Cloning and structural analysis of the calf prochymosin gene

197

Gene, 43 (1986) 197-203 Elsevier GENE

1605

Cloning and structural analysis of the calf prochymosin gene (Recombinant DNA; chymosin; cDNA; exon-intron junction; abomasum; >WES * 1B and IZL47AB vectors; nucleotide sequence; aspartyl protease)

Makoto Hidaka *, Katsutoshi Sasaki, Takeshi Uozumi and Teruhiio Beppu Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113 (Japan) Tel. (03)812-2111, ext. 5123 (Received

July 23rd, 1985)

(Revision

received

(Accepted

February

February

5th, 1986)

19th, 1986)

SUMMARY

Calf prochymosin (cPC) gene was cloned from calf abomasum DNA by using a cloned cPC cDNA as a probe. The cPC gene spans approx. 10.5 kb and consists of 9 exons and 8 introns. The positions of exon-intron junctions coincide completely with those in the human pepsinogen gene. Analysis of 5’4lanking region sequence and S 1 nuclease mapping revealed that the transcriptional start point was located 25 bp upstream from the start codon and that the putative transcriptional promoter sequences (TATA box and CCAAT box) were located in -30 and -90 regions, respectively. Some distinctive sequences possibly functioning as regulatory signals for gene expression are present at the 5’-flanking region.

INTRODUCIION

Chymosin is the predominant milk-clotting aspartyl protease in the fourth stomach of calf. Synthesis of chymosin in the stomach mucosal tissue occurs

* To whom

correspondence

and

reprint

requests

should

be

addressed. Abbreviations:

aa, amino acid(s); bp base pair(s); cDNA,

complementary bromide;

to mRNA;

cPC,

calf

kb, 1000 bp; nt, nucleotide(s);

prochymosin;

Pipes,

0378-I 119/86/$03.50

PC;

EtdBr,

PA, polyacrylamide;

1,4-piperazinediethanesulfonic

0 1986 Elsevier

Science Publishers

DNA

ethidium PC,

acid.

B.V. (Biomedical

only during the neonatal period, which is followed by synthesis of pepsin as the predominant aspartyl protease in the gastric juice of adult cattle. The aa sequence of PC shows striking similarities to that of pepsinogen (Foltmann et al., 1979). These observations suggest the possibility that switching of gene expression from PC to pepsinogen occurs during development, as observed with expression of a-fetoprotein and albumin genes (Sala-Trepat et al., 1979). We previously reported cloning and sequence analysis of cPC cDNA (Nishimori et al., 1981; 1982a). In the present paper, we report cloning of PC gene from calf abomasum DNA and structural analysis of the gene and its 5’4lanking region. Division)

198

MATERIALS

AND METHODS

(a) Enzymes and special materials The restriction enzymes, Escherichia coli alkaline phosphatase, T4 polynucleotide kinase and Sl nuclease were purchased from Takara Shuzo Co. and New England Biolabs. T4 ligase was obtained from Boehringer Mannheim. [a-32P]dCTP (ap400 Ci/mmol) and prox. 3000 Ci/mmol and [ y-32P]ATP (7000 Ci/mmol) were purchased from Amersham and New England Nuclear, respectively. A nick-translation kit was from Amersham, and an Ml3 sequencing kit was from Takara Shuzo Co. (b) Southern blotting of calf genomic DNA High M, DNA was extracted from the mucosal tissue of calf abomasum as described by Blin and Stafford (1976) and purified by CsCl-EtdBr densitygradient centrifugation. The DNA (15 pg) was digested with 50 units of either EcoRI or Hind111 at 37 “C for 2 h, fractionated on a 0.8% agarose gel and blotted onto a nitrocellulose filter for Southern (1975) blot analysis. As the probes, cPC cDNAs carried by pCRlO1 and pCR301 (Nishimori et al., 1982b) were digested with BamHI and Hinfl, respectively, and 32P-labeled by nick-translation.

1979). The recombinant phages of QtWES * J,B were plated with E. coli LE392 and those of &47AB were plated with E. coli WL95 metB supE supF hsdR, ton4 trpR(P2), respectively. The obtained phages were screened by the procedure of Benton and Davis (1977) with the nick-translated HinfI -cleaved cDNA of pCR301.

RESULTS

AND DISCUSSION

(a) Detection of cPC gene sequence To detect PC gene in calf DNA preparation, we first examined the hybridization patterns of the total calf DNA digested with EcoRI or Hind111 by using [ 32P]cDNA as the probe (Fig. 1). The Hi&-cleaved fragment (1208 bp) obtained from the insert of pCR30 1 corresponds to the major part (94 %) of the cPC cDNA and was designated as ‘PC-probe’. The cDNA insert in pCRlO1 was separated into two large fragments and two small fragments by BamHI digestion. One of the large fragments (474 bp) coding the N-terminal portion of PC from 5th Arg to 163rd Gly was designated as ‘5’-probe’, and another fragment (525 bp) extending from 163rd Gly to 338th Trp was called ‘3’-probe’.

(c) Recombinant phage construction and plaque screening Calf DNA was digested with EcoRI or Hind111 as described above and electrophoresed on a 0.7% agarose gel. The EcoRI-cleaved fragments (about 4-20 kb in length) and HindIII-cleaved fragments (about 7-20 kb) were recovered by the method of Chen and Thomas (1980). Phage DNAs were prepared from AgtWES * LB and IZL47AB grown on E. coli LE392 (Maniatis et al., 1982). The left and right arms of EcoRI-cleaved >WES . IB DNA and HindIII-cleaved K47AB DNA were separated from the stuffer fragments on 0.7 % agarose gels and recovered by electroelution. Two kinds of recombinant DNAs were prepared by ligation of the EcoRIfragments of calf DNA with Igt WES * IB arms and by ligation of the HindIII-fragments with ;1L47AB arms, and these recombinant DNAs were packaged as viable phage particles (Enquist and Steinberg,

c ab

Fig. 1. Southern cDNA

probes.

hybridization

ab

ab

ab

of calf genomic

Calf DNA was digested

Hind111 (lane b) and blot-hybridized

DNA with PC

with EcoRI

derived from different parts of the cDNA designated (B), 5’-probe stained

agarose

(C) and 3’-probe gel.

(lane a) or

with nick-translated

(D). (A) Photograph

probes

as pc-probe of EtdBr-

199

By hybridization of the EcoRI digest with the PC-probe, four EcoRI-fragments (7.7 kb, 5.8 kb, 4.4 kb and 1.3 kb) were detected. Among these four fragments, the 7.7-kb and 1.3-kb fragments hybridized only to the 3’ probe, and the 4.4-kb fragment hybridized only to the 5’ probe. In contrast, the 5.8-kb fragment hybridized to both the 5’ and 3’ probes. However, in cPC cDNA, the EcoRI site locates only 10 bp upstream from the BumHI site which is used to divide the 5’ and 3’ probes. Therefore, any fragments produced by complete digestion of genomic DNA with EcoRI should not hybridize to both the 5’ and 3’ probes. From this consideration, the 5%kb fragment was assumed to be the partially digested product composed of the 4.4-kb and 1.3-kb fragments. On the other hand, the Hind111 digest gave two positive bands (12 kb and 6.1 kb) with the pc-probe among which the 12-kb fragment mainly hybridized to the 5’ probe and the 6.1-kb one hybridized to the 3’ probe. Weak hybridization of the 12-kb fragment with the 3’ probe was also observed.

(b) Isolation of phage clones carrying genomic cPC gene A shotgun cloning of the EcoRI-fragments of calf DNA was performed by using AgtWES *AB and recombinant phages were screened for the PC gene by the plaque hybridization with the PC-probe. Seven clones out of 9 x 10’ recombinant phages were isolated. Three of them (1CR84, ICR91 and ;1CR180) contained a 4.2-kb fragment, two (;1CR72 and ICR177) contained a 7.3-kb fragment and 1CR42 contained the 5.3-kb fragment composed of the 4.2-kb and an additional l.l-kb fragments. Cloning of the Hind111 fragments using AL47AB gave one clone (KRL22) out of about 2 x lo5 recombinants, which carried a lo-kb Hind111 fragment. (c) Alignment and sequence analysis of the cPC gene The restriction map of each cloned fragment is shown in Fig. 2. Sequencing of the fragments cloned

A 0

5

t

E

H

c

1

I I

1

E

ss

I

ACR42

I

XCR84

I

ACR91

,

1

ACRl80

,

A

w

B

’ V

B

F

EHCEC

I I I GG B

I

I

0 -

I

7G8

BGG

Hind111 fragment The structure and introns

map and nt sequencing

strategy

cloned in Jgt FWS. 1B phages described

in RESULTS

of the PC gene is illustrated are numbered

for genomic

cPC DNA fragments.

and of a fragment

AND DISCUSSION, schematically

cloned in IL47AB

I

0

0

-

indicate

arrows

(1980) or of Sanger

of EcoRI (E) and Hind111 (H) sites arrow represents

ofthe cPC gene and strategy and flanking

the 5.5-kb

for sequencing. regions).

Exons

(B), EgZII (G), EcoRI (E) and Hind111

AvuI (V), Ban1 (A), Hinfl (F), MfI (M), NcoI (N), PsrI (P),

orientations

et al. (1980).

1

_

(A) Location

in the order 5’ to 3’. All the BarnHI determination,

and Gilbert

_

I

with black boxes (exons) and solid lines (introns

1 to 9 and A to H, respectively,

ScaI (C) and SmaI (S), are shown. The horizontal of Maxam

d

phage. Double-headed

section c. (B) Organization

(H) sites are shown. Only those sites used for nt sequence by the procedure

I

B -_

-_

H

F

I

II

I

“9

AN

4-W Fig. 2. Restriction

I

ACR177

I

5E6

V

hCR72

c

I

2’3’4 EP

cc-

of six fragments

I

E

H

I

PM I I

I

EH 4

MxL22

18kb

15

‘P I

1

and approximate

length of each sequence

determined

-150

-100

CCCGGGGTGGTGGAACCCGTGGCCCTTATCAGAGTGGGTGTTG~GTAGTCTGG~T~TATCTT~ATGTGTTTG~~AGTG~~~~TTAG~TATCTCT~A~TT~AG -50

+1 . .__________ 1~~~~~~.,.~~~~~~~-_~_

50

CCAGAG‘GGAGCCCCGTACCTTC~GCTG~GGAAGTGG~~~TG~TACA~A~G~T~A~~~AGATCCAAGATGA~GTGTCT~T~GT~TACTT~TGTCTT~~T~T~TCC MetdrgCys~UValValLeuLeuAlaValPhedl~~uSer

-10

-16

CAiGGC~CTGAGiTCACCAGGTGAGTGTCA----- n-itxm A -----CTCTCCTCAGGAT~CCTCTGTACAAAGGCAAGTCTCTGAGGAAGGCJXTGAAGGAGCATGGGCTTCTG GlnClyThrGluIleThrdr(g) -1 +1

(dr~gIleProLeuTyrLySClyLysSerLeud~gLysdl~~uLySClUHisGlyLeuLeu 10 20

(3.0 kb)

GAGGACTTCCTGCAGAAACAGCAGTATGGCATCAGCAGCATTCGGGGAGGTGGCCAGCGTGCCCCTGACCAACTACCTGGAT

GTGAGTGGTT---- nmxm

G1udspPhel~uGlnLy~GlnGlnTyrGlyIleSerSerLysTyrSerGlyPheGlyGluValdlaSerValProLeuThrdsnTyrLeudsp 40 50

B -

(0.7 kb)

----CTGTCCTCAGAGTCAGTACTTTGGGAAGATCTACCTM;GGACCCa;CCCCAAG SerGln~yrPheG1yLysIleTyr~uG1yThrProProGlnGluPheThrValLeuPhedspThrGlySerSerdspPheTrpVa1ProSerIleTyrCysLys 80 90 60 70

AGCAATGCCTGCA GTGAGTGACA---- Intmn C -----TGTGTTCCAG AAAACCACCAGCGCTTCGACCCGAGAAAGTCGTCCACCTTC~GA~CTG~AA~CCCTGTCT SerdsndlaCysL(ysl

(1.0 kb)

(L)y~snHisGlndrgPhedspProdrgLysSerSerThrPheGlndsn~uGlyLysProLeuSer 100 110

ATCCACTACGGGACAGGCAGCATGCAGGGCATCCTGGGCTATGACACCGTCACT GTGAGTGGAG---- Inem IleHisTyrGlyThrGlySerMetGlnG1yIle~uGlyTyrdspThrValThr 120 130

D ----TCTCTTGCAG GTCTCCAACATTGTGGACATCCAG ValSerdsnIleValdspIleGln 140

(1.6 kb)

CAGACAGTAGGCCTGAGCACCCAGGAGCCa;GGGACGTCTTCACCTATGC~AATT~A~GGATCCTGG~AT~CTACCCCT~T~CCTCA~GTACT~ATA~~TGTTT~C GlnThrValGlyLeuSerThrGlnGluProGlydspValPheThrTyrdlaGluPhedspGlyIleLeuGlyMetdlaTyrPsoSer~euAlaSerG1uTyrSerIleProValPhedsp 150 160 170 180

AACATGATGAACAGGCACCTGGTGGCCCAAGACCTGTTCTCGGTTTACAT~ACAG GTAGGAGCTG----- 1ra-m E -----CTGGTTTCAGGAATGKCAGGAGAGCATGCTC dSnMetMetdsndrgHisLeuValdl~GlnAspLeuPheSerValTyrMetdspdr(g) 190 200

(dr)gdsnGlyGlnGluSerMetLeu

(0.5 kb)

ACGCTGGGGGCCATCGACCCGTCCTACTACACAGM;TCCCT~ACTG~T~C~TGACAGT~A~AGTACT~AGTTCACTGTGGACAG

GTGGGCGAGG---

ThrLeuGlydlaIledspProSerTyrTyrThrG1ySerLeuHisTrpValProValThrValGlnGlnTyrTrpGlnPheThrV~ldspSefr~ 210 220 230

Intmn F -(1.5 kb)

---CCCTCTGCAG TGTCACCATCAGCGGTGTGGTTGTGECCTGTGAGGGTGGC (Se)rValThrIleSerGlyValValValdlaCysG1uG1yG1yCysGlnAlaIleLeuAspThrGlyThrSerLysLeuValG1yProSerSerdspIleLeuAsnIle 240 250 260 270

CAGCAGGCCATTGSAGCCACACAGAACCAGTACGGTGAG GTGAGCCCAG---

Intmn G ----TGTTCTTTAG TTTGACATCGACTGCGACAACCTGAGCTACATGCCCACT

GlnGlndlaIleGlydlaThrGltisnClnTyrGlyGlu 280

(0.5 kb)

PhedspIledspCysdspdsnLeuSerTyrMetProThr 290 300

GTGGTCTTTGAGATCAATGGAAAATGTACCCACTGACCCCCTCCGCCTATACCA~~G GTATGCATCT----- Inem Va1ValPheGluIledsnGlyLysMetTyrProLeuThrProSerdlaTyrThrSerCln 310 320

H ----CCTTTCCCAG GACCAGGGCTTCTGTACC

(0.6 kb)

AspClnGlyPheCysThr

AGTGGCTTCCAGAGTGAAAATCATTCCCAGAAATGGATCCTGG~GATGTTTTCATC~AGAGTATTACA~GTCTTTGACAGG~CAACAACCTCGTGG~TG~CAAAGC~TCTGA SerClyPheGlnSerG1udsnHisSerGlnLysTrpIleLeuGlyAspValPheIleArgCluTyrTyrSerValPheAspArgAlaAsnAsnLeuValG1yLeuAlaLysAlaIle 330 360 340 350

“..

Fig. 3. Nucleotide sequence of the coding regions and of the 5’- and 3’-flanking regions of the cPC gene. The aa sequence deduced from the nt sequence is shown underneath. Numbering of aa of mature PC and of a signal peptide is with positive and negative numbers, respectively. The approximate size of each intron is given in parentheses. Position + 1 is the transcriptional start point and the arrow adjacent to the + 1 indicates the direction oftranscription. The CCAAT and TATA boxes in the 5’-flanking region are marked by chains of squares and dots, respectively. Two inverted repeats are indicated by solid and dotted arrows. The AATAAA sequence in the 3’-untranslated region is underlined. The putative 3’ ends of the transcribed region are indicated by closed triangles. The asterisk (nt 74) represents the position of the base conversion observed in the present study (see RESULTS AND DISCUSSION, section c).

201

in LCRL22, rZCR42 and KR72 revealed nine exons covering the whole coding sequence for cPC (Fig. 2B). We failed to obtain a clone overlapping both KR42 and KR72 inserts. However, direct alignment of these two inserts (Fig. 2A) seems to be highly probable since three HindIII-restriction sites in this construction yield two HindIII-fragments of lo-kb and 5.5-kb (represented by a double-headed arrow in Fig. 2), the sizes of which coincide well with those of the 12-kb and 6.1-kb Hind111 fragments, respectively, detected by Southern hybridization (Fig. 1). The EcoRI fragments detected by Southern hybridization also coincide well with the fragments in this construction. Fig. 3 shows the sequences of nine exons and those of the exon-intron junction points. The exons consisted of 87, 151, 118, 119,200, 114, 145,99 and 275 bp in the order of 5’ to 3’. The exon sequences are identical with those of the corresponding regions of the cDNA (Nishimori et al., 1982a; Harris et al., 1982; Moir et al., 1982) except that the nt 74 (G, in the case of cDNA) was converted into A. By this conversion, the encoded aa Ala at the N terminus of PC was changed to Thr in the genomic gene, which may be due to the allelic variation. Both sides of all exon-intron splicing junctions matched reasonably well with the consensus sequences of AG/ GT%GT------(&N$AG/G (Breathnach and Chambon, 1981). (d) Structure of the 5’ - and 3’ -flanking regions The 5’- and 3’~flanking sequences are also shown in Fig. 3. The transcriptional start point was determined by Sl nuclease mapping. As shown in Fig. 4, one major protected fragment was detected along with some minor fragments, which corresponds to the T-specifically cleaved fragment migrating oneand-a-halfnt faster than this fragment (Fig. 4, lane c) as described by Soldier-Webb and Reeder (1979). Thus the transcriptional start point was determined to the nt A, 25 bp upstream from the start codon. Two conserved sequences involved in the promotion of eukaryotic gene transcription by RNA polymerase II were found in the 5’-flanking region. The so-called TATA box, TATAAAA, is located about 30 bp upstream from the start point (positions - 3 1 to -25) and the sequence CGAAT, which is homologous to the consensus CCAAT box, is pres-

abc

d

Fig. 4. Determination of the 5’ terminus of cPC mRNA by Sl nuclease mapping (Berk and Sharp, 1977). cPC mRNA was purilied from the mucosal tissue of abomasum of newborn calf essentially according to the procedure of Uchiyama et al. (1980). The mRNA-coding strand of the D&I-PstI fragment extending from - 67 to + 75 was 5’-end-labeled with 3zP. The fragment (3000 cpm) was heat-denatured, hybridized with cPC mRNA (10 pg) in 4 ~1 of 80% formamide, 0.4 M NaCl, 0.04 M Pipes, pH 6.4 and 1 mM EDTA at 46°C for 21 h and then treated with Sl nuclease (350 units) in 100 ~1 of 30 mM Na. acetate, pH 4.6, 50 mM NaCI and 1 mM ZnSO, at 30°C for 50 min. The digest was analyzed by 8% PA-8 M urea gel electrophoresis (lane a). The same DNA fragment was chemically cleaved by the method of Maxam and Gilbert (1980) and electrophoresed in parallel as chain-length markers. The products of G + A- and T + Cspecific degradation were electrophoresed in lane b and lane c, respectively, and nt of mRNA-like strand corresponding to each product were shown in lane d. The arrow represents the start point and the direction of transcription of cPC mRNA.

202

ent at nt - 90 to - 86. An 8-bp inverted repeat is present in the region immediately upstream from the TATA box (-63 to -56 and -48 to -41) and another 13-bp inverted repeat, including 3-nt mismatch, was also observed at the transcriptional start point. The 3’ end of the cPC gene was assessed by comparing the sequence of the 3’untranslated region of the gene with that of cDNA containing the complete 3’-noncoding region (Nishimori et al., 1982a). The potential polyadenylylation signal AATAAA was found about 15 bp upstream from the putative poly(A) addition sites. Several CA clusters were detected in the 3’-untranslated region. (e) Conclusions Comparison of the cPC gene sequence with that of the human pepsinogen gene (Sogawa et al., 1983) showed a striking similarity. The cPC gene spans approx. 10.5 kb and is separated into nine exons by eight introns of various sizes. The human pepsinogen gene also has nine exons (Fig. 5), all of which show significant homology with those of cPC gene (in the order of exon 1 to 9: 59%, 59%, 73x, 67x, 612, 66%) 66%) 55 % and 65 % nt sequence homology, respectively). Furthermore, all exon-intron junction points of both genes coincide completely even in the

nt sequence. This perfect coincidence of exon-intron structures suggests that both genes belonging to an aspartyl protease family originated from a single ancestral gene. The TATA and CCAAT boxes probably involved in the correct initiation of transcription by RNA polymerase II are present upstream from the transcriptional start point. The characteristic inverted repeat structures observed just before the TATA box and at the transcription initiation site may represent recognition sites for some cellular proteins as suggested by Jilley (1980) and possibly enhance the role of the TATA box functioning as a transcription initiation signal. Analysis of gastric enzymes of pig during development revealed that chymosin production in the gastric mucosa is replaced by pepsin production during several weeks of the neonatal period (Foltmann et al., 1981), suggesting conversion of expression from chymosin gene to pepsin gene. A similar conversion of gene expression was observed between mouse cl-fetoprotein and albumin genes (Tilghman and Belayew, 1982) and significant homology of the 5’-flanking sequences was observed between them (Scott and Tilghman, 1983). When the 5’-flanking region sequence of cPC gene is compared with that of human pepsinogen gene (Fig. 6), no extensive similarities are observed, but two conserved 7-bp sequences of (F)GGA(g)CC are found slightly upstream from the TATA box and initiation codon in both genes. In the case of cPC gene, these sequences are included in the inverted repeats described above. It is unclear so far whether these conserved 7-bp sequences represent regulatory sig-

CPC

hPE

‘Dz ;:m;

::A:~~~~:,:g:::~~~~Ir~:E:~~~:O;~::~ +I

hPPE

cPC

C~GCAGCGGC~AGnTccnnGXiEaGG

hPE

CXCCTTCCTCCCGTCTTGCCTTCTCCCTCGAGTTGGGACCCGGGAAGAACC~~~AAG

Yl”g:18~~~l~~~::gr~~::Oi~~~:~~~~~B:B:~ 360

CPPC

GCTCCCAGCCAGAGGGAGCCCCGTACCTTC~~~~~~GCTGTGGAAGTGGGCCCTGGCTA . .. . . . . TCAGAGGCCGATAAGGCGGGACCCAACTTGTATATAAGGGCAGCTCATGCTGCTGCTCTG

5LNHSOKWILGD”FIRE

1111

380

310

aGMNLPTESGLL~~~~~~~~~“~

0

YS

FDRAN

50

L

GL

KA

Fig. 5. Alignment of aa sequences of calf preprochymosin (cPPC) and human prepepsinogen (hPPE). Locations of splice junctions of the cPC and the human pepsinogen genes are indicated by arrows. Common aa residues are boxed.

Fig. 6. Comparison of the 5’-flanking region sequences between cPC and human pepsinogen (hPE) genes. Both sequences are aligned with respect to the TATA sequences. TATA sequences, transcriptional start point and start codons are marked by tilled-in circles, triangles and squares, respectively. The 7-bp sequences conserved in both genes are underlined by bold lines.

203

nals for switching gene expression according to the developmental stage. Further investigation of the sequences of pepsin and chymosin genes of other mammals will be necessary to assess the significance of the possible regulatory structure.

ACKNOWLEDGEMENTS

We would like to thank Dr. M. Obata for providing AL47AB phage, and Dr. Y. Fujii-Kuriyama for generous gift ofE. coli WL95 and for valuable advice.

REFERENCES Benton, W.D. and Davis, R.W.: Screening Igt recombinant clones by hybridization to single plaques in situ. Science 196 (1977) 180-182. Berk, A.J. and Sharp, P.A.: Sizing and mapping of early adenovirus mRNA by gel electrophoresis of Sl endonucleasedigested hybrids. Cell 12 (1977) 721-732. Blin, N. and Stafford, D.W.: A general method for isolation of high molecular weight DNA from eukaryotes. Nucl. Acids Res. 3 (1976) 2303-2308. Breathnach, R. and Chambon, P.: Organization and expression of eucaryotic split genes coding for proteins. Am~u. Rev. Biochem. 50 (1981) 349-383. Chen, C.W. and Thomas Jr., C.A.: Recovery of DNA segments from agarose gels. Anal. Biochem. 101 (1980) 339-341. Equist, L. and Sternberg, N.: In vitro packaging of lDam vectors and their use in cloning DNA fragments. Methods Enzymol. 68 (1979) 281-298. Foltmann, B., Pedersen, V.B., Kauffman, D. and Wybrandt, G.: The primary structure of calf chymosin. J. Biol. Chem. 254 (1979) 8447-8456. Foltmann, B., Jensen, A.L., Lonblad, P., Smidt, E. and Axelsen, N.H.: A developmental analysis of the production of chymosin and pepsin in pigs. Comp. Biochem. Physiol. 68B (1981) 9-13. Harris, T.J.R., Lowe, P.A., Lyons, A., Thomas, P.G., Eaton, M.A.W., Millican, T.A., Patel, T.P., Bose, CC., Carey, N.H. and Doel, M.T.: Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin. Nucl. Acids Res. 10 (1982) 2177-2187.

Jilley D.M.: The inverted repeat as a recognizable structural feature in supercoiled DNA molecules. Proc. Natl. Acad. Sci. USA 77 (1980) 6468-6472. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Maxam, A.M. and Gilbert, W.: Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65 (1980) 499-560. Moir, D., Mao, J., Schumm, J.W., Vovis, G.F., Alford, B.L. and Taunton-Rigby, A.: Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin. Gene 19 (1982) 127-138. Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T.: Cloning in Escherichiu coli of the structural gene of prorennin, the precursor of calf milk-clotting enzyme rennin. J. Biochem. 90 (1981) 901-904. Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T.: Nucleotide sequence of calf prorennin cDNA cloned in Escherichiu coli. J. Biochem. 91 (1982a) 1085-1088. Nishimori, K., Kawaguchi, Y., Hidaka, M., Uozumi, T. and Beppu, T.: Expression of cloned calf prochymosin gene sequence in Escherichia coli. Gene 19 (1982b) 337-344. Sala-Trepat, J.M., Dever, J., Sargent, T.D., Thomas, K., Sell, S. and Bonner, J.: Changes in expression of albumin and a-fetoprotein genes during rat liver development and neoplasia. Biochemistry 18 (1979) 2167-2178. Sanger, F., Coulson, A.R., Barrell, B.G., Smith, J.H. and Roe, B.A.: Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143 (1980) 161-178. Scott, R.W. and Tilghman, SM.: Transient expression of a murine a-fetoprotein minigene: deletion analyses of promoter function. Mol. Cell. Biol. 3 (1983) 1295-1309. Sogawa, K., Fujii-Kuriyama, Y., Mizukami, Y., Ichihara, Y. and Takahashi, K.: Primary structure of human pepsinogen gene. J. Biol. Chem. 258 (1983) 5306-5311. Sollner-Webb, B. and Reeder, R.H.: The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. 1uevi.r.Cell 18 (1979) 485-499. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975) 503-517. Tilghman, S.M. and Belayew, A.: Transcriptional control of the murine albumin/a-fetoprotein locus during development. Proc. Natl. Acad. Sci. USA 79 (1982) 5254-5257. Uchiyama, H., Uozumi, T., Beppu, T. and Arima, K.: Purification of prorennin mRNA and its translation in vitro. Agric. Biol. Chem. 44 (1980) 1373-1381. Communicated by H. Yoshikawa.