Isolation of a Cdna Clone for Human X-Linked 3

Home , Phosphotransferase, Reverse transcriptase

Proc. Nati Acad. Sci. USA Vol. 80, pp. 802-806, February 1983 Genetics

Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe (X-chromosome inactivation/recombinant DNA/colony hybridization/chemical DNA synthesis/mixed-probe method) JUDITH SINGER-SAM*, ROBERT L. SIMMER*, DOUGLAS H. KEITH*, LOUISE SHIVELY*, MARLYN TEPLITZ*, KEIICHI ITAKURA*, STANLEY M. GARTLERt, AND ARTHUR D. RIGGS* *City of Hope Research Institute, Duarte, California 91010; and tDepartments of Medicine and Genetics, University ofWashington, Seattle, Washington 98195 Communicated by A. H. Doermann, October 27, 1982

ABSTRACT We have obtained a cDNA clone encoding most PGK protein ofhuman X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate EC mRNA amino ac 1-phosphotransferase, 2.7.2.3). Total position:ii1 291-29)6 310-313 417 was prepared from human adenocarcinoma-derived cell line NH21 I ICOOH LS174T and used for cDNA preparation. Double-stranded cDNA Z was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled Lys -Phe-Asp -Glu -Asn -Ala Gly-Trp- Met-Gly oligodeoxyribonucleotides. A pool of hexadecamers complemen- poss UUU GAA AAU 5 GGN UGG AUG GGN3 tary to all 32 possible sequences encoding amino acids 291-296 of 5 AAt GAC GCN3 X-linked PGK was used for the initial screen. One clone among complement I3 TTT AAA CTA CTT TTG C 5] CN ACC TAC CC5 2,500 gave a strong positive signal. Plasmid DNA from this clone 16 - mer 11-mer was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consist- FIG. 1. Oligonucleotide probes used in the isolation and charac- ingofa mixture offour sequences. The cloned fragment hybridizes terization of a cDNA clone containing PGK sequences. N = A, C, G, preferentially to DNA from human cells with five X chromosomes. or T(U). DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH the only X-linked enzyme or protein whose amino acid se- terminus. quence had been determined (12). Synthetic oligonucleotides have been extremely valuable for About 5% oftotal mammalian DNA is contained in the X chro- recombinant DNA work (13, 14), but the use of a mixture of mosome, but a much higher percentage of identified genetic oligodeoxyribonucleotides as colony hybridization probes is markers, including hereditary defects, are X-linked. The reason only now beginning to emerge as a reliable technique (15-20). for this overrepresentation is the functional hemizygosity ofX- When only the amino acid sequence of a protein is known, it linked genes in mammalian cells. This is true even for female usually is not possible to determine a unique nucleotide se- cells, because of X-chromosome inactivation (1-3). Unfortu- quence for a probe because of the degeneracy of the genetic nately, despite the preponderance of genetically and develop- code. Therefore, we have used the "mixed-probe" method as mentally interesting genes on the X chromosome, X-linked developed by Wallace and co-workers (17, 18). Fig. 1 illustrates genes have been difficult to clone, probably because most, per- that, for PGK, the most favorable site required that 32 different haps all, of these genes are expressed only at low levels, be- sequences be synthesized to make a tween 0.01% and 0.1% ofthe total protein. Thus, even the pro- 16-base-long probe. For- teins have been difficult to purify and characterize. The tunately, the different sequences can be made simultaneously isolation ofonly two X-linked genes-those for glucose-6-phos- in one (or two) "mixed syntheses," in which more than one tri- phate dehydrogenase (G6PD) (4) and hypoxanthine phosphori- nucleotide or dinucleotide is added to the condensation mixture bosyltransferase (HPRT) (5, 6)-has thus far been reported. at positions of ambiguity (15, 21). Because these mixed Primarily because we are interested in the molecular basis syntheses and the use of mixed probes are not yet a widely ac- of X-chromosome inactivation (2), we have embarked on a proj- cepted method, a significant aspect of the work reported here ect to clone X-linked genes, using synthetic oligodeoxyribonu- is to further document that the mixed probe method is reliable cleotides as hybridization probes for specific detection of the and rapid and can be used as the sole means of screening for desired clones. In this paper, we report the isolation of an X- desired genes. linked gene by this method, the gene for human X-linked 3- phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1- MATERIALS AND METHODS phosphotransferase, EC 2.7.2.3). Sources of Materials. (dT)12-18 and DNA polymerase I Kle- X-linked PGK is an enzyme that has been extensively studied now fragment A were from Boehringer; reverse transcriptase from genetic (7), developmental (1, 3, 8), and biochemical (9, (RNA-dependent DNA polymerase) was a gift from J. Beard. 10) points ofview. Some alleles ofPGK are associated with he- Terminal deoxynucleotidyl transferase and DNA polymerase I molytic anemia (11). When we began this project, PGK was also were from Bethesda Research Laboratories. DNase I was from Sigma. Restriction enzymes were from Boehringer Mannheim, The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: kb, kilobase pair(s); PGK, 3-phosphoglycerate kinase; ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. NaCl/Cit, 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.2. 802 Downloaded by guest on October 1, 2021 Genetics: Singer-Sam etaL Proc. Natl: Acad. Sci. USA 80 (1983) 803 Bethesda Research Laboratories, or New England BioLabs and temperatures for anempirical determination ofthe temperature were used as directed by each supplier. Oligo(dT)-cellulose giving best discrimination against imperfect matches. [The ap- (Ty7) was from P-L Biochemicals. parent melting point (ta) of a perfect.match is estimated for 6X [3 P]ATP (3,000 Ci/mmol) and deoxynucleoside [32P]tri- NaCl/Cit or its equivalent by summing 20C for each AKT base phosphates (600X800 Ci/mmol), were from New England Nu- pairand 40C for each G-C base pair (33). Hybridization was done clear (1 Ci = 3.7 x 1010 becquerels). at about 15'C below the average estimated td. ] Synthesis of a Mixed Oligonucleotide Probe. The 16-base Hybridization of Probes to Plasmid DNA. Plasmid DNA in and 11-base probes shown in Fig. 1 were synthesized on a solid large quantities was purified by the high-yield cleared lysate support and purified by high-performance liquid chromatog- method of Norgard (34). The method of Ish-Horowicz and raphy as described (15, 21). The four 11-base oligodeoxyribonu- Burke (35) was used for rapid preparation of small amounts of cleotides were prepared in on-e mixed synthesis. The 16-base plasmid. The 32P-labeled probes were hybridized to restriction- oligodeoxyribonucleotides, consisting of32 species, were made enzyme-digested plasmid DNA on dried agarose membranes in two mixed syntheses: (S. G. S. Tsao, C. F. Brunk, and R. E. Pearlman, personal communication). Gels stained with ethidium bromide were soaked 5'-C-A-T-TTTCA-T-CA-A-AT-T-T first in 500 mM NaOH/150 mM NaCl for 20 min at room temperature, then in 500 mM Tris'HCI, pH 8.0/150 mM NaCl for and 20 min at 40C. The gels were placed on Whatman 3 MM paper 5 ' --G-T-T T-T-CA-T-CA-A-AT-T-T. and dried under reduced pressure at 60'C for 1-2 hr. The dried gel, now a membrane, was separated from the filter paper sup- These two pools differ only at the underlined nucleosides. port by floating it briefly on water. The buffers used for hy- Isolation of mRNA. Cells from the human adenocarcinoma- bridization and washes were the same as those described above, derived cell line LS174T (22) were the source of mRNA. except that the prehybridization step was omitted. Twenty-four hours prior to lysis, cycloheximide was added to DNA Sequence Determination. Portions of the pGK824 se- the culture medium (modified Eagle's medium) to a final con- quence were determined by the method of Sanger (36) after centration of 50 -,ug/ml (23). Lysis of cells and RNA isolation subeloning in phage M13 (37) offragments produced by cleav- were done as described by Daskal et aL (24), except that cyclo- age with Sau IIIA, Hpa II, or by double digestion with Pst I and heximide was present in all buffers at 50 jig/ml and the con- Sau IIIA. The sequences ofother regions were determined by centration ofIvory detergent was 0.1%. mRNA was purified by the method of Maxam and Gilbert (38). oligo(dT)-cellulose column chromatography (25). Southern Blot Analysis. Human DNA was isolated (39) from Isolation ofcDNA Clones. mRNA (5 Ag) was used as a tem- karyotypically normal male primary fibroblasts (1X) and female plate-for oligo(dT)-primed cDNA synthesis, followed by syn- fibroblast cells carrying five-X chromosomes (5X) but otherwise thesis ofthe complementary strand by use of DNA polymerase karyotypically normal (a gift from Steven Funderburk). These I Klenow fragment A and addition of nuclease S1, all as pre- iX and 5X DNAswere digested to completion with BamHI; 30 viously described (26). cDNA greater than 1,000 base pairs long ,4g of each DNA was electrophoresed in agarose, stained with was selected by electrophoresis on a 6% polyacrylamide gel, ethidium bromide, and blotted onto nitrocellulose (40). The electroeluted, and "tailed" with dC residues at the 3' terminus isolated Pst-I fragment of pGK824 [1.2 kilobase pairs (kb)] was by the use of terminal deoxynucleotidyl transferase (27). After nick-translated with [a-32P]dCTP (800 Ci/mmol) as specified annealing with Pst I-cut and dG-tailed plasmid pBR327 (kindly by Amersham. Hybridization was in a mixture containing 6X supplied by Y. Takahashi and J. Rossi) (28), the DNA was used NaCl/Cit, lx Denhardt's solution, -sonicated denatured salmon to transform Escherichia coli strain LS1, a lac' derivative of sperm DNA at 20 ug/ml, and 10% dextran sulfate at 650C for RR1 (pro, leu, thi, rpsL2O, hsdR, hsdM) (29) by the procedure 8 hr. Washes were in 0.2X NaCI/Cit/10-mM Tris-HCl, pH 8.0/ of Hanahan as described in ref. 30. Clones were selected on L 1 mM EDTA/0. 1% sodium dodecyl sulfate at 650C for 2 hr (41). agar plates with tetracycline added at 10 gg/ml. Screening of cDNA Clones. Clones were screened by use RESULTS ofa filter paper method (31). Colonies (about 2,500) were trans- Screening Colonies with at Mixed Oligonucleotide Probe. ferred in an ordered array to fresh L agar/tetracycine plates The target site for the hexadecamer probe was selected to be (320colonies perplate) and incubated at370C until the diameter (i) near the COOH-terminus so that full-length cDNA would of the colonies was 3-4 mm. The plates were overlaid with not be required, (ii) where degeneracy of the genetic code was Whatman no. 541 paper for 2 min. The filters were transferred minimal, and (iii) not extremely G+C or A+T rich. The best to fresh L agar plates containing chloramphenicol (250 Ag/ml), site (amino acids 291-296, Lys-Phe-Asp-Glu-Asn-Ala) required incubated for 42 hr, and then treated with alkali (31). The oli- 32 different sequences to cover all possibilities for a 16-base gonucleotide probes were labeled with 32P by use of polynu- probe. Two mixtures of 16 sequences were synthesized and cleotide kinase and [32P]ATP and were purified on Whatman then combined to make one probe mixture containing 32 se- DE-52 DEAE-cellulose columns (32). After filtration (0.2-gm- quences (see Fig. 1). pore Nalgene filters), the probes were used directly for hy- mRNA from a human adenocarcinoma-derived cell line was bridization at a concentration of0.2 ng/ml per species ofprobe used to construct a cDNA library of 2,500 transformants. Col- (about 107 total cpm). onies were lysed, fixed to Whatman filterpaper, and hybridized Hybridization was done as described (18). Filters were pre- to the 32P-labeled hexadecamer probe mixture. After three 20- hybridized in 6X SET (SET = 0.15 M-NaCl/0.001 M EDTA/ min washes at room temperature and -three 5-min washes at 0.015 M Tris HCl, pH 7.5) containing sonicated salmon sperm 37°C, one colony gave .a strong autoradiographic signal, espe- DNA at 100 Ag/ml and 0.5% Nonidet P-40. They were hy- cially when compared to colonies in its immediate vicinity (Fig. bridized to the probe at room temperature in the same buffer 2). The colony was still positive after an additional 4-min wash except that yeast RNA at 250 ,g/ml was substituted for salmon at 420C. sperm DNA. As detailed further in Results, filters were washed Screening by Gel Hybridization or Southern Blots. All col- in 6x NaCl/Cit (NaCI/Cit = 0.15 M NaCl/0.015 M sodium onies giving a significant signal were picked, purified, and citrate, pH 7.2) and autoradiographed at successively higher grown in 5-ml cultures. Plasmid DNA was partially purified by Downloaded by guest on October 1, 2021 804 Genetics: Singer-Sam et al. Proc. Natl..Acad. Sci. USA 80 (1983)

9v 1AR 1 2 3 4 5 6

A B FIG. 2. Hybridization of cDNA clones to a 32P-labeled mixed hexa- FIG. 3. Hybridization of 32P-labeled mixed hexadecamer probe to decamerprobe forPGK. Arrow indicates the clone containing pGK824. pGK811, pGK824, and pBR327. Plasmids were digested with restric- Filters were incubated with the probe for 2 hr and washed for 20 min tion enzymeEcoR[ for 1 hr at 3700 andelectrophoresed on a 1.2% agar- three times at room temperature then for 5 min three times at 3700. ose gel; the gel was dried and DNA in it was hybridized to the 32P-la- The wash buffer was 6x NaCl/Cit. beled 16-base probe (107 cpm) overnight. Hybridization and three 20-min washes in 6x NaCl/Cit were done at room temperature. (A) Ethidium-bromide stained gel. (B) Autoradiogram. Lanes: 1, pGK811, a rapid method (35), cut with restriction endonuclease EcoRI, uncut; 2, pGK811, cut with EcoRI; 3, pGK824, uncut; 4, pGK824, cut and electrophoresed in a 1.2% agarose gel. The gel was dried with EcoRI; 5, pBR327, uncut; 6, pBR327, cut with EcoRI. and the DNA in the resulting agarose membrane was hybridized with the 32P-labeled hexadecamer probe mixture. After three serves as an internal control for the amount of DNA present in 5-min washes at 37TC, only one plasmid (designated pGK824) each lane. showed a strong autoradiographic signal. This result confirms the preliminary data obtained at the colony screen level. DISCUSSION Pure pGK824 DNA was prepared for the next level ofscreen- Genes on the X chromosome seem to be expressed at low levels ing. Discrimination between perfect matches and close matches (4, 6, 12). Most methods for cloning such, genes depend on a is remarkable whenidentical amounts ofsimilar sizelinear DNA sensitive immunoassay for an in vitro translation product. One are electrophoresed on the same gel and hybridized with oli- enriches for the desired mRNA and screens clones by hybrid- gonucleotides. Earlier we had obtained a plasmid (pGK811) selected translation (4, 42). We were not able to use methods pairing with a single purine-purine mismatch with two of the based on immunoprecipitation, because PGK is a poor immu- sequences in our hexadecamer mixture. Thus, we could use nogen. Rabbits, chickens, and gninea pigs did not yield antisera pGK811 as a standard for comparison with pGK824. The result with adequate affinity for PGK after repeated immunization of this comparison is shown in Fig. 3, where it is readily ap- attempts. The poor immunogenicity of PGK may be the result parent that pGK824 gives a much stronger signal. We also hy- of its highly conserved amino acid sequence (7, 9). bridized Hpa II-cut DNA with the undecamer mixed probe Fortunately, the mixed-probe method that we used is in- directed against a different region of the gene (see Fig. 1) and dependent ofantibody production and requires only that amino found significant hybridization only with the insert DNA of acid sequence information be available for at least a segment pGK824. of the protein. The mixed-probe method should be generally Characterization of pGK824 by DNA Sequence Analysis. useful, especially in view ofprotein microsequencing methods DNA sequence analysis of the region corresponding to amino allowing amino acid sequences to be determined for proteins acid positions 201-240 revealed total compatibility with the available only at microgram levels (43). known amino acid sequence of PGK. These sequence data are We have isolated a cDNA clone containing a 1.2-kb insert shown in Fig. 4. Preliminary DNA sequences compatible with in the Pst I site of pBR327. Characterization of the clone by the amino acid sequence of PGK were also obtained corre- hybridization to two sets of mixed oligonucleotide probes and sponding to amino acid positions 120-200, 240-253, and 330- by nucleotide sequence analysis of DNA segments correspond- 373. The only possible discrepancy we have found thus far is ing to several regions of PGK provide convincing evidence that at amino acid position 250, where the DNA sequence codes for pGK824 codes for amino acids 120-417 of PGK, as well as for asparagine rather than aspartate (12). a portion of the 3' untranslated region. Hybridization ofthe pGK824 Insert to DNA Containing One There is an autosomal PGK that is expressed only during X Chromosome and Five X Chromosomes. The 1.2-kb Pst I spermatogenesis (44). We believe that we have cloned the X- fragment of pGK824 was isolated, nick-translated, and hybrid- linked PGK rather than the autosomal isozyme for the following ized to human DNA containing either one or five X chromo- reasons: (i) The adenocarcinoma cell line used as a source of somes. Fig..5 shows that three genomic BamHI fragments, ap- mRNA expresses only X-linked PGK (M. E. Sparkes & R. proximately 9, 7, and 5 kb, bind more strongly in DNA con- Sparkes, personal communication). (ii) Preliminary sequence taining five X chromosomes than in DNA containing one X chro- analysis ofmore than 40% ofthe pGK824 insert shows complete mosome. The 9-kb fragment binds less strongly than the other sequence compatibility with the published amino acid se- two. A fourth fragment, approximately 1.5 kb, hybridizes with quences ofX-linked PGK (12). (iii) The pGK824 insert hybrid- the probe to an equal extent in 1X and 5X DNA. This 1.5-kb izes more strongly with DNA from human cells having five X band could represent fortuitous hybridization to autosomal se- chromosomes than with DNA from male fibroblast cells (Fig. quences or might be autosomal PGK. In either case, the band 5). Downloaded by guest on October 1, 2021 Genetics: Singer-Sam et aL Proc. Nati Acad. Sci. USA 80 (1983) 805

210 220 ... .Leu - Glu - Ser - Pro - Glu - Arg - Pro - Phe - Leu - Ala - Ile - Leu - Gly - Gly - Ala - Lys - Val - Ala - Asp - Lys - 5'. . . TTG GAG AGC CCA GAG CGA CCC TTC CTG GCC ATC CTG GGC GGA GCT AAA GTT GCA GAC AAG

230 240 Ile - Gln - Leu - Ile - Asn - Asn - Met - Leu - Asp - Lys - Val - Asn - Glu - Met - Ile - Ile - Gly - Gly - Gly - Met ... ATC CAG CTC ATC AAT AAT ATG CTG GAC AAA GTC AAT GAG ATG ATT ATT GGT GGT GGA ATG ... 3' FIG. 4. Sequence of pGK824 corresponding to amino acids 201-240 of X-linked PGK. The sequence was determined by the method of Sanger (36), using two phage M13 clones containing inserts 5' and 3' to the Sau IIA site at amino acid position 221 (underlined). Numbers refer to amino acid positions.

Wechose adenocarcinoma-derived cells as a source ofmRNA probes should be used for colony hybridization. For probes 14 after a more obvious choice proved unsuccessful. We first iso- nucleotides or longer, the signal-to-noise ratio is acceptable, lated mRNAfrom human uterine cells, because we found mouse even though the complexity of the sequence may be much muscle to have relatively elevated levels of PGK (0.2% of sol- greater. uble protein compared to 0.05% for liver cells). However, when Our results (Fig. 2) show that even a mixture of 32 hexade- we screened over 10,000 cDNA clones obtained by either mixed camers allows detection of the desired clone. In the colony undecamer-primed or oligo(dT)-primed cDNA synthesis, we screening described here, only one strongly positive clone was did not find a PGK-coding clone. observed, and indeed it did contain PGK -sequences. In pre- When we began this work, it was not clear what length of vious screenings, we have occasionally observed "false posi- oligodeoxyribonucleotide would be required for reliable detec- tives," strongly hybridizing colonies that did not hybridize to tion by colony hybridization. Short oligonucleotides have the the same probe after partial plasmid purification. We also de- .advantage that only 'a few possible sequences must be synthe- tected sequences closely related to the probe. For example, sized. For example, there are only four possibilities for the un- plasmid pGK811 was detected as a strongly hybridizing colony, decamer shown in Fig. 1. Even'though undecamers are ade- and it hybridized with a G-A or G-G mismatch to 2 of the 32 quate for hybridizations on nitrocellulose or dried agarose species ofthe probe. Therefore, it is important to use a second membranes, we found, as did Suggs et aL (17), that longer screening method once potential colonies are selected. One approach is to determine the sequence ofonly the plasmid that gives the strongest signal after hybridization to the iX 5X probe on nitrocellulose or gel membranes. At this level, hybridization with oligodeoxyribonucleotides can easily distin- guish single base differences, especially if washes are done at a series of temperatures. Fig. 3 shows that a single mismatch (pGK811) shows only a faint hybridization signal with the mixed probe under conditions in which a perfect match (pGK824) 9 shows a very strong signal. A better procedure (which we also used) is to use a second probe directed against a different site in the protein. DNAs from our putative clones were screened -7 with the 11-base-long mixed probe shown in Fig. 1, and only pGK824 showed a positive hybridization signal. The final proofthat the desired clone has been obtained must -5 come from comparison of the DNA sequence with the protein A- sequence. Even at this step, the mixed probes can be useful, allowing identification of the fragment carrying the DNA sequence complementary to the probe and serving as primers in DNA sequence analysis by the method of Sanger (unpublished observation). We thank R. Bruce Wallace, Gerald Forrest, and Yvonne Santana for their help. This work was supported in part by National Institutes ofHealth Grants GM25825 and GM31263 to A.D.R., Grant GM25658 to K.I., and.Grant GM15253 to S.M.G. 1. Gartler, S. M. & Andina, R. J. (1976) Adv. Human Genet. 7, 99- 140. 2. Riggs, A. D. (1975) Cytogenet. Cell Genet. 14, 9-25. 3. Martin, G. R. (1982) Cell 29, 721-724. 4. Persico, M. G., Toniolo, D., Nobile, C., d'Urso, M. & Luzzatto, L. (1981) Nature (London) 294, 778-780. -1.5 5. Jolly, D. J., Esty, A. C., Bernard, H. U. & Friedmann, T. (1982) Proc. NatL Acad. Sci. USA 79, 5038-5041. 6. Brennand, J., Chinault, A. C., Konecki, D. S., Melton, D. W. & Caskey, C. T. (1982) Proc. Nati Acad. Sci. USA 79, 1950-1954. FIG. 5. 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