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Cloning of α2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs
Gene, 13 (1981) 145-152 Elsevier/North-ttolland Biomedical Press
145
Cloning of ~2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs (Recombinant DNA; rat liver; hormonal control)
David T. Kurtz and Christopher F. Nicodemus ('old Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 11724 (U.S.A.)
(Received October 14th, 1980) (Accepted December 4th, 1980)
SUMMARY An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200-500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA for a2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of C~2uglobulin mRNA in male and female rats.
INTRODUCTION The advent of molecular cloning techniques has resulted in a much deeper understanding of the mechanisms regulating gene expression in eukaryotes. Methods for cloning specific cDNAs have depended, to a large extent, on the fact that the cDNA to be cloned was prepared from an mRNA population in which the specific messenger RNA under study represented a high percentage of the total mRNA. Cloning of cDNAs has been effected by using either homopolymeric tailing (Villa-Kamaroff et al., 1978) or by ligating a synthetic oligonucleotide linker Abbreviations: bp, base pairs; cDNA, DNA complementary to mRNA; DBM, diazobenzyloxymethyl; ds, double stranded; kb, kilobase pair; RI, EcoRI; SDS, sodium dodecyl sult'ate.
onto the cDNA and inserting the resultant molecule into a single restriction site in pBR322 (Fiddes and Goodman, 1979). The efficiencies that have been reported, using either method, are in the range of 1 - 4 0 recombinants per ng of ds cDNA (Fiddes and Goodman, 1979; Ordahl et al., 1980). For the study of low-abundance mRNAs, a high cloning efficiency would be desirable. We have developed a method for cloning cDNAs that results in approx. 200-500 recombinants per ng of ds cDNA. ds cDNA is ligated with equal amounts of EcoRI and SalI linkers. On average, 50% of the molecules in the cDNA preparation will have one EeoRI end and one SalI end. Plasmid BR322 is digested with EcoRI and SalI, and the 3.7 kb fragment, containing the ampicillin-resistance gene, is purified free of the smaller fragment. A large excess of the RI-Sal-cleaved pBR322 fragment is ligated to
0378-1119/81/0000 0000/$02.50 ©Elsevier/North-Holland Biomedical Press
146 the cDNA preparation. The RI-Sal-ended pBR fragment recycles with extremely low efficiency. Thus, a large excess can be used, and the level of background colonies never exceeds 5% of the total number of colonies. Using this technique, we have cloned a cDNA for the male rat liver protein, c~2uglobulin. The synthesis of this protein, which represents 1% of hepatic protein synthesis (Sippel et al., 1976), is under complex hormonal control in vivo, involving the participation of sex steroids, glucocorticoids, thyroid hormone, and growth hormone (Kurtz and Feigelson, 1977; Kurtz et al., 1976; 1978b).
MATERIALS AND METHODS
(a) Preparation of double-stranded cDNA Messenger RNA was extracted from rat liver as previously described (Kurtz et al., 1976a). 32P-labeled cDNA (5 X l0 s cpm/~g) was synthesized using the conditions outlined by Kacian and Myers (1976), except that pyrophosphate was omitted. After 1 h incubation at 37°C, the reaction was brought to 10 mM EDTA, 0.1% SDS, and 0.3 M NaOH, left at 37°C for a further 4 - 6 h, neutralized, and then extracted with phenol/chloroform. The cDNA was separated from unincorporated nucleotides using Sephadex G-50 chromatography. The cDNA was precipitated with no carrier, and resuspended in 50 mM potassium phosphate, pH 7.4, 5 mM MgCl2, 1 mM mercaptoethanol, containing 50 /~M of each of the four dNTPs. The reaction volume was 120 /.cl/#g of cDNA. Klenow fragment of F.. coli polymerase I (Boehringer) was added at a concentration of 10 units/~g of cDNA, and the mixture was incubated at 37°C for 30 min. (This short incubation time, and the use of the Klenow fragment, seemed to prevent the homopolymet formation that can occur when polymerase I is used to synthesize the second strand.) The reaction was phenol/chloroform-extracted, and passed over Sephadex G-50. Typically, 85-95% of the first strand was rendered Sl-resistant using this procedure. The ds cDNA was rendered blunt-ended using S1 endonuclease (Sigma), 100 units//~g of ds cDNA. The cDNA was phenol/chloroform-extracted and ethanol-precipitated.
(b) Cloning by the double-linker method 1 /ag of ds cDNA was resuspended in 50/A 50 mM Tris, pH 7.5,8 mM MgC12, 10 mM ~-mercaptoethanol. 1 mM ATP, and 0.2 mM of each of the 4 dNTPs. One unit of Klenow fragment was added, and the mixture was incubated for 15 min at 12°C, to convert "ragged" ends to blunt ends. The reaction was stopped by incubating at 65°C for 10 15 min. l # g each of IScoRI and SalI oligonucleotide linkers (Collaborative Research), which had been treated with polynucleotide kinase (Seeburg et al., 1977), were added. The reaction was made 1 mM ATP, 1 mM spermidine, and 10 units T4 DNA ligase (BRL) and 5 units T4 RNA ligase (New England Biolabs) were added. The reaction was allowed to proceed for 2 4 h at 4°C. The reaction was stopped by incubation at 65°C for 15 min, and brought to 0.I M NaC1.50 units of F.coRI and 50 units SalI were added and the mixture was incubated for 8-12h at 37°C. The reaction was extracted with phenol/chloroform and passed over Sephadex G-50. Plasmid pBR322 was cleaved simultaneously with EcoRI, BamHI and Sail, using conditions described by the supplier (BRL) for the BamHI enzyme. The reaction was extracted with phenol/chloroform, and the 3.7 kb fragment, containing the ampicillin-resistance gene, was separated from the smaller fragments using a 10-40% sucrose gradient in 10 mM Tris pH 8.0, 10 mM EDTA, 1.0 M NaCl, in an SW41 rotor, 40000 rev./min for 40 h. Typically, 100 #g of DNA were loaded onto one gradient. The gradients were collected from tile bottom, in 0.5 ml fractions, and 10 gl of each fraction was run on a 0.75% agarose gel to detect the presence of the 3.7 kb fragment. Fractions containing the fragment were pooled, diluted 4-fold to reduce the sucrose concentration, and ethanol-precipitated. To ensure that the 3.7 kb RI-Sal fragment was not contaminated with uncut or singly cut pBR322, 1 #g was incubated with 5 units T4 DNA ligase, using the conditions described by the supplier (BRL). The DNA was ethanol-precipitated and used to transform E. coli strain MM294, as described below. Only 500 bacterial colonies resulted, a value 10 -4 that found with uncut or singly cut pBR322. Colonies that did grow in ampicillin were found to contain a plasmid with an EcoRI-Sall fusion (data not shown).
147 (c) Bacterial transformation 200 ng of ds cDNA containing the linkers was mixed with 5 ~g of the RI.Sal fragment of pBR322 in a volume of 3 ml of 66 mM Tris • HC1, pH 7.5, 6.6 mM MgC12, 10 mM dithiothreitol, 0.4 mM ATP. 10 units of T4 DNA ligase (BRL) was added, and the reaction was incubated for 2 4 4 8 h at 4°C. 2 ml of 5 M ammonium acetate was then added, followed by 10 ml of ethanol. The DNA was collected by centrifugation and resuspended in I00 gl sterile water. 25 gl aliquots were used directly to transform E. coli strain MM294, using the transformation technique described by Dagert and Ehrlich (1979). The resultant colonies were screened using the high-density screening protocol of Hanahan and Meselson (1980). (d) Preparation of specific cDNA probes a2u globulin cDNA, labeled with 32p at a specific activity of 5 × 107 cpm/#g, was prepared as previously described (Kurtz and Feigelson, 1977). Rat albumin cDNA was prepared essentially as described by Yap et al. (1977). These cDNAs were used to screen the rat-liver cDNA library. (e) Identification of specific clones by hybridizationtranslation Chimeric plasmids suspected to contain the C~2u globulin or albumin cDNAs were linearized with EcoRI, phenol/chloroform-extracted, and ethanolprecipitated. 20 #g of plasmid DNA, in 20 /j1 1 M Na.acetate, pH 4.5, was spotted onto a 0.5 X0.5 cm square of diazo-benzyloxymethyl cellulose ("Northern paper"), which had been prepared as described by Alwine et al. (1977). The DNA was spotted onto the paper in 4/al aliquots, and the DBM paper was left at room temperature for 4 - 5 h. The square was then incubated in 50% formamide, 5 × SSC, 10 mM EDTA 1 ×Denhardt's (1966)solution, 0.1% SDS, 0.5 ~g/ml calf thymus DNA at 37°C for 10-12 h. The paper was then incubated with 100 /ag of male rat liver mRNA in the above solution minus the DNA and Denhardt's solution. The hybridization was performed in a 4 ml polypropylene tube, using just enough solution to cover the square of DBM paper (about 300 /.d). Hybridization was allowed to proceed for 10-12 h. The square was then
washed 10 12 times with 2 ml 50% formamide, 5 X SSC, 10 mM EDTA, 0.1% SDS. Washing was done by vortexing, and centrifuging the square to the bottom of the tube following each wash. The paper was then washed 2 - 3 times with 10 mM EDTA 0.1% SDS. Hybridized RNA was eluted by boiling the square for exactly 60 s, in 500 /.tl sterile water. The tube was placed into ice immediately, and then centrifuged. The water was removed, made 0.3 M in LiC1 and the RNA was precipitated with 2 vols. ethanol. The RNA was recovered by centrifugation in an SW60.1 rotor, 50 000 rev./min for 30 min. The tube was inverted, allowed to dry, and the components of the rabbit reticulocyte translational system, containing [3SS]methionine (New England Nuclear), were added directly to the tube (25 /al final volume). Translation was allowed to proceed for 60 rain and the entire translation reaction was brought to 2% SDS, 5% /3-mercaptoethanol, 10% glycerol, and 0.0625 M Tris, pH 6.8, boiled, and subjected to SDS gel electrophoresis as described by Laemmli (1970). The gels were dried and subjected to autoradiography. (f) Other methods Gel electrophoresis of RNA under denaturing conditions and transfer to DBM paper ("Northern blotting") was performed as described by Rave et al. (1979). Labeling of DNA by nick-translation and Southern blot analysis was done as described by Weinstock et al. (1978).
RESULTS The double-linker method of cDNA cloning described above is extremely efficient: approx. 2 0 0 400 recombinant colonies per ng of cDNA can be routinely obtained. A large excess of the EcoRI-SalI fragment of pBR322 can be used to drive a high percentage of the cDNA into recombinant plasmids, and the level of background colonies (i.e. bacteria harboring a plasmid containing an RI-Sal fusion) never exceeds 5% of the total number of colonies. In one experiment, 200 ng of rat liver ds cDNA was cloned using this technique and resulted in approx. 50 000 bacterial colonies. These colonies were subjected to the high-density screening protocol of Hanahan and
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Fig. 1. Northern blot analysis using pal76. 1 ;zg of male rat liver (lane 1) or female rat liver (lane 2) mRNA was denatured and subjected to gel electrophoresis and Northern blot analysis, as described in the text. Plasmid pc~176 was nicktranslated with [a2P]dNTPs and used as probe. Lane 3: SV40 DNA cleaved with ttindIIl and 5'-end-labeled with polynucleotide kinase. The fragment sizes are 1768, 1169, 1101, 526, 447, and 215 bp long. In these gels, DNA standards may be used to estimate RNA length (McMaster and Carmichael, 1977).
Meselson (1980), using either 32P-labeled a2u globulin or albumin cDNA as probes. A large number of positive colonies were detected. These were grown in 1 ml cultures, and plasmid DNA was extracted and subjected to gel electrophoresis as described by Birnboim and Doly (1979). The largest recombinant plasmids were then grown up in mass culture for further study. One putative C~2u globulin plasmid, pcd76, containing an insert of 750 bp, was nick-translated and used as a probe in Northern blotting vs. male and female rat liver mRNA; as shown in Fig. 1, a single
mRNA band, approx. 1 kb in length, is visible in the male liver mRNA track, while no detectable hybridization occurs with female mRNA. To positively identify pc~176 as a2u globulin, the plasmid was linearized, bound to DBM paper, and hybridized to male rat liver mRNA as described in MATERIALS AND METHODS. The RNA that hybridized to pcd 76 was translated in a rabbit reticulocyte cell-free system containing [aSS]methionine. A single labeled polypeptide, which migrates with authentic a2u globulin, was detected (Fig. 2). (The largest albumin plasmid, pRSA35, was used in similar analysis to positively identify it as being rat albumin; Fig. 2b.) The level of hepatic C~2uglobulin mRNA in vivo is under extremely complex hormonal control: no translatable C~2u mRNA is normally present in adult female rats, but it can be induced in ovariectomized females by either androgens or glucocorticoids (Kurtz et al., 1978a). Also, a2u globulin mRNA synthesis can be depressed in adult male rats by chronic estrogen administration (Kurtz and Feigelson, 1977). Hepatic mRNA was isolated from rats following various hormonal treatments, and subjected to Northern blot analysis vs. pcd76 (Fig. 3). No bands are detectable in female or ovariectomized female mRNA, while mRNA from ovariectomized females treated with dexamethasone or androgen contain an mRNA species identical in size to that found in male liver. Hepatic mRNA from a male rat treated with estrogen contains no detectable C~2uglobulin mRNA (Fig. 3). pc~176 was mapped with various restriction enzymes (Fig. 4). An Aval site was found approx. 400 bp from the EcoRl site. pc~176 was cleaved with AvaI, which cuts in the insert as described, and also cleaves pBR322 at a site approx. 800 bp downstream from the SalI site. The DNA was then ligated at extremely low concentration (0.5 /.tg/ml) to allow only cyclization to take place. The resultant RI-AvaI plasmid was designated p~A pal (Fig. 4). The 5'-+ 3' orientation of pcd76 was determined by cleaving simultaneously with EcoRI, AvaI and Sail. The resultant fragments were subjected to Southern blot analysis, using either male rat liver [a=P]cDNA, or [32p]cDNA made with chain-terminating dideoxynucleotides present at 1/10 the concentration of the deoxynucleoside triphosphates. The resultant 3'-specific cDNA was found to hybridize only to the 350 bp AvaI-SalI fragment, while both the RI-AvaI and the RI-SalI fragments hybridized to
149
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Vig. 2. Identification of pal76 as a2u globulin. A rabbit-reticulocyte translational system containing [3 s S] methionine was used to translate mRNA. The products were subjected to SDS-gel electrophoresis (Laemmli, 1976). The bands represent [35S]protein autoradiograms. (A) Lane 1-translation products from total feinale rat liver mRNA; lane 2-immunopreeipitate t'rom lane 1, using anti~2u globulin;lane 3 total male rat liver mRNA translation product;lane 4-immunoprecipitate from lane 3, using antie~2u globulin; lane 5 no added mRNA (bands represent endogenous translation products of the reticulocyte lysate); lane 6-translation product of mRNA which hybridizes to pal76. (B) Lane 1-total male liver mRNA translate: lane 2-immunoprecipitate from lane 1 using anti-rat albumin (provided by Dr. L. Chasin); lane 3-no added mRNA; lane 4 translation product of mRNA which hybridizes to pRSA35.
full-length cDNA (data not shown). The AvaI-SalI fragment thus represents the 3'-end o f the cDNA. p a l 7 6 (approx. 750 bp) does not represent a full length a2u cDNA, which is approx. 1000 bp; ifEcoRI or Sall sites were present in aZu cDNA, a full-length clone would not be obtainable using this cloning method. The presence Of an EcoRI site was suspected, because four other aZu globulin clones were isolated, all approx. 7 0 0 - 7 5 0 bp in length, which were found to have AvaI, HinclI and PvuII sites at exactly the same distance from the EcoRI site as found in p a l 7 6 . Further, several very small clones ( 2 0 0 - 2 5 0 bp in length), had been isolated in the initial screening. One clone, p a l 1, of approx. 240 bp
was found to give the same pattern of hybridization to the mRNAs, as shown in Fig. 3, but did not hybridize to p a l 7 6 . Full-length male-liver cDNA hybridized weakly to p a l l , and the 3'-specific cDNA did not hybridize at all. It was concluded that there is indeed an EcoRI site in azu cDNA, and that p a l 1 represents the 5'-end of the cDNA. The total length of p a l 7 6 and peril thus represents >95% of full length a2u globulin mRNA. The plasmids p a l l , p a A V A I , and p a l 7 6 represent, respectively, 5'-, middle-, and 3'-specific probes. It was reported by Hastie et al. (1979) that the m R N A for rat azu globulin cross-hybridizes with the cDNA for the corresponding male mouse liver pro-
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Fig. 3. Hormonal control of the level of hepatic C~2u globulin mRNA. 5 ~tg of hepatic mRNA obtained from rats in various endocrine states was subjected to Northern blot analysis using pal76 as probe. Lane 1-male rat liver; lane 2 female; lane 3-ovariectomized female; lane 4-ovariectomized female treated with dexamethasone; lane 5-ovariectomized female treated with dihydrotestosterone; lane 6 male treated with estradiol 17/3. Fig. 5. Cross-hybridization of rat C~2u globulin cDNA and mouse major urinary polypeptide (MUP) mRNA. 5 #g of mRNA was subjected to Northern blot analysis using pod76 as probe. Lane 1-male rat liver mRNA; lane 2-female rat liver mRNA: lane 3-male mouse liver mRNA; lane 4-
female mouse liver mRNA. The higher molecular weight bands visible in lane 1 represent a2u globulin mRNA precursors (D. Kurtz, manuscript in preparation). The only crosshybridization between pal76 (rat a2u globulin eDNA) and MUP mRNA is seen in lane 3.
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po(176 ~ Fig. 4. Restriction enzyme map of C~2u globulin cDNA plasmids. The cDNA is not cut by BamHI, PstI, Xhol, Xbal and HhaI. Arrows represent the lengths and locations along the eDNA of plasmids pal 1, pal76, and paAval.
151 tein, designated MUP. Plasmid pc~176 was used as a probe in Northern blot analysis of male and female mouse liver mRNA (Fig. 5). A weakly hybridizing band can be seen in male mouse liver mRNA, while no detectable hybridization occurs with female mouse liver message.
reported for cloning RNA-DNA hybrids using homopolymeric tailing (Zain et al., 1979). This double-linker method of cDNA cloning should prove to be of value for studies of low abundance messenger RNAs which may be developmentally regulated (Ordahl et al., 1980). The availability of the C~2u globulin cDNA clone will make it possible to explore the molecular mechanisms underlying the hormonal control of the synthesis of this protein.
DISCUSSION The cloning procedure described above has proven to be extremely efficient in constructing several cDNA "libraries". We have used it to clone a cDNA for hamster dihydrofolate reductase (Lewis et al., 1981), and to clone a cDNA for herpes thymidine kinase from an m R N A population in which this message represented approx. 0.1% o f the total m R N A population. The use of two linkers increases the likelihood of cleaving the cDNA. (As described above, the ~2u globulin cDNA was found to contain an EcoRI site.) We have explored the possibility of protecting EcoRI sites in the cDNA, using EcoRI methylase. Methylation of the ds cDNA was found to protect it from EcoRI digestion, and did not seem to alter the size distribution, but, surprisingly, methylation was found to lower transformation efficiency almost 10-fold. It is possible that the R1 methylase used in these studies (New England Biolabs) was contaminated with exonuclease or phosphatase activity, thus lowering the efficiency of linker addition. However, methylation of plasmid sequences has been found to lower the efficiency of transformation in other systems (D. Hanahan, personal communication). We have also a t t e m p t e d to clone RNA-DNA hybrids using this technique. First-strand cDNA was prepared with the inclusion of pyrophosphate in the reaction t o inhibit the RNase H activity of the reverse transcriptase (Kacian and Myers, 1976). The reaction was phenol/chloroform-extracted, passed over Sephadex G-50, and treated with S1 endonuclease. The hybrids were then treated exactly as ds cDNA in the cloning procedure described above. Bacterial colonies containing recombinant plasmids were obtained, but the inserts were found to be very small ( 2 0 0 - 3 0 0 bp), and the overall efficiency was very low (approx. 3 recombinant colonies/ng of RNA-cDNA hybrid). This is similar to the transformation efficiency
ACKNOWLEDGEMENTS We are indebted to Dr. J.W. Beard, Life SciencesResearch Laboratories, for supplying AMV reverse transcriptase as part of the NIH-NCI program. This work was supported in part by a grant from the National Institutes of Health, AM 26968-01.
REFERENCES Alwine, J.C., Kemp, D.J. and Stark, G.R.: Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74 (1977) 53505354. Birnboim, H.C. and Doly, J.: A rapid alkaline procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7 (1979) 1513-1524. Dagert, M. and Erlich, S.D.: Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6 (1979) 23-28. Denhart, D.T.: A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23 (1966) 641-646. Fiddes, J.C. and Goodman, H.M.: Isolation, cloning, and sequence analysis of the cDNA for the ct-subunit of human chorionic gonadotropin. Nature 281 (1979)351 355. Hanahan, D. and Meselson, M.: Plasmid screening at high colony density. Gene 10 (1980) 63-67. Hastie, N.D., Held, W.A. and Toole, J.T.: Multiple genes coding for the androgen-regulated major urinary proteins of the mouse. Cell 17 (1979) 449-457. Kacian, D.L. and Myers, J.C.: Synthesis of extensive, possibly complete DNA copies of poliovirus RNA in high yields at high specific activities. Proc. Natl. Acad. Sci. USA 73 (1976) 2191-2195. Kurtz, D.T. and Feigelson, P.: The multihormonal induction of hepatic C~2u globulin mRNA as measured by hybridiza-
152 tion to complementary DNA. Proc. Natl. Acad. Sci. USA 74 (1977) 4791 4795. Kurtz, D.T., Chart, K.-M. and Feigelson, P.: Glucocorticoid induction of hepatic O~2uglobulin synthesis and messenger RNA level in castrated male rats in vivo. J. Biol. Chem. 253 (1978a) 7886-7890. Kurtz, D.T., Chan, K.-M. and Feigelson, P.: Translational control of hepatic C~2u globulin synthesis by growth hormone. Cell 15 (1978b) 743-750 Kurtz, D.T., Sippel, A.E. and Feigelson, P.: Effect of thyroid hormone on the level of the hepatic mRNA for C~2uglobulin. Biochemistry 15 (1976) 1031 1036. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-695. Lewis, J., Krutz, D.T. and Melera, P.: Molecular cloning of Chinese hamster dihydrofolate reductase specific CDNA. Nucl. Acids Res. (1981) in press. McMaster, G.K. and Carmichael, G.C.: Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74 (1977) 4835-4838. Ordahl, C.P., Kioussis, P., Tilghman, S., Ovitt, C.E. and Fornwald, J.: Molecular cloning of developmentally regulated, low abundance mRNA sequences from embryonic nmscle. Proc. Natl. Acad. Sci. USA 77 (1980) 4519-4523. Rave, N., Crkvenjakov, R. and Boedtker, H.: Identification of procollagen mRNAs transferred to diazobenzyloxymethyl
paper from formaldehyde agarose gels. Nucl. Acids Res. 6 (1979) 3559-3568. Seeburg, P.H., Shine, J., Martial, J.A., Baxter, J.D. and Goodman, H.C.: Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone. Nature 270 (1977) 486-494. Sippel, A.E., Kurtz, D.T., Morris, H.P. and Feigelson, P.: Comparison of in vivo translation rates and messenger RNA levels of a2u globulin in rat liver and Morris bepatoma 5123D. Cancer Res. 36 (1976) 3588- 3593. Villa-Komaroff, L., Efstratiadis, A., Broome, S., Lomedico, P., Tizard, R., Naber, S.P., Chick, W.L. and Gilbert, W.: A bacterial clone synthesizing proinsulin. Proc. Natl. Acad. Sci. USA 75 (1978) 3727-3731. Weinstock, R., Sweet, R., Weiss, M., Cedar, tI. and Axet, R.: Intragenic DNA spacers interrupt the ovalbumin gone. Proc. Natl. Acad. Sci. USA75 (1978) 1299 1303. Yap, S.H., Strait, R.K. and Shafritz, D.A.: Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization. Proc. Natl. Acad. Sci. USA 74 {1977) 5397-5401. Zain, S., Sambrook, J., Roberts, R.J., Keller, W., Fried, M. and Dunn, A.R.: Nucleotide sequence analysis of the leader segnrents in a cloned copy of adenovirus 2 fiber mRNA. Cell 16 (1979) 851 861.
Communicated by A.I. Bukhari.
145
Cloning of ~2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs (Recombinant DNA; rat liver; hormonal control)
David T. Kurtz and Christopher F. Nicodemus ('old Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 11724 (U.S.A.)
(Received October 14th, 1980) (Accepted December 4th, 1980)
SUMMARY An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200-500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA for a2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of C~2uglobulin mRNA in male and female rats.
INTRODUCTION The advent of molecular cloning techniques has resulted in a much deeper understanding of the mechanisms regulating gene expression in eukaryotes. Methods for cloning specific cDNAs have depended, to a large extent, on the fact that the cDNA to be cloned was prepared from an mRNA population in which the specific messenger RNA under study represented a high percentage of the total mRNA. Cloning of cDNAs has been effected by using either homopolymeric tailing (Villa-Kamaroff et al., 1978) or by ligating a synthetic oligonucleotide linker Abbreviations: bp, base pairs; cDNA, DNA complementary to mRNA; DBM, diazobenzyloxymethyl; ds, double stranded; kb, kilobase pair; RI, EcoRI; SDS, sodium dodecyl sult'ate.
onto the cDNA and inserting the resultant molecule into a single restriction site in pBR322 (Fiddes and Goodman, 1979). The efficiencies that have been reported, using either method, are in the range of 1 - 4 0 recombinants per ng of ds cDNA (Fiddes and Goodman, 1979; Ordahl et al., 1980). For the study of low-abundance mRNAs, a high cloning efficiency would be desirable. We have developed a method for cloning cDNAs that results in approx. 200-500 recombinants per ng of ds cDNA. ds cDNA is ligated with equal amounts of EcoRI and SalI linkers. On average, 50% of the molecules in the cDNA preparation will have one EeoRI end and one SalI end. Plasmid BR322 is digested with EcoRI and SalI, and the 3.7 kb fragment, containing the ampicillin-resistance gene, is purified free of the smaller fragment. A large excess of the RI-Sal-cleaved pBR322 fragment is ligated to
0378-1119/81/0000 0000/$02.50 ©Elsevier/North-Holland Biomedical Press
146 the cDNA preparation. The RI-Sal-ended pBR fragment recycles with extremely low efficiency. Thus, a large excess can be used, and the level of background colonies never exceeds 5% of the total number of colonies. Using this technique, we have cloned a cDNA for the male rat liver protein, c~2uglobulin. The synthesis of this protein, which represents 1% of hepatic protein synthesis (Sippel et al., 1976), is under complex hormonal control in vivo, involving the participation of sex steroids, glucocorticoids, thyroid hormone, and growth hormone (Kurtz and Feigelson, 1977; Kurtz et al., 1976; 1978b).
MATERIALS AND METHODS
(a) Preparation of double-stranded cDNA Messenger RNA was extracted from rat liver as previously described (Kurtz et al., 1976a). 32P-labeled cDNA (5 X l0 s cpm/~g) was synthesized using the conditions outlined by Kacian and Myers (1976), except that pyrophosphate was omitted. After 1 h incubation at 37°C, the reaction was brought to 10 mM EDTA, 0.1% SDS, and 0.3 M NaOH, left at 37°C for a further 4 - 6 h, neutralized, and then extracted with phenol/chloroform. The cDNA was separated from unincorporated nucleotides using Sephadex G-50 chromatography. The cDNA was precipitated with no carrier, and resuspended in 50 mM potassium phosphate, pH 7.4, 5 mM MgCl2, 1 mM mercaptoethanol, containing 50 /~M of each of the four dNTPs. The reaction volume was 120 /.cl/#g of cDNA. Klenow fragment of F.. coli polymerase I (Boehringer) was added at a concentration of 10 units/~g of cDNA, and the mixture was incubated at 37°C for 30 min. (This short incubation time, and the use of the Klenow fragment, seemed to prevent the homopolymet formation that can occur when polymerase I is used to synthesize the second strand.) The reaction was phenol/chloroform-extracted, and passed over Sephadex G-50. Typically, 85-95% of the first strand was rendered Sl-resistant using this procedure. The ds cDNA was rendered blunt-ended using S1 endonuclease (Sigma), 100 units//~g of ds cDNA. The cDNA was phenol/chloroform-extracted and ethanol-precipitated.
(b) Cloning by the double-linker method 1 /ag of ds cDNA was resuspended in 50/A 50 mM Tris, pH 7.5,8 mM MgC12, 10 mM ~-mercaptoethanol. 1 mM ATP, and 0.2 mM of each of the 4 dNTPs. One unit of Klenow fragment was added, and the mixture was incubated for 15 min at 12°C, to convert "ragged" ends to blunt ends. The reaction was stopped by incubating at 65°C for 10 15 min. l # g each of IScoRI and SalI oligonucleotide linkers (Collaborative Research), which had been treated with polynucleotide kinase (Seeburg et al., 1977), were added. The reaction was made 1 mM ATP, 1 mM spermidine, and 10 units T4 DNA ligase (BRL) and 5 units T4 RNA ligase (New England Biolabs) were added. The reaction was allowed to proceed for 2 4 h at 4°C. The reaction was stopped by incubation at 65°C for 15 min, and brought to 0.I M NaC1.50 units of F.coRI and 50 units SalI were added and the mixture was incubated for 8-12h at 37°C. The reaction was extracted with phenol/chloroform and passed over Sephadex G-50. Plasmid pBR322 was cleaved simultaneously with EcoRI, BamHI and Sail, using conditions described by the supplier (BRL) for the BamHI enzyme. The reaction was extracted with phenol/chloroform, and the 3.7 kb fragment, containing the ampicillin-resistance gene, was separated from the smaller fragments using a 10-40% sucrose gradient in 10 mM Tris pH 8.0, 10 mM EDTA, 1.0 M NaCl, in an SW41 rotor, 40000 rev./min for 40 h. Typically, 100 #g of DNA were loaded onto one gradient. The gradients were collected from tile bottom, in 0.5 ml fractions, and 10 gl of each fraction was run on a 0.75% agarose gel to detect the presence of the 3.7 kb fragment. Fractions containing the fragment were pooled, diluted 4-fold to reduce the sucrose concentration, and ethanol-precipitated. To ensure that the 3.7 kb RI-Sal fragment was not contaminated with uncut or singly cut pBR322, 1 #g was incubated with 5 units T4 DNA ligase, using the conditions described by the supplier (BRL). The DNA was ethanol-precipitated and used to transform E. coli strain MM294, as described below. Only 500 bacterial colonies resulted, a value 10 -4 that found with uncut or singly cut pBR322. Colonies that did grow in ampicillin were found to contain a plasmid with an EcoRI-Sall fusion (data not shown).
147 (c) Bacterial transformation 200 ng of ds cDNA containing the linkers was mixed with 5 ~g of the RI.Sal fragment of pBR322 in a volume of 3 ml of 66 mM Tris • HC1, pH 7.5, 6.6 mM MgC12, 10 mM dithiothreitol, 0.4 mM ATP. 10 units of T4 DNA ligase (BRL) was added, and the reaction was incubated for 2 4 4 8 h at 4°C. 2 ml of 5 M ammonium acetate was then added, followed by 10 ml of ethanol. The DNA was collected by centrifugation and resuspended in I00 gl sterile water. 25 gl aliquots were used directly to transform E. coli strain MM294, using the transformation technique described by Dagert and Ehrlich (1979). The resultant colonies were screened using the high-density screening protocol of Hanahan and Meselson (1980). (d) Preparation of specific cDNA probes a2u globulin cDNA, labeled with 32p at a specific activity of 5 × 107 cpm/#g, was prepared as previously described (Kurtz and Feigelson, 1977). Rat albumin cDNA was prepared essentially as described by Yap et al. (1977). These cDNAs were used to screen the rat-liver cDNA library. (e) Identification of specific clones by hybridizationtranslation Chimeric plasmids suspected to contain the C~2u globulin or albumin cDNAs were linearized with EcoRI, phenol/chloroform-extracted, and ethanolprecipitated. 20 #g of plasmid DNA, in 20 /j1 1 M Na.acetate, pH 4.5, was spotted onto a 0.5 X0.5 cm square of diazo-benzyloxymethyl cellulose ("Northern paper"), which had been prepared as described by Alwine et al. (1977). The DNA was spotted onto the paper in 4/al aliquots, and the DBM paper was left at room temperature for 4 - 5 h. The square was then incubated in 50% formamide, 5 × SSC, 10 mM EDTA 1 ×Denhardt's (1966)solution, 0.1% SDS, 0.5 ~g/ml calf thymus DNA at 37°C for 10-12 h. The paper was then incubated with 100 /ag of male rat liver mRNA in the above solution minus the DNA and Denhardt's solution. The hybridization was performed in a 4 ml polypropylene tube, using just enough solution to cover the square of DBM paper (about 300 /.d). Hybridization was allowed to proceed for 10-12 h. The square was then
washed 10 12 times with 2 ml 50% formamide, 5 X SSC, 10 mM EDTA, 0.1% SDS. Washing was done by vortexing, and centrifuging the square to the bottom of the tube following each wash. The paper was then washed 2 - 3 times with 10 mM EDTA 0.1% SDS. Hybridized RNA was eluted by boiling the square for exactly 60 s, in 500 /.tl sterile water. The tube was placed into ice immediately, and then centrifuged. The water was removed, made 0.3 M in LiC1 and the RNA was precipitated with 2 vols. ethanol. The RNA was recovered by centrifugation in an SW60.1 rotor, 50 000 rev./min for 30 min. The tube was inverted, allowed to dry, and the components of the rabbit reticulocyte translational system, containing [3SS]methionine (New England Nuclear), were added directly to the tube (25 /al final volume). Translation was allowed to proceed for 60 rain and the entire translation reaction was brought to 2% SDS, 5% /3-mercaptoethanol, 10% glycerol, and 0.0625 M Tris, pH 6.8, boiled, and subjected to SDS gel electrophoresis as described by Laemmli (1970). The gels were dried and subjected to autoradiography. (f) Other methods Gel electrophoresis of RNA under denaturing conditions and transfer to DBM paper ("Northern blotting") was performed as described by Rave et al. (1979). Labeling of DNA by nick-translation and Southern blot analysis was done as described by Weinstock et al. (1978).
RESULTS The double-linker method of cDNA cloning described above is extremely efficient: approx. 2 0 0 400 recombinant colonies per ng of cDNA can be routinely obtained. A large excess of the EcoRI-SalI fragment of pBR322 can be used to drive a high percentage of the cDNA into recombinant plasmids, and the level of background colonies (i.e. bacteria harboring a plasmid containing an RI-Sal fusion) never exceeds 5% of the total number of colonies. In one experiment, 200 ng of rat liver ds cDNA was cloned using this technique and resulted in approx. 50 000 bacterial colonies. These colonies were subjected to the high-density screening protocol of Hanahan and
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Fig. 1. Northern blot analysis using pal76. 1 ;zg of male rat liver (lane 1) or female rat liver (lane 2) mRNA was denatured and subjected to gel electrophoresis and Northern blot analysis, as described in the text. Plasmid pc~176 was nicktranslated with [a2P]dNTPs and used as probe. Lane 3: SV40 DNA cleaved with ttindIIl and 5'-end-labeled with polynucleotide kinase. The fragment sizes are 1768, 1169, 1101, 526, 447, and 215 bp long. In these gels, DNA standards may be used to estimate RNA length (McMaster and Carmichael, 1977).
Meselson (1980), using either 32P-labeled a2u globulin or albumin cDNA as probes. A large number of positive colonies were detected. These were grown in 1 ml cultures, and plasmid DNA was extracted and subjected to gel electrophoresis as described by Birnboim and Doly (1979). The largest recombinant plasmids were then grown up in mass culture for further study. One putative C~2u globulin plasmid, pcd76, containing an insert of 750 bp, was nick-translated and used as a probe in Northern blotting vs. male and female rat liver mRNA; as shown in Fig. 1, a single
mRNA band, approx. 1 kb in length, is visible in the male liver mRNA track, while no detectable hybridization occurs with female mRNA. To positively identify pc~176 as a2u globulin, the plasmid was linearized, bound to DBM paper, and hybridized to male rat liver mRNA as described in MATERIALS AND METHODS. The RNA that hybridized to pcd 76 was translated in a rabbit reticulocyte cell-free system containing [aSS]methionine. A single labeled polypeptide, which migrates with authentic a2u globulin, was detected (Fig. 2). (The largest albumin plasmid, pRSA35, was used in similar analysis to positively identify it as being rat albumin; Fig. 2b.) The level of hepatic C~2uglobulin mRNA in vivo is under extremely complex hormonal control: no translatable C~2u mRNA is normally present in adult female rats, but it can be induced in ovariectomized females by either androgens or glucocorticoids (Kurtz et al., 1978a). Also, a2u globulin mRNA synthesis can be depressed in adult male rats by chronic estrogen administration (Kurtz and Feigelson, 1977). Hepatic mRNA was isolated from rats following various hormonal treatments, and subjected to Northern blot analysis vs. pcd76 (Fig. 3). No bands are detectable in female or ovariectomized female mRNA, while mRNA from ovariectomized females treated with dexamethasone or androgen contain an mRNA species identical in size to that found in male liver. Hepatic mRNA from a male rat treated with estrogen contains no detectable C~2uglobulin mRNA (Fig. 3). pc~176 was mapped with various restriction enzymes (Fig. 4). An Aval site was found approx. 400 bp from the EcoRl site. pc~176 was cleaved with AvaI, which cuts in the insert as described, and also cleaves pBR322 at a site approx. 800 bp downstream from the SalI site. The DNA was then ligated at extremely low concentration (0.5 /.tg/ml) to allow only cyclization to take place. The resultant RI-AvaI plasmid was designated p~A pal (Fig. 4). The 5'-+ 3' orientation of pcd76 was determined by cleaving simultaneously with EcoRI, AvaI and Sail. The resultant fragments were subjected to Southern blot analysis, using either male rat liver [a=P]cDNA, or [32p]cDNA made with chain-terminating dideoxynucleotides present at 1/10 the concentration of the deoxynucleoside triphosphates. The resultant 3'-specific cDNA was found to hybridize only to the 350 bp AvaI-SalI fragment, while both the RI-AvaI and the RI-SalI fragments hybridized to
149
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Vig. 2. Identification of pal76 as a2u globulin. A rabbit-reticulocyte translational system containing [3 s S] methionine was used to translate mRNA. The products were subjected to SDS-gel electrophoresis (Laemmli, 1976). The bands represent [35S]protein autoradiograms. (A) Lane 1-translation products from total feinale rat liver mRNA; lane 2-immunopreeipitate t'rom lane 1, using anti~2u globulin;lane 3 total male rat liver mRNA translation product;lane 4-immunoprecipitate from lane 3, using antie~2u globulin; lane 5 no added mRNA (bands represent endogenous translation products of the reticulocyte lysate); lane 6-translation product of mRNA which hybridizes to pal76. (B) Lane 1-total male liver mRNA translate: lane 2-immunoprecipitate from lane 1 using anti-rat albumin (provided by Dr. L. Chasin); lane 3-no added mRNA; lane 4 translation product of mRNA which hybridizes to pRSA35.
full-length cDNA (data not shown). The AvaI-SalI fragment thus represents the 3'-end o f the cDNA. p a l 7 6 (approx. 750 bp) does not represent a full length a2u cDNA, which is approx. 1000 bp; ifEcoRI or Sall sites were present in aZu cDNA, a full-length clone would not be obtainable using this cloning method. The presence Of an EcoRI site was suspected, because four other aZu globulin clones were isolated, all approx. 7 0 0 - 7 5 0 bp in length, which were found to have AvaI, HinclI and PvuII sites at exactly the same distance from the EcoRI site as found in p a l 7 6 . Further, several very small clones ( 2 0 0 - 2 5 0 bp in length), had been isolated in the initial screening. One clone, p a l 1, of approx. 240 bp
was found to give the same pattern of hybridization to the mRNAs, as shown in Fig. 3, but did not hybridize to p a l 7 6 . Full-length male-liver cDNA hybridized weakly to p a l l , and the 3'-specific cDNA did not hybridize at all. It was concluded that there is indeed an EcoRI site in azu cDNA, and that p a l 1 represents the 5'-end of the cDNA. The total length of p a l 7 6 and peril thus represents >95% of full length a2u globulin mRNA. The plasmids p a l l , p a A V A I , and p a l 7 6 represent, respectively, 5'-, middle-, and 3'-specific probes. It was reported by Hastie et al. (1979) that the m R N A for rat azu globulin cross-hybridizes with the cDNA for the corresponding male mouse liver pro-
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Fig. 3. Hormonal control of the level of hepatic C~2u globulin mRNA. 5 ~tg of hepatic mRNA obtained from rats in various endocrine states was subjected to Northern blot analysis using pal76 as probe. Lane 1-male rat liver; lane 2 female; lane 3-ovariectomized female; lane 4-ovariectomized female treated with dexamethasone; lane 5-ovariectomized female treated with dihydrotestosterone; lane 6 male treated with estradiol 17/3. Fig. 5. Cross-hybridization of rat C~2u globulin cDNA and mouse major urinary polypeptide (MUP) mRNA. 5 #g of mRNA was subjected to Northern blot analysis using pod76 as probe. Lane 1-male rat liver mRNA; lane 2-female rat liver mRNA: lane 3-male mouse liver mRNA; lane 4-
female mouse liver mRNA. The higher molecular weight bands visible in lane 1 represent a2u globulin mRNA precursors (D. Kurtz, manuscript in preparation). The only crosshybridization between pal76 (rat a2u globulin eDNA) and MUP mRNA is seen in lane 3.
O~2U Globulin cDNA HincTT
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po(176 ~ Fig. 4. Restriction enzyme map of C~2u globulin cDNA plasmids. The cDNA is not cut by BamHI, PstI, Xhol, Xbal and HhaI. Arrows represent the lengths and locations along the eDNA of plasmids pal 1, pal76, and paAval.
151 tein, designated MUP. Plasmid pc~176 was used as a probe in Northern blot analysis of male and female mouse liver mRNA (Fig. 5). A weakly hybridizing band can be seen in male mouse liver mRNA, while no detectable hybridization occurs with female mouse liver message.
reported for cloning RNA-DNA hybrids using homopolymeric tailing (Zain et al., 1979). This double-linker method of cDNA cloning should prove to be of value for studies of low abundance messenger RNAs which may be developmentally regulated (Ordahl et al., 1980). The availability of the C~2u globulin cDNA clone will make it possible to explore the molecular mechanisms underlying the hormonal control of the synthesis of this protein.
DISCUSSION The cloning procedure described above has proven to be extremely efficient in constructing several cDNA "libraries". We have used it to clone a cDNA for hamster dihydrofolate reductase (Lewis et al., 1981), and to clone a cDNA for herpes thymidine kinase from an m R N A population in which this message represented approx. 0.1% o f the total m R N A population. The use of two linkers increases the likelihood of cleaving the cDNA. (As described above, the ~2u globulin cDNA was found to contain an EcoRI site.) We have explored the possibility of protecting EcoRI sites in the cDNA, using EcoRI methylase. Methylation of the ds cDNA was found to protect it from EcoRI digestion, and did not seem to alter the size distribution, but, surprisingly, methylation was found to lower transformation efficiency almost 10-fold. It is possible that the R1 methylase used in these studies (New England Biolabs) was contaminated with exonuclease or phosphatase activity, thus lowering the efficiency of linker addition. However, methylation of plasmid sequences has been found to lower the efficiency of transformation in other systems (D. Hanahan, personal communication). We have also a t t e m p t e d to clone RNA-DNA hybrids using this technique. First-strand cDNA was prepared with the inclusion of pyrophosphate in the reaction t o inhibit the RNase H activity of the reverse transcriptase (Kacian and Myers, 1976). The reaction was phenol/chloroform-extracted, passed over Sephadex G-50, and treated with S1 endonuclease. The hybrids were then treated exactly as ds cDNA in the cloning procedure described above. Bacterial colonies containing recombinant plasmids were obtained, but the inserts were found to be very small ( 2 0 0 - 3 0 0 bp), and the overall efficiency was very low (approx. 3 recombinant colonies/ng of RNA-cDNA hybrid). This is similar to the transformation efficiency
ACKNOWLEDGEMENTS We are indebted to Dr. J.W. Beard, Life SciencesResearch Laboratories, for supplying AMV reverse transcriptase as part of the NIH-NCI program. This work was supported in part by a grant from the National Institutes of Health, AM 26968-01.
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152 tion to complementary DNA. Proc. Natl. Acad. Sci. USA 74 (1977) 4791 4795. Kurtz, D.T., Chart, K.-M. and Feigelson, P.: Glucocorticoid induction of hepatic O~2uglobulin synthesis and messenger RNA level in castrated male rats in vivo. J. Biol. Chem. 253 (1978a) 7886-7890. Kurtz, D.T., Chan, K.-M. and Feigelson, P.: Translational control of hepatic C~2u globulin synthesis by growth hormone. Cell 15 (1978b) 743-750 Kurtz, D.T., Sippel, A.E. and Feigelson, P.: Effect of thyroid hormone on the level of the hepatic mRNA for C~2uglobulin. Biochemistry 15 (1976) 1031 1036. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-695. Lewis, J., Krutz, D.T. and Melera, P.: Molecular cloning of Chinese hamster dihydrofolate reductase specific CDNA. Nucl. Acids Res. (1981) in press. McMaster, G.K. and Carmichael, G.C.: Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74 (1977) 4835-4838. Ordahl, C.P., Kioussis, P., Tilghman, S., Ovitt, C.E. and Fornwald, J.: Molecular cloning of developmentally regulated, low abundance mRNA sequences from embryonic nmscle. Proc. Natl. Acad. Sci. USA 77 (1980) 4519-4523. Rave, N., Crkvenjakov, R. and Boedtker, H.: Identification of procollagen mRNAs transferred to diazobenzyloxymethyl
paper from formaldehyde agarose gels. Nucl. Acids Res. 6 (1979) 3559-3568. Seeburg, P.H., Shine, J., Martial, J.A., Baxter, J.D. and Goodman, H.C.: Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone. Nature 270 (1977) 486-494. Sippel, A.E., Kurtz, D.T., Morris, H.P. and Feigelson, P.: Comparison of in vivo translation rates and messenger RNA levels of a2u globulin in rat liver and Morris bepatoma 5123D. Cancer Res. 36 (1976) 3588- 3593. Villa-Komaroff, L., Efstratiadis, A., Broome, S., Lomedico, P., Tizard, R., Naber, S.P., Chick, W.L. and Gilbert, W.: A bacterial clone synthesizing proinsulin. Proc. Natl. Acad. Sci. USA 75 (1978) 3727-3731. Weinstock, R., Sweet, R., Weiss, M., Cedar, tI. and Axet, R.: Intragenic DNA spacers interrupt the ovalbumin gone. Proc. Natl. Acad. Sci. USA75 (1978) 1299 1303. Yap, S.H., Strait, R.K. and Shafritz, D.A.: Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization. Proc. Natl. Acad. Sci. USA 74 {1977) 5397-5401. Zain, S., Sambrook, J., Roberts, R.J., Keller, W., Fried, M. and Dunn, A.R.: Nucleotide sequence analysis of the leader segnrents in a cloned copy of adenovirus 2 fiber mRNA. Cell 16 (1979) 851 861.
Communicated by A.I. Bukhari.