Nucleic Acids Research September 1978 5lubr9Spebr178NcecAisRsac

Volume 5 Number 9 Vlum

Cloni,ng of chicken lysozyme structural gene sequences synthesized in vitro Albrecht E.Sippel, Hartmut Land, Wemer Lindenmaier, M.Chi Nguyen-Huu, Tilmann Wurtz, Kenneth N.TimmTis, Klaus Giesecke and GUnther Schuitz l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 63-73, D-1000 Berlin 33, GFR Received 14 July 1978 ABSTRACT Double-stranded chicken lysozyme cDNA was synthesized from an oviductwmRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.

INTRODUCTION The application of genetic engineering methods for studies of gene expression has recently brought new insights into the molecular mechanisms of the early events of mRNA synthesis in higher cells. A comparison of the sequence organisation of the mouse B-globin gene with that of its primary RNA transcript and of the final mRNA product has revealed a new step in gene expression (1). To study the regulatory principles of specific gene activity, it is, however, necessary to use model systems in which specific mRNA synthesis can be readily controlled. The chicken oviduct is an attractive system of this kind. In the magnum part of the oviduct steroid hormones coordinatedly regulate the synthesis of the major egg white proteins ovalbumin, conalbumin, ovomucoid and lysozyme (2,3,4). The steroid-controlled rate of egg white protein synthesis is closely correlated to the cellular concentration of the respective mRNA sequences (5-9). Using isolated oviduct nuclei for the analysis of specific RNA synthesis and methods to C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

3275

Nucleic Acids Research distinguish newly synthesized RNA from preexisting molecules, we have shown that the increased concentration of cellular mRNAs for these proteins following estrogen induction is primarily due to a transcriptional activation of the respective genes (10-12). Since we expect that the basis of the coordinated control of synthesis of the major egg white proteins is partly reflected in the organisation of the structural genes and their neighbouring sequences in the genome DNA, we are currently interested in the isolation of the egg white protein genes. Bacterial clones containing the ovalbumin structural gene (13,14) have been of great use in studies of the organisation of the ovalbumin gene in cellular DNA (15-17) and for its isolation (18). We describe here the cloning, identification, characterization ana partial sequencing of a synthetic DNA copy of chicken lysozyme mRNA, the least abundant of the four egg white protein mRNAs. MATERIALS AND METHODS 1. Enzymes Avian myeloblastosis virus reverse transcriptase was provided by Dr. J.W. Beard through the Office of Program Resources and Logistics, National Cancer Institute of the U.S.A.; Endonuclease S1 from Aspergillus oryzae was Type III enzyme from Sigma, St.Louis; DNA polymerase I was a gift from Dr. K. Geider, Heidelberg; T4 DNA ligase was obtained from Bethesda Research Laboratories, Rockville; alkaline phosphatase grade 1 from calf intestine was from Boehringer, Tutzing; T4 DNA polymerase and T4 polynucleotide kinase were from P-L Biochemicals, Milwaukee. Restriction endonucleases BamHI, EcoRI, HindIII, HpaII and PstI were obtained from I Boehringer, Tutzing; AluI, HaeIII, HhaI, HincII, HinfI and were from New England BioLabs, Beverly; TagI was a gift from Dr. H. Mayer, Stockheim. DNA digestions with restriction endonucleases were performed in 10 mM Tris-HCl, pH 7.5, 6mM MgCl2, 10 mM NaCl, 6mM dithiothreitol except that for EcoRI the NaCl concentration was increased to 0.1 M. 2. Isolation of mRNA Lysozyme and ovomucoid mRNA, used for preparation of cDNA hybridization probes were isolated in high purity by immunoadsorption of specific polysomes to matrix-bound antibodies and electropho3276

Nucleic Acids Research retic size fractionation as described earlier (19). The 8S-12S fraction enriched for lysozyme and ovomucoid mRNA was prepared gel electrophoretic size fractionation (19) of total polysomal poly(A)-containing RNA from laying hen oviducts (9).

by

Synthesis of ds-cDNA and addition of chemically-synthesized DNA-linker molecules cDNA was synthesized in the presence of actinomycin D and 0.4 mM t32P]dCTP (0.1 Ci/mMol) from the RNA fraction enriched for lysozyme mRNA and ovomucoid mRNA. The cDNA was isolated as previously described (9), except that no carrier DNA was added and that the cDNA was recovered by lyophilization after Sephadex G-50 chromatography in 10 mM NH4HC03. The yield of full length cDNA was between 10 and 30% of the original mRNA template. 0.5 ig of single-stranded cDNA was incubated in a 100 pl reaction mixture containing 50 mM Tris-HCl pH 8.2, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM each of dATP, dGTP, dCTP, and dTTP and 18 units reverse transcriptase. After incubation for 2 h at 460C, the reaction mixture was extracted with phenol containing 20% 1 M Tris-base, chromatographed on Sephadex G-50 in 10 mM NH 4HCO3 and lyophilized to dryness. The resulting DNA product was digested with 8 units endonuclease S1 in 150 pl 0.3M NaCl, 3 mM ZnSO4 and 0.03 M sodium acetate, pH 4.5, for 15 min at 370C. After addition of 20 pg E.coli tRNA, the reaction mixture was phenol-extracted and the ds-cDNA precipitated with 2.5 volumes of ethanol. Approximately 0.5 pg of S1 resistant ds-cDNA was recovered. In order to increase the number of blunt ends, the ds-cDNA was incubated for 10 min at 12.50C with 1 unit of E.coli DNA polymerase I (20) or 1 unit of T4 DNA polymerase (21) in 20 pl containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol and 0.25 mM each of dATP, dGTP, dCTP and dTTP. 200 pmoles of the chemically synthesized duplex DNA decamer containing a BamHI restriction endonuclease cleavage site (Collaborative Research, Waltham) was phosphorylated at its 5'-ends with 3 units (22) of T4 polynucleotide kinase for 3.5 h at 25°C in 10 pl of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP or [y-32 P]-ATP (10 Ci/mMol) and was added to the ds-cDNA solution. The reaction volume was increased to 40 pl, adjusted to 50 mM Tris-HCl, pH 7.5, 10 mM MgC12, 10 mM dithiothreitol, 1 mM ATP and 3.

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Nucleic Acids Research 3 units (23) of T4 ligase were added. After incubation for 8 h at 12.50C, the enzymes were inactivated by heat-treatment at 800C for 5 min. The reaction volume was brought to 200 pl containing a final concentration of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM NaCl, 5 mM dithiothreitol and 10 units of restriction endonuclease BamHI. After digestion for 2 h at 37°C, 20 pg of E.coli tRNA was added, the reaction mixture was phenol-extracted and chromatographed in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl and 1 mM EDTA at room temperature on a 10 ml BioGel A-5m, mesh 200-400 (BioRad) column made up in a 10 ml plastic pipette. The excluded material was collected by ethanol precipitation and dissolved in 20 pl H20. The final yield was 0.1 -0.2 ig of linker-ligated ds-cDNA. 4. Construction of recombinant DNA plasmid Recombinant DNA consisting of plasmid pBR322 (24) and ds-cDNA derived from a chicken oviduct mRNA fraction enriched for lysozyme and ovomucoid mRNA was constructed by incorporation of the in vitro synthesized eukaryotic DNA into the BamHI site of the plasmid. 5 ag pBR322 was linearized by digestion with 10 units BamHI in 100 pl. To remove Mg2+ ions, the reaction mixture was poured through 0.1 ml of recycled and H20-suspended Chelex 100 (200-400 mesh, BioRad) ion exchange resin in a siliconized Pasteur pipette. 5'-terminal phosphate groups were removed by incubation for 30 min at 37°C in 500 pl containing 1 mM EDTA and 2 units alkaline phosphatase. 10 pg of E.coli tRNA was added to the reaction mixture before heat-inactivation of the phosphatase for 2.5 min at 65°C and phenol extraction. Residual phenol was carefully removed by five successive ether extractions and the plasmid DNA was collected by alcohol precipitation. 0.1 vg linker-terminated ds-cDNA was then ligated to 2 .g of phosphatase-treated linear plasmid DNA in 40 pl containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP and 2 units T4 DNA ligase by incubation for 1 h at 200C. Recombinant DNA was recovered by alcohol precipitation. 5.

Transformation of X1776 and selection for transformants

E.coli K12 X1776 was transformed with recombinant plasmid DNA using a procedure similar to that of Manske, Wall and Salser (personal communication). 100 ng ds-cDNA ligated to 2 pg phospha-

3278

Nucleic Acids Research tase-treated pBR322 DNA in 100 pl of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA was added to 5 cells of E.coli strain X1776 made competent in 200 p1 10mM Tris-HCl, pH 7.0, 140 mM NaCl and 75 mM CaCl2. The mixture was kept at 00C for 15 min, heat-shocked at 250C for 4 min and then returned to 00C for 30 min. 150 p1 aliquots were plated on 1% agar L-broth plates supplemented with 50 pg/ml thymidine, 100 pg/ml diaminopimelic acid and 20 pg/ml ampicillin and incubated at 370C for 40 h. 20 transformants were obtained and were replated on ampicillin and tetracycline (10 pg/ml) containing plates. 18 colonies proved to be tetracycline-sensitive. When 0.33 pg supercoiled pBR322 DNA was used in a parallel transformation experiment, we obtained 4.5 x 104 transformants (1.35 xlO5 per pg). The viability of X1776 was reduced approximately 5-fold by the transformation procedure.

x108

6.

Colony screening

gor

recombinant DNA plasmids

Colonies were transferred from agar plates onto nitrocellulose filters by contact and 'grown overnight at 370C by layering the filters colony-side up on top of an agar plate. After growth the filters were placed on 3 MM paper and soaked with 0.5 M NaOH for 4 min, placed for 4 min twice consecutively into 1 M Tris-HCl, pH 7.4 and twice into 1 M Tris-HCl, pH 7.4, 1.5 M NaCl, dipped briefly twice into absolute ethanol, air dried and baked under vacuum for 2 h at 800C. To screen for specific recombinant DNA with highly purified lysozyme and ovomucoid [32 P]cDNA (5-107 1.108 cpm-pg 1), filters were pretreated, hybridized and washed as described for the blotting experiment. For comparison, colonies on filters were made visible by staining with 0.04% methylene blue (25) in 1% acetic acid, 2% Na-acetate. 7. Preparation of plasmid DNA E.coli HB101 carrying plasmid pBR322 was grown in L-broth containing 20 pg/ml ampicillin and E.coli X1776 carrying recombinant plasmids were grown in ampicillin containing L-broth supplemented with 50 pg/ml thymidine and 100 pg/ml diaminopimelic acid to 3 x 108 cells/ml. After the addition of chloramphenicol to 12.5 pg/ml, the cultures were incubated 5 -16 h for plasmid amplification. Bacteria were collected by centrifugation for 5 min at 8000 g. For large scale preparations plasmid DNA was isolated similar to

3279

Nucleic Acids Research the cleared-lysate method described by Sidikaro and Nomura (26). For small scale plasmid preparations the method described by Meagher et al. (27) was used.

8.

Gel electrophoresis and isolation of specific DNA fragments

Slab gel electrophoresis was carried out either in 1.5% agarose in Tris-phosphate buffer (28) or in 5% polyacrylamide gels in Tris-borate buffer (29). For the isolation of specific DNA fragments after gel electrophoresis in polyacrylamide, DNA was either detected by staining with ethidium bromide or by autoradiography. Appropriate gel bands were cut out, crushed in 3 volumes of 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and eluted for 10 h at 45 0C. The eluate was cleared from gel pieces by centrifugation through cotton wool, extracted with phenol and chloroform and the DNA was recovered by ethanol precipitation. 9. Hybridization to DNA on nitrocellulose filters Nitrocellulose filters (Schleicher and Schuell) carrying DNA transferred from gels (30) or from lysed bacterial colonies were pretreated, hybridized, washed and autoradiographed as described by Jeffreys and Flavell (31), except that 5 ng/ml specific [32p1cDNA (1 x 108 cpm /pg) was used for hybridization in sealed plastic bags.

10. DNA sequence analysis DNA sequencing of fragments derived from lysozyme DNA by digestion with restriction endonucleases was performed according to Maxam and Gilbert (32) as modified by Gray et al. (33). 11. Safety considerations Cloning experiments and growth of recombinant DNA plasmids were carried out under L3/B2 conditions as specified by "Richtlinien zum Schutz vor Gefahren durch in vitro rekombinierte Nukleinsauren" of the BMFT of the Federal Republic of Germany. The safety containments are similar to the conditions of P3 + EK2 containment required for these experiments by the NIH Guidelines for Recombinant DNA Research in the U.S.A. RESULTS 1.

Construction of the recombinant DNA and bacterial transformation

The isolation to high purity of nucleic acid 3280

probes carrying the

Nucleic Acids Research for a specific protein is prerequisite for the anaof a specific gene. Although we recently sucexpression of lysis ceeded to isolate all four of the major chicken egg white protein mRNAs to high purity (9,19), the supply of large quantities of these mRNAs and their DNA complements in high purity and of homogeneous size is still a considerable problem. We therefore have prepared plasmids, in which a double-stranded DNA copy of the ovomucoid and lysozyme mRNA has formed a recombinant with plasmid pBR322. We used poly(A)-containing polysomal mRNA fractions moderately enriched for lysozyme and ovomucoid mRNA for the construction of the recombinant DNA. Transformants carrying lysozyme or ovomucoid DNA were identified by colony hybridization with highly specific cDNAs. The general scheme used for the construction of recombinant plasmids containing the coding sequence for lysozyme is shown in Fig. 1. The procedure follows the protocol used by Ullrich et al. (34) and Seeburg et al. (35) for cloning rat insulin and rat growth hormone sequences with minor modifications as described in Materials and Methods. 8S -12S RNA obtained by size-fractionation of polysomal poly(A)-containing RNA was used as template for the reverse transcriptase. This RNA contained approximately 15% lysozyme mRNA and 60% ovomucoid mRNA as judged by the A260-profile in a gel (data not shown). cDNA was synthesized in the presence of actinomycin D and reisolated after alkaline digestion of the template RNA. Reisolation of the cDNA proved to be advantageous for the extent of the 5'-hair pin dependent second strand synthesis (36), when compared with other procedures (37). As Si nuclease is contaminated with endonuclease activity and is strongly inhibited by dNTPs (38), we used the minimal amount of Si nuclease to completely open the 5'-hair pin loop of the ds-cDNA. This was achieved by omitting carrier from the ds-cDNA preparation and by removal of the dNTPs before Si nuclease treatment. Efficient ligation of chemically synthesized DNA linkers containing cleavage sites for restriction enzymes to both ends of the double strand structural gene DNA is dependent on the presence of a high number of blunt ends. As suggested by Seeburg et al. (35), we used the gap-filling and the 3'-exonuclease activity

mRNA

sequence

3281

Nucleic Acids Research mRNA

A

A

A

A

A

(dTh2-s

RT

dNTP 0

cDNA

3'

c

RT

(G

dNTP

(2)

GSO

C

Si

TTT

Tel = SolI Salm HI I*-S

I -.-* PstPtI_|Amp'

pBR322

TT5

'

sNA 5'

T4-Ligose () Boan HI

BomH 3'

HindN

14 TTT S AA

haor pin

EcoRI

pH 14

(2) G5O

I

AA 3' Linker

Sam HI BomNI

(T)

FPhosphatose Bio Gel

|())

(2)

(h0 OD

A-5 rm

_

J.

chelex

(hocit

3,

%D

3'

3'

anneol TL-Ligose

(

Schematic diagram of the construction of recombinant plasmids containing eukaryotic mRNA sequences. Numbers indicate separate biochemical steps between the reisolation of the products byeither lyophilization or ethanol precipitation. RT, reverse transcriptase; SI, S1 nuclease; Ampr, B-lactamase gene; Tcr, tetracycline resistance gene. Fig. 1

of E.coli DNA polymerase I, or later T4 DNA polymerase, to increase the number of blunt ends in the synthesized DNA. We know from a restriction map of in vitro synthesized lysozyme DNA that BamHI does not cleave this ds-cDNA (Nguyen-Huu, M.C. et al., in preparation). We therefore used the DNA linker containing a BamHl restriction site to generate cohesive ends on the ds.-cDNA. The efficiency of synthesis of ds-cDNA containing BamHI cohesive ends was judged from the appearance of 32P]ds-cDNA in position of plasmid-sized DNA bands after ligation of the synthesized DNA to plasmid DNA and subsequent agarose gel electrophoresis of the ligation products. About 30 -50% of the ds-cDNA was ligated to

plasmid DNA (data not shown). The integration of foreign DNA sequences into the restric3282

Nucleic Acids Research a) methylene blue

bi lysozyme

c) ovomucoid

4Ebe. w ,

Fig. 2 Colony screening of E.coli X1776 transformants for lysozyme and ovomucoid recombinant plasmid DNA. Colonies identified by antibiotic selection were grown on nitrocellulose filters. After lysis the filters were hybridized to pure lysozyme and ovomucoid [32p]cDNA and radioautographed. For comparison all colonies were made visible by methylene blue staining. The colonies of the top and bottom row are E.coli X1776 carrying pBR322. tion endonuclease BamHI site of pBR322 DNA leads to inactivatAon of the tetracycline resistance gene (24), while the ampicillin resistance gene is not affected. This insertional inactivation of one antibiotic resistance gene and the suppression of recircularization of pBR322 by prior removal of 5'-terminal phosphate groups with alkaline phosphatase facilitated the screening for recombinants (34). Transformation of E.coli X1776 with 0.1 pg of ds-cDNA ligated with a 20-fold excess of phosphatase-treated pBR322 DNA yielded 20 ampicillin-resistant transformants, 18 of which were tetracycline-sensitive. These transformants were screened by colony hybridization with cDNA probes made from highly purified lysozyme mRNA and ovomucoid mRNA (Fig. 2). 4 colonies gave a positive signal with lysozyme cDNA, 9 colonies with ovomucoid cDNA, and 1 colony gave a signal with both cDNAs. The high proportion of colonies carrying lysozyme and ovomucoid recombinant DNA (14 our of 18) reflects the content of lysozyme and ovomucoid mRNA in the original RNA fraction used as a template for the in vitro synthesis of the structural gene sequences. The purity of the labeled cDNA hybridization probes used for colony screening can be judged by the fact that with one exception no cross-hybridization of ovomucoid and lysozyme colonies has been seen (Fig. 2).

2. Length determination of the inserted structural gene sequence

Plasmid DNA was isolated from all four of the colonies which had 3283

Nucleic Acids Research given a positive signal with lysozyme cDNA. The length of the linearized plasmid DNA after digestion with restriction endonuclease HindIII, when compared to pBR322, should give a rough estimate of the length of the inserted DNA sequence. One of the recombinant DNA plasmids, termed pls-1, was chosen for further characterization because it had the longest DNA insertion. Fig. 3 shows the electrophoretic gel pattern of DNA fragments produced when pls-1 and pBR322 DNA were digested with the restriction endonucleases HindIII, HincII and BamHI. When plasmids were linearized with HindIII, pls-1 was seen to be longer than pBR322. To obtain more precise size determinations, we performed digestions with HincII and BamHI. HincII cuts pBR322 twice, producing two DNA fragments of the size of 2.95 and 1.05 kb (24). When we digested pls-1 with HincII, the smaller of these fragments had increased in size to about 1.61 kb, whereas the length of the larger fragment was unaltered. Hybridization with lysozyme [ 32P]cDNA showed that the smaller HincII fragment contained lysozyme DNA sequences (Fig. 3). This is consistent with the fact that the BamHI-site used for the integration is located on the smaller of the two DNA fragments (24). Cleavage of pls-1 with BamHI gave the expected unit length pBR322 2.6 megadalton DNA fragment and an additional 0.36 megadalton fragment, the latter hybridizing to lysozyme cDNA (Fig. 3). We concluded from these results that plasmid pls-1 contains an insertion of approximately 560 base pairs in the BamHIsite of pBR322 which hybridizes to lysozyme cDNA. 3. Restriction endonuclease cleavage map of the lysozyme structural gene The lysozyme DNA fragment excised from pls-1 by cleavage with BamHI was digested with a variety of restriction endonucleases to obtain a restriction endonuclease cleavage map. Consistent with the map which we have independently determined for in vitro synthesized duplex lysozyme cDNA (Nguyen-Huu, M.C. et al., in preparation), there are no restriction endonuclease cleavage sites for HindIII, HincII, BamHI (Fig.3), EcoRI (Fig.6) or for PstI (data not shown) in the cloned lysozyme DNA fragment. A single cleavage site exists for HinfI, HaeIII, TagI (Fig.4) and KpnI and MboII (data not shown), two cleavage sites for HhaI and HpaII (Fig.4) and three for AluI and MboI (data not shown). The DNA 3284

Nucleic Acids Research

Fig. 3 Length determination of the lysozyme DNA f ragment in pls-1. Plasmids pls-1 and pBR322 were digested separately with the restriction endonucleases HindIII, HincII and BamHI and the DNA fragments were separated by electrophoresis on 1.5% agarose slab gels. After taking a photograph of the ethidium bromide stained gel, the DNA fragments were transferred onto nitrocellulose filters (30), the filte'rs were hybridized to lysozyme [32p]cDNA and radioautographed. The radioautogram is not quite in scale with the gel. Marker DNA fragments were derived from EcoRI plus HindIII-digested X DNA (44) and HaeIII-digested X dvl DNA (45) . fragments obtained were ordered with respect to the single Hinf I site by double digests as shown in Fig. 4. Hinf I cuts the lysozyme fragment asymmetrically into a 195 and a 360 base pair DNA fragment. Sequencing data (see chapter 4) unambiguously locate the HinfI site closer to the 5'-end (orientation according to the mRNA sequence). This was confirmed by Hinf I digestior of in vitro synthesized ds-cDNA (Nguyen-Huu et al., in preparation). In a double digest, HaeIII cuts the larger 3'-terminal Hinf I f ragment into a 240 and a 120 base pair fragment. Since the 120 base pair fragment also occurs after digestion with HaeIII alone, the HaeIII site must be located 240 base pairs from the HinfI site in and the SII 3K-direction (Fig. 4). Similarly the two HhaI and single Kpn site have been located by HinfI/HhI, HaeIII/HpII 3285

Nucleic Acids Research Adv Is-fragment rc

Adv ls- fragment c

kb

1.50 ; 88r;U .4 rJ 1

'5 034S

2

CT-Tcr

-

-r I

0-

1:

0

J

oI) co C

°

I II

I :1

== m

T a:

Fig. 4 DNA fragments produced by digestion of the isolated lysozyme DNA fragment of pls-1 with various restriction endonucleases. 0.2 pg of lysozyme DNA excised from pls-1 by BamHI restriction endonuclease was digested as indicated, the products were stained with ethidium bromide after slab gel electrophoresis in 5.2% polyacrylamide and the gels were photographed. The HinfI digestion and the HpaII/HaeIII double digestion were not complete. Marker fragments were derived from digestion of X dv1 DNA (45).

.

12 0.08

and HhaI/KpnI double digests (Fig. 4). Results are summarized in the restriction endonuclease cleavage map shown in Fig. 5. The map contains some additional data for which evidence is not presented in this communication. The asymmetric location of the HinfI site was also used to determine the orientation of the cloned lysozyme DNA fragment within pBR322. A map of the HinfI sites on pBR322 has not yet been published. We therefore determined the locations of the HinfI sites closest to the BamHI site in pBR322 by digestion of pBR322 with HinfI, HinfI/EcoRI and HinfI/BamHI. Fig. 6A shows that the largest of the HinfI fragments is cut by EcoRI into a 990 and a 615 base pair fragment and by BamHI into a 1360 and a 245 base pair fragment. This localizes the two neighbouring HinfI sites 990 base pairs to the left of the EcoRI site and 245 base pairs to the right of the BamHI site, using the map of pBR322 published by Bolivar et al. (24). When, for comparison, pls-1 was digested with the same combinations of restriction endonucleases (Fig. 6A), the lysozymeDNA-specific HinfI site was found to be 720 nucleotides to the 3286

Nucleic Acids Research 0

100

300

200

I a

0

Sequences GA GANTC

-J

3'

135

tI

195

1 Hin I

GAAGANNNNNNNN'

e

Mbol

GCGC GGTAC^C OCGG G GCC

HaI

1> HhoI

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EcoRI. BamHI. Hindm, PstI.

Fig. 5

435 325 370 420 445

Toq

At*T

600

500

400 I

---

5'

Hincd

555

~bese ~ ~ ~ ~pairs

ofsites

1 1 1 2 1 2 1 3 3 0

Restriction endonuclease cleavage map of the lysozyme DNA

fragment in pls-1. Data were taken from experiments shown in Figs. 3, 4 and 6, except for MboII, AluI, MboI and PstI (data not shown). The fragment sizes have been determined in a number of experiments and agree with those shown in Figs. 3, 4 and 6 within 10 base pairs. right of the EcoRI site. 370 of the 720 nucleotides belong to the unaltered distance between the EcoRI and BamHI site in pBR322 and the residual 360 nucleotides originate from the larger BamHI HinfI part of the inserted DNA. This shows that the 3'-terminal part of the lysozyme DNA is closer to the EcoRI site than the 5'terminal part (Fig. 6B). Localization of the coding sequence by sequencing parts of the inserted DNA The amino acid sequence of chicken lysozyme has been known for 15 years (39) and was recently extended by the determination of the sequence of 18 NH2-terminal amino acids preceding this sequence in the primary translation product, pre-lysozyme (40). DNA sequencing data from the cloned DNA fragment can therefore prove that it contains lysozyme structural gene sequences. It also allows the determination of the 5'-3'-polarity of the coding strand and the localization of the coding region within the mRNA. For sequence analysis we digested the lysozyme DNA fragment isolated from pls-1 with HinfI and labeled the 5'-ends with T4polynucleotide kinase. The two HinfI fragments were redigested with TaqI and HaeIII, respectively. Consistent with the map shown in Fig. 5, we obtained 4 labeled fragments, which were separated on polyacrylamide gels and subjected to DNA sequencing according to Maxam and Gilbert (33). The protocol allows the determination 4.

3287

Nucleic Acids Research Fig. 6 Orientation of the lysozyme DNA fragment in pls-1. A. DNA fragments produced by digestion of pls-1 DNA with the enzymes indicated were separated on 4.5% polyacrylamide gels and stained with ethidium bromide. B. Map of cleavage sites around the inserted lysozyme DNA fragment.

A

B

."-. , ".. -,-Yj !.

L.

.,- -L'

t

.:

t

"I

I

of sequences starting in both directions from the HinfI site as well as from both ends of the cloned DNA fragment. The sequence derived from the HinfI/HaeIII fragment (Fig. 7) is completely consistent with that predicted for the coding region from amino acid 55 to amino acid 77 of chicken lysozyme (39). The HinfI cleavage point was found between nucleotide 215 and 216, the first base of the AUG codon of prelysozyme being counted as nucleotide 1. The sequence derived from the TaqI/Hinf I fragment conformed to the sequence predicted for the sequence of amino acids from 42 to 53. The DNA sequence, which covers the coding sequence for 35 amino acids, unequivocally established the 5'- 3'-polarity from the TagI site in the direction of the HaeIII site and localized the coding region of the lysozyme mRNA in the cloned DNA fragment. The 5'- and 3'-end of this DNA have been determined by partially sequencing the 135 base pair BamHI/TaqI fragment and the 120 base

3288

Nucleic Acids Research GA C C A 'GI T FC

Autoradiogram of a 20% acrylamide - 7M urea gel used for sequence analysis of the HinfI/HaeIII fragment of cloned lysozyme DNA. The sequence is derived from the HinfI/ HaeIII fragment starting from the HinfI site. The fragment was subjected to the base-specific cleavage reactions according to Maxam and Gilbert (32,33) and the cleavage products were analyzed on a 20% acrylamide - 7M urea gel. The nucleotide sequence which can be read from the autoradiogram is consistent with that from chicken lysozyme mRNA from codon 55 to 68.

Fig. 7

A-

.

.__

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|

-

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pair HaeIII/BamHI terminal

These

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11

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amino

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contain

not

DNA

Sequence

(40).

the

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data

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consistent

pre-lysozyme

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6

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of

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of

for

region

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is

that

sequence

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ds-cDNA

and

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to

gene

DNA.

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+2

sequence of

the

lysozym'

coding

for

the

5'-terminus and

the

probably

the

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ends

at

5'-end

the

synthetic

codons

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unknown

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missing.

3'-end

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the

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3289

Nucleic Acids Research 5'-END FRAGMENT

(GG GCG GGA) CUG CCU CUG GCU GCU CUG GGG AM GT LINKER

UNKNOWN SOURCE

leu pro leu ala ala leu gly lys val -7 -6 -5 -4 -3 -2 -1 1 2

ALU I A GCU ACA MC CGU MC ACC GAU GGG AGU

HINFI SITE

gln ala thr asn arg asn thr asp gly ser

CARRYING FRAGMENT

41

45

50

MBOI

HINFI

ACC GAC UAC GGA AUC UUA CAG AUC AAC AGC CGC UGG UGG UGU MC GAU GGC AGG ACC CCA thr asp tyr gly ilu leu gln ilu asn ser arg trp trp cys asn asp gly arg thr pro

51

55

60

65

70

GGC UCU AGG MU CUG UGU M gly ser arg asn leu cys asn

71

3'-END FRAGMENT

75

CUCC POLYA (CC) n= 33 LINKER

Fig. 8 Partial chicken lysozyme mRNA sequence. The mRNA sequences were determined by DNA sequencing of parts of the isolated lysozyme DNA fragment of pls-1. Sequences read from the autoradiograms are presented in the RNA form of the coding strand of lysozyme DNA. 5'-end: BamHI TaqI fragment; middle part: TaqI -HinfI and HinfI HaeIII fragments; 3'-end: HaeIII v-BamHI fragment. Nucleotides 214 -218 were not directly sequenced but inferred from the existence of the HinfI site and the amino acid sequence, respectively. Sequences derived from the BamHI linker and those of unknown origin are bracketed.

showed that the synthetic BamHI linker DNA was coupled to a tail of 33 dA:dT base pairs (Fig. 8) which could only have been derived from the poly(A) moiety of the original mRNA. The lysozyme DNA fragment in pls-1 must therefore contain sequences corresponding to the complete 3'-non-coding region of the lysozyme mRNA.

DISCUSSION The use of synthetic DNA linker molecules containing a recognition sequence for a restriction endonuclease for the integration of ds-cDNAs into vector DNA facilitates subsequent excision of the cloned DNA fragments from the plasmid. The cloning of the entire structural gene with a DNA linker is, however, only possible, if the restriction enzyme which recognizes the linker molecule does not cut the sequence to be cloned. We knew from 3290

Nucleic Acids Research digestions of full-sized ds-cDNA, prepared from highly purified lysozyme mRNA, that BamHI does not cleave within the lysozyme riRNA sequence (Nguyen-Huu et al., in preparation). Since ovomucoid ds-cDNA contains a BamHI site, other linkers must be used for cloning of this structural gene in its entirety. The length of lysozyme mRNA was found to be approximately 640 -650 base pairs (19,41). Assuming an average poly(A) length of 30 -40 residues and taking into account that 10 base pairs of the cloned lysozyme DNA are contributed by the linker molecules, it can be estimated that approximately 80 -100 nucleotides at the 5'-end of the mRNA have not been cloned in pls-1. It had previously been observed that the 5'-terminal sequence of various eukaryotic.mRNAs were not represented in the cloned DNA, possibly because of the construction of the double-stranded gene sequences via a 5'-terminal hairpin formation and subsequent Sl-nuclease treatment (42,34,35,13). Indeed, we observed a reduction in the length of the lysozyme ds-cDNA during Sl-nuclease digestion (Nguyen-Huu et al., in preparation). Alternatively, incomplete synthesis or partial degradation of the double-stranded structural gene may be responsible for the lack of the 5'-terminal nucleotides in pls-1 DNA. The length determinations of lysozyme DNA and the partial sequence data allow length-estimations of the 3'-terminal and 5'terminal noncoding region of the lysozyme mRNA. Pre-lysozyme with 147 amino acids (including 18 amino acids for the pre-sequence) requires a coding region of 441 nucleotides. About 40 residues of the mRNA represent the poly(A) region. Thus, approximately 170 nucleotides remain for the untranslated parts of the mRNA. We located the HinfI cleavage site 360 nucleotides to the left of the 3'-end of the cloned DNA and by DNA sequencing between nucleotide 215 and 216 of the coding region. It can thus be estimated that 100 bases represent the 3'-non-coding region and approximately 70 bases the 5'-non-coding part. As shown in the restriction endonuclease cleavage map in Fig. 5, the HaeIII site is located about 240 nucleotides from the HinfI site in the direction of the 3'-end. Therefore, the HaeIII site marks the very first part of the 3'-non-coding region. It is noteworthy that this region contains a cluster of GC-rich restric3291

Nucleic Acids Research tion endonuclease recognition sequences. The mRNA sequence determined for 7 of the 18 amino acids of the leader portion of pre-lysozyme confirms the protein sequence recently determined by Palmiter et al. (40). The amino acid sequence of the pre-sequence is very characteristic since 16 out of 18 amino acid residues are hydrophobic. The nucleotide sequence of the seven codons we have sequenced is also very peculiar; it does not contain a single adenine residue (15 out of 21 bases are G and C). A similar lack of A's has been found in the sequence of the last 23 bases of the pre-region of a XII light chain (43); this sequence precedes a splicing point for a 93 nucleotide long intervening sequence (43). We have recently used the highly purified, cloned lysozyme DNA of pls-1 as a hybridization probe to map the lysozyme structural gene sequences in chicken genome DNA. We have found strong evidence for at least 3 intervening gene sequences, one 5'-terminal of the HinfI site, one between the HinfI and the HaeIII site and one 3'-terminal to the HaeIII site (manuscript in preparation). We are currently attempting to isolate the lysozyme gene sequences from cellular DNA in order to compare in detail the sequence organisation of the gene with its final mRNA product. ACKNOWLEDGEMENTS We are grateful to Dr. P.-H. Hofschneider for giving us the possibility to use the L3 facilities and for his generous support during the course of the cloning experiments. We thank Drs. N.E. Hynes, A. Efstratiadis and L. Villa-Komaroff for help in the initial phase of the project, Drs. R. Thompson and M. Meyer for help with DNA sequencing, Drs. K. Geider, J.W. Beard and H. Mayer for enzymes, S. Jeep and M. Stratmann for expert technical assistance, and I. Schallehn and E.M. Philippi for secretarial assistance. We thank Dr. J. Collins for his help to localize the lysozyme coding region in the ds-cDNA and Drs. R. Thompson and M. Achtman and S. Molgaard for critically reading the manuscript.

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