Molecular cloning and sequence analysis of cDNA for human transferrin

BIOCHEMICAL

Vol. 119, No. 1, 1984 February 29, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 273-281

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF cDNA FOR HUMAN TRANSFERRIN Georges Claude

Uzar~(l’~),

Besmond(l), Mario

Monique

GBrard

Frainc3),

Maessen(l),

M. Zakin(*)

Insoo Park(*), Jo& Sala T~+pat(~)

and Axe1 Kahn(l)

MolBculaire, INSERM U. 129, CHU COCHIN 75674 Paris Cedex 14, France * Unit6 de Biochimie Cellulaire, Institut Pasteur, 28 rue du Dr. Roux 75724 Paris Cedex 15, France 3 Laboratoire d’Enzymologie du C.N.R.S. 91110 Gif sur Yvette, France

Received

1 Institut

de Pathologie

41nstitut

de Pathologie Cellulaire, INSERM U. 143 Hdpital 94270 Le Kremlin Bicetre, France

December

de Eic&tre

7, 1983

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3’ non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome. Transferrin

is the

species. It is composed together weight

with

major

the two N-linked

of 79 570 (of which

Transferrin

iron-binding

belongs

about

to a family

Interest

of a one gene expressed Burkitt

lymphoma

transferrin. essential

growth

factor

reported

to stimulate

activity

(5,6). Finally

partial

homology

which

exhibits

by the glucidic e.g. lactoferrin

lymphomas

growth

of lymphocytes

a melanoma

surface

of avian

egg

by the discovery in human

transferrin

(3). Transferrin

of

is an

has also been

of its iron

has been identified

transport

as sharing

a

(7).

as in large

has been reported

(1,2).

to the amino terminus

independently

antigen

moiety)

protein

and transport,

for cells in serum free medium

molecular

(31, and perhaps

sequence

binding

which

(or lactotransfer-

been stimulated

a homologous in iron

of vertebrate residues,

gives a calculated

is the iron binding

B cell

to its role

with transferrin

in man as well polymorphism

proteins,

has very recently

in chicken

(41, which

In addition

chains

6 % is contributed

of related

in these proteins

in the plasma

chain of 679 aminoacid

oligosaccharide

rin) found in milk and ovotransferrin white.

protein

of a single polypeptide

number

(8,9) ; about

273

of animal 20 human

All

species variants

a transferrin are known,

Copyright 0 I984 righrs of reproduction

genetic some of

0006-291X/84 $1.50 by Acudemic Press. Inc. in or,?: form reserved.

Vol. 119, No. 1, 1984 them with

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a frequency

variants

with

transferrin

of 1 % or more in the population.

chromosomal

markers

gene to chromosome

mia was reported

3 (10-14).

the

One family

in 1961 (15). Such a multiplicity

or transferrin-related of studying

has enabled

proteins

regulation

prompted

of the

of some transterrin

probable with

assignment

congenital

of phenomena

us to clone

gene(s)

Linkage

expression

under

atransferrine-

involving

transferrin

cDNA

normal

of the

transferrin with

and

the goal

pathological

conditions. MATERIALS All the methods used for RNA purification, construction of a human liver dscDNA recombinant plasmid library and RNA selection on nitrocellulose filters have been described in earlier papers (18-23). Differential screening of the cDNA library : Each of 10 different microtitration plates (containing 96 clones per plate) was incubated on 5 different agar plates, then transfered to nitrocel Hose BA 85 filters. The filters corresponding to each plate were hybridized with ( P) labeled cDNA reverse transcribed from 5 different mRNA preparations : lo-14 S, 16-17 S and 18-20 S sucrose gradient fractions of human adult liver mRNAs ; 18-20 S sucrose gradient fraction of human muscle RNA (19) and, finally, very pure rat sery albumin mRNA (21). The hybridization was performed for 48 h in presence of 5 x 10 cpm of labeled probe per ml. All the clones hybridizing with the human muscle and rat albumin cDNA probes were eliminated. Those remaining clones that displayed a more intense hybridization signal with the liver 18-20 S probe than with the probes corresponding to lighter mRNAs were selected : they represented 20 of the 960 screened clones. cDNA sequence : The nucleotide of Maxam and Gilbert (24).

sequences

were determined

Electrophoresis of RNA in agarose-formaldehyde on nitrocellulose filters (27,28) and hybridization inserts (29) have been previously described. High molecular slightly modified

weight cellular from reference

by the chemical

method

(25,261, transfert of RNA and DNA with nick-translated, purified cDNA

DNA (30).

was purified

tested

with

from

circulating

leukocytes

as

probes

as described

in

RESULTS Positive

hybridization-selection

20 out of the 960 clones methods with

were

albumin

preselected

: they corresponded

and muscle-specific

complementary

to 18-20

mRNA

These

species.

the 5 ss-cDNA to sequences

probes and hybridized

S liver

mRNAs

20 clones

were

than with tested

which

did not hybridize

more strongly

probes

by positive

with

complementary

ss-cDNA to lighter

hybridization-selection.

3

corresponded to Acu and 1 to BP fibrinogene chains (Uzan et al, in preparation), 1 gave a polypeptide of 76 000-80 OOO-Mr and another a polypeptide of 72 000-75 OOOMr (fig.

1) which

prothrombin

not cross-react

either

with

anti-fibrinogen

or with

anti-

antisera.

The clone candidate

did

encoding

for containing

was designated

the 72 000-75 transferrin

cDNA

OOO-Mr polypeptide sequences

as Tf 66 G2. 274

was considered

and was therefore

a good

sequenced.

it

Vol.

119,

No.

BIOCHEMICAL

1, 1984

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

m

w

Positive Plasmid

hybridization-selection analysis. DNA was bound to nitrocellulose filters, then hybridized with poly Acontaining human liver mRNA as indicated in “materials and Methods”. Eluted mRNA was translated in 25 ul of a reticulocyte cell free system, 5 ul of the translation re being dissociated and analyzed by SDS-polyacrylamide gel electrophoresis. ?“?I labeled plasma fibrinogen 2 to 6 : translation products obtained under direction of RNA eluted from filters bound to non-recombinant pBR 322 (21, plasmid 5 B8 (31, 60 El (4). 62 G12 (5) and 66 G2 (6). 0 : translation blank, without. addition of exogenous RNA. Plasmid 5 BEI contained cDNA for the B p fibrinogen chain and 60 El for the A Q fibrinogen chain. Plasmid 62 G12 insert has not yet been identified.

Sequence (fiq.

of

a cDNA

clone

sequence

contains

a 1.000

established

that

aminoacid

403

precedes

Kb

insert

the

1001 the

a 3’-non-coding AATAAA

tract.

The

results

of

Mac

instead

of

an Asp

(31)

of

the

human

et

transferrin

of a Gln

PstI

; 653,

site

166

in the

the

same

The

sequence

for

322.

structural

the

of a Thr

qene

from UAA

poly-adenylation

the

the

poly(A)

protein

instead

finally

also

codon

6 positions

a Thr

this

transferrin

24 bp from

with

that

obtained

canonical

following

and

results

termination

at minus

modification,

showed

human

precisely

; 542,

G.2

The

encodes

agrees

except

instead

66

translation

nucleotides.

cDNA

Tf

of pi3R

The

sequence al (2)

clone

fragment

of

a Pro

cDNA

terminus.

aminoacid

; 539,

the

nucleotide COOH

is present

Gillivray

of

in the

region

predicted

instead

position

sequence

: 417,

an

of a Pro

a Gly

instead

Asn

; 572,

a

of Glu

in

672.

Characterization Tf

of transferrin

66

G2

characterizing species

plasmid specific

was

fibrinogen

After

part

analysis

through

signal

mRNA

encoding

2). Nudeotide

Glu

clone

detected,

chain was

a long

more

mRNA abundant

autoradiographic

mRNA.

was mRNA of

labelled in adult about

(fig.

nick

and

2400

3). in

by

fetal

bases,

In contrast

fetal

(4

exposure,

translation

to

months

liver. i.e.

of

275

In both similar

ACX intra-uterine

a supplementary

and

used

as

tissues to

fibrinogen

the

a probe

a single length

mRNAs, life)

heavy

than

mRNA

for major

of

ACY

transferrin in

aduit species

liver. of

Vol. 119, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

410 GLY LEU VAL PRO VAL LEU ALA GLU ASN TYR ASN LYS SER ASP ASN CYS GLU ASP THR PRO T GGT CTG GTG CCT GTC TTG GCA GAA AAC TAC AAT AAG AGC GAT AAT TGT GAG GAT ACA CCA

430 GLU ALA GLY TYR PHE ALA VAL ALA VAL VAL LYS LYS SER ALA SER ASP LEU THR TRP ASP GAG GCA GGG TAT TTT GCT GTA GCA GTG GTG AAG AAA TCA GCT TCT GAC CTC ACC TGG GAC 450 ASN LEU LYS GLY LYS LYS SER CYS HIS THR ALA VAL GLY ARG THR ALA GLY TRP ASN ILE AAT CTG AAA GGC AAG AAG TCC TGC CAT ACG GCA GTT GGC AGA ACC GCT GGC TGG AAC ATC

470 PRO MET GLY LEU LEU TYR ASN LYS ILE ASN HIS CYS ARG PHE ASP GLU PHE PHE SER GLU CCC ATG GGC CTG CTC TAC AAT AAG ATC AAC CAC TGC AGA TTT GAT GAA TTT TTC AGT GAA 490 GLY CYS ALA PRO GLY SER LYS LYS ASP SER SER LEU CYS LYS LEU CYS MET GLY SER GLY GGT TGT GCC CCT GGG TCT AAG AAA GAC TCC AGT CTC TGT AAG CTG TGT ATG GGC TCA GGC

510 LEU ASN LEU CYS GLU PRO ASN ASN LYS GLU GLY TYR TYR GLY TYR THR GLY ALA PHE ARG CTA AAC CTG TGT GAA CCC AAC AAC AAA GAG GGA TAC TAC GGC TAC ACA GGC GCT TTC AGG

530 CYS LEU VAL GLU LYS GLY ASP VAL ALA PHE VAL LYS HIS GLN THR VAL PRO GLN ASN THR TGT CTG GTT GAG AAG GGA GAT GTG GCC TTT GTG AAA CAC CAG ACT GTC CCA CAG AAC ACT 550 GLY GLY LYS ASN PRO AS? PRO TRP ALA LYS ASN LEU ASN GLU LYS ASP TYR GLU LEU LEU GGG GGA AAA AAC CCT GAT CCA TGG GCT AAG AAT CTG AAT GAA AAA GAC TAT GAG TTG CTG 570 CYS LEU ASP GLY THR ARG LYS PRO VAL GLU GLU TYR ALA ASN CYS HIS LEU ALA ARG ALA TGC CTT GAT GGT ACC AGG AAA CCT GTG GAG GAG TAT GCG AAC TGC CAC CTG GCC AGA GCC 590 PRO ASN HIS ALA VAL VAL THR ARC LYS ASP LYS GLU ALA CYS VAL HIS LYS ILE LEU ARG CCG AAT CAC GCT GTG GTC ACA CGG AAA GAT AAG GAA GCT TGC GTC CAC AAG ATA TTA CGT 610 GLN GLN GLN HIS LEU PHE GLY SER ASN VAL THR ASP CYS SER GLY ASN PHE CYS LEU PHE CAA CAG CAG CAC CTA TTT GGA AGC AAC GTA ACT GAC TGC TCG GGC AAC TTT TGT TTG TTC

630 ARG SER GLU THR LYS ASP LEU LEU PHE ARG ASP ASP THR VAL CYS LEU ALA LYS LEU HIS CGG TCG GAA ACC AAG GAC CTT CTG TTC AGA GAT GAC ACA GTA TGT TTG GCC AAA CTT CAT

650 ASP ARG ASN THR TYR GLU LYS TYR LEU GLY GLU GLU TYR VAL LYS ALA VAL GLY ASN LEU GAC AGA APC ACA TAT GAA AAA TAC TTA GGH GAP GAA TAT GTC AAG GCT GTT GGT AAC CTG

670 ARG LYS CYS SER THR SER SER LEU LEU GLY ALA CYS THR PHE ARG ARG PRO AGA AAA TGC TCC ACC TCA TCA CTC CTG GGA GCC TGC ACT TTC CGT AGA CCT TAA

AAT CTC

AGA GGT AGG GCT GCC ACC AAG GTG AAG ATG GGA ACG CAG ATG ATC CAT GAG TTT GCC CTG

GTT TCA CTG GCC CAA GTG GTT TGT GCT AAC CAC GTC TGT CTT CAC AGC TCT GTG TTG CCA

TGT GTG CTG AAC AAA AAA TAA AAA TTA TTA TTG ATT TTA T-poly

A

Fig.

Nucleotide and deduced amino acid sequences of the human transferrin clone. The given nucleotide sequence is that of the noncoding strang. The deduced amino acid sequence for the correct reading frame is showed. numbers correspond the residue number in the human serum transferrin protein.

66 G2 cDNA

276

to

Vol.

119,

No.

BIOCHEMICAL

1, 1984

AND

BIOPHYSICAL

Fiq. liver

3. Northern blot analysis of mRNA mRNA with different cDNA probes. 2pg poly A-containig RNA were deposited 1 : human adult liver ‘2 : human fetal liver,(4 months of gestational

3 : rat adult liver to the transferrin probes -t’(61

are 69)

about

5000

liver

mRNA,

bases

for

the

could

three

human

in each

fibrinogen

be detected.

detecting

adult

and

COMMUNICATIONS

fetal,

human

and

rat

slot.

age) liver (17 days) of gestational age). 66 G2 corresponds in this paper ; the three other recombinant plasmid

; 4 : rat fetal probe studied

specific

from

RESEARCH

Tf

a mRNA

chains

66 G2

species

with

: Ac~(60

plasmid

El,

Bfl(5

hybridized

similar

68)

and

slightly

molecular

with

weight

to

rat

human

transferrin. Screeninq

of human

Among

the

transferrin albumin

(about

2-3

Hybridization

probe

similar

changed 65O

C for

for

SSC

for

the

gave

some

other

3 with

Eco When

of 214,

364

of

clones,

be

compared

fibrinogen

genomic

analysis

cDNA library

fig. 4

4.

The

the

Tf 66 G2 insert.

2 hybridized to

clones

used.

strongly

the

with

percentage

(l-l.5

In rat,

%

we

found

of

the serum

according

to

2 positive

clones

the

final

washing

in 0.1 in

high

2 x

x SSC SSC,

patterns,

with

did

in at

both

at

9 to 10 bands

Tf

or

not

significantly (2 x SSC

non

65O)

66 G2

enzymes

stringent

65O)

weight

the

restriction

and

performed

molecular

with

9 different

samples,

were

washing

fragments

with

DNA

washing

predominant complex

4). restriction

obtained

leukocyte final

final

a single

(fig. DNA

pattern

individual

hybridization,

more

DNA

of genomic

hybridization,

ones

cDNA

cDNA

with

stringent

(6 x

conditions.

fragment

(such

with

HI,

Barn

at

Some as

XbaI),

4 with

PstI,

RI. the

insert

and

423

nick-translation, uncleaved

and

hybridization,

enzymes

human can

human

human

in

when

libraries

screened.

blot

is shown

was

%)

with

Southern

cDNA

proportion

in the

colonies

liver

screened

This

chains)

of 576

rat

1000

probe.

different out

and

then probe

gave

was bases.

cut These

hybridized 3 Eco

by

PstI

and

fragments to

the

RI fragments,

Hind were

Southern

III,

three

fragments

individually blots.

of 8, 4 and

purified While

1.6

were

k bp,

and

hybridization the

423bp

generated, labeled

by

with

the

3’ Hind

III-

Vol. 119, No. 1, 1984

L:;b,4.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Southern

blot

analysis

of human

leukocyte

DNA

with

the Tf 66 G2 insert

as a

10 ug DNA were digested to completion with the various restriction enzymes indicated under the figure, then electrophoresed in a 0.7 % (w/v) agarose gel for 14 hours) at 1.5 volts/cm. After transfer to nitrocellulose, the blot was hybridized with the nick-translated insert, as indicated in Materials and Methods 10 cpm/ml, 18 hours hybridization). Washing was performed under stringent conditions (0.1 % SSC + 0.5 % SDS at 65O C). Exposure time : 36 hours at - 80° C with intensifying screens.

PstI

fragment

detected

bp PstI-Hind way,

III

out

kbp

of

the

the

fragment the

only

with

and 4 and

hybridizing

detected

(not

8 kbp only

fragments

were

5’ probes

the

detected

4 PstI

fragments

with

mainly

fragments

with the

the

1.6

the

5’ 214

kbp

bp PstI-PstI

fragments.

uncleaved

3’ probe

and

probe,

the

4.2

and

and

364

in

the

same

the

6.8

and

3.8

3

fragments

shown).

DISCUSSION To mRNAs 18-20

screen

the

expressed S, albumin

medium

mRNA

abundancy

plasminogen, clones

we have for

insert

revealed

Bfi

such

direction

of

been

unidentified.

as those

mRNA to

more

mRNA-specific and

etc... able

value

cDNA

probes for

72

with

this a

this

000-75

approach the

: among

7 fibrinogen

16-17

group

3 clones

was

000-80 was

confirmed with

these

clones

we

chains

(Uzan

et

of

band

Mr

were al,

A fith

1 G2

clone, remains

studying

the

clones

or

heavier

liver

lighter able

and

Tf66

under

polypeptide, by

the

III,

ACX

the

synthesized

pretransferrin. 000

of

of 20 such

for

determination

S than

278

mRNAs antithrombin

a first

Mr

to around

liver-specific

From

000

plasmid 76

corresponding coefficient

prothrombin,

group.

sequence

the

encoding

with

this

clones

that

fibrinogen, to

Nucleotide that

of

those

a sedimentation

by hybridization-selection

chains.

strongly

4 clones

encoding

to identify

mRNA

selected with

It is expected

belong

hybridizing a

we

in muscle,

excluded.

unambiguously

The

hybridizing

library,

not

being

fibrinogen

corresponding

60

cDNA but

transferrin,

clone

for

human in liver

to identify

in preparation),

4 clones plus

clones

Vol.

119,

No.

BIOCHEMICAL

1, 1984

corresponding

to

five

as

yet

AND

unidentified

BIOPHYSICAL

RESEARCH

liver-specific

proteins

COMMUNICATIONS

(Mr

from

32 000

to

44 000). In

most

selection For

cases,

with

the

we have

this seric

sequence

of

accounted being

for Pro

instead correspond

either errors

Gillivray

to

in the

paper

coding

(2).

therefore

of

Taking

be of the Transferrin

(about

relative

(16-19).

antibodies

raised

(not

G2

shown),

with

the

6 differences.

as

of

well

in

position

the be

residue

542 ;

for

and

they

or

published

to that

Gly could

transferrin,

sequence

as compared

at could

deduced

understand

described The

which

(the

Pro to

protein

Three,

change,

differences

difficult

modified

66

- Asn

instead

during

by

reported

to

Mac

in their

that

observed

the

probe

a single,

was

the

5’ part

679

3’ non-

aminoacid

sequence

liver

of

likely

should

a fetus

at

explanation

oestrogens

of

4

is the

(32-33),

or a low

to

those

number

which

which

in that

or

are

these

related

members

different

hybridizing

observed B or L-type of the 279

for

a same

transferrin

gene

stringent

bands

could probes

fragments,

sites

inside

of a gene

family.

observed amount

copies

one when

Moreover,

kinase

G2

than

non

different

to

66

change

different

bands

pyruvate

Tfl

more

or

with

the

parallel

not

genes.

of restriction

libraries

the

used, did

by cleavage

the

with

stringent

hybridized

cDNA

is roughly

bands

either

insert

rat

fragments

these

pseudo-

of

aldolase

and

enzymes

of

the

intensity

similar

albumin,

hypothesis

with

chains,

for

performed

to be generated

the

the

in human

restriction

hybridizing

by hybridization

fibrinogen

the

against

3’ or

the

coding

most

by

clones

intensity were

cross

therefore than

all

relative

washing

Quantitatively,

that

transferrin

almost

argues

short

pregnancy. of

restriction

of

suggest

induced

DNA

which

to

synthesis

in

The

genomic

The

for

5’ non

abundant

(34-35).

with

coding the

liver.

proteins

to partially

probes

more

human

of these

correspond

transferrin

be

rate

conditions,

rather

to

synthesis

the

a relatively

bases.

of

of

with

bases

A tract,

1:lO

and

seem

long,

2037

poly

in adult

level

band.

constituted

and the

seems than

bases

is about

hybridization

gene

Tf

reveals

determination.

of 150-200

frequency

revealed

which

nucleotides account

transferrin

Hybridization

recognized

166

of

observed

3 %)

probe

Thr

2400-2500

into

age

at a high The

is about

mRNA

stimulation

secreted

clone

other

more

already

the

transferrin

or Asp

polymorphism,

was

order

gestational

known

(2)

three

are

sequence

(2)

al

539,

672)

products

because

of

The

protein

RNA

sequence

months

et

position

neosynthesized

hybridization-

(1).

Transferrin

residues

sequence

reasons. in

by

(16).

a Glu-Gln

a protein

et al in 1983

1982

the

Thr

the

clones

neosynthesized

involves

position

preselected

successful

pre-prothrombin

Gillivray

653,

of in

not

nucleotide

by technical

Glu

of

was

Mac

and

instead of

minor

by

572

identification

recognized

for

the

published 417,

the

poorly

shown

Comparison

characterize

approach

protein

previously

positions

first

immunological

transferrin,

against

we

of

(not

a

single

with

the

DNA

shown)

exist.

with which

It appears

Vol. 119, No. 1, 1984 therefore different result

that,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at least

members

with

does not preclude

genomic

transferrin

availability regulation

the cDNA

probe

of the transferrin-related

of human mechanisms

the possible

DNA

for

and rat

utilization

identifying transferrin

of the transferrin

used in this work

protein some cDNA

family

in the future related probes

gene expression

the genes for the

are not recognized. of some subclones

genes. will

permit

in response

In addition, to anafyse to hormones

This of the the and

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