Molecular Cloning, Expression, and ... - Semantic Scholar

Vol.

6, 1659-

1664,

December

Cell Growth

1995

Molecular Cloning, Expression, Characterization of the Murine Gene Rb2/p 1301

Paolo Pertile,2 Alfonso Babdi,2’3 Antonio Luigi Bagella, Laura Virgilio, M. Michele Antonio Giordano4 Department

of Microbiology/Immunology,

A. B., A. D. L., L. B., L. V., A. C.), and Cell Biology, Jefferson Medical EM. M. P.1, Philadelphia,

Pennsylvania

Jefferson

De Luca, Pisano, and Cancer

Institute

IP. P.,

and Department of Pathology, Anatomy College, Thomas Jefferson University 19107

Introduction The

mole of tumor

of cell proliferation

suppressor

genes

has become

in the

negative

more and more

control

intriguing

within the last decade. Because inactivating mutations in these genes are associated with deregulated growth in tumom development, their gene products are likely to play important moles in the regulation of cell growth.

Received 9/1 4/95; accepted 1 1/1/95. 1 This work was supported by Sbarro Institute for Cancer Research and Molecular Medicine, NIH Grant ROl CA60999-OiAi, the Council for Tobacco Research (to A. C.), and by Grants RCDA DE00348 and R29 DEl 0323 (to M. M. P.). A. D. L. is supported by a fellowship from University of Ban (Dottorato di Ricerca in Morfologia Umana e Sperimentale), and L. B. is supported by a fellowship from AIRC. 2 P. P. and A. B. contributed equally to this study. 3 On leave of absence from the Department of Anatomic Pathology, School of Medicine, University of Naples “Federico II,” Naples, Italy. 4 To whom requests for reprints should be addressed, at Sbarro Institute for Cancer Research and Molecular Medicine and Departments of Microbiology/ Immunology and Pathology, Jefferson Cancer Institute, Thomas Jefferson University, B.L.S.B., 233 South 10th Street, Philadelphia, PA 19107. Phone: (21 5) 955-0781 ; Fax: (21 5) 923-9626; E-mail: [email protected].

1659

and Developmental Retinoblastoma-related

Retinoblastoma is a childhood cancer bi-allelic inactivation of a gene located on some i 3q1 4 (1 , 2). pRb5 is a nuclear protein as a tumor suppressor, acting as a critical cell cycle (3, 4). Inactivation of Rb occurs tions

or by binding

proteins, such papilbomavirus

Abstract The product of the retinobbastoma-related human gene Rb2/p130 is highly homologous with the product of the retinoblastoma tumor suppressor gene (pRb) and Rbrelated p1 07. This homology is shared mainly in the pocket domain, a region that seems to play a key role in the functions of these proteins. Here we report the molecular cloning and initial characterization of the cDNA encoding the murine homobogue of the human Rb2/p130 gene product. The 4.8-kb cDNA encodes a protein of 1 1 25 amino acids that shows 90% identity to that of the human protein. The Rb2/p130 mRNA is found to be expressed in all of the adult mouse tissues examined, with the highest level being detected in kidney and skeletal muscle. For the protein characterization, we used a polycbonal antibody raised against the COOH terminus of the human Rb2/pl 30 protein that also recognizes the mouse protein. In developing mouse embryos, the Rb2/pl 30 protein is expressed as early as day 1 0 of gestation and reached a peak of expression around day 1 3 of gestation, implying a developmental regulation of the Rb2/p130 gene in murine ontogeny.

& Differentiation

pressive

function

scniption

factors

the binding

to a number

of DNA

induced by the human chromothat

functions

regulator of the either by muta-

tumor

viral

onco-

as adenovimus Ei A, SV4O I antigen, E7 (5-8). Rb seems to exert its growth by inhibiting

the

activity

of the

that bind in the same region

with

the viral

oncoproteins.

This

and sup-

E2F tran-

responsible region,

for called

the “pocket region,” is defined by two conserved regions, A and B, which are separated by a nonconserved spacer. The pocket region is also present in another El A-binding protein named piO7 (9, iO). The homology between Rb and p1 07 in the pocket region enabled us to clone another Rb-related gene, termed Rb2/ p130 (1 1). As is the case for Rb and p107, pRb2/p130 is capable of interacting with the adenovirus EiA-transforming protein suppressive

within domain 2 (1 1) and is able to exert growthactivities when transfected into certain cell (i 2). Moreover, Rb2/p130 maps to human chrorno-

lines some i 6q1 2.2, a region that has been frequently found altered in many human neoplasias (i 3). Recently, we have found that the Rb2/pi 30 protein displays a cell cycle-dependent phosphomylation pattern, and the highest kinase activity associated with pRb2/pl 30 occurs at the G1-S phase transition ofthe cell cycle” (14-16). The ectopic expression of Rb2/p130 arrested cells in the G1

phase i of the cell cycle, suggesting role of this protein in the regulation the cell

cycle.

These

results

a possible functional of the early phases of

are further

strengthened

by the

observation that the newly characterized E2F family membems, E2F-4 and E2F-5, have been shown to interact in vivo with the pRb2/pi 30 protein, primarily in the G0-G1 phase of the cell cycle (1 7, 1 8). Moreover, it has been shown that the complexes of E2F that contain the pRb2/pi 30 protein are regulators of the exit of cells from the cell cycle; this implies

Here

a mole in terminal

differentiation

for pRb2/pi

30 (i 9).

we report

the cloning and charactemization of the munine Rb2/pl3Ogene. The protein showed high homology to human pRb2/p130. It also demonstrated developmental regulation and to have activities characteristic of the previousby described human pRb2/pi 30 protein. first step in the development of an animal

p1 30-associated

tumonigenesis.

The abbreviations used are: retinoblastoma. 6 p, p. Claudio, A. De Luca, C. E. J. Firpo, M. C. Paggi, and A. growth-suppressive properties in 5

for publication.

This study is a model for Rb2/

pRb,

retinoblastoma

M. Howard, A. Baldi, Giordano . The Rb2/pi a cell cycle-dependent

gene

product;

Rb,

N. Sang, W. Yuan, 30 protein exhibits manner, submitted

1660

Molecular

Cloning

of the Murine

Rb2/p130

Gene

Results Isolation and Characterization cDNA. To isolate the mouse

of the Mouse

Rb2/p130 homobogue of the human Rb2/pl3Ogene, a mouse brain cDNA library was screened at low stringency with a 3.5-kb human Rb2/p130 cDNA fragment (i i). Three positive clones were obtained and found to contain the same cDNA sequence, although the

size of the inserts varied. The sequences were compiled and revealed an open contained

the

followed

full-length

cDNA,

including

by a 3’ untranslated

region

tail. This indicated that additional ing. Using the clone containing

probe,

an overlapping

from a mouse ditional 460

sented

cDNA

clone

embryo 5’-stretch nucleotides at the

initiation

a stop

harboring

codon

a poly(A)

5’ sequences were the most 5’ sequence

missas a

of 586 bp was isolated

library that contained ad5’ end and likely repre-

most on all of the 5’ mRNA

translation

of the three clones reading frame that

sequences,

including

the

site.

The complete sequence of mouse Rb2/p130 cDNA is depicted in Fig. 1 The length of the cDNA is 4806 bp, with a unique open reading frame from nucleotide 20 to a stop codon at nucleotide 3394, and has 78.8% similarity to the human Rb2/p13OcDNA. Itencodes i125 amino acids of

Immunobbot Analysis of pRb2/pl 30 Protein Levels during Murine Development. Based on the above-described results, wherein we showed that the Rb2/p130 gene is differentially expmessed in adult munine tissues, we decided to investigate ifthis protein could be differentially expressed in a similar manner during the course of mumine development. pattern

To define the of the Rb2/pi

relative 30 gene

spatial-temporal product during

expression mumine on-

togeny, protein extracts were prepared from the heads and bodies of mouse embryos/fetuses at various times of gestation

from

days

1 0 through

1 7 of development.

seen in Fig. 5, the pRb2/pl detectable

mouse pressed

bevels

30 protein

in developing

is indeed mid-

to

As can

be

expressed

at

late-gestation

embryos/fetuses. The pRb2/pl 30 protein at low levels as early as day 1 0 of gestation

embryonic/fetal

heads

and

bodies,

with

is exin both

a marked

increase

in expression on day 1 3 of gestation in both the developing head and body. Subsequent to day i 3 of gestation, pRb2/ pi 30 protein bevels in the mouse remained elevated and relatively invaniate throughout the remainder of gestation.

.

the mouse Rb2/pi3O weight of about Mr

predicted protein showing

amino is highly a high

protein, i 30,000.

acid

with a predicted As depicted in

sequence

molecular Fig. 2, the

of the mouse

homologous to the human degree of conservation.

Rb2/pl

protein

30

(20, 2i),

Comparison of the amino acid sequences of the pRb2/ pi 30 proteins of mice and humans shows that the human pRb2/pl 30 protein has i 4 additional amino acids. In total, 99 of i i i 1 amino acids are different between mouse and human identical.

Rb2/pi3O

Expression analyze in adult

proteins,

of the Rb2/p130

the expression mouse tissues,

on filter-immobilized multiple

making

adult

tissues

proteins

(Fig.

90%

Tissues.

To

mouse Rb2/p130 gene Northern blot analysis

polyadenylated as a 4.7-kb

two

Gene in Mouse

pattern ofthe we performed

munine

gene was expressed

the

RNA (2 tg/lane) 3). The

mRNA

mouse

species

Rb2/p

from 130

in essentially

all tissues investigated. Although Rb2/p130 mRNA expression appeared to be ubiquitous, steady-state Rb2/p 130 mRNA levels were higher in some tissues such as skeletal muscle and kidney, as opposed to tissues such as heart, brain, lung, liver, and testis. Interestingly, the level of mRNA transcript in spleen was almost undetectable.

Mouse serum

pRb2/p130

against

COOH-terminal

Protein.

a synthetic

A polycbonal

obigopeptide

rabbit immune

corresponding

to the

of the human Rb2/pi 30 protein was gennamed ADLi It was tested for its reactivity by

erated and . immunoprecipitation with nadiobabeled in vitro-translated forms of human and mouse Rb2/pl3OcRNAs. The immune serum specifically precipitated both in vitro-translated proteins, thus confirming its ability to detect the human and mouse forms of Rb2/pi 30 protein (data not shown). The subunit molecular weight of the mouse Rb2/pl3O protein was detected in lysates of 135S]methionine-labeled

NIH3T3

cells by immunoprecipitating

with

the ADL1

anti-

body. A single polypeptide of Mr “l 30,000 was recognized by lysates of the NIH3T3 mouse cell line (Fig. 4). The molecular weight was similar to the size determined for human pRb2/pi3O (20, 21), confirming the molecular homobogy between the human and the mouse Rb2/pl 30.

Discussion In this

report,

we

describe

the

molecular

cloning

of the

mouse Rb2/p130 cDNA. If the Rb2/p130 gene has a fundamental role in growth control, the gene should be mepresented

in a very

similar

form

in other

mammalian

species,

as it happens for other critical human genes that are evolutionamiby conserved. Our data indicate that the mouse Rb2/pi

tein, the

30 protein

showing one

is highly

90%

obtained

homologous

of identity. with

the

to the human

This result

mouse

pro-

is very similar

Rb protein

that

to

shows

a

9i % identity with the human protein (22). Moreover, based on known syntenic relationships among humans, rats, and mice, it is likely that the Rb2/p130 gene maps on mouse chromosome 8 (13). Northern blot analysis of steady-state levels of Rb2/p 130 mRNA in adult mouse tissues demonstrates is expressed constitutively in many tissues.

that regulation sion,

occurs

bevel. Adult were found

of Rb2/pl

30 expression,

that Rb2/pi 30 This indicates

as with

at a posttranscniptionab

or

Rbi expres-

posttranslational

tissues, such as skeletal muscle and to express high levels of Rb2/p130

kidney, mRNA,

while tissues such as heart, brain, lung, and liver expressed significantly lower Rb21p130 mRNA levels, and levels in testis and spleen were barely detectable. This relative tissue-specific pattern of Rb2/p130 mRNA expression is mark-

edly

different

pression

from

in adult

that pattern mouse

reported

tissues

(22).

for Rb mRNA Adult

mouse

ex-

brain,

kidney, spleen, thymus, and lung were found to have exceptionally high levels of Rb mRNA, while muscle, heart, liver,

testis,

and

uterus

demonstrated

significant

but

com-

paratively bower levels of Rb mRNA. Members of the metinoblastoma gene family encode nuclear phosphoproteins, which are hypothesized to constrain normal cell proliferation by regulating cell cycle progression (i 0). Moreover, it has been suggested that the retinoblastorna phosphoprotemns integrate cell cycle progression with terminal try into the

population fementiation Increasing

cell differentiation G0 phase of the

cell

by facilitating cellular encycle, creating a stable

of postmitotic cells that is required of many cell types (23, 24). experimental

evidence

phosphopmoteins have a biological Undeniably, much ofthis evidence

indicates

for the difthat

the

Rb

role in development. has centered on the mole

Cell

Growth

& Differentiation

1661

100

1

HASGGNQSPPPPPAAAASSEEEEEDGD 101

QccGcaGAocGcGcAccccccaooGccccGaccATcAGATccAocAGcGGr1vGAoQAGTroTocAGcccccTcAAcATaGAcGAQGcoGcGcocG AADRAQPAGSPSHQ IQQPFEELCSRLNMDEAARA

200

201

300 EAWSSYRSMSESYTLECNDLHWLACALYVAc

RK

301

400 SVPTVSXGTAEGNYVSLTR

I

I

LRCSEQS!.

EFFN 500

401

MKWEDMANLP 501

PH

FRERTERLERNFTVSAV

ILK

AGAAATA2AACCCA1?1NAAGACA1?1?1’MATATCCCCAAGAAGA.ACAOCCTCGCCAGCAAAGAOQAAGMAACAGACOcGACAOCCCmTACCAC

KYEP

I

FQD

I

PKY

600

PQEEQPRQQRGRXQRRQ

PCTT

601

700

SKI 701

800 CALDLVYGNALQCSNRKELVHPHFKGLSEDcHPK

801

900 DSKASSDP

PCV

I

EKLCSLHDGLVLEAKG

I

KEH

F

901

1000 WXPY

IRKLFEKKr.LKGKEENLTOFLEPONFOES

1001

1100 FXAVNKAYEEYVLPAOHLDERVFLOEDAEEEVGT

1101

1200

1.s

RCLSAA

SOTESAERTQMKD

I

Z.QQ

H

!.DKSXAL

1201

1300 RVCTPrTGVRYVQENS

PCVTPVSTAAH

S

S

RLH

1301

1400 TMLSOLRNAPSEKLER

I

LRSCSRDPTQA

I

ADOLK

1401

1500 ENVE

IYSQHFQPDENPSNCAKEHLYYKVLESV

I

1501

1600 EQEQKRLGDMDLSGVLEHDAFHRSLLACC

LEVV 1700

1601

AFSHKPPGHFPFIAEI

FDVPHYHFYKVI

EVF

IRA

1701

1800 EDGLCREVVKHLNQIEEQILDHLAWXTKSPLWD

1801

Fig. 1. otid s

cDNA sequence uen eofth murin

and nudeRb1 13#{176}

ageo

strands

were

sequenced

an aver-

reetmes.

lAGS

P

1901

2000 LTPRRVGEVRADAGGLGRS

Both

1900

CAGAATAGAGATAATGAAAACACAGCCTACTAAGAOGCACCACCTCAAAACCTAGAGAGAACAGATGAAA1TrACA3’COCTCGCTCTCCC K I RDNENRVPTCEEVHPPQHLERTDEIY

I

TS

PTTLY

2001 VSTTRR 2101

H

LFENDS

PS

DRYSS

2100

I

EGSTSGR

PT

P

PQ

P

LVNAV

P 2200

CC1CAGATGTACC1OQGAGAC11’1TACACCAC11’CCTGGACAGACCTYOGTCACCATOOCAACAGCCACTGTCACOGCCAACAAWGACAA VQNVPGKTVSVTPV PGQTLVTNATATVTANPOGQ

2201

2300 TVT

I

PVQG

I

AHEHGG

I

TPFPVQVHVGGQAQAVAG

2301

2400 I

S 2401

Q

PLSAQALAGS

LSSQQVTGTTLQV

PG

PVA

I

Q 2500

ACAGMTrCCCCTOGTOGACAACAGCAGAACCCAOOCCAGCCACTAACCAOCAGCAGTATCCGGCCGCGGAAGACTAGCTCCTrAOCGCTGTrCTrrAGA Q ISPGGQQQNPGQPLTSSS I

2501

2600 XVYYLAGVRLRDLC

IKLDISDELRKK

IWTCFEFS

2601

2700

I

I

QCTELMMDRH

LDQLLMCA

I

YVMAKVTXEDRS

2701

2800 FQH

IMRCYRTQPQARSQVY

RSVL

I

KOKRRNSGS

2801

2900 S

ESRSHQNS

PTELNTDRASRDSS

PVMRSNSTLPV

2901

3000 PQPSSAP

PTPTRLTGASS

DVEEEKRGDL

I

Q

FY

H

3001

3 100 N

I

Y

RKQ

I

QAFAHKY

SQAHAQTDTPPLS

PT

P

FVR 3200

3 101

TGSPRRVQLSQSHP

IT

ISPHNNEAMPSPREK

I

3201

3300 Y

FSHS

I

PSXRLRE

HSH

I

RTGETPTKK

3301

KG

I

LLDD 3400

GOES

PARR

I

CPENHSALLRRLQDVAHDRGSQ

3401 3501 3601 3701 3801. 3901 4001 4101

3500 3600 3700 3800 39t0 4000 4100 4200

4201

4300

4301

4400 4500 4600 4700 4800 4806

4401 4501 4601 4701 4801

of the Rb nuclear phosphoprotein. and Rb2/pi 30 in cell differentiation

FT

AAAAAA

The function of p1 07 and development have

yet to be examined to the extent that Rbi has. Northern blot analysis of steady-state Rb mRNA expression in mouse embryos/fetuses demonstrated that Rb is

expressed examined),

as early as day 9.5 of gestation (the first stage with maximal expression apparent around day

i 3 of gestation (22). p1 30 protein in the similar to that of pRb

The expression pattern of the pRb2/ developing mouse was found to be in that expression is apparent as early

1662

Molecular

Cloning

mouse

of the

Murine

Gene

...

1 MASGGNQS

97

PPPPPAAAASSEEEEEDGDAADRAQPAGSPSHQIQQRFEELCSRLNMDEAARAEAWSSYRSMSESYTLEGNDLHWLACALYVACRKSVP

1:1 II:II human

Rb2/p130

IIIIIIIIII.III

fI:I.I I..

11.1 IIIll:lllllllllllll:lll.l 1111111 IIIIIIIIIIIIIII IIIIIIll I

1 MPSGGDQSPPPPPPPPAAAASDEEEEDDGEAEDAAPSAESPTPQIQQRFDELCSRLNMDEAARPEAWDSYRSMSESYTLEGNDLHWLACALYVACRKSVP

mouse

197

TVSKGTAEGNYVSLTRILRCSEQSLIEFFNKNKKWEDMANLPPHFRERTERLERNFTVSAVIFKKYEPIFQDIFKYPQEEQPRQQRGRKQRRQPCTTSEI

98

I I I III. I I III I III I I:

101

human

100

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I . III

TVSKGTVEGNYVSLTRILKCSEQSLIEFFNKMKKWEDMANLPPHFRERTERLERNFTVSAVIFKKYEPIFQDIFKYPQEEQPRQQRGRKQRRQPCTVSEI

200

FHFCWVLFIYAKGNFPMISDDLVNSYHLLLCALDLVYGNALQCSNRKELVNPNFKGLSEDCHPKDSKASSDPPCVIEKLCSLHDGLVLEAKGIKEHFWKP

297

mouse

1

human

2 01

mouse

298

397

human

3 0 1 YIRLYEKKLLKGKEJLTGFLEPGNFGESFKAINKAYEEYVLSVGNLDERIFLGEDAEEEIGTLSRCLNAGSGTETAERVQMKNILQQHFDXSKALRIS

400

mouse

3 9 8 TPLTGVQENSPCVTPVSTAAHSLSRLHTMLSGLRNAPSEKLERILRSCSRDPTQAIADRLKEMYEIYSQHFQPDENFSNCAK

human

401

mouse

4 8 8 KVLESVIEQEQKRLGDMDLSGVLEHDAFHRSLLACCLEVVAFSMKPPFPFIAEIFDVPHYHFYKVIEVFIRAEDGLCREVVKHLNQIEEQILDHLAWK

human

501

8

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII III111111 III111111 I111111 1:1111:1 IIIII:IIIIllllllllllllllllll III FHFCWVLFIYAKGNFPMISDDLVNSYHLLLCALDLVYGNALQCSNRKELVNPNFKGLSEDFHAKDSKPSSDPPCIIEKLCSLHDGLVLEAKGIKEHFWKP

300

.

mouse

588

human

601

487

EMLYY

IIIIIIII:.IIlIlIIlIlIl.IIIIIIIIII.Illllllllll.lII.IIIIlIIIlI:IIlll:IIlIIlIlIII:IIlIlI

11111

TPLTGVRYIKENSPCVTPVSTATHSLSRLHTMLTGLRNAPSEKLEQILRTCSRDPTQAIANRLKEMFEIYSQHFQPDEDFSNCAKEIASKHFRFAEMLYY

I I I I I I I I I I I I I I I I I I I I I: I I

500

587

I I I I I I I. I I. I I I I I I I I I. I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I III

:

KVLESVIEQEQKRLGDMDLSGILEQDAFHRSLLACCLEWTFSYKPPGNFPFITEIFDVPLYHFYKVIEVFIRAEDGLCREVVKHLNQIEEQILDHL.AWK

..

IIII

IIIIIIIIIIIIIIIIIIIIII II

:

.

600

.

ENDSPSEGSTSGRIPP

TKSPLWDRIRDNENRVPTCEEVMPPQNLERTDEIYIAGSPLTPRRVGEVRADAGGLGRSITSPTTLYDRYSSPTVSTTRRRLF

686

IIIIIIIIIII IIII1.1 IIIIIIIIIIIIIIIIIII.. IIIIIIII IIIIII: I: I. II:II .

PESPLWEKIRDNENRVPTCEEVMPPQNLERADEICIAGSPLTPRRVTEVRADTGGLGRSITSPTTLYDRYSSPPASTTRRRLFVENDSPSDGGTPGRMPP mouse

687

human

701

mouse

7 87

700 786

I I I I I I I I I I I I . I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I . I I I I I I I I I I I I I I I I I I I I I I I III 800

VPGPVAIQQISPGGQQQNPGQPLTSSSIRPRKTSSLALFFRKVYYLAGVRLRDLCIKLDISDELRKKIWTCFEFSI

III.IIIIIIIIIIIII..l

1:11

II

I liii

human

801

VPGQVAIQQISPGGQQQKQGQSVTSSSNRPRKTSSLSLFFRKVYHLAAVRLRDLCAKLDISDELRKKIWTCFEFSI

mouse

887

KVTKEDRSFQNIMRCYRTQPQARSQVYRSVLIKG

human

90 1 KVTKEDKSFQNIMRCYRTQPQARSQVYRSVLIKGKRKRRNSGSSDSRSHQNSPTELNKDRTSRDSSPVMRSSSTLPVPQPSSAPPTPTRLTGANSDMEEE

IIIIII

985

mouse

IQCPELMMDRHLDQLLMCAIYVMA

900

..

984

I I I I I I I I: I I II I I I I I I I 1.1 1.1 I I I I I I II I. I I I I I I I I I I I I I I I I I I I I I I I :1 II .

ERGDLIQFYNNIYRKQIQAFANKYSQANAQTDTPPLSPYPFVRTGSPRRVQLSQSHPIYISPHNNEANPSPREKIFYYFSNSPSKRLREINSMIRTGETP

II IIIIIIIIIII III.. IIIIIIIII RGDLIQFYNNIYIKQIKTFANKYSQAN

human

1001

mouse

1085

TKKRGILLDDGSESPAKRICPENHSALLRRLQDVANDRGSQ

human

1099

TKKRGILLEDGSESPAKRICPENHSALLRRLQDVANDRGSH

1084

I.IIIIIIIllIII Illl:Illl.ll:IllIl.ll.l

III IIIIIIIIIIIIIIIIIIIIIIIIIIII

MDAPPLSPYPFVRTGSPRRIQLSQNHPVYISPHKNETMLSPREKIFYYFSNSPSKRLREINSMIRTGETP

Fig. 2.

Comparison of the amino acid sequences indicate the absence of amino acid in that

C .

ci) ci)

.0

Cl)

.t (

..

1000

1098

1125

I IIIIIIIIIIIIIIIIII:

III IIII:

Dots

886

RNSGSSESRSHQNSPTELNTDRASRDSSPVMRSNSTLPVPQPSSAPPTPTRLTGASSDVEEE

IIIIIIIIIIIIIIIIIIIIIIII

:

IQCTELMMDRHLDQLLMCAIYVMA

I II.lIIIlII.II:IIIIIIl

0)

1139

of the mouse and particular position.

human

Rb2/pl

30. A vertical

bar represents

amino

acid

identity

between

the two

proteins.

(.)WcI) -

(1)

C

C’) .

.2

E

.

7.5*

4.4-

Fig. 3. contains length

Expression 2 of the

200

pattern

of the Rb2/p130

of polyadenylated Rb2/pl 30 transcript

RNA is

gene in adult mouse. from various tissues. The 4.7 kb.

Each lane estimated

Rb2/pl

()

C’)

z

z

K-

30-’

I I

as approximately

pRb2/pl

day

30 expression

iO of gestation,

with

an increase

on day i 3 of gestation.

mRNA expression, which peaks and declines tion, pRb2/pi 30 protein expression remains

Unlike

116K-

in

Rb

at mid gestaat an elevated

Fig. 4. NIH3T3 a Mr

_______

Expression pattern of the Rb2/pi 30 protein product in the mouse fibmoblastic cell line and in several organs. Rb2/pi 30 is expressed as 130,000 protein.

Cell

gestation (days) - head 10

11

12

13

14

15

Materials

16

10 Rb2/pl Fig. 5.

17

12

13

14

body

15

16

17

30 -‘4 Expression

of mouse

11

and Methods

Cbontech. of pRb2/pl

embryos

& Differentiation

Materials. The T-Iyrnphoblastoid mumine cell line NIH3T3 was obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in DMEM supplemented with 1 0% fetal bovine serum and L-glutamine (2 mM) at 37#{176}C and 5% CO2. The antibody anti-Rb2/pi 30 (ADL1) is a rabbit polycbonab antibody raised against a peptide corresponding to the camboxy terminus of Rb2/pi 30 of human cell origin. Adult mouse Northern tissue blot was purchased from Cbontech (Palo Alto, CA). The mouse brain cDNA library was purchased from Stmatagene (La Jolla, CA), while the embryo 5’-stmetch cDNA library was from

Rb2/p130-.j

gestation (days) -

Growth

30 protein in heads times of gestation.

at various

(top)

and bodies

cDNA screened

Library Screening. The libraries were plated and with standard protocols (28). To isolate the mouse homobogue of the human Rb2/p130 gene, a mouse brain cDNA library (Stratagene) was screened with the 4.3-kb fragment of the human Rb2/p130 cDNA (i i Additional 5’ sequences were generated by screening a mouse embryo

(bottom)

).

level until late in gestation. Early postnatal bevels of pRb2/ p1 30 protein expression were not examined. Although tissue-specific differences in Rb mRNA expression have been

reported

in mid gestation

(day

i 2.5) mouse

embryos

(22),

with the highest levels observed in liver, brain, and spinal column and lower levels in other viscera, cranium, and developing limbs, no differences were noted in the expression of Rb2/pi 30 mRNA in the developing cranial region versus the body, although the experimental design used

does not allow us to discriminate between specific differences in Rb2/pi 30 expression

precise tissueduring munine

development. Through the use of a number of gene engineering technobogies, the Rb gene has been found to be essential for normal embryonic development (25-27). Although targeted disruption of a single Rb allele does not lead to an overt developmental phenotype, disruption of both alleles of Rbi in mice is lethal in utero at approximately day i 4 of gestation. These results indicate that Rb does not have a mea-

surable essential cell cycle function in early embryonic development, nor does it have an essential role in the ontogeny

pleted events

of most

tissues,

which

have

by and

barge

corn-

their development and terminal cell differentiation by day i 4 of gestation. Since the levels of pRb2/pl 30

protein were

determined

to remain

markedly

elevated

until

at least day 1 7 of gestation, targeted disruption of the Rb2/ p130 gene may result in a more pronounced aberrant phenotype for populations of cells that differentiate relatively bate in gestation, such as those of the neural lineages. Although recent functional studies of pi 07, pRb2/pi 30, and pRb indicate that the Rb phosphoprotein family members are capable of complementing each other, the Rbfamily proteins are not functionally identical (1 0). Although

pRb2/pi

30 has been

cells through in constraining

cell

differentiation

known.

shown

to inhibit

the G3 phase ofthe cell proliferation

A thorough

pi 30 in cellular

during

development

understanding

growth

control

the progression

of

cell cycle, its potential mole and promoting terminal

is at present

of the function

and

un-

of pRb2/

momphogenesis

will

undoubtedly evolve as gene engineering technologies are used to manipulate expression of the Rb2/p130 gene. The availability of the mouse Rb2/p130 cDNA and antimouse antibody will serve to facilitate future investigations into the function of the pRb2/pi 30 protein in growth control and development.

5’-stmetch

cDNA

library

(Cbontech)

using

the 5’ 1-kb

nude-

otides of the clones isolated previously as a probe. Nucleotide Sequence Analysis. The nucleotide sequences of recombinant clones were determined by automated DNA sequencing using the dideoxy terminator meaction chemistry for sequence analysis on the Applied Biosystems Model 373A DNA sequencer. Northern Blot Analysis. Filter-immobilized polyadenybated RNA (2 pg/bane) from multiple adult munine tissue (Cbontech) was hybridized to a 32P-labeled mouse cDNA probe corresponding to the fragment from nucleotide 728 to nucleotide 1 i 7i , according to the manufacturer’s instructions. In Vitro Transcription and Translation. For in vitro transcniption, the coding region of the mouse Rb2/p130 cDNA was cloned into pBlueScnipt SK (Stratagene), and cRNA was transcribed using T7 RNA polymerase. Translation was performed in vitro using the TNT rabbit reticubocyte bysate kit (Pmomega) with [35Sjmethionine (translation grade; Dupont).

Cell Labeling and Immunoprecipitation. Cells were inat 37#{176}C in medium without rnethionine for 10 mm prior to addition of any labeling materials. The cells were labeled for 4 h at 37#{176}C with 500 Ci of [35Sjmethionine (Dupont) per 35-mm culture dish in 2 ml of methionine-free

cubated

medium. The labeled cells were collected and lysed on ice for 30 mm in bysis buffer (50 mr’i Iris, 5 mri EDTA, 250 mri NaCI, 50 mM NaF, 0.i% Triton X-lOO, 0.1 mt.i Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 0 leupeptin).

Immunoprecipitation

was

ously

of the

(29).

Reactivity

performed polycbonal

as described antibody

pmeviprepama-

tion against the mouse Rb2/pl 30 protein was confirmed by immunopmecipitation using the in vitro translated form of the mouse Rb2/pl 30 cDNA. Tissue Preparation and Immunoblot Analysis. Protein extracts were prepared from adult mouse tissues and from the heads and the bodies of embryos/fetuses. Mature male and female ICR mice (Ace Laboratories, Boyertown, PA) were housed in rooms with a 1 2-h alternating light and dark cycle and maintained on Purina mouse chow and water ad Iibitum. Timed pregnancies were obtained by mating a single mature male with two nulbiparous females for 4 h in the morning (early in the light cycle). The presence of a vaginal plug was considered evidence of mating, and the time was considered as 0 days, 0 h of gestation. Mouse

1663

1664

Molecular

Cloning

of the

Rb2/p130

Murine

Gene

10. Sang,

embryos were identified according to their developmental stage, following the staging method oflheiler (30). Embryo/ fetuses and tissues were homogenized at 4#{176}C in 250 mt’i

and

is

The homogenates mm at 13,000 x

extracts

were cleaned g at 4#{176}C, and

was determined.

by boiling in electrophoresis

2X

Fifty g

by centnifugation total protein

of protein

Laemmbi sample buffer in a 7% SDS-polyacrylamide

by ebectrophonetic dene difluonide

transfer membrane

1 2. Claudio,

for in the

separated by gel, followed

of the proteins to a polyvmnybi(Millipore) in 3-Ecycbohexyl-

aminol-i-propanesulfonic

acid

buffer

(iO

mtvi

3-[cycbo-

hexylaminol-l-pmopanesulfonic acid and 20% methanol, pH i i ). The membrane was then blocked with 5% milk in lBS-I buffer (2 mM Iris, i3.7 mtvt NaCI, and 0.1% Iween 20,

pH

7.6)

incubated

and

washed

in lBS-I.

with the membrane

Primary

antibody

was

in 3% milk and then washed

in lBS-I. Rabbit antimouse ish pemoxidase was incubated

antibody coupled to horseradwith the membrane and then

washed in lBS-I. The presence of bound to the membrane was detected (Dupont NEN). Other. Protein concentration was Bradford assay reagent from Bio-Rad BSA as standard.

secondary antibody using the ECL system determined using a (Hercules, CA), using

We gratefully acknowledge Dr. H. Alder (Thomas Jefferson University, Philadelphia, PA) for automated DNA sequencing and Dr J. L. Rothstein (Thomas Jefferson University) for the kind gift of the mouse libraries. We

Rockland,

Inc. (Cilbertsville,

PA) for its support

in the preparation

the antibody used in this project. We also thank all the laboratory for helpful discussion and M. Cadden for expert tance

in the preparation

of mouse

tissue

members technical

of

of the assis-

samples.

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