Vol. 185, No. 3, 1992 June 30, 1992

BIOCHEMICAL

The cDNA cloning, intracellular

AND BIOPHYSICAL

nucleotide

protein

Hitoshi

sequence

tyrosine

Miyasaka

Institute

and Steven

of

National Research Triangle

and expression

phosphatase

Laboratory National

RESEARCH COMMUNICATIONS Pages 818-825

of

from

S.-L.

of

mouse

an

testis

Li*

Genetics

Environmental

Health

Institutes of Health Park, North Carolina

Sciences 27709

Received Nay 8, 1992

The PTP-1 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase) was isolated and sequenced from a mouse testis cDNA library. This PTP-1 cDNA was found to contain an open reading frame of 1,296 nucleotides as well as 5' (83 nucleotides) and 3' (289 nucleotides) non-coding regions. The deduced sequence of 432 amino acids of mouse PTPase-1 exhibited 93% and 83% identity to that of rat PTPase-1 and human PTPase-lB, respectvely. Thus, this PTP-1 is a mouse homologue of human PTP-1B and rat PTP-1. Northern blot analysis indicated that PTP-1 mRNAs were most abundant in testis, and were detected in sizes of 4.4Kb, 2.4Kb and 2.2Kb, 2.OKb. The PTP-1 transcripts of 4.4kb and 2.OK, but not 2.4Kb and 2.2Kb, were also present in kidney, spleen, muscle, liver, heart and brain. Genomic blot analysis showed that a single copy of the PTP-1 gene is contained in the mouse genome and that introns are present in mammalian PTP-1 genes. 0 L99.?Academic mess, 1°C.

Protein the

tyrosine

control

of

phosphorylation

cell

growth, proliferation, The level of cellular (l-2).

transformation

is determined by the relative kinases (PTKases; EC2.7.1.112) (PTPase;

EC3.1.3.48).

that

not

simply

* To whom

only

correspondence

Abbreviations used are: tyrosine PTKase, protein 0006-291

Copyright All rights

X/92

Inc. reserved.

Recent

reverse

should PTPase, kinase.

$4.00

0 1992 by Academic Press, of reproduction in any form

implicated

and neoplastic phosphotyrosine

activities of protein and protein tyrosine

phosphatases PTPases

has been

818

the

evidence effects

tyrosine indicates of PTKases

be addressed. protein

tyrosine

phosphatase;

in

Vol.

185,

No.

3,

BIOCHEMICAL

1992

by dephosphorylating

the

important

roles

PTPase-1B

was purified

its

acid

amino

in

AND

protein

BIOPHYSICAL

substrates,

cellular

regulation

to

homogeneity

sequence

RESEARCH

determined

but

(3-4). from

also

A low

play Mr

human placenta With

(5-6).

COMMUNICATIONS

the

and

use of

oligonucleotide

probes/primers derived from this protein cDNAs encoding low Mr PTPases have been cloned from sequence, human placenta (PTP-lB), human T-cells (TC-PTP), human HeLa human breast carcinoma (PTP-lC), rat brain cells (PTPHI), (PTP-l),

rat

spleen

We are

studying

expression PTK genes

during such

and rat

(PTP-S), the

developmental

mammalian

as c-abl,

striatum regulation

spermatogenesis

pim-1

and

(STEP)

ferT

of

(15-16). have

been

(7-14). gene Several

reported

exhibit stage-specific expression during spermatogenesis 21) . To investigate PTP gene expression during spermatogenesis, we have isolated cDNAs encoding low Mr intracellular the nucleotide full-length Northern

to (17-

PTPases from mouse testis. This paper describes sequence and deduced amino acid sequence of a

cDNA, designated and Southern blot

as PTP-1, analyses.

and the

results

of

Materials:

The rat PTP-1 cDNA probe was kindly provided by Drs. K. cDNA library was Guan and J. Dixon (8). The lambda gtll prepared by oligo(dT) priming of mRNAs from a mixture of germ cells isolated from adult CD-l male mice, and was generously The evo blot of given by Drs. J. Welch and E. M. Eddy (22). mammalian DNAs was purchased from BIOS Corporation. I I Isolationand: The EcoRI-HincII DNA fragment containing the proteincoding sequence of rat PTP-1 cDNA was used as a probe to screen the mouse germ cell cDNA library using standard procedures (23). The Genescreen membrane was hybridized at 60 C overnight in 1M NaCl/ 1% SDS/ 10% dextran/ 50mM Tris-HCl, pH7.5/ 200 ug/ml salmon sperm DNA, and washed twice with 2X SSC (0.3M sodium chloride/ 0.03M sodium citrate, pH7) at room temperature. The cDNA insert from the plaque-purified lambda gtll phages was isolated and subcloned in pBluescript phagemid. The nucleotide sequence of cDNA inserts was determined by the dideoxy chain-termination method using a T7 DNA polymerase (24). Oligonucleotides synthesized according to the newly determined sequence were used as primers to obtain additional sequence. Both strands of the cDNA inserts were completely sequenced. Northern heart

blot

analvsis:

Tissues including testis, and brain, of CD-1 mice

spleen, muscle, (six weeks old) 819

liver, kidney, were quickly

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185,

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1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

frozen in liquid nitrogen, and total RNAs were extracted by the guanidinium thiocyanate-phenol-chloroform method using the RNAzol B kit (Biotecx Laboratories, Inc.). Poly(A)+RNAs were purified by oligo(dT) cellulose using the FastTrack kit (Invitrogen Corp.). Ten micrograms of poly(A)+RNAs were electrophoresed on a 1.2% agarose/2.1% formaldehyde gel, transferred to a Nytran membrane (Schleicher & Schuell) by capillary blotting with 10X SSC (1.5M sodium chloride/O.l5M sodium citrate, pH7), and UV-cross-linked as described (23). The Northern blot was hybridized overnight at 42 C in 50% Formamide/ 5X Denhardt's solution/ 5X SSPE (0.75M sodium chloride, 50mM sodium phosphatate, 5mM EDTA, pH7.4)/ 1% SDS/ salmon sperm DNA (2OOug/ml) with randomly 32P-labelled cDNA probes of PTP-1. The filter was rinsed twice at room temperature for 15 min each and washed twice at 65 C for 15 min each with 1X SSPE/ 0.1% SDS. Autoradiograms were obtained by exposing the blot to Kodak XAR-5 film with intensifying screen at -70 C for various length of times. lot

e:

The Southern blot containing EcoRI fragments of mammalian genomic DNAs was hybridized using 32P-labelled cDNA probes corresponding to either catalytic domain or COOHterminal and 3' noncoding region of PTP-1 according to the procedure recommended by the supplier.

tion

and

Several and partially

sequence

-is

strongly

of

positive

characterized

PTP-1

cDNA:

cDNA clones from

a library

were

identified

prepared

from

germ

cells of male mice by screening with the rat PTP-1 probe (8). The cDNA inserts from two of these strongly positive clones were

completely

sequenced

(Fig.

1).

Clones

my254 and mY201

200 bp -

PTP-1 mY254

H

I

7

i mY201

Fig.

1. Sequencing strategy of mouse PTP-1 CDNA clones. The protein-coding sequence is shown by open box. The 5' and 3' non-coding regions are indicated by solid lines. Clones mY254 and mY201 were isolated from a lambda gtll library prepared with mixed germ cells from male mice (22). The direction and length of each sequencing run are indicated by horizontal arrows. The sequence of the complementary strands of the cDNA inserts from clones mY254 and my201 were determined completely. 820

Vol.

BIOCHEMICAL

185, No. 3, 1992

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 2. The nucleotide and deduced amino acid sequences of mouse PTP-1 cDNA. The complete nucleotide sequence of mouse PTP-1 (clone and the deduced amino acid sequence is mY254) is presented, given below its nucleotide sequence.

contained

cDNA

respectively. reading frame

of

inserts

nucleotides)

(Fig. 2). The 5' was truncated at

sequence to that of

change

codon

Tyr

by Asp.

clone

T to

G at

ended

The deduced

mY254 exhibited

PTP-1 clones

at

nucleotide

nucleotides,

no.

48 resulting

sequence 93% and

homologue of rat has a calculated of:

no.

1647

of

of the cDNA insert clone mY254, except of

in

432 amino

83% identity

(8) and human PTP-1B (6, mY254 and mY201 contained

composition

1,426

sequence of the cDNA insert from clone my201 codon no. 46 (nucleotide no. 222) and its 3'

non-coding sequence mY254. The nucleotide mY201 was identical of

and

1,668

Clone mY254 was found to contain an open of 1,296 nucleotides as well as 5' (83 and 3' (289 nucleotides) non-coding regions

to

clone

from for

clone a

replacement acids that

g-10), respectively. cDNAs encoding

the

of

from of

rat

Thus, mouse

PTPase-1 and human PTPase-1B. Mouse PTPase-1 Mr of 49,585, and a deduced amino acid 23 Ala,

26 Arg,

12 Asn,

22 Asp,

11 Cys,

17

Gln, 42 Glu, 22 Gly, 16 His, 16 Ile, 39 Leu, 27 Lys, 15 Met, 17 Phe, 24 Pro, 37 Ser, 20 Thr, 9 Trp, 12 Tyr, and 25 Val. ession

of

PTP-1

The results of isolated from mouse

volv(A)+RNAS: Northern testis,

blot analysis spleen, muscle, 821

of poly(A)+RNAs liver, kidney,

Vol.

185, No. 3, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12345678 kb

_b $7’e: _::

Fig.

3. Northern blot analysis of mouse PTP-1 poly(A)+RNAs. Lanes 1, testis; 2, spleen; 3, muscle; 4, liver (3pg); 5, kidney; 6,heart; 7, brain; 8, testis (5pg). Ten micrograms of poly(A)+RNAs were loaded except where indicated. The blot was hybridized with the cDNA probe containing COOH-terminal and 3' non-coding region of PTP-1.

heart

and brain

most abundant 2.4Kb, 2.2Kb 2.OKb,

but

muscle,

are

given

in testis, and 2.OKb. not

2.4Kb

liver,

in

Fig.

and 2.2Kb,

kidney,

heart

mY254 contained

length,

including

was not

found.

spleen of

were

Rat PTP-1 4.3Kb

cDNA of

in

in

The PTP-1

spleen,

mRNAs of

splicing germ cells.

1,668

4.4Kb, and

and/or Mouse

nucleotides

in

AATAAA polyadenylation

mRNAs from

and 2.OKb

mRNAs were sizes of of 4.4Kb

present

alternative in male

the

in

brain,

length

(8).

kidney,

signal liver

and

Human PTP-1

mRNAs

4Kb and 3.5Kb

were found in liver, heart, lung, placenta (9). Thus, mouse PTP-1 mRNAs of 4.4Kb was probably presence of a much longer 3' non-coding region as

and kidney due to the the

but

also

and brain.

a PTP-1

14As,

The PTP-1

detected transcripts

were

2.4Kb and 2.2Kb might be due to the extension of the poly(A)-tail clone

3.

and were The PTP-1

case

of

Southern

human

blot

and rat

PTP-1

(8-9).

analysislian

genomes:

The EcoRI fragments of mammalian genomic DNAs were analyzed using cDNA hybridization probes corresponding either the catalytic coding region (Fig. a single hybridization non-coding

probe

as a single genomic

copy

DNAs

cDNA probe

to

domain or the COOH-terminal and 3' non4). Mouse and rat genomic DNAs exhibited band using the COOH-terminal and 3'

of mouse

PTP-1.

in

and rat

mouse

exhibited

corresponding

multiple

to

Thus,

PTP-1

genomes. hybridization

catalytic 822

domain

gene

All

is

present

mammalian bands

of

using

mouse

the

PTP-1.

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185,

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1992

1 2 3 4 5 6 7 8

AND

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BIOPHYSICAL

12

3

4

RESEARCH

5

6

7

6

COMMUNICATIONS

91011

Fig. 4. Southern blot analysis of mammalian qenomes using mouse PTP-1 cDNA probe. Lanes 1, dog; 2, cat; 3, rabbit; 4, cow; 5, sheep; 6, mouse; I, rat; 8, hamster; 9, pig; 10, marmoset; and 11, human. The same Southern blot containing EcoRI fragments of 8 pq each mammalian DNA was sequentially hybridized with cDNA probes of either catalytic domain (left) or COOH-terminal and 3' non-coding region of PTP-1 (right).

This latter PTP-1 gene

result suggested of mouse as well

absence

EcoRI

of

sites

in

the presence of as other mammals the

cDNA sequence

introns because

in of

of mouse

the the

PTP-1.

The human PTP-1 gene was also found to be present in a single copy in the genome, and mapped to the long arm (q13.1-q13.2) of

chromosome

no.

20

(10).

ACKNOWLEDGMENTS We thank Drs. K. Guan and J. Dixon for providing rat PTP-1 cDNA probe, Drs. J. Welch and E. M. Eddy for giving mouse germ cell cDNA library, Drs. F. Johnson, E. M. the manuscript and making Eddy, and J. Welch for reading helpful suggestions, and Ms. N. Gore for preparing the manuscript.

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