Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

Gene, 19 (1982) 127-138 Elsevier Biomedical

Press

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin (Recombinant

DNA;

bacteriophage

f 1 as cloning

vector;

gel-transfer

hybridization;

bovine

protease;

rennin)

Donald Moir, Jen-i Mao, James W. Schumm, Gerald F. Vovis, Bernadette L. Alford and Alison TauntonRigby Department of Molecular Genetics, Collaborative Research, Inc., Waltham, MA 02154 (U.S.A.) (Received

May IOth, 1982)

(Accepted

June Ist, 1982)

SUMMARY

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an fl bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched (abomasum) and immunoprecipitation with antichymosin antiserum (probably

preprochymosin

judging

from that tissue. Gel-transfer

from the &-value)

hyb~dization

is the major

RNA from the calf fourth stomach revealed that a form of chymosin in vitro translation

of restriction

endonuclease-cleaved

with labeled cDNA probes indicated that the two known two different alleles of a single chymosin gene.

forms of chymosin,

Chymosin (EC 3.4.23.4), also known as rennin, is the major proteolytic enzyme in the fourth (abomasum)

of the unweaned

calf (Folt-

mann, 1970). Its biological function appears to be the specific cleavage of the k-casein molecule of milk resulting in the coagulation of milk protein (Kaye and Jolles, 1978). In addition to aiding the suckling calf in the digestion of its diet, this activAbbreviations: kb, kilobase

AMV, avian myeloblastosis pairs;

SDS, sodium

0378-l f 19/82/~-~/$02.75

dodecyl

DNA

A and B, must be products

of

Chymosin (M, 36000) is synthesized in vivo as a zymogen precursor called prochymosin (M, 41000) which is activated by proteolytic release of a 42 amino acid fragment from the amino terminal end (Pederson and Foltmann, 1975). Two chromatographically different forms, A and B, of the

virus; bp, base pairs; sulfate.

0 1982 Etsevier Biomedical

chromosomal

of RNA

ity makes chymosin commercially useful for the production of cheese. Many other proteolytic enzymes, including pepsin, chymotrypsin, and papain will coagulate milk, but these enzymes degrade milk proteins more extensively than does chymosin (Tang et al., 1959) and are therefore less suitable for cheese production.

INTRODUCTION

stomach

bovine

product

Press

12X

enzyme amino

and

its zymogen

acid sequence

determined

et al., 1977;

1979). Com-

(Foltmann

of this sequence

quence

of pepsinogen

ogy (about

55%); both

inactivated

by chemical

1979).

aspartate

Consequently,

classified

with the amino

A reveals pepsin

extensive

modification

homol-

have

of

et al.. been

of the same acid protease

family.

contain

report,

we describe

copy of the chymosin

Nucleotide

sequencing

the cloning A coding

of the cloned

of a

sequence.

gene has al-

lowed us not only to determine for the first time the complete prochymosin A amino acid sequence. but also to predict that the protein is synthesized in vivo as a “preprochymosin” molecule containing a 16 amino acid “signal sequence” at its amino terminus. Further, we describe experiments, using radioactively labeled cloned cDNA as probes, which indicate in the bovine

that there is a single chymosin gene genome, and therefore, that the A

and B forms must represent alleles of the same polymorphic

MATERIALS

products

Minimal

medium

was

by J. Miller (1972). modified

0.5% casamino

to

acids.

(b) Enzymes and synthetic oligonucleotides Restriction purchased under

enzymes

from

New

the conditions

AMV

Biochemicals, reverse

Life Sciences,

and T4 DNA England

ligase were

Biolabs

recommended

plier. T4 polynucleotide P-L

In this cDNA

and 60 mg NaOH.

M9 as described

are

of either

(Foltmann

enzymes

glucose,

acid se-

and chymosin

residues these

as members

the entire B has been

parison

two essential

are known:

of prochymosin

and

kinase was obtained

and pepsin

transcriptase

used

by the supfrom

was from Sigma.

was purchased

Inc. Sl nuclease

from

and E. coli DNA

polymerase I Klenow fragment ringer-Mannheim. E. coli DNA

were from Boehpolymerase I was

the gift of S. Scherer (Stanford University). Oligo(dT)-cellulose. oligo(dpT),,_,,, synthetic oligonucleotide Hind111 linkers (CCAAGCTTGG) and micrococcal nuclease-treated rabbit reticulocyte lysate were products of Collaborative Research. Inc. (c) Preparation of antiserum

of different

gene.

AND METHODS

(a) Strains and media Escherichia coli strain JM 101 (F’traD36 proAB lacZZAM15 in a A( lac pro)supE thi- background) as described by J. Messing (1979) was used as the f 1 host. Transformations and transfections were done using E. co/i strain BNN45 (hsdR hsdM supE44 supF met- thi-) of Davis et al. (1980). Phage CGF4, containing a unique Hind111 restriction site in the intergenic space, was constructed from f 1 phage R229 (Boecke, 1981) by restriction with EcoRI, incubation with E. coli polymerase I Klenow fragment plus all four deoxynucleoside triphosphates to fill in the cohesive termini, and ligation with Hind111 synthetic oligonucleotide linkers (CCAAGCTTGG). Rich medium (TY) for growth of E. coli infected with fl bacteriophage contained, per liter: 10 g Bacto tryptone, 8 g NaCl, 1 g yeast extract, 1 g

Partially purified chymosin (Sigma) was purified to apparent

(EC 3.4.23.4) homogeneity on

DEAE-cellulose (Foltmann, 1970). About 500 pg was injected into each of two New Zealand white rabbits in complete Freund’s adjuvant followed by injections every 14 days of 250 pg chymosin in incomplete adjuvant until a high titer of specific anti-chymosin antiserum was achieved. (d) Isolation of poly(A)-enriched

RNA

Total RNA (45 mg) was isolated from 21 g of mucosal tissue from the calf fourth stomach essentially by the method of Deeley et al. (1977). Poly(A)-enriched RNA (1 mg) was obtained by one passage over a 4 ml oligo(dT)-cellulose column as described by Desrosiers et al. (1975). (e) Preparation cDNA

and cloning

of double-stranded

About 16 pg of double-stranded cDNA was synthesized from 20 pg of calf stomach poly(A)containing RNA (Wickens et al., 1978). Digestion with Sl nuclease and separation of the double-

129

stranded enzyme

cDNA

from the nucleotides

were performed

Wahl et al. (1979a). To insure double-stranded was treated radioactively

cDNA

DNA

ligase

After

treatment

nuclease were

that the ends of the

running

and

MacDonald,

Hind111

released

synthetic

from

high-M,

DNA

on Biogel A-5 m (Sippel (0.3

by

in a 2% agarose

0.37 M Tris-glycine buffer).

rate of about NaOH

The gel was run (about

5 cm/h.

Following

for 1 h, rinsing

and examined

in 0.2 M

in water, and neutralization

in 0.5 pg/ml under

and

3 h) at a

soaking

in 0.5 M Tris - HCl, (pH 7.4) for about gel was stained

gel con-

pH 9.5 (gel buffer

ultraviolet

15 min, the

ethidium

bromide

illumination.

(h) In vitro translation and immunoprecipitation

chro-

et al., 1978).

Hind111

to electrophoresis

endo-

oligonucleotides

matography

Erg) bearing

1979).

restriction

The

DNA

I and

(2 pg) were

cDNA (1.5 pg) using T4

with

separated

the DNA

polymerase

HTindIII linkers

(Goodman

the

as described

would be blunt,

ligated to the blunt-ended

jected taining

with E. coli DNA labelled

and the Sl by

exactly

cohesive

termini was ligated to phage CGF4 doublestranded DNA (0.2 pg) which had been cut with Hind111 endonuclease and treated with calf intestinal alkaline phosphatase (Goodman and MacDonald, 1979). Hind111 linkers were chosen for

In

vitro

translations

were

performed

as de-

scribed by Pelham and Jackson (1976) in a 25-~1 final volume containing 10 ~1 of micrococcal nuclease-treated rabbit reticulocyte of [ 35S]t.-methionine (100 Ci/mmol, Nuclear),

and

20 pg/ml

lysate, 50 FCi New England

poly(A)-enriched

RNA

from the calf abomasum. A portion (10 ~1) of the translation reaction was immunoprecipitated with

insertion of the double-stranded cDNA into the vector because analysis of the known amino acid

chymosin

sequence of prochymosin predicted there would be no Hind111 sites within the DNA sequence encod-

10 pg of unlabelled chymosin or pepsin essentially as described by Gordon et al. (1977) except goat

ing chymosin. Cells of E; colt strain BNN45 were transfected with the ligated DNA and plated with

anti-rabbit serum was added as a second antibody. The washed immunoprecipitates were subjected to

a seed culture of strain (0.7% in TY medium).

electrophoresis in a discontinuous polyacrylamide gel containing 0.1% SDS according to the method of Laemmli and Favre (1973).

JMlOl

in soft top agar

antiserum

in the presence

or absence

of

(f) Plaque hybridization Plaque hybridization was carried out essentially as described by Benton and Davis (1977). Under these conditions of hybridization, the labelled cDNA concentration should be limiting.

(i) Hybridization

selection

Hybridization selection was carried out essentially as described by Miller et al. (1980) using either 5 pg of purified

recombinant

fl phage dou-

(g) Sizing of intact fl phage in agarose gel

ble-stranded DNA or a mixture of 5pg each of four recombinant phage DNAs. Eluted RNA was

Intact filamentous phage subjected to electrophoresis in agarose gels under the following condi-

translated

tions migrate at a rate inversely proportional to the size of the packaged DNA (Beaudoin, 1970). The method is essentially as described by Nelson et al. (1981). Samples of intact phage (5 X lO*/ml) picked directly from the transfection plate were amplified by growth overnight at 37°C on strain JMlOl ( IO5 phage into 1 ml of culture at A,, = 0.1) in TY medium and harvested by centrifugation. Aliquots of the supernatant (20 ~1 containing 1X 10” phage) were mixed with 5 ~1 of a solution of 40% sucrose and 0.02% bromphenol blue and sub-

in vitro

and

the translation

products

were subjected to electrophoresis in a 10% polyacrylamide gel as described above.

tj) DNA isolation Plasmid DNA was prepared essentially by the method of Clewell and Helinski (1969). Doublestranded replicative form I DNA was isolated from bacteriophage fl infected bacterial cultures by the method of Model and Zinder (1974). Sequencing was by the method of Maxam and Gilbert ( 1980).

(k) DNA gel transfer

hybridization

Three nick-translated the method DNA

either

probe”), single

from

or from HincII

the

entire

fragments

were prepared phage

et al., 1975) after separation Isolated

were isolated

by the freeze-squeeze

a 0.6% agarose

the

and “5’-terminal”

These DNA fragments

to labeling

from

et al.. 1978) to

Kpn site in the preprochymosin

insert (the “3’-terminal”

probes).

by

(“complete

extending

site in fl (Horiuchi

the single internal cDNA

probes

of Rigby et al. (1977) using R118/37

method

prior

(Thuring

by electrophoresis

in

gel.

calf thymus

DNA (72 pg) was cut with

2530 units of the appropriate restriction enzyme in a 90-~1 volume for 5 h at 37°C. Each restriction enzyme

reaction

was divided

into three equal por-

tions, and the DNA was subjected to electrophoresis in three lanes of a 0.6% agarose gel. DNA fragments of known size were included in another lane. After electrophoresis, the DNA was blotted onto a nitrocellulose filter (Schleicher and Schuell) according to the methods of Southern (1975). and the filter was cut into three pieces to be hybridized with the three different probes. The nitrocellulose was pretreated, hybridized with 2-3 X 10h cpm/lane

of nick-translated

essentially

as described

probe,

and

washed

by Wahl et al., (1976b).

Fug.I. In

I) or

hybridizations

lksate

phoresis in a 0.6% agarose gel according to McMaster and Carmichael (1977). The RNA was transferred to nitrocellulose, hybridized with lo6 cpm of ‘*P-labeled nick-translated R207 DNA and washed exactly as described by Thomas ( 1980).

RESULTS

(a) Polyadenylated encodes

RNA

from

the calf abomasum

chymosin

Poly(A)-enriched calf fourth stomach determine whether

RNA was isolated from the as described in METHODS. To this RNA preparation con-

translation

III

ucts

\ubJect

the

pepsin (lane 6X000). and

to

methods.

\Itrc,

from

(lane 4).

The

In the

or

of

P-lactoglohulin

four

(lane

IO p(g of

( M,

43000).

(M,

1X000)

2)

was

in-

retuxloc\ protan

proteins

mRNA

v+err

chymoaln uith

of bo\lnc

em-

(lmr te

prod-

sutoradiographq

antiserum.

unlabelled

immunoprecipitated positions

The

lanes.

\tomach

,tnd Buffer

rabbit

and

last

anti-chymosin

mlgratwn

ovalbumin

RiXA

-mrthiomnr.

the

calf

mRNA

.mtiserum.

electruphorrsis

with

6).

stomach

nuclrase-treated

of [“S]-I

m

presence

calf

poly(A)-rnrxhed

prewvx

munoprecipitated in

of

ant]-chymosln

micrococcal

the

were

III

with

stomach

with

described

or

Poly(A)-enriched calf stomach RNA (10 pg) was treated with glyoxal and subjected to electro-

calf

cuhated

aired

(I) RNA gel-transfer

vitro

munclprrcipitation

Lib synthe-

either

alone

(lane

preimmunr serum

chymosin

( M,

are shown

at left.

albumln 36000.

1n1-

(lane

3)

5) or xrum

( M, “c”‘)

tained messenger RNA encoding chymosin. it was translated in a rabbit reticulocyte lysate system (see METHODS). When the [-7sS]methionine-labeled translation products were analyzed on a 10% polyacrylamide gel containing SDS, a single major protein product with an M,-value of about 43000 was observed (Fig. 1, lane 2). Immunoprecipitation of the translation products using rabbit antiserum prepared against purified chymosin (see METHODS) and analysis of the precipitate on the same gel (Fig. 1, lane 3) revealed that the major protein was precipitated by the anti-chymosin serum. Specific-

ity was confirmed purified

by adding

chymosin

munoprecipitation not pepsin, binding

or

competed

by excess

and unlabeled

sin, or, alternatively, translation

imbut

protein

for

in vitro translation,

than chymosin, was

chymosin.

after

specifi-

This minor

product

a smaller

starting

was also

competed

species may be a degradation from

the

chymosin,

(Fig. 1, lanes 5 and 6). A during

smaller

immunoprecipitated

during

purified

with the labeled

species produced

which is slightly cally

pepsin

reaction:

to the antibody

minor

excess nonradioactive

of chymo-

protein

resulting

the normal

ATG

initiation codon. It is clear from these results that chymosin, in one form or another, is the major in vitro

translation

poly(A)-enriched

product

from

the calf stomach

RNA.

(b) Identification

of cDNA

clones

bearing

the

chymosin structural gene

Fig. 2. Identification

of cDNA

DNA by the technique

The apparent high abundance of prochymosinencoding mRNA suggested a way to screen for cDNA clones likely to contain chymosin-coding sequences. The method involves the use of a limiting amount of a radioactive cDNA probe derived from the total polyadenylated RNA to screen candidate clones by hybridization. Under these conditions, only the cloned sequences representing the abundant

mRNA

species should hybridize

nificant amounts of probe. A library of about 2000 cDNA

sig-

poly(A)-enriched

RNA (lane “C’)

cellulose membranes

containing

or from a recombinant reticulocyte

products

were

DNA from phage CGF4

lysate

subject

phage

(“mix”)

was translated

(see METHODS). to electrophoresis

gel and the dried gel was exposed

18 h. The

migration

tains

(arrow

position

products

in a

The

translation

in an

SDS-poly-

to X-ray film for

of ovalbumin

(M,

43000)

at left, “43 K”). The last lane (“-RNA”)

translation

coding

Calf stomach

or RNA eluted from nitro-

bound

acrylamide shown

chymosin

phage (R68, RI 18, R143, and R207) or

from all four recombinant rabbit

clones bearing

of hybridization-selection.

seen in the absence

is

con-

of any added

RNA.

clones in an fl

phage vector (see METHODS) was screened by plaque-filter hybridization using limiting con-

(CGF4)

centrations of 32P-labeled cDNA as probe. Intact phage from 85 of the most intensely hybridizing

RNA which yielded an M, 43000 product upon translation. One recombinant, R68 (Fig. 2), gave

plaques agarose

an intermediate result, but subsequent experiments involving hybridization with an authentic chymosin-encoding DNA probe confirmed that it does not carry chymosin sequences; presumably, the intermediate result was due to difficulty in washing away the unbound chymosin mRNA which represents a major portion of the total poly(A)enriched RNA. Restriction endonuclease cleavage analysis of DNA from two of the phage which appear to

were subjected gels to determine

to electrophoresis in the approximate size of

the DNA insert (see METHODS). Double-stranded replicative form I DNA was isolated from cells infected with eight different phage having inserts on the order of 1 kb. Inserts bearing sequences coding for chymosin were identified by the technique of hybridization-selection as described in METHODS. The results with four of the cloned cDNAs are shown in Fig. 2. Three of the four and R143) clearly hybridized (R118, R207, chymosin mRNA as judged from the appearance of the M, 43000 product revealed by translation of the eluted mRNA. Fig. 2 also shows that fl vector

DNA

with

no insert

did not

bind

any

contain chymosin cDNA inserts (R207 and R118) was carried out. Comparison with the distribution of restriction endonuclease cleavage sites predictable from the amino acid sequence suggested that

132

these two phages the

chymosin

analysis

might carry

coding

also indicated

two Hind111 inserts length.

all or nearly

sequence.

The

that phage RI 18 contained approx.

1000 and

phage

chymosin The inserts

(R118/37)

sequences DNA

in phage

termined

The

R118/37

R118/37

analysis.

and revealed

the coding

shown

in Fig. 3 along

quence

analysis.

were

protein

coding

and Gilbert

untranslated

that

nucleotides

sequence

phage

for all of

ment

the strategy

information

for se-

from both

and R207 was combined

this map: together

de-

with

Sequence

phages R118/37 cDNA

R207

of Maxam

sequences

contains

and the to contain

of the chymosin

by the method

(1980).

was shown

by restriction

sequences

A map displaying restriction endonuclease cleavage sites in the chymosin-encoding cDNA is

1200 bp in

The larger of these was subcloned

resulting

(c) Nucleotide sequence of the preprochymosin gene

all of

restriction

to make

the two phages carry the entire

sequence

plus 20 nucleotides

sequence

as well

of 3’ untranslated

of the cDNA

as at

sequence.

inserts

in both

of 5’

least

100

Every segrecombinant

preprochymosin (see below) except for the first 24 nucleotide residues and that phage R207 contains

phage was sequenced twice, usually from different, labeled ends. The nucleotide sequence is given in

the missing sequence. The total length of unique consecutive chymosin sequence that was cloned, combining the infor-

Fig. 4. Two unusually gross features of the cDNA insert in phage R707 are shown in Fig. 3. First. about 180 nucleotides of sequence from the middle of the gene (nucleotides 391-566 in Fig.4) were duplicated in an inverted orientation at the beginning of the R207 insert. Second, about 100

mation from phage R118/37 and R207. is about 1300 bases. When the calf stomach poly(A)enriched RNA was denatured and subjected to RNA gel-transfer

hybridization

cloned

cDNA sequences discrete RNA

chymosin

METHODS),

using radioactively

1500 nucleotides shown).

nucleotides of coding sequence (nucleotides 644757) are deleted from phage R207 but are present in phage R118/37. Artifactual, inverted repetitions at the 5’ end of cDNA clones have been

as probe (see species about

detected

(data

not

It thus appears that there is a single major mRNA species in the calf stomach cells,

and that the clones tion in that mRNA.

c

contain

in the literature

.

a

*

c

e-1

U

endonuclease

cleavage

sites in the cDNA

inserts of recombinant

endonuclease

and

the open

box denotes

sites shown in parentheses

were used for DNA restriction

above the full length

fragment

sequencing. labeled

the sequence are present

The arrows

at the site marked

below

codon. The direction

R207 and R118/37.

Hind111 site at the 3’ end of the R207 insert is not shown here. but is more than 100 nucleotides The heavy arrows.

ATG initiation

phage

linker-derived R207,

*

.

beginning

phage

with the A of the preprochymosin

I

are numbered

site in RI 18/37.

*

I

.

reverse

of a “loop-back”

-

*

e

Fig. 3. A map of restriction

arise during

due to formation

c

*

(Chan et al., 1979; O’Hare

et al., 1979); they apparently

most of the

transcription

/---+7 1

reported

line representing missing

from

at additional, the full-length

by the vertical

the cDNA

inserts,

R207 but present

unmarked

locations

line denote

line associated

denote

in RI IS/37 in the cDNA

the length

with each arrow.

Nucleotides

is 5’ to 3’. left to right. The synthetic

of DNA

3’ to the corresponding

the inverted

repeat

(see RESULTS).

present

insert but these particular sequence

in

Restriction

determined

sites from

a

133

,‘dT

CGCTCCACCCACATCCAAG -52‘

ARG

CYS

LEU

YAL

VAL

LEU

LEU

ALA

b$L

PHE

ALA

LEU

30 ARC ILE

PRO

LEU

TYR

LYS

SER

GLN

CLY

ALA

FLU

ILE

@R

TG AGG TGT CTC CTC GTG C A CTT GCT GTC TTC GCT CTC CC CAG GGC CCT GAG ATC AC TO a* P SO

CLY

LYS

SER

LEU

40 ARC

LYS

ALA

LEU

LYS

GLU HIS

CLY

LEU

LEO

GLIJ

ASP

PHE

LEU

CLN

LYS

CLN

ACC

ATC CCT CTG TAC AAA G C AAG TCT CTG AGG AAG GCG TG AAG GAG CAT CCC CTT CT GAG GAC TTC CTG CAC AAA CAG Ii0 10 f 10 E 140

CLN

TYR

SO GLY

ILE

CA1; TAT

GGC

ATC ACC ACC

PXE

LYS

ILE

SER

SER

LYs

TYR

SER

GLY

Pm

cLY

60 ixu

vAL

ALA

SER

TYR

PRO

LEU

THR ASN

TYR

70 LEU

ASP

SER

GLN

TYR

G TAC TCC GGC TTC GGG GA GTG GCC AGC GTG CCC CTG A C AAC TAC CTG GAT AGT CAG AC E 2T 0 10 %" 10 2c 0

80 GLY

VAL

LEO

90 CLY

THR

PRO

PRO

GLN

GLU

PXG

TXR

VAL

LEU

100 PHE

ASP

THR

GLY

SER

SER

ASP

PHZ

TRP

VAL

PRO

TTT WC

AAG ATC TAC CT OGGG ACC CCG CCC CAG GAG ; ; ACC GTG CTG TTT CAC ACT GC TCC TCT GAC TTC TGG CT CCC c 30 s T 28 0

SER

TYR

110 ILE

CYS

TCT ATC TX

LYS

SER

ASN

ALA

CYS

120 LYS

ASN

HIS

GLN ARC

PHE

ASP

PRO

ARC

LYS

SER

SER

THR

PHE

GLN ASN

LEO

CLY

TGC AAG A C AAT CCC TGC AAA AAC CAC AG CGC TTC GAC CCG AGA AA TCG TCC ACC TTC CAG AAC C G GCC 30 ! 30 Ii: 30 s 30 H 140

330 SER

ILE

HIS

TYR

GLY

TXR

GLY

SER

MET

150

LYS

PRO

LEU

GLN

CLY

!L*(;

CCC

CTC TCT ATC CAC TAC GGG ACA GGC AGC ATG CAG GGC ATC CTG GGC TAT GAC ACC GTC ACT GTC TCC AAC ATT GTG 460 420 440 4bo

ASP

ILE

GLN

(;AC

ATC

CAG CAG ACA GTA GGC CTG AK 480

160 GLN

THR

ILE

LEU

GLY

TYR

PRO

SER

CLY

LEO

SER

THR

GLN

LEO

ALA

SER

SER

VAL

TYR

VAL

THR

GLU

PRO

GLY

ASP

TYR

SBR

ILF

MET ASP

TCG

ATA 5h

ARG ASN

GLY

220 GLN

PHE

TRP

THR

TYR

ALA

GLU

PHE

T(‘C

CT<: CAC

Kf,““;

ALA

CYS

GLY

CCC

TIX GAG

‘;L#

ASP

CLY

ILE

LEU

CLY

CLU

PHE

ASP

ASI

MET MET ASN

ARG

HIS

LEU

VAL

ALA

CCC

GTG

TTT

GAC

AAC

A;

AAC

AGG

CAC

CTG

GTG

G C CAA 6f 0

GAC

ILE

230 ASP

PRO

SER

TYR

TYR

CLY

OATG s

SER

MET

LEU

THR

LEV

GLY

ALA

Tee TAC

6II 0 250

VAL

VAL

VAL

660

T HIS

ASM ILE

PRO

280 LEU

SER

180 VAL

crc, TTC ‘rc(; Gr~6 ;c ATG cAc AGG AAT GGC c.4G GAG AGC ATG cTc ACG CTG G G GCC ATC GAC cm

SER

VAL

200 GLU

F

PHE

THR

ACC CAG C@ CCC GGG GAC GTC TTC ACC TAT CCC GAA TTC GAC GCG AT CTG GGG sso 56: 0 500

ATC GCC TAC CCC ; ; CTC GCC TCA GAG TAC

LEU

ASP

l?O VAL

190 MET ALA

TYR

GLN ASP

THR

AC ACA GGG 7F 0

260

PRO

VAL

THR

VAL

GLN

GLN TYR

TRP

GLN

PHE

THR

VAL ASP

SER

VAL

THR

ILE

SER

GLY

VAL

VAL

VAL

CCC

GTG

RCA

GTG

CAG

CAG 760

TAC

TGG

CAG

TTC

ACT

GTG

AC 7P 0

AGT

GTC

ACC

ATC

AGC

GG

GTG

GTT

GTG

CYS

270 GLN ALA

ILE

LEU

ASP

THR

CLY

THR SER

LYS

280 LEU

VAL

CLY

PRO

SER

SER

ASP

ILE

LEU

ASN

290 ILE

GGC TGT CAC GCC ATC

CTG8

;C

ACG

GGC

ACC

TCC

AAG

CT

GTC

GGG

CCC

AGC

AGC

GAC

TYR

ASP

GLU PHE

ASP

ILE

ASP

CYS

ASP

310 ASN

GLY

E 300 GLN

:L1/

CTB

ALA

ILE

CLY

ALA

THR

GLN ASN

LEU

SER

TYR

MET

PRO

THR

VAL

Cht;

C.\G

GCC

ATT 8bO

CGA

GCC

ACA

CAG

AAC

CA TAC 9s0

GAT

GAG

TTT

GAC

ATC

GAC 910

TGC

GAC

AAC

CTG

AGC

TAC

TG 9 %0

CCC

ACT

GTG

'IALPHE GLLI ILE

ASN

GLY

LYS

MET

TYR

PRO

THR

330 PRO

SER

ALA

TYR

THR

SER

GLN

ASP

GLN

GLY

340 PHE

CYS

TXR

SER

GLY

320 LEU

crc TTT G.\ ATC AAT GGC AAA ATG TAC c A CTG ACC ccc TCC CCC TAT ACC ACC CAG GAC CAG GGC TT TGT ACC AGT GGC 1s 20 90 E Id00 90 : GLU

ASN

HIS

370

360

350 PiiE GL:I SER

SER

GLN

LYS

TTC CAG A,T oh\ ;\AT CAT Tee CAG AAA

1s10

TRP

ILE

LEU

GLY

ASP

VAL

PRE

ILE

ARG

CLU

TYR

TYR

SER

VAL

PHE

ASP

ARC

ALA

GG ATC cTc GGG GAT GTT TT ATC CGA GAG TAT TAC AGC G c TTT GAC AGG wc 1T00 1s 80 18 60

380 AS0 AS:1 LEU &\C

MC

VAL

GLY

LEU

ALA

LYS

ALA

TC <;TG GGG CTC CCC AAA GC

ILE

ATC TGA

TCACATCGCTGACC

AGAACCTCACTGTCCCCA

1P 60

Fig.4. The nucleotide

sequence

phage R207 and R118/37. Nucleotides nucleotides

are numbered

and the derived ammo acid sequence

The ammo acids are numbered as in Fig. 3 beginning

391 to 566 present

of preprochymosin

consecutively

beginning

with the A of the signal

at the 5’ end of the cDNA

insert of phage

ACACCTGCACACACACAT;$;A$AcATGTA

1f 80

encoded

by the cDNA

with the initial methionine

sequence

initiation

R207 is not shown.

codon.

inserts in recombinant of the signal sequence.

The inverted

repetition

of

134

stabilized by a short region of homology between a segment near the 5’ end of the mRNA and an

crepancy,

internal

sequence

at position

region

of homology

(644-650

(Weaver

et al., 198 1). A short

between

and 758-764,

two internal

see Fig. 4) is probably

the cause of the observed

deletion

but in this case, the mechanism occurring

after the transfection

the bacteria1

cell. In any event,

tion is relatively cDNA tions

also

in phage R207,

may involve events of the DNA

into

in this region as judged

chymosin

of the chymosin

coding

by the sizes of restriction

translated amino acid sequence using the only reading frame free of termination codons for sufficient length to code for chymosin. The only ATG initiation codon suitable for initiation of translation lies 48 nucleotides 5’ to the beginning of the known amino acid sequence of prochymosin (the alanine residue number 17 encoded by nucleotides 49-51). Therefore, the initial product of translation in vivo must be a “preprochymosin” molecule “signal sequence”. This

secreted. The amino chymosin chymosin

with

is an extracellular acid

B molecule A molecule

the fact

protein

sequences

forms

(Foltmann

et al.. is the

result of deamidation

during

termination.

protein

In any case, the sequence that

is reported

this discrepancy

that

suggests

to the differences

the prochymosin

sequence

A and

differ by only one or two amino

de-

comparison B forms

acids out of 365.

that

pro-

which must be

of the entire

(d) Analysis of bovine genomic DNA by gel-transfer hybridization with cloned chymosin cDNA

se-

(data not shown).

chymosin

218 of both

1979). It is possible

frag-

The nucleotide sequence of the entire protein coding region is shown in Fig. 4 together with the

with a 16 amino acid conclusion is consistent

is unrelated

B forms since aspartate

this kind of dele-

rare. since three other

clones (R45, R118 and R143) had no dele-

quences ments

regions

however,

in the A and

pro-

and about half the prohave been reported previ-

ously (Foltmann et al., 1977; 1979). The amino acid sequence obtained by translation of the cloned cDNA gene agrees with the published prochymosin B sequence with two exceptions. First the codon beginning with nucleotide 652 (corresponding to amino acid residue 218 of Fig. 4 or residue number 204 of Foltmann et al. (1979)) specifies asparagine instead of aspartate. Second, the codon beginning with nucleotide 904 (amino acid residue 302 of Fig. 4 or residue number 290 of Foltmann et al. (1979)) specifies aspartate instead of glycine. The latter difference involves the single amino acid which is known to differ in prochymosins A and B (Foltmann et al., 1977; 1979); prochymosin A has an aspartate at position 302 in place of glycine. Therefore, the cloned DNA apparently represents the gene for preprochymosin A. The first dis-

The two forms of chymosin,

A and B, could be

the products of different genes of a gene family or they could be different alleles of the same gene. To distinguish between these possibilities, gel-transfer hybridization experiments were done by the method of Southern (1975). DNA from the thymus of a single calf was digested to completion with restriction endonucleases, subjected to electrophoresis through an agarose gel. transferred to a nitrocellulose membrane, dioactive probes derived sin cDNA. one

Three

(EcoRI)

chymosin

restriction

which

cDNA

and hybridized with rafrom the cloned chymoenzymes

recognizes

sequence

sites

were used: within

the

and two which do not

(Sac1 and BglI). Enzymes which do not cut the cloned cDNA might, however, cut within the chromosomal copy of the gene in the event that the chromosomal gene contains intervening sequences (“introns”). To discriminate between fragments produced from many genes and fragments produced from a single gene containing intervening sequences, probes derived by nick-translation of different parts of the cDNA carried by phage R118/37 were used (see METHODS). One (called the 5’-terminal probe) extended from the single HincII site in bacteriophage fl (Horiuchi et al., 1978) to the single KpnI site early in the gene and includes the 5’ end of the chymosin cDNA insert (see Fig. 3); another (called the 3’-terminal probe) extended from the KpnI site to the single HincII site in fl at the other end of the cDNA and includes the 3’ end of the cDNA insert; the third probe (called the complete probe) consisted of the entire recombinant phage DNA. The results are shown in Fig. 5. Sac1 and BglI, which do not cut in the cloned cDNA sequence, gave similar results (lanes S and B in Fig. 5, A, B,

135

and C). The 3’ terminal probe

hybridized

probe

to three

while the 5’-terminal

probe

hybridization expected

. If chymosin

placed

(more if there

inte~ening

that both

a single restriction

genes,

probe would be

to yield at least two bands

The observation

to only a

of two different

with either terminal

were appropriately

case,

with one of the three

using the other two probes

A and B were the products

contain

in each

hybridized

single band which comigrates observed

and the complete

bands

sequences).

Sac1 and Bg/I digests fragment

hybridizing

to

the 5’-terminal is only

probe

one bovine

restriction

strongly chymosin

endonuclease

suggests gene.

cleavage

that there

Conceivably,

might

produce

fragments of similar size from the 5’ terminus of two different chymosin genes, but the fact that two different

enzymes

a coincidence chymosin terminal

give the same result makes such

unlikely.

If there

is only

gene, then the observation probe hybridized

ment indicates

chymosin

that the 3’-

to more than one frag-

that there are intervening

in the genomic

a single

gene.

sequences

It appears

that

there must be at least two intervening

sequences

since

Sac1 and

three

bands

are seen with both

BgEI. The results obtained with EcoRI, a restriction enzyme which cuts the cloned cDNA sequence, are shown in lane E of Fig. 5, A, B, and C. Cleavage of the genomic DNA with EcoRI produced two bands which hybridized to both the complete and 3’terminal probes; only one of these bands hybridized to the 5’-terminal probe. There is only one EcuRI site in the cloned cDNA sequence and it is about 240 nucleotides to the 3’ side of the KpnI site used to make the 5’ and probes. In the absence of intervening containing

additional

EcoRI

sites,

3’-terminal sequences a

single

chymosin gene should, after cleavage with EcoRI, yield two bands that hybridize to the 3’-terminal and complete probes, and only one of these bands should also hybridize to the 5’-terminal probe. This is exactly what was observed. Thus these results also support the hypothesis of a single chymosin gene and further predict the absence of EcoRI sites in any intervening sequences. In this case, the singularity of the gene is indicated not only by the presence of the one band detected by the Y-terminal probe, but also by the observation that the 3’-terminal probe detects the expected two bands. In conclusion, the results of the gel-transfer experiments with digests of bovine genomic DNA Fig. 5. Gel-transfer

hybridization

cleaved bovine genomic derived

from the cloned

chromosomal

DNA

of restriction

DNA with radioactively preprochymosin

was digested

endonucleaselabeled probe

cDNA

gene. Bovine

to completion

with EcoRI

(lane E), Sac1 (lane S), or BglI (lane B), subjected phoresis hybridized

in an agarose

gel, transferred

with nick-translated

of the preprochymosin (see METHODS

probes

and

derived from the 5’ end

gene (B), or the entire DNA

for experimental

to electro-

to nitrocellulose,

details).

gene (C)

are most consistent with the existence of a single bovine chymosin gene containing at least two intervening sequences. Digestion with three different enzymes supports the idea that there is a single chymosin gene, and the results with two of the three enzymes provides evidence for the hypothesis that this gene contains at least two intervening sequences. Therefore, it seems most likely that the

136

highly homologous

enzymes

the products

of different

in the bovine

population.

chymosin

A and B are

alleles of the same gene

their intense

hybridization

made from total poly(A~-containing

unweaned that DISCUSSION

this

chymosin. chymosin

The

enzymology

mann,

have

and

been

1970), but relatively

the molecular

protein

studied

chemistry

extensively

little was known

biology of the mRNA

of

Pepsin,

to be the major

and the gene(s)

a typical

sig-

However,

judging

2) and a long

produce active chymosin, and raises the question, currently under investigation, of the ability of prokaryotic or other eukaryotic organisms to carry out these processing events. Reinspection of the chymosin amino acid sequence in light of recent research on the attachto proteins

(Hubbard

is

enzyme

in the

and

Ivatt, 1981) reveals that chymosin contains two regions with the consensus sequence for addition of asparagine-linked oligosaccharides. These are Asn-Leu-Ser (amino acids 310-312 of Fig. 4) and Asn-His-Ser (amino acids 349-3.51 of Fig.4). Whether or not these acceptor sites are actually used for attachment of oligosaccharides is presently under study. Our data support the notion that chymosin mRNA must be an abundant species in calf fourth stomach cells. A chymosin-related protein (preprochymosin, judging from the molecular weight) appears to be the major product of in vitro translation of poly(A)-enriched mRNA, and cDNA clones containing chymosin sequences are found at high frequency among clones that are selected by

from

the

in vitro

translation

results, pepsin must be only a minor product in the unweaned calf’s stomach cells. This apparent developmental switch from chymosin synthesis to pepsin synthesis may be worth further study. The nucleotide sequence data suggest strongly that the gene we have cloned corresponds to the prochymosin A of Foltmann et al. (1977; 1979). since

at position

prochymosin

302 aspartate

A) as opposed

(asparagine

of carbohydrates

to

acid sequence,

after

stretch of hydrophobic amino acids. This means at least two protein processing events are required to

ment

of

1970).

amino

at position

proteolytic

source

homologous

the calf is shifted to a grain diet (Foltmann,

in the B form. published amino

(arginine

is quite

at the level of amino

nal sequence (Steiner et al., 1980), contains the customary positively charged amino acid near the terminus

is a rich commercial which

of the

with the fact

about

synthesized in vivo is “preprochymosin”, a form of prochymosin containing 16 additional amino acids at the amino terminus. This sixteen amino acid to constitute

tissue

is consistent

same tissue at a later stage of development,

new information. Inspection of the nucleotide sequence of the cloned chymosin cDNA indicates that the protein

which appears

calf, which

is the major

of the abomasum

(Folt-

coding for the enzyme. The cloning of a cDNA copy of the chymosin mRNA has provided some

peptide,

known

product

cDNA

RNA.

Thus, it seems likely that chymosin in vivo protein

chymosin

to radiolabelled

is encoded

to the glycine

(as in found

The other difference from the acid sequence of prochymosin B

at position

2 18 instead

of the aspartate

found in the amino acid sequence of both prochymosins A and B) most likely represents a sponianeous deamidation during protein sequencing. However, the possibility that our cloned gene represents a third form of prochymosin has not been rigorously excluded. It should be noted that each of the differences that have been found can be accounted for by single nucleotide substitutions, and the differences do not involve the two aspartate residues known to be important for chymosin activity, aspartate 92 and aspartate 274 (78 and 261 by the numbering of Foltmann et al. ( 1979)). During the course of this work, Harris et al. ( 1982) reported the nucleotide sequence of a cDNA clone coding for calf preprochymosin B. The sequence differs from that of preprochymosin A reported here at six nucleotide positions. One of these differences, A to G at position 905 (*4 in preprochymosin A and G in preprochymosin B). results in an aspartate to glycine change at amino acid 302. This is the known difference between prochymosin A and B determined by Foltmann et al. (1979). Four of the other five differences (G to A at positions 45, 432, and 1008, and C to T at

137

position

817) involve

modified

nucleotides

as part of EcoRII

Since these differences

which would

recognition

appear

to be in regions

the DNA in which only one strand by Harris

et al. (1982),

are

likely

more

residues

(1982) rather

A and

to asparagine

may be an additional A and B forms; Foltmann

by Harris

B. The

G to A at position

aspartate

cytosine

than true allelic differences

preprochymosins ference,

changes

modified

which were misidentified

between difin an

acid change

allelic difference

however,

et al.

additional

688, results

amino

of

was sequenced

these nucleotide

to represent

be

sequences.

and

between

the

it was not observed

by

A and B apparently

differ by

only one or two base changes, then the genes must be very homologous. A probe derived from one of the

chymosin

genes

should

hybridize

with

any

other. When we used our cDNA as a probe in gel-transfer hybridization experiments with bovine genomic

W.D.

clones

and

DNA we detected

only a single chymosin

gene which contains at least two intervening sequences. Therefore chymosins A and B apparently represent different alleles of a single locus; if the aspartate to asparagine change is not a protein sequencing artifact, then the third form would represent a third allele. In any case, the gel-transfer results indicate that the chymosin gene is not a member of an extensive family genes encoding similar proteins.

of homologous

Davis,

R.W.:

by hybridization

Screening

to single

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196 (1977) 180-182. Boecke,

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B.E., Agarwal,

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full length

rat insulins

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fl. Mol. Gen. Genet.

Construction

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assistance,

Tom

Susan

and MacDonald, a mixture

Gravius Hayes

for expert

and Joya

technical

Minicucci

for

preparation of the manuscript. We thank David Botstein, Gerald Fink, Ronald Davis, and David Baltimore for helpful discussions and David Botstein, Gerald Fink, and Chris Goff for critically reading the manuscript. This investigation was supported by a contract from Dow Chemical Company.

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