * * * CTGCAGGAC CCGCGCTATG GCAATCCATT GCCAAGTTCG TTCCTCCCAT GGGCCGCCGT GCCGAGCCGTC CGAGATGGC GTCGGTCATC -240 -230 -220 -210 -200 -190 -180 -170 -160 * * * * * * * * * GCCTTTTTGA TGAGCCCGGC CGCAAGCTAT GTGCATGGCG CGCAGATCGT CATTGATGGC GGCATTGATG CGGTGATGCG CCCGACACAG -150 -140 -130 -120 -110 -100 -90 -80 -70 * * * * * * * * * TTCTGACCTC TCATGTGGCG CTTTGCCAGA GGGGCCTGCG CCTTCCCCCC CTCGCTGTGC GGGAGGGGGA AGCGGCCTCG CTGCAAGGCT -60 -50 -40 -30 -20 -10 1 10 * * * * * * * * TTTTTTTGTT CACCGCCCCC GTTTGGCATT GCCGGTTTTC AACGGCGCCT GCTAGAAGGA TGA TTT GAG ATG AAT ACC CCA GAA Met Asn Thr Pro Glu 20 30 40 50 60 70 80 90 * * * * * * CAT ATG ACC GCC GTG GTA CAG CGC TAT GTG GCT GCG CTC AAT GCC GGC GAT CTG GAC GGC ATC GTC GCG CTG TTT His Met Thr Ala Val Val Gln Arg Tyr Val Ala Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe 100 110 120 130 140 150 160 * * * * * * * GCC CAT GAG GGG ACG GTG GAA GAC CCC GTG GGT TCC GAG CCC AGG TCC GGT ACG GCT GCG ATT CGT GAG TTT TAC Ala Asp Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr Ala Ala Ile Arg Glu Phe Tyr 170 180 190 200 210 220 230 240 * * * * * * * * GCC AAC TCG CTC AAA CTG CCT TTG GCG GTG GAG CTG ACG CAG GAG GTA CGC GCG GTC GCC AAC GAA GCG GCC TTC Ala Asn Ser Leu Lys Leu Pro Leu Ala Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe 250 260 270 280 290 300 310 * * * * * * * GCT TTC ACC GTC AGC TTC GAG TAT CAG GGC CGC AAG ACC GTG GTT GCG CCC ATC GAT CAC TTT CGC TTC AAT GGC Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala Pro Ile Asp His Phe Arg Phe Asn Gly 320 330 340 350 360 370 380 390 * * * * * * * * GCC GGC AAG GTG GTG AGC ATG CGC GCC TTG TTT GGC GAG AAG AAT ATT CAC GCT GGC GCC TGA AG AGAGTCAGGC Ala Gly Lys Val Val Ser Met Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Gly Ala *** 400 410 420 430 440 450 460 470 480 * * * * * * * * * GCTTGACTGC TCCTGATATG GAAGCTGTCT TCGCCTGATT CAATTGAGTT TCAAATACAT TTTGATCTGA AATACATTGA AATCATGCGT 490 500 510 520 530 540 550 560

* * * * * * * * TGAATGCTAT TGATCTTGCT GTTCAAGGCA GTGCAATGAG CACTCCCGGC CTGAGCGCTT ACAGATGGAA GTGTCCGGCC G

FIG. 3. Nucleotide and deduced amino acid sequence ofthe open reading frame ofthe isomerase gene. The nucleotide sequence ofthe sense strand flanking and including the isomerase gene is shown. The Shine-Dalgarno ribosome binding site (30) is underlined. The sequence numbering begins at the Pst I site 338 bases upstream from the isomerase initiator methionine and extends to the Eag I site 561 bases downstream from the initiation site. The in-frame termination codons are labeled ***. Because of secondary structure in the DNA, giving rise to an ambiguous region, an additional G may be present between bases -44 and -45. Downloaded by guest on September 25, 2021 8896 Biochemistry: Kuliopulos et al. Proc. NatL Acad. Sci. USA 84 (1987) When grown overnight in the presence ofthe thiogalactoside, the E. coli/pAK 1370 strain produced isomerase at a level of 10% of total cytosolic cell protein as determined by specific activity measurements and contained a major protein band that migrated on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with the isomerase subunit (Fig. 4). The specific activity of the isomerase in uninduced cells grown under the same conditions was 31% of that of induced cells. Furthermore, by storing the cell pellets overnight at 40C and washing them with 50 mM Tris HCI buffer (pH 7.4), it was possible to extract -50% of the total isomerase activity at a purity of 40% (Fig. 4). Crystalline isomerase was then prepared by gradual addition of a saturated ammonium sulfate solution at 40C (Fig. 4). The crystalline enzyme had a final specific activity of56,000 units per mg ofprotein, which was comparable to that ofthe native enzyme (9). Large crystals ranging from 0.2 to 0.4 mm in length were grown from 40% saturated neutral ammonium sulfate at 250C in hanging drops and showed the typical morphology ofthe monoclinic crystal form ofisomerase (Fig. FIG. 5. Monoclinic (Upper) and hexagonal (Lower) crystal forms of 5). Furthermore, the enzyme was also crystallized at low pH recombinant isomerase. Isomerase was obtained from E. coli JM 101 in the hexagonal crystal form (Fig. 5), which is also charac- containing pAK 1370 and grown in the presence of 0.67 mM teristic of P. testosteroni isomerase (6). The Km value of the thiogalactoside. The crystals were obtained in hanging drops (20-1LI vol) recombinant enzyme for 5-androstene-3,17-dione by the vapor diffusion method at 250C. (Upper) Isomerase (18.2 Aig) crystalline equilibrated with 40%o saturated ammonium sulfate in 50 mM TrisHCl was 340 uM, in close agreement with that of pure isomerase (pH 7.5). (Lower) Isomerase (7.6,ug) equilibrated with 30%o saturated from P. testosteroni (33). The Ki value for 19-nortestosterone ammonium sulfate, pH 5.5/50 mM Tris-HCl. (Bars = 0.1 mm.) was 11.9 ,uM, similar to the values determined for isomerase from P. testosteroni (9). Various lines of evidence indicate that the recombinant steroid-induced P. testosteroni: (i) the DNA sequence of the enzyme obtained by overexpression in E. coli is identical, or coding region agrees in length and base sequence with the at least closely similar, to the native isomerase isolated from amino acid sequence of the native enzyme with the excep- tions noted above; (ii) the recombinant enzyme crystallizes in A B C D E the two crystal forms characteristic ofthe native enzyme; (iii) the specific activity, the Km value for 5-androstene-3,17- dione, and the Ki value for 19-nortestosterone all agree with those previously determined for the enzyme obtained from P. testosteroni; and (iv) the subunits of the native and recom- binant enzymes have similar electrophoretic mobility on denaturing gels. The availability of cloned isomerase of known sequence and 43.0- confirmed identity with the native enzyme now permits the use of the technique of site-directed mutagenesis (34) to identify 25.7- amino acid residues involved in the catalytic mechanism, the 18.4- binding of steroid substrates, and the maintenance of the 14.3- tertiary and quaternary structures of this interesting protein.

6.2- We are most grateful to several colleagues for their contributions to these studies. Dr. Albert S. Mildvan provided much encourage- ment and many important and perceptive suggestions. Dr. James M. Ntambi advised on cloning procedures. Drs. Mark Molliver and Mary Blue photographed the crystals. These studies were supported by National Institutes of Health Grants AM 07422 (to P.T.), GM FIG. 4. Polyacrylamide gel electrophoresis in the presence of 34171 (to D.S.) and AM 28616 (to Albert S. Mildvan). A.K. was sodium dodecyl sulfate of isolated and recombinant crystalline supported by National Institutes ofHealth Medical Scientist Training isomerase and offractions ofE. coli containing the plasmid encoding Grant GM 7309. the isomerase gene. The gels containing 17% polyacrylamide and 0.1% sodium dodecyl sulfate were stained with Coomassie blue R 1. Talalay, P. & Wang, V.-S. (1955) Biochim. Biophys. Acta 18, 250. Molecular weights (x 10-3) are indicated on the left. Lanes: A, 300-301. crystalline isomerase isolated from P. testosteroni; B, centrifuged 2. Benson, A. M., Suruda, A. J. & Talalay, P. (1975) J. Biol. cell pellet of E. coli JM 101 grown in presence of 0.67 mM Chem. 250, 276-280. thiogalactoside and containing the unmodified pUC 19 vector was 3. Benson, A. M., Jarabak, R. & Talalay, P. (1971) J. Biol. boiled in 0.1% sodium dodecyl sulfate; C, similar preparation as in Chem. 246, 7514-7525. lane B but prepared from E. coli JM 101 containing pUC 19 with the 4. Kawahara, F. S. & Talalay, P. (1960) J. Biol. Chem. 235, 1370-bp insert (pAK 1370); D, centrifuged wash obtained from cells PC1-PC2. (described in lane C) that had been stored overnight at 40C; E, crystalline isomerase obtained from E. coli JM 101 containing pAK 5. Westbrook, E. M., Sigler, P. B., Berman, H., Glusker, J. P., 1370 and induced with the thiogalactoside. Note that the pure Bunick, G., Benson, A. & Talalay, P. (1976) J. Mol. Biol. 103, migrate with a calculated subunit molecular weight of 665-667. 10,300, whereas the calculated molecular weight obtained from the 6. Westbrook, E. M. (1976) J. Mol. Biol. 103, 659-664. sequence is 13,399. The abnormally rapid electrophoretic mobility of 7. Westbrook, E. M., Piro, 0. E. & Sigler, P. B. (1984) J. Biol. the subunit of this enzyme in the presence of sodium dodecyl sulfate Chem. 259, 9096-9103. (Mr, 11,000 ± 500) has been observed and discussed (2). 8. Kuliopulos, A., Westbrook, E. M., Talalay, P. & Mildvan, Downloaded by guest on September 25, 2021 Biochemistry: Kuliopulos et al. Proc. Natl. Acad. Sci. USA 84 (1987) 8897 A. S. (1987) Biochemistry 26, 3927-3937. 20. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, 9. Batzold, F. H., Benson, A. M., Covey, D. F., Robinson, 193-197. C. H. & Talalay, P. (1976) Adv. Enzyme Regul. 14, 243-267. 21. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 10. Malhotra, S. K. & Ringold, H. J. (1965) J. Am. Chem. Soc. 87, 1513-1523. 3228-3236. 22. Marcus, P. I. & Talalay, P. (1956) J. Biol. Chem. 218, 661-674. 11. Buki, K. G., Robinson, C. H. & Talalay, P. (1971) Biochim. 23. Southern, E. (1975) J. Mol. Biol. 98, 503-517. Biophys. Acta 242, 268-277. 24. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 12. Batzold, F. H. & Robinson, C. H. (1975) J. Am. Chem. Soc. 103-119. 97, 2576-2578. 25. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. 13. Martyr, R. J. & Benisek, W. F. (1973) Biochemistry 12, 2172- Acad. Sci. USA 74, 5463-5467. 2178. 26. Talalay, P. & Boyer, J. (1965) Biochim. Biophys. Acta 105, 14. Martyr, R. J. & Benisek, W. F. (1975) J. Biol. Chem. 250, 389-392. 1218-1222. 27. Kawahara, F. S., Wang, S.-F. & Talalay, P. (1962) J. Biol. 15. Bounds, P. L. & Pollack, R. M. (1987) Biochemistry 26, 2263- Chem. 237, 1500-1506. 2269. 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 16. Penning, T. M. & Talalay, P. (1981) J. Biol. Chem. 256, 29. Penning, T. M., Westbrook, E. M. & Talalay, P. (1980) Eur. J. 6851-6858. Biochem. 105, 461-469. 17. Penning, T. M., Heller, D. N., Balasubramanian, T. M., Fen- 30. Shine, J. & Dalgarno, L. (1975) Nature (London) 254, 34-38. selau, C. C. & Talalay, P. (1982) J. Biol. Chem. 257, 12589- 31. Ogez, J. R., Tivol, W. F. & Benisek, W. F. (1977) J. Biol. 12593. Chem. 252, 6151-6155. 18. Talalay, P., Dobson, M. M. & Tapley, D. F. (1952) Nature 32. Linden, K. G. & Benisek, W. F. (1986) J. Biol. Chem. 261, (London) 170, 620-621. 6454-6460. 19. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 33. Wang, S.-F., Kawahara, F. S. & Talalay, P. (1963) J. Biol. Cloning: A Laboratory Manual (Cold Spring Harbor Labora- Chem. 238, 576-585. tory, Cold Spring Harbor, NY). 34. Shortle, D. & Botstein, D. (1985) Science 229, 1193-1201. Downloaded by guest on September 25, 2021