Molecular cloning and characterization of a cDNA clone for a protein specifically expressed in embryo as well as in a chemically induced pancreatic B cell tumor of rat

Vol. 124, No. 1, 1984 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15, 1984

Pages

164-l 71

MOLECULAR CLONING AND CHARACTERIZATION OF A cDNA CLONE FOR A PROTEIN SPECIFICALLY EXPRESSED IN EMBRYO AS WELL AS IN A CHEMICALLY INDUCED PANCREATIC B CELL TUMOR OF RAT Gen-Ichiro Department

Received

August

Soma,*

Namiko

Kitahara,and

of Hygienic Chemistry, 1-22-1 Yato-cho, Tanashi,

30,

Toshiwo

Andoh

Meiji College of Pharmacy, Tokyo 188, Japan

1984

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were A plasmid cDNA library consistpreviously demonstrated not to be expressed. ing of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32 P-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/ acidic residues being 1.63. 0 1984 Academic press, IX.

The wide

variety

transformed

and normal

strate

that

complex

Thus,

although

of normal embryo

(7),

human

and the

however,

haps

1,000 of

have

reported

viruses

has been

and their same cells hepatoma recently

specifically

Augenlicht expressed

found

that

line

between

(6),

involving and Kobrin

the

(3) demon-

tranformation. mRNA populations

counterparts with

activation

between

differences

accompany

transformed cell

differences

as antigenic

SV40-transformed

may be more extensive,

new transcripts.

sequences

as well

biochemical

in gene expression

homology

fibroblasts

and other

(1,2)

alterations

and a minimun-deviation

transforming

ing

cells

extensive

fibroblasts

liver

of enzymatic

bezopyrene Groudine of host the

and Weintraub cell

genes

Copyright All rights

$1.50

0 1984 by Academic Press. Inc. of reproduction in any form reserved.

by

of per-

succeeded

in clon-

tumors.

*To whom all correspondence and reprint requests should be addressed. Abbreviations used: cpm, counts per minute; SSC, standard saline citrate, M NaCl, 0.015 M sodium citrate. 0006-291X/84

hamster

(5) and rat

appearance

(8) have

in a mouse colon

(4),

0.15

Vol. 124, No. 1, 1984

BIOCHEMICAL

Furthermore, are expressed liver

in various

during

embryonal

and carcinoembryonic

sed in

transformed of the

analyzed,

and were

one)

(11-17).

normal

(2).

cells

cells

related of

to normal

PDGF, platelet

respectively. sion

In

of genes

specifically ing

normal

rat

pancreatic

paper embryo

growth

order

to elucidate

differentiated B cell for

met these

cells. tumor the

role

to search

as well To this

time

been

in rat

as in embryos

isolation

normal of

exprestrans-

oncogenes

(v-

detected

development found

sis

to be closely

grcwth

embryo not

expres-

the

genes

in correspond-

chemically

B cells.

two such

for

part

factor,

oncodevelopmental

but

in

(23,

and erb B being

end we employed

and corresponding first

prenatal

of such

are

has been

and EGF, epidermal

the

in fetal

malignant

of viral

e.g.

which

and extensively

genes

recently (251,

factor,

we attempted

c-one during

factors

derived

in tumors

these

for

isolated

homologues

have

tracts

responsible been

especially

growth

in general

we describe which

cellular

expressed

recently

gene products

genes (9)

gastrointestinal

genes

of some of (18-22),

24) ; some of the c-one

in

to be cellular

Transcription

vertebrate

(10)

have

some of the

such as a-fetoprotein

Cellular

(c-one)

revealed

of malignancies

development

antigen

cells

formation

forms

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

induced

In the clones

from

present rat

criteria.

MATERIALS --AND METHODS Northern and Southern Blot Hybridization: Pregnant Wistar rats were purchased from Tokyo-. Jikken Dohbutsu Co., Ltd., Tokyo. At day 12 of prenatal development embryos were isolated and polyadenylated (poly(A)+) RNA was prepared as described previously (26). Similarly poly(A)+ RNAs were isolated from rat Langerhans islet B cell tumor induced by streptozotocin and nicotinamide (2729) and normal adult islets which consist mainly of B cells (29). 32P-labelled cDNA probes were synthesized as described previously (26) by incubation at 42OC for 90 min in 20 ~1 of reaction mixture containing 50 mM Tris-HCl, pH 8.3; 30 mM NaCl; 10 mM MgC12; 0.5 mM dithiothreitol; 4 mM Na-pyrophosphate; dATP, dGTP and dTTP at 1 mM each; 10 uCi of [a-32P]dCTP (410 Ci/nnnol, Radio1 unit of avian myeloblastosis virus reverse transchemical Center, Amersham); criptase (EC 2.7.7.49) purchased from Seikagaku Kogyo, Inc.; 50 ng poly(A)+ RNA. The specific activity was aboy; 1 x 108 cpm/ug. Electrophoresis and P-labelled probe were carried out as hybridization of poly(A)+ RNA with RNA dissolved in water were glyoxalated in described by Thomas (30): Poly(A)+ 1 M glyoxal in 50 % dimethylsulfoxide containing 10 mM phosphate buffer, pH 7.0 for 1 hr at 50°C, and 100 ng each of the RNA was subjected to electrophoresis for 3 hr at room temperature on 1.2 % agarose gel in 10 mM phosphate buffer, pH 7.0. After electrophoresis RNA was transfered to nitrocellulose paper. After baking for 1 hr at 80°C the blot was prehybridized for at least 4 hr at 42OC in hybridization buffer containing 5 x SSC (Standard Saline Citrate); 50 mM phosphate buffer, pH 6.5; 1.0 x Denhard's solution; 250 ug/ml of sonicated 165

Vol. 124, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Subsequently the blot was and denatured calf thymus DNA in 50 % formamide. hybridized for 18 hr at 42OC with 5 x lo5 cpm of 32P-cDNA probe per ml of hybridization buffer except that the concentrations of all components were After hybridization decreased to 0.8 x and contained 10 % dextran sulfate. the blot was washed three times in 2 x SSC at 42OC for 0.5 hr and exposed to X-ray films (Kodak X-AR) with intensifying screen at -8OOC. Plasmid DNAs isolated from cDNA clones of E. coli and one pg each of the DNA was electrophoresed and transfered to nitrocellulose paper as described by Southern (31). Blots were hybridized with 32P-cDNA probes prepared from normal islet 5 cells or B cell tumor. Preparation of cDNA Clones: Construction of cDNA library from rat embryo was --carried out according to the method of Goodman and MacDonald (32) with some modifications (to be described elsewhere). Five )ig of poly(A)+ RNA from embryo was used for the preparation of cDNA. By ligating 240 ng of double-stranded d(C)-tailed cDNA into d(G)-tailed PstI site of pBR322 DNA and transfecting E. coli with the recombinant molecules we obtained 4.8 x lo4 tetracyclin resistant and ampicillin sensitive transformants. The transformants were colonyhybridized (33) with 32 P-cDNA probes prepared as described above. Nucleotide Sequence Analysis: Nucleotide sequence was determined by the chemical degradation method of Maxam and Gilbert (34). Restriction enzyme fragments were labelled at the 3' end with calf thymus terminal deoxynucleotidyl transferase and [s-32 PldideoxyATP (500 Ci/mmol, Radiochemical Center, Amersham) or labelled at the 5' end with T4 polynucleotide kinase and [Y-~~P]ATP (1,000 Ci/mmol, Radiochemical Center, Amersham). Single end-labelled DNAs were prepared for sequence analysis by secondary cleavage with another restriction endonuclease.

--RESULTS AND DISCUSSION RNAs were

Poly(A)+ streptozotocin sist

of B cells.

electrophoresed

on an agarose with

32P-cDNA

in tumor

cell

poly(A)+

however,

we could

ion

cell

blotted

we carried

one having

the

other

slightly

from

0.8

poly(A)+

tumor

not

RNA species

Mr of 0.8 smaller

slightly

experiment with

cells.

Mr and hybridizing 166

only

and

embryos.

As detected

of the bases

further

filter,

only

Figure

in which

2 clearly

poly(A)+ with

only

tumor with

in

confirmatfilter

32 P-cDNA synthesized

among embryonic

kb and hybridizing

For

con-

RNAs were filter

less

shown).

a reciprocal

or normal

these

exposure

by

which

was specifically

a longer

with

induced

islets

RNA from

(kb)

Under

(data out

pancreatic

kilobases

transcript

tumor

to nitrocellulose

RNA was hybridized

either

are two prominent

with

another

poly(A)+

RNA from

prepared with

B cell

by glyoxalation

transfered

RNA population

results

embryonic

poly(A)+ there

poly(A)+

of these

isolated

RNA preparation.

detect

pancreatic

denaturation gel,

probe

1, a transcript

rat

and from

After

shown in Fig.

normal

from

and nicotinamide

mainly

hybridized

isolated

from

shows that

RNA populations, probe

normal

and the probe,

confinn-

Vol. 124, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.3kb-m 0.8kb-

4-0.7

kb

O.Bkb-

02

0

1

Figure 1. Tumor-specific expression of genes which are also expressed in embryos. Poly(A)+ P.NAs were isolated from rat Langerhans islet B cell tumor, normal adult islets and embryos at day 12 of prenatal development. PO&'(A)+ l?NAs from normal islets and islet B cell tumor were glyoxalated and 100 ng each of the ENA was electrophoresed on agarose el and transfered to nitrocellulose filter. The filter was hybridized with 92 P-cDNA probe synthesized from RNA of embryos, and autoradiographed. poly(A)+ Lane 1, B cell tumor poly(A)+ RNA; lane 2, normal islet poly(A)+ RNA. 'The arrow indicates the position of RNA with approximately BOO bases which was determined using glyoxalated HaeIII digests of $X174 replicative form DNA. Figure 2. Expression in embryos of genes which are specifically expressed in B cell tumor. Poly(A)+ RNA from embryos was2electrophoresed, transfered to nitrocellulose filter, and hybridized with P-cDNA prepared from B cell tumor or normal islet poly(Aj+ RNA, as described in Figure 1 and Materials and Methods. Lane 1, embryo poly(A)+ RNA hybridized w'th tumor 32P-cDNA; lane 2, embryo poly (A)+ RNA hybridized with normal B cell 31 P-cDNA.

ing

the

result

possesses

Mr of about These

that in

are

results

the

thousand

of

these

were

We found

embryo

probe,

that indicating

from all

that

are

as well

embryonal

from

embryo,

obviously

selected B cell

possessed

167

RNA species

such genes

as in B cell

qenome

tumors

but

as well

a plasmid

RNA of

in rat

importance

development them from

selected CDNA inserts,

to elucas in tumor

12 day embryos.

and normal were

not

cDNA library

and colony-hybridized tumor

which

probes.

of great

messenger

transformants they

there

in

randomly

of the

both

is

to isolate

a third

with

It

constructed

synthesized

RNAS.

genes

end we attempted

transformants

that

in embryos

B cells.

tranfonnants

cDNA probes

we detected

suggest

expressed

function

48,000

In addition

strongly

normal

To this

cells.

1.

1.3 kb and hybridizes

specifically

corresponding

idate

of

of Figure

B cell

One

with

32P-

poly(A)+

positive and 150 out

with of

1,000

Vol. 124, No. 1, 1984 were

positive

normal

with

and tumor

only

were

blot

probes.

found

(data

shown).

not

two has been out

using

of

the

cDNA insert

confirm

the

were

the plasmid

specific

expression

800 bases

ly demonstrated in both

one of the encoded a key (23)

cloned

that

that

required

those

for

tumor

genes

embryonal

not

related

were

not

expressed,

hybridize

with

the

above

the

function

of the

acid

composition

pare

its

sequence

oncogene

products,

clone

and further

pRET2

and sequence with

oncogenes

those

a-fetoprotein

it

of the

is

the

of

cDNA

the

findings

it

This

plasmid.

as v-myc,

that

the

interest product

or carcinoembryonic

pRRT2 gene

further

al.

during

strongly

sug-

are also

revealed v-src,

play

et

expressed

cDNA clone

of great

of oncodevelopmentally

pRET2 being

may possibly

result

analysis

about

unequivocal-

carcinogenesis

to get

RNAs

Milller

were

to

expressed

the

However,

and abl

to

In order

cDNA clone

such

putative

and blot-

pRHT2 mRNA with

What role

with

the

was car-

poly(A)+

insert

In order

168

of

mRNAs specifically

Preliminary

v-oncogenes.

tumor

These

tissues.

associated

development.

and v-kis

as fos

in normal

insert

long,

as in carcinogenesis.

such

intimately

some virally

RNA.

genes.

as well

probe

mRNA corresponding

the

to be elucidated.

some oncogenes but

that

and thus

expressed

remains

development

the

32P-cDNA

tumor

rat,

tumor

analysis

800 bases

one of the

of

in embryogenesis

revealed

gested

we have

plays

the

only

blot

to

two plasmid

longer

mRNA in B cell

with

in B cell

oncodevelopmentally

function

the

demonstrated

tumor

Finally

32P-nick-translated

was approximately of

probe

RNA was electrophoresed with

with

and subjected

with

3 shows that

only

and B cell

protein

embryonal

cell

that

embryo

Figure

4 clearly

was detected

the

tumor

therefrom,

Northern

embryo:

rescreened

with

as pRRT2 harboring analyses.

and probed

shown in Fig.

positive

reaction

and hybridized

pRHT2.

were

probes.

positive

further

filter

blotted

clones

and normal

denoted

of

similarly

Results

tumor

RNA from

plasmid

150 clones

DNAs were prepared

clearly

to

poly(A)+

the

candidate

One clone

to nitrocellulose

insert

Forty

gave

subjected

These

probe.

using

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tumor

and plasmid

analysis

DNAs were

ted

B cell

selected

Southern

ried

BIOCHEMICAL

that

in B

v-yes,

v-mos

pRET2 did

not

insight

into

to know the

amino

of the

gene and to com-

expressed

genes

antigens.

For

such

as

this

pur-

Vol. 124, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

2 *

O.Okb

Figure 3. Northern blot hybridization to detect in embryo the mRNA corresponding to the clone pRET2. Hundred ng of poly(A)+ RNA from rat embryo was glyoxalated and electrophoresed on agarose gel fo lowed by transfer to nitrocellulose filter. The filter was hybridized with 32P-nick-translated insert DNA of was done as described in Figure 1. pRET2. The size estimation Fi ure 4 Expression of pRET2 gene in B cell tumor of rat. Fifty ng of poly +-(A) RNAs -- from B cell tumor (lane 1) and normal B cells (lane 2) were glyoxalated and electrophoresed on agarose gel followed by transfer to nitrocellulose filter. The filter was hybridized with 32P-nick-translated insert DNA of pRBT2.

poses

we determined

PRET2S

Figure

poly(A)

tract

entire ing

codon

was temporarily

arginine histone

was compared

with

spanning the

nucleotide

H2B in amino those

Although

the

5'

deduced

acid

plus

upstream

number is

of glutamic composition.

of known oncogenes,

169

there

basic, acid

excluding

tract

2 and 3),

no possible

longest

360. i.e. plus

When the

If

almost

appraoch-

Therefore,

to the

clone

covers

poly(A)

1 through

highly

the

insert

region.

according

of

cDNA insert

pRET2 mRNA (see Figs.

in the

vs 16 residues

cDNA insert

of 615 nucleotide

determined

protein

of the

615 nucleotides

for

ATG was found

ment was correct,

resembles

sequence tails.

estimated

frame

plus

whole

of pRRT2 mEWA, i.e.

alignment

lysine

sequence

and poly(dG).(dC)

800 nucleotides

open reading

nucleotide

5 shows the

region

initiation acid

the

amino possible

this

align-

26 residues aspartic nucleotide

was no homology

of

acid,

and

sequence between

them,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 1, 1984

1 30 Ser Ser Ser Ser Ser CZy Ser Asp Ser Asp Ser Clu VaZ Glu Lys Lys Leu Lys Aq

60 Lys Lye Gln VaZ

5'UjG-GGGTTTCTTCGW:TCITCAGGCAGT69TTCIGACAU:69Am64AA9APAGTTAPAGAGGAAAIW;W\m 90

Val Pm GZu Lys Pm Val Lys Lys Gin Lys Pm Ser CZu Ser Ser Aq

120 AZu Lou AZa Ser Ser Lys Cln Ser

mccAGAGAAACCTGTGPdGA9GU\AAAGCCTAGTWIGAGTTCTAGAGCA~GCATU:TU:APGWjAGT 150 180 210 Ser Ser Ser Atg Asp Asp Am Met Phe GZn IZe Gly Lys Met Arg TOP VaZ Ser VaZ Arg Asp phe ‘ys GZy

PU:~PU:AGAGATGATA4CATG~CICATTGGA~ATGAGATATGTCAGImcu;GACmAAAGGA Lys IZd Leu IZe Asp Ite

240 Arg GZu Tyr TV Met Asp Ser CZu CZy Gtu Met 4s

270 Pm Arg Arg Lys GZy Ile

~AATTcTAATTGATATTpGAGAATATTGGATGGAcT~~u;TGc\AATG~w\~AGGw\AIv\AATT 330 Lys GZu Gin IZe Ser Asp IL

300 Ser La, Am Met GZu GZn Trp Ser GZn ku

360 Asp Asp AZa VaZ Arg Lys Leu Term

TcT~AApcATGw\cAATffiAu:wj~AAGc4Aw;ATCTcTGAcATAu\T64cGo\GTAw\ApGcTGT~

Figure 5. Nucleotide sequence of NucleotTde residues are numbered idues of TCT triplet encoding the putative protein. The predicted nucleotide sequence.

suggesting induce

that

the

a wide

clonedgenes

spectrum

pRET2 cDNA insert and its amino acid sequence. in 5' to 3' direction begining with the respredicted first amino acid serine of the amino acid sequence is displayed above the

aredistinct

of malignancies

from

including

those

oncogenes

sarcomas,

known

to

carcinomas,

lymph-

omas and leukemias.

It sion

has been widely

and hence

Our results isolating

likely

lend

support which

as in malignant

such genes the

to this should

cloned

genes

of various

that

pattern

of malignant

transformation

contention

are hitherto involved

of cells. Although

uncharacterized in prenatal of tumor 170

various

roles

gene expresincluding

of

cells

a general in

possibility oncogenes,

like

(l-6). method

prenatal

We should

development cells.

of

enzymes

and establish

some important

genes".

properties

in the of

transformation

genes

change

constitution

play

as "oncodevelopmental

be among those

manifestation

of the

as a consequence

genes

as well

that

alteration

ensues

isozymes

accepted

of

development to refer

still they as well

to

remains could

more

as in

the

Vol. 124, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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