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Proceedings C)F THE

OCTOBER 1985

VOLUME 82

NUMBER 19

National Acaden1y of Sciences

This material was~:u~iei:l

at the, NLM ani:! may De Subje-ct US Copyright Laws

PFIZER EX. 1042 Page 1

Proceedings OF THE

National Academy of Sciences OF THE UNITED STATES OF AMERICA

Officers of the Academy

Editorial Board of the Proceedings

ROBERT A. ALBERTY PAUL BERG RONALD BRESLOW EDWARD E. DAVID, JR. STUART A. KORNFELD

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ANNOUNCEMENT

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PFIZER EX. 1042 Page 3

Proceedings OF THE

National Academy of Sciences

OF THE UNITED STATES OF AMERICA

October 1985 Volume 82, Number 19 pp. 6367-6722

Table of Contents

AUTHOR INDEX

Physical Sciences

CHEMISTRY

Peptide mixture sequencing by tandem Fourier-transform mass spectrometry

Biological Sciences

APPLIED BIOLOGY

Brood care by male bumble bees

BIOCHEMISTRY

Apolipoprotein multigene family: Tandem organization of human apolipoprotein AI, CIII, and AIV genes

Structure of canine pulmonary surfactant apoprotein: eDNA and complete amino acid sequence

The "fl-like-globin" gene domain in human erythroid cells

Mutagenesis of avian carcinoma virus MH2: Only one of two potential transforming genes (8ga!?-myc) transforms fibroblasts

iii

Robert D. Cody, Jr., I. Jonathan Amster and Fred W. McLafferty '

Sydney A. Cameron

Sotirios K. Karathanasis

Brad Benson, Samuel Hawgood, James Schilling, John Clements, Deborah Damm, Barbara Cordell, and R. Tyler White

Dorothy Tuan, William Solomon, Qiliang Li, and Irving M. London

Ren-Ping Zhou, Nancy Kan, Takis Papas, and Peter Duesberg

ix

6367

6371

6374

6379

6384

6389

PFIZER EX. 1042 Page 4

I

Contents

Cloning and characterization of two cDNAs coding for human von Willebrand factor

Preferential rearrangement of the immunoglobulin K chain joining region 1,1 and J , 2 segments in mouse spleen DNA

Replication of phage ()29 DNA with purified terminal protein and DNA polymerase: Synthesis of full-length ()29 DNA

Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1

DNA sequence of the lactose operon: The /acA gene and the transcriptional termination region

Activation of retinoic acid by coenzyme A for the formation of ethyl retinoate

Crystallographic structure of an active, sequence-engineered ribonuclease

RNase Tis responsible for the end-turnover of tRNA in Escherichia coli

Inhibition of chymotrypsin by heparin cofactor II

Binding of protein kinase C to neutrophil membranes in the presence of Ca2 +

and its activation by a Ca2+ -requiring proteinase

Phorbol 12-myristate 13-acetate and insulin increase the concentration of fructose 2,6-bisphosphate and stimulate glycolysis in chicken embryo fibroblasts

Cleavage of cruciform DNA structures by an activity from Saccharomyces cerevisiae

Isolation of the human insulin-like growth factor genes: Insulin-like growth factor II and insulin genes are contiguous

Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70

Site-specific cleavage of left-handed DNA in pBR322 by A­tris(diphenylphenanthroline) cobalt(III)

Sequence of a second human asialoglycoprotein receptor: Conservation of two receptor genes during evolution

Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo chromatin structures

Replication timing of the H4 histone genes in Plzysarum polyceplzalum

A developmentally regulated mRNA from 3T3 adipocytes encodes a novel serine protease homologue

Gene expression of pigment-binding proteins of the bacterial photosynthetic apparatus: Transcription and assembly in the membrane of Rhodopseudomonas capsulata

The structure of caltrin, the calcium-transport inhibitor of bovine seminal plasma

Aflatoxin metabolism in humans: Detection of metabolites and nucleic acid adducts in urine by affinity chromatography

A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1 /epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma

iv

J. Evan Sadler, Beverly B. Shelton­Inloes, James M. Sorace, John M. Harlan, Koiti Titani, and Earl W. Davie

Miyuki Nishi, Tohru Kataoka, and Tasuku Honjo

Luis Blanco and Margarita Salas

Kenneth A. Thomas, Mari Rios­Candelore, Guillermo Gimenez­Gallego, Jerry DiSalvo, Carl Bennett, John Rodkey, and Susan Fitzpatrick

Matthias A. Hediger, David F. Johnson, Donald P. Nierlich, and Irving Zabin

Duncan A. Miller and Hector F. DeLuca

Hope C. Taylor, Akira Komoriya, and Irwin M. Chaiken

Murray P. Deutscher, Christopher W. Marlor, and Richard Zaniewski

Frank C. Church, Claudia M. Noyes, and Michael J. Griffith

E. Melloni, S. Pontremoli, M. Michetti, 0. Sacco, B. Sparatore, F. Salamino, and B. L. Horecker

Lisardo Bosca, Guy G. Rousseau, and Louis Hue

Stephen C. West and Ann Korner

Graeme I. Bell, Daniela S. Gerhard, Noel M. Fong, Ray Sanchez-Pescador, and Leslie B. Rail

Clayton Hunt and Richard I. Morimoto

Jacqueline K. Barton and Adrienne L. Raphael

Martin Spiess and Harvey F. Lodish

Mark J. Solomon and Alexander Varshavsky

R. Jalouzot, B. Toublan, M. L. Wilhelm, and F. X. Wilhelm

Kathleen S. Cook, Douglas L. Groves, Hye Yeong Min, and Bruce M. Spiegelman

Gabriele Klug, Norbert Kaufmann, and Gerhart Drews

Randolph V. Lewis, Jovenal San Agustin, William Kruggel, and Henry A. Lardy

John D. Groopman, Paul R. Donahue, Jiaqi Zhu, Junshi Chen, and Gerald N. Wogan

Kentaro Semba, Nobuyuki Kamala, Kumao Toyoshima, and Tadashi Yamamoto

6394

6399

6404

6409

6414

6419

6423

6427

6431

6435

6440

6445

6450

6455

6460

6465

6470

6475

6480

6485

6490

6492

6497

PFIZER EX. 1042 Page 5

Contents

Location of cis-acting regulatory sequences in the human T-cellleukemia virus type I long terminal repeat

Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF

Aleurain: A barley thiol protease closely related to mammalian cathepsin H

The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: Its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma

Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA

BIOPHYSICS

Visualization of oriented hemoglobin S in individual erythrocytes by differential extinction of polarized light

Molecular packing and area compressibility of lipid bilayers

A molecular dynamics simulation of double-helical B-DNA including counterions and water

BOTANY

Active auxin uptake by zucchini membrane vesicles: Quantitation using ESR volume and .1.pH determinations

Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection

CELL BIOLOGY

Peroxisomal organization in normal and cerebrohepatorenal (Zellweger) syndrome fibroblasts

Identification of a nuclear localization signal of a yeast ribosomal protein

Do agonists promote rapid internalization of ,8-adrenergic receptors?

Repeating modular structure of the fibronectin gene: Relationship to protein structure and subunit variation

Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells

Analyzing the components of microtubules: Antibodies against chartins, associated proteins from cultured cells

DEVELOPMENTAL BIOLOGY

Growing Xenopus oocytes have spare translational capacity

GENETICS

Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA

v

Craig A. Rosen, Joseph G. Sodroski, and William A. Haseltine

Frederick Esch, Andrew Baird, Nicholas Ling, Naoto Ueno, Fred Hill, Luc Denoroy, Robert Klepper, Denis Gospodarowicz, Peter Bohlen, and Roger Guillemin

John C. Rogers, Duff Dean, and Gregory R. Heck

Hidemitsu Matsuura and Sen-itiroh Hakomori

Tetsuji Yamada, Curtis J. Palm, Bob Brooks, and Tsune Kosuge

William Mickols, Marcos F. Maestre, Ignacio Tinoco, Jr., and Stephen H. Embury

Stephen H. White and Glen I. King

G. L. Seibel, U. C. Singh, and P. A. Kollman

TerriL. Lomax, Rolf J. Mehlhorn, and Winslow R. Briggs

M. Gurevitz, C. R. Somerville, and L. Mcintosh

Allan M. Showalter, John N. Bell, Carole L. Cramer, John A. Bailey, Joseph E. Varner, and Chris J. Lamb

Manuel J. Santos, Jose Manuel Ojeda, Jorge Garrido, and Federico Leighton

Robert B. Moreland, Hong Gil Nam, Lynna M. Hereford, and Howard M. Fried

Lawrence C. Mahan, Harvey J. Motulsky, and Paul A. Insel

Erich Odermatt, John W. Tamkun, and Richard 0. Hynes

Pamela A. Maher, Elena B. Pasquale, Jean Y. J. Wang, and S. J. Singer

Margaret Magendantz and Frank Solomon

Mark A. Taylor, Andrew D. Johnson, and L. Dennis Smith

Andrea D. Branch, Bonnie J. Benenfeld, and Hugh D. Robertson

6502

6507

6512

6517

6522

6527

6532

6537

6541

6546

6551

6556

6561

6566

6571

6576

6581

6586

6590

PFIZER EX. 1042 Page 6

Contents

Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family

Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line

Distribution of initial and persistent 2-acetylaminofluorene-induced DNA adducts within DNA loops

Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD

UV-induced mutagenesis of phage Sl3 can occur in the absence of the RecA and UmuC proteins of Escherichia coli

Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia

One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

IMMUNOLOGY

In vivo prevention of thyroid and pancreatic autoimmunity in the BB rat by antibody to class II major histocompatibility complex gene products

Leukotriene C4 production by murine mast cells: Evidence of a role for extracellular leukotriene A4

Purification and characterization of a human tumor necrosis factor from the LuKII cell line

Phorbol ester induces a differential effect on the effector function of human allospecific cytotoxic T lymphocyte and natural killer clones

Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes

Monoclonal antibodies against human Ia antigens stimulate monocytes to secrete interleukin 1

The human Thy-/ gene: Structure and chromosomal location

MEDICAL SCIENCES

Glial cells metabolically cooperate: A potential requirement for gene replacement therapy

Visualization of fJ.! opiate receptors in rat brain by using a computerized autoradiographic subtraction technique

Detection of benzo[a]pyrene diol epoxide-DN A adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers

Chloride uptake into cultured airway epithelial cells from cystic fibrosis patients and normal individuals

vi

Steven R. Tronick, Nicholas C. Popescu, Marc S. C. Cheah, David C. Swan, Suzanne C. Amsbaugh, CaroleR. Lengel, Joseph A. DiPaolo, and Keith C. Robbins

Wayne Frankel, Terry A. Potter, Naomi Rosenberg, Jack Lenz, and T.V. Raj an

Ramesh C. Gupta, Nutan R. Dighe, Kurt Randerath, and Harold C. Smith

John T. Mulligan and Sharon R. Long

Irwin Tessman

S. Hitotsumachi, D. A. Carpenter, and W. L. Russell

Miroslava Protic-Sabljic and Kenneth H. Kraemer

Christian Boilard, Sara Michie, Peter Serrurier, Geoffrey W. Butcher, Audrey P. Larkins, and Hugh 0. McDevitt

Clemens A. Dahinden, Robert M. Clancy, Muriel Gross, Jacques M. Chiller, and Tony E. Hugli

Berish Y. Rubin, Sylvia L. Anderson, Susan A. Sullivan, Barbara D. Williamson, Elizabeth A. Carswell, and Lloyd J. Old

Armand Bensussan, Beatrice Tourvieille, Li-Kuang Chen, Jean Dausset, and Marilyne Sasportes

Louis D. Falo, Jr., Kathleen Sullivan, Baruj Benacerraf, Matthew F. Mescher, and Kenneth L. Rock

Ronald Palacios

Tetsunori Seki, Nigel Spurr, Fumiya Obata, Sanna Goyert, Peter Goodfellow, and Jack Silver

H. E. Gruber, R. Koenker, L. A. Luchtman, R. C. Willis, and J. E. Seegmiller

Robert R. Goodman and Gavril W. Pasternak

Curtis C. Harris, Kirsi Vahakangas, Mark J. Newman, Glennwood E. Trivcrs, Abulkalam Shamsuddin, Nuntia Sinopoli, Dean L. Mann, and William E. Wright

M. Jackson Stutts, Calvin U. Cotton, James R. Yankaskas, Elaine Cheng, Michael R. Knowles, John T. Gatzy, and Richard C. Boucher

6595

6600

6605

6609

6614

6619

6622

6627

6632

6637

6642

6647

6652

6657

6662

6667

6672

6677

PFIZER EX. 1042 Page 7

Contents

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B­and T-lymphocyte cell lines, and monocyte-macrophages

Correlation of cell-surface phenotype with the establishment of interleukin 3-dependent cell Jines from wild-mouse murine leukemia virus-induced neoplasms ·

c-src is consistently conserved in the chromosomal deletion (20q) observed in myeloid disorders

Polyneuropathy with monoclonal gammopathy: Glycolipids are frequently antigens for lgM paraproteins

NEUROBIOLOGY

Benzodiazepine/ y-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil

Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry

Quantitative in vivo receptor binding Ill: Tracer kinetic modeling of muscarinic cholinergic receptor binding

Calcium binding in pigmented and albino eyes

Corrections

BIOCHEMISTRY

In vitro expression of chloroplast genes in Jysates of higher plant chloroplasts

IMMUNOLOGY

Antigen conformation determines processing requirements for T-cell activation

NEUROBIOLOGY

Precursor forms of substance P (SP) in nervous tissue: Detection with antisera to SP, SP-Giy, and SP-Giy-Lys

William N. Valentine, Donald E. Paglia, Steven Clarke, Bruce H. Morimoto, Misae Nakatani, and Richard Brockway

Kevin L. Holmes, Edmund Palaszynski, Torgny N. Fredrickson, Herbert C. Morse III, and James N. Ihle

Michelle M. LeBeau, Carol A. Westbrook, Manuel 0. Diaz, and Janet D. Rowley

Amjad A. Ilyas, Richard H. Quarles, Marinos C. Dalakas, and Roscoe 0. Brady

Richard W. Olsen, James K. Wamsley, R. Tyler McCabe, Randall J. Lee, and Peter Lomax

Bertrand Bloch, Theodora Popovici; Mariano J. Levin, David Tuil, and Axel Kahn

K. A. Frey, R. D. Hichwa, R. L. E. Ehrenkaufer, and B. W. Agranoff

Ursula C. Drager

Julie Bard, Don P. Bourque, Mark Hildebrand, and David Zaitlin

Howard Z. Streicher, Ira J. Berkower, Mark Busch, Frank R. N. Gurd, and Jay A. Ber;wfsky

Richard M. Kream, Thomas A. Schoenfeld, Robert Man~uso, Andrew N. Clancy, Walid El-Bermani, and Foteos Macrides

6682

6687

6692

6697

6701

6706

6711

6716

6721

6721

6722

PFIZER EX. 1042 Page 8

Proc. Nat/. Acad. Sci. USA Vol. 82, pp. 6497-6501, October 1985 Biochemistry

A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1 /epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma

(src gene family /kin:tse family/ gene amplification/ cancer)

KENTARO SEMBA*, NOBUYUKI KAMATA*t, KUMAO TOYOSHIMA*, AND TADASHI YAMAMOTO*

•The Institute of Medical Science, The University of Tokyo 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan; and tTokyo Medical and Dental University, f'aculty of Dentistry 1-5-45, Yushima, llunkyo-ku, Tokyo 113, Japan

Communicated by Takashi Su~:imura, June /0, 1985

ABSTRACT From a human genomic library, we obtained six v-erb/J-related DNA clones. A DNA probe prepared from one of the clones, >..107, hybridized to EcoRI fragments of 6.4 and 13 kilobase pairs of human DNA. Neither of these fragments was amplified in A431 vulva carcinoma cells, in which the gene encoding the epidermal growth factor receptor is amplified. In addition, the probe from >..107 hybridized with a single, 4.8-kilobase poly(A)+ RNA species and did not react with EGF receptor mRNA. Thus, we conclude that clone >..107 represents a v-erb/J-related gene (c-erb/J-2) that is distinct from the EGF receptor gene. In contrast, the other five clones were shown to represent the EGF receptor gene (c-erb/J-1). Partial nucleotide sequence analysis of the >..107 insert showed that this clone contained at least seven putative exons and that six of them could encode the kinase domain characteristic of protein products of the src oncogene family. Southern blot analysis showed close similarity of the restriction patterns of the rat c-erbJJ-2 gene and the rat 11eu oncogene, suggesting possible involvement of c-erblJ-2 in human cancer. In fact, =30-fold amplification of c-erbll-2 was observed in a human adenocar­cinoma of the salivary gland.

At least 19 genes have been identified as rctroviral oncogenes that arc responsible for inducing tumors in vivo and trans­forming cells in vitro (l). Ten of them apparently encode transforming proteins that share a kinase domain homologous to that of pp60"", a tyrosine-specific protein kinase. The cellular cognate, encoded by the c-src gene, also exhibits tyrosine-specific kinase activity. Of particular interest is the fact that tyrosine-specific kinascs arc also encoded by the genes for several receptors for polypeptide growth factors, including the receptors for epidermal growth factor (EGF) (2), platelet-derived growth factor (PDGF) (3), insulin (4), and insulin-like growth factor I (5). This implies a possible link between the action of the growth factor-receptor complex and the oncogene product with tyrosine-specific kinase activity. In fact, recent analysis of the v-erhB gene and the EG F receptor gene indicated that the v-erhJJ gene is a part of the EGF receptor gene and codes for the internal domain and transmembrane portion of the receptor (6-8). These findings, together with the extensive identity of the amino acid sequences of the v-sis protein and platelet-derived growth factor (9, 10), suggest that some viral oncogene products mimic the action of the polypeptide growth factor-receptor complex in activating a cellular pathway involved in cell proliferation.

'I he publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "mh•erti.H'IIll'llt" in accordance with lH U.S.C. §1734 solely to indicate this fact.

6497

We examined details of the relation between the v-erbB gene and the EGF receptor gene and the possible involve­ment of this gene in human cancer. During this study we identified two v-erbB-related genes, c-erbB-1 and c-erbB-2, in the human genome.

MATERIALS AND METHODS

Cells and Tissues. A431 vulva carcinoma cells and human embryo fibroblasts were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. K562 chronic myelogenous leukemia cells and MT2 adult T-cellleukemia cells were maintained in RPMI 1640 medium with 10% fetal calf serum. Mouse FM3A cells were from M. C. Yoshida (Hokkaido University) and primary tur 1rs were provided by K. Rikimaru (Tokyo Medical and Dernal University).

Isolation of Clones. A human genomic library was con­structed from placental DNA as described (11). The library was screened for the v-erbS-related sequence by plaque­hybridization as described (12) in 30% (vol/vol) formamide/ 4x NaCI!Cit (lx is 0.15 M NaCl/15 mM sodium citrate)/50 mM Hcpes, pH 7.0/denatured salmon sperm DNA (20 f.tg/ml)/lOX Denhardt's solution (1 x is 0.02% polyvinyl­pyrrolidone/0.02% Ficoll/0.02% bovine serum albumin) at 42°C for 16 hr. After hybridization, the filters were washed with 2x NaCI/Cit/0.1% NaDodS04 at room temperature and then with 0.6x NaCI/Cit/0.1% NaDodS04 at 50°C. Plaques were purified by successive plaque-hybridization. The probe used was a 1.7-kilobase-pair (kbp) Sst I-Stu I DNA fragment that represents the v-erbB gene of avian erythroblastosis virus strain H (6); it was labeled with [a-32P]dCTP by nick-translation (13) to a specific activity of 2 x 108 cpm/ f.tg of DNA. Phage DNAs were prepared as described (14).

Nucleotide Sequence Analysis. The nucleotide sequence was determined by the procedure of Maxam and Gilbert (15) and the dideoxy chain-termination method (16, 17) in con­junction with bacteriophage M13 mp19 (18).

Blot-Hybridization Analysis of DNA and RNA. High mo­lecular weight DNAs were prepared from chicken blood, human embryo fibroblasts, mouse FM3A cells, rat spleen, and primary tumors. The DNAs (10 f.tg per lane) were digested with restriction endonucleases under the conditions recommended by suppliers (Takara Shuzo) and fractionated by electrophoresis in 1% agarose gels. The 1ragments were subjected to Southern blot hybridization (19) at 42°C for 16 hr in 4X NaCI/Cit/50 mM Hepes, pH 7.0/10x Denhardt's solution/denatured salmon sperm DNA (20 J.Lg/ml)/30% (relaxed conditions) or 50% (stringent conditions) formam­ide. After hybridization, the filters were washed with 2x

f\bbrcviati~ns: EGF, epidermal growth factor; kb, kilobasc(s); kbp, kilobasc patr(s).

PFIZER EX. 1042 Page 9

6498 Biochemistry: Semba et a/. Proc. N at/. A cad. Sc i. USA 82 ( 1985)

a c

kbp 2 4 6 2 4 6

23-

* 94-6.6- * * 44- - * -- -- -- -2.3- • -2.0- - -0. 5 -

b S E S s X EK s s s X I I [ I ¥ I ]I I :>. 107 1---i 1 kbp

FIG. 1. Identification and cloning of the v-erbB-related sequence that is di stinct from the EGF receptor ge ne . (a ) Hybridi zation with the v-erbB probe. DNAs prepared from A431 cell s (lanes 1, 3, and 5) and pl acenta Oanes 2, 4, and 6) were digested with Lco RI Oanes 1 and 2),Sac I (lanes 3 and 4), or Pvu II (lanes 5 and 6). Because the placental DNA was contaminated with RNA , sampl es in l a ne~ 2, 4, and 6 cont amed less DNA than those in lanes 1, 3, and 5. The digests were subjected to Southern blot hybridi zation using a v-erb/3 probe under the relaxed conditions described in Materials and Methods . Autoradiographs obtained by longer ex posures of lanes 2, 4, and 6 arc shown to the right of the respective lanes. Arrowheads indicate positions of fragments of Hind III-digested :>. DNA used as standard s. Stars indica te bands that were not amplified in the A431 samples. (b) Restriction map of the :>.107 in sert. E, K, S, and X represent restriction sites of L ea R I, Kpn I, S ac I, and Xba l , respectively. The thick line indicates the Kpn 1- Xba I fragment (KX fragment) used as a spec ific probe of the c-erb/3-2 gene. (c) Hybridization with the KX fragment. The same filter represented in a was washed and then hybridi zed with th e KX probe und er the stringent conditions described in Materials and Methods.

NaCI/Cit/0 .1% NaDodS04 and then with either 0.2 x NaCI/Cit/0.1% NaDodS04 (s tringent) or 0.6 x NaCI/Cit/ 0.1% N aDodS04 (relaxed). RN As were prepared by the guanidinium isothiocyanate/cesium chloride method (20) . Poly(A) + RNA selected by oligo(dT)-cellulose (P-L Biochem­icals type 7) column chromatography was denatured with 50% formamide/2.2 M formaldehyde and 5 JLg of each RNA sample was subjected to electrophoresis in 1% agarose gel containing 2 .2 M formaldehyde (21) . RNAs on the gel were transferred direct ly to a nitrocellulose filter and subjected to blot hybridization under stringent conditions, as de scribed (22). The DNA probes used for blot hybridization were v-erbB DNA (described above), an EG F receptor eDNA clone (pE7) (22) , and c-erbB-2-specific DNA (described below).

RESULTS Isolation of a Human v-erbB-Related Sequence Distinct from

the EGF Receptor Gene. High molecular weight DNAs prepared from A431 cells and human placenta were dige sted with either EcoRI , Sac I, or Pvu II , and the dige sts were analyzed by Southern blot hybridization under relaxed con­ditions with the v-erbB probe, which covers almost 90% of the v-erbB gene. Most of the v-erbB-related sequences were amplified in A431 cells at least 20-fold compared with those in placenta (Fig. 1a). These sequences were observed as 8.1- , 5.9-, 5.4-, and 3.5-kbp EcoRI fragments; 20- , 4.7-, 3 .3- , 1.7- , 0.9- , and 0.5-kbp Sac I fragment s ; a nd 6.6- , 3.3- , 2.9- , 2.3- , 1.9- , and 0.9-kbp Pvu II fragment s . The amplified sequences were of the EGF receptor gene as reported (8 , 23) . However , we observed several restriction fragments that hybridized to the v -erbB probe but that were not amplified in A431 cell s 03-and 6.4-kbp EcoRl fragment , a 6.9-kbp Sa c I fragment, and a 5 .1-kbp Pvu II fragment , marked with s tars in Fig . 1a) . This suggested that besides the EGF receptor gene, there may be

anothe r v-erbB-rela ted ge ne, from w hi c h th e una mplified restri ction fragments a re gene ra ted .

We search ed a huma n geno mi c library fo r v-e rbB-re la ted sequences, us ing the v-erbB probe, unde r the re laxed hy­bridiza tion a nd washin g conditions, a nd iso la ted s ix indepe n­dent clones . Restri cti o n ma p ana lys is s ho wed th a t all the cloned inserts except th e A- 107 inse rt re pre se nted one ge ne, c-erbB-1 . In these clones, we ide ntifi ed four £co R I fragme nt s as e xon-conta ining seque nces by th e c rite rion tha t the y hybridized to the v-erbB probe. A ll th e se fragme nts we re

a b 2 3 4 5 2

-- 4.8 kb

4

kb -to.o - 5.6

- 2.9

FIG. 2. Express ion of the EG F receptor gene and the c-erbB-2 gene in human cell s. Duplicate samples of pol y(A)+ RN A were subjected to blot hybridization with the 32P-labeled KX fragment (a ) or with the 32P-I abeled EGF receptor e DN A clone pE7 (22)(b). Stzes of RN A species that hybridi ze with th e probes are given. Chicken ribosomal RN As (28S, about 4.8 kb , and 18S , about 2.0 kb) and Rous sarcoma virus RNA 09S, about 10.0 kb) served as size standards. RNAs were isolated from human pl acenta (lanes 1), A431 cell s (lanes 2), human embryo fibroblasts (l anes 3), MT2 ce lls il anes 4), and K562 cell s (lanes 5).

PFIZER EX. 1042 Page 10

Bioc hemi stry: Semba eta/. Proc. Nat/. A cad. Sci. USA 82 ( 1985)

L E

I I 5' --------+1--tllll+l-tll---+111-- 3' ;>..107

b

c-L~rbJJ -2

1-:Gf.'H

c-t.· 1 bl3- L EGl·'H

, . -t.·J l.;U- 2 I;G FI<

r__- -c I l>B- L I·' FCH

c-er bD-L CcT·n

c:-t:rb U-2 t:G'TH

c-nrhB-2 t:GFH

c-e r bB-2 EGPK

c-e rbB-2 EGFH

c:-erb B-2 EGFH

c-erbB-2 'EG"FH

30 60 ~ly ll ~ 'I' J pi l~Pr ·o A S pGlyGluAS ilV~iLy s ll c Pr o VdLAlall c Ly ~Vd lLeuAr~GluAs n'rhrScr P ro Ly sAlaAsnL y s

gGGCNI'C'I'Gt ;IIT<'C<' T G/\ 'I'GGGG/\G,\,\TG' I'G/\ ,\IIII TTCCAG 'I'GGCC/\ 'I'C AA/\GTGTTG/\GGGA/\IIAC ACC T CCCCCA AAGCCAIICAAA

yO ,\1. II ,\ T 1\ T I ~ O C 'l' G 1\1\ ,\ II GC 1\ l ~ O '!' G G

f; 1 u I I t.~L12UI\sp G 1 u/\ 1 o'l' y 1 V,Jl Mc-LA lac.; 1 Yvu lGl yS e ,- Pr o 'l'y rVa l ScrArg Lcu Lc uG ly I leC y s Le u Th r Se r G/\1\/\ 'I'C 'I"I',\C/\<' y L----- .lq GI\ ,\ GC/1 '1'1\CC'I'G,\' I'GGCT GG 'I' (,;TGGGC TCCCC,\ '1' 1\ 'I'G'l'C 'l'CCC GCC' I'TCT GGGCAT CT GCC 'l'GACI\TCC

C C d) 0 C C A 21' 0 1\ 1\A CC C G c; c; 2 4 0 C C

Th' Vd l t; 1 n L t.~ ~, Vol Til r C 1 n I. ~,.•uf'olt• L P r~ o 'l'y r C.;l y c ys Lc ~Lc u/\ s p!lt s vul/\rgGl u/\ s n ArgGl yAn3Le u Gl y SerG lnAspLeu IICGC'I'CC/\l;C TGC 'I 'CIIC IIC ,\GC ' ! "I'I\TGCCl' 'l' ~. ·l·c c;c TGCC 'I'C 'I'T IIGI\CC,\ 'l'G 'I'CCGGGAI\A ,\CCGCGGACGCC 'l'GGGC 'l'CCC AGGACCTG

1\ Cl\ l' G C ·n: C G T C A/\A AC/\AT A T T !70 300 330

Lc u/\ ~ ,,·r r pC y~ M~LLlrl ll cAldLys GlyMeLSer'ryrLe ~Gl u A s pva l ArgLeuVal Hi sArgAs pLe ~Al aA l aArg C T GII/\f" I'GG T G'l'IITl ;C IIGNI"I 'GC< . AI\Gg L----- agGGGIIT GAGC 'I'IICC' I'GG,\CGA' I'GTGCGGC T CG T AC ACAG GGACTTGGC CGC T CGG

l' G C r, C A '!' CCG T CT G G C C C A C A ]60 390 '120

AsnVal L~u V~lLy sSe rPJ- 6As r l il lSVdlLyslle'l'hrA spPhcG ly L c ~AlaArgLc uLc uAsp il eAspG lu1'h rG l~Ty r Hi sA l a IIACG T GC 'I'GGT C ,\AGAG' I'CCCAACC ,\'I'GTCAIIIIII TTAC ,\ G/\C TTCGGGC T GGC T CGGC T CC T GGACIITTGACGAGAC AGAG T ACCATGCA

,\ G 1\ C ll GC' G G C 'I' '!' C AliA G'I'GCG A II A 4 50 4 80 510

As pGlyGlyLy s ValPro Tl~Ly s 1' rpMetAld LcuGluScr ll c Le uAr~ArgArg Phe 'I'h r H isGl nSer AspVal 'r r ~ GATGGGGGC IIIIG'J L---- - agCTGCCC NI'C IIAGT GG ,\'I'GGCGCT GG,\G T CC I\TTCT CCGCCGGCGG 'I"I'C IICCC ACC AGAGT GATGTGTGG

II ,\ II T , 111' A A 'I' ,\ A 1\ AA T C A'!' C ~ ~ o s1o

Scr 'l'y, G 1 yV<J I 'l'h t· Va l TrpGlu LcuMc~ Th r PheGl y lllaLysPro'l'y r ,\ spG ly IleProAlaArqClu I leProAsp ,\(,; ']'']' ll'l'G<j L-- - -- agGTGTGI\C 'I'GTGT GCG AGCT GII T GACT'I'TTGGc;GcC AAACC'l"I' I\Cc;,;T GGGATCCC /\GCCCGGGAGATC CCT GAC

C C G C T T C 11 '1' G A T C A T A C 'I' CT C

GUO 630 6 ?0

L~ uLc uGI~Ly sG lyGlullrg LeuProG lnP roPro !l 6CysThrll eAspValTyr Met ll eMet ValLy sC y s T rpMet C TGCTGGIIIIIIIIGGGGGIIGCGGC T GCC CC ,\GCCCCCC Nl'C T GCIICCA TTGATG T CTIICAT GAT CII T GGT CAAATg t ---- -agGTTGGATG

,\ C G ,\ II A C C 'I' A II T C G 7 50 C 690 720

ll ellsp ScrG lu C y s Ar~ProllrqPhe ArgGluLc uValSerGluPheSerllrg MetAlaArgAspP roG lnArgPhe Va lValll eG ln IITTGIIC '!'C T G,\ATGT C GGCC AIIGATTCCGGG AGTTGGTG T CTGAATTCT CCCGC ATGGCCAGGGACCCCC AGCGCTTTGTGGTCATCCAG

II G II Til C ,\G 780

1' II C ATC Mil 8 1 0

C 1\ !ICC 'I' 8!

0 AsnGlulls pLeuG lyP r~l\laSerPro Le ull s p Ser 'l'hrP h eTy rAr~Ser Leu LeuGluAspAspAspMetG lyAs~Leu

gt-----agiiiiT CIIGGAC TTGGGCCCAGCCAGTCCC TTGGIICIIGC IICCT TCTIICCGCT CAC T GC TGGAGGACG ATGAC ATGGGGGACCTG GGGG IIAGAII C ATTTGC A T AC A TC A T G C A T A A AC G

870

ValAspAla Gl u Gl uTyrLe uValPr~Gl nGl nGlyP he Phe GT GGAT GCTGAGGAGTATC T GGTACCCC IIGC AGGGC TTCTTCT--

C C C C A C II A

6499

FI G. 3. Nuc leotide sequence of the exons for the kinase domain ofc-erbB-2. (a) Putative exons in the /..107 clone are indicated by thick vertical ba r>. T'he two EcoR I IE) sit es are shown by vertica l lines. (b) Nucleotide seq uence of the putat ive exons. Restriction fragments that hybridized o v-erh/J DNA were sequenced. The exons we re defined by the splicing consensus seq uence and by the high homology of the deduced amino

ac id sequ ences to th ose of th e EGF receptor ge ne EGFR. The nucleotide sequence of putative exons of c-erbB-2 (in capital letters) is compared it h that of th e EGF receptor gene (8). Onl y nonidentical nucleotides are shown for the EGF receptor gene. lntron sequences at the splicing

~ it es th at flank th e exons are shown in lowercase lett ers. The splicing donor site of the last exon was not identified. The predicted amino acid ~ e qu e n ce is shown above the nu cleotide sequence.

ampl il~ ed in A431 ce ll s (data not shown) , suggesting that the c-erb/3-1 ge ne is the same as the EG F receptor gene .

A re striction map of the A.l07 DNA inse rt is shown in Fig. lb. T he 440-bp Kpn 1-Xba I fragment (KX fragment) was used as a probe in the following hyb ridizat ion experiments . T hi s fragment did not cont ain huma n repetitive sequences a nd hyb ridi zes with the v-erbB probe under the relaxed co nditi o ns . The v-erbB probe on the filter represented in Fig. lo was washed off a nd the filter was rehybridized with the 32P -Iabe led KX fragme nt. Fig. l c s hows that the KX probe reac ted wit h the re striction fragment s (13- and 6.4-kbp EcoRI frag me nts, 6.9-kbp Sac I fragment , and 5.1-kbp Pvu II frag me nt) tha t were not amplifi ed in A431 ce ll s, suggesting tha t th e A.l07 insert does not rep resent the EG F receptor ge ne. Therefore, we tentatively concluded that the Al07 i n ~e rt represe nted a v-erbB-related gene (c- erbB-2) tha t d iffers from the EG F receptor gene .

Th e n we exa mined the express io n of the v-erbB-related ' e nes . RNA s were prepared from human cell lines (K562 , MT2 , a nd A431 ce ll s) , huma n embryo fibrobla sts , and human p lace nta . Hybridization was carri ed ou t under stringent co ndition s (Fig . 2 o a nd b). Poly( A) ' RNA samples were ~ ubjec t ed to blot-hybridi zation ana lysis . As reported previ-

ously , most transcripts detected with the EGF receptor eDNA probe were of 10 kilobases (kb) and 5.6 kb and were apparently overproduced in A431 cells (Fig. 2b and refs. 8 and 23) . In addition, variant 2.9-kb mRNA was detected in A431 cells, as reported previously (8, 23). On the other hand the mRNA that hybridized with the KX probe was a sing!~ species of about 4 .8 kb (Fig. 2a). The level of expression of thi s mRNA was virtually the same in A431 cetls as in placenta and was slightly higher in human embryo fibroblasts than in other cells. Interestingly , transc ription of the EGF receptor gene a nd c-erbB-2 genes was not detected in the leukemic cell lines K562 a nd MT2. The above results strongly indicate that the human genome contains two v-erbB-related genes, the c-erbB-1 /EGF receptor gene and the c-erbB-2 gene.

c-erbB-2 Encodes a Protein with a Kinase Domain. We determined the nucleotide sequence of the A.l07 insert that hybridized with the v-erbB DNA and identified seven puta­tive exons flanked by a consensus sequence of splicing junctions (data not shown) . Fig. 3 shows that the nucleotide sequences of all seven exons (total, 885 bp) were highly homologous with the corresponding regions of the eDNA clone for the EGF receptor (74% ). Only one reading frame deduced from putative exons was not interrupted by termi-

PFIZER EX. 1042 Page 11

6500 Biochemistry: Semba et a/. Proc. N at/. Acad. Sci. USA 82 (1985)

c - e rbB-2 EGFR

GIW I PDGEN VKI PVA I KVLRENTSPKANKEILDEAYV MAGVG SPYVS RLLGI CLTSTVQL- 60 L E K E A S DN fl C

c - e rbB- 2 EGFR

VTQLMPYGCLLDHV RENRGR LGS QDLLNWCMQ IAKGMSYLEDVRLV~IRDLAARNVL VKSP -1 2 0 I F Y HKDN I Y V N R T

c - erbB- 2 EGFR

NHVKr TDFGLARLLD I DETEYHADGGKVP I KwMP.d:s r LRRRFT HQ s ovwsYGVTVWELM -18 o Q K GA E K E H I Y

c - e rbB-2 EGFR

TFGAKPYDGIPAREIPDLLEKGERLPQP PICTI DVY MI MVKCWM IDSECRPRF RELVSEF-2 40 S S SS I ADS K I I

c - erbB- 2 EG FR

SRMARDPQRFVVIQ-NEDLGPASPLDSTFYR SLLEDDDMGD LVDAEEY LVPQQGFF K YL GO RMHLP 1' N A MDEE D V D I

FIG. 4. Comparison of the deduced amino ac id sequences of c-erbB-2 and the EGF receptor (EGFR). The standard one-letter abbrev iations are used. Only nonmatching amino acids are shown for the EGF rece ptor. The kinase domain of EGF receptor is shown as the amino acid sequence between positions 705 and 937 (8). Asterisks indicate residues common to the protein products of the v-ab/ , v-erbB , v-fgr, v ~fins , v-fp s , v-ros, v-src, and v-yes genes.

nation codons. The amino acid sequence deduced from th is open-reading frame suggested that these exons in c-erbB-2 could encode a polypeptide characteristic of the kinase domain (residues 1-233 in Fig. 4) that showed high homology with the EGF receptor kinase domain (82%). The amino acid sequence of the kinase domain of the c-erbB-2 product is highl y homologous to that of the v-erbB product and di stantly related to those of the protein products of other members of the src gene family (Table 1).

Conservation of the c-erbB-2 Gene in Vertebrates. High molecular weight DNAs were prepared from chicken blood , mouse FM3A cell s , and rat spleen. The DNAs were digested with EcoRI and subjected to Southern hybridization with the 32P-Iabeled KX probe. Since the hybrid ization conditions were stringent enough to avoid detection of the EGF receptor gene with the KX probe (see F ig. Sa, lane 1), a ll the fragments shown in Fig. 5 are specific to the c-erbB-2 gene . Thus, we conclude that c-erbB-2 is conserved in vertebrates.

The possible re lat ion between c-erbB-2 and the recently di scovered neu oncogene was examined by Southern blot hybridization. The 32P-Iabeled KX probe hybridized with a rat EcoRI fragment of more than 23 kbp and with a 4.4-kbp fragment generated by digestions of the DNA with EcoRI and BamHl (Fig. 5). These fragments are the same sizes as the respective restriction fragment s of the neu oncogene (24).

Association of Amplification of the c-erbB-2 Gene with a Primary Human Tumor. Next we examined whether the c-erbB-2 gene is amplified in human cancers. High molecul ar weight DNAs were isolated from human placenta, A431 cells , and several primary tumors (one neuroblastoma , two epidermoid cell carcinomas, and one adenocarcinoma of the salivary gland) . These DNAs were digested with EcoRI and analyzed by Southern blot hybrid ization with the KX probe. Two EcoRI fragment s (13 and 6.4 kbp) were identified in all the DNA samples and were amplified about 30-fold in the adenocarcinoma of the salivary gland (UY adenocarcinoma) , indicating poss ible invol vement of c-erbB-2 gene expression in this cancer (Fig. 6a) . We do not know whether the entire c-erbB-2 gene is amplified in thi s tumor , since the KX probe detects o nl y part of the c-erbB-2 gene. When the same filters were hybridized with the EGF receptor eDNA probe, am­plification of the EGF receptor gene was observed in A431 DNA , as has been reported (8, 23), but not in the UY adenoca rcinoma. Owing to the limited amou nt of tissue available, RNA of the UY adenocarcinoma could not be analyzed. We have detected amplification and enhanced

ex pression of the c-erbB-2 ge ne in a ce ll line (MKN-7) estab li shed from human gastri c cance r (data no t shown).

DISCUSSION

T here are two v-erbB-rela ted ge nes in the human ge nome: the c-erbB-1 /EGF receptor ge ne and the c-erbB-2 gene. The c-erbB-2 gene is a ppa rently not a pseudo gene of the EG F receptor gene because it consists of both exons a nd introns and is transcribed in a ce ll -type-specific ma nne r. We could not find any termination codon interrupting the reading frame of the putati ve kinase domain coded by c-erbB-2 . In addition , we have shown that the c-erbB-2 gene is conserved in chickens , mice, rats, and huma ns , indicating that thi s gene is not confined to the huma n ge no me and probab ly fu lfill s a n indi spensable fun ction. Obviou sly, efforts to ide ntify a pro­tein product of the c-erbB-2 gene are required .

The amino acid seque nce deduced from the nucleotide seq uence of c-erbB-2 exons in A107 suggests that the c-erbB-2 gene may code for a tyros ine-specific prote in kinase. The putative kinase doma in e ncoded by the c-erbB-2 gene shows 82% homology with tha t of the EG F receptor and the v-erbB protein . The eight protein products of the SIT fami ly show tyrosine kinase activ ity in vitro. All the a mino acid res idues that are common to the se e ight proteins a re a lso conserved in the c-erbB-2 ge ne product , strongly supporting the idea that thi s gene cou ld e ncode a prote in kinase. O ur nucleotide sequence data further show that the c-erbB-2 protein carries an amino acid seque nce (residues 234-295) that is 65% ho mologous to the corresponding portio n o f the EGF recep­tor. Since the deduced amino ac id seque nce did not conta in a puta tive tra nsme mbrane portion , it is not known whether c-erbB-2 encodes a receptor-like prote in o r only a po lypep­tide similar to the intrace llula r po rtion of the EG F receptor. Analys is of the a mino-terminal portio n o f the protein is necessary to determine whether it ha s a trans me mbrane seq ue nce a nd a domain that recogni zes so me growth fac­tor(s) .

Recently , the ne 11 oncoge ne was ide ntifi ed , by gene­transfer techniques , in the ge nomes o f four neuro/gliob las­toma cell lines derived from c he micall y induced ra t tumors (24). The neu gene is re lated to the v-erbB gene a nd e ncodes a tumor antigen with a molecul ar we ight of 185 ,000 (p185). pl85 is sero logicall y rela ted to, but di s tinct from , the EGF receptor (25). Our data show that the rat c-erbB-2 ge ne yields restri ction fragme nts of the same s izes as does the rat ne11 oncogene when digested with EcoRI o r EcoRI plus Bam HI.

Table l. Amino acid sequence homology between the c-erbB-2-encoded kinase domain and those encoded by various viral oncogenes

Viral oncogene: % homology:

erbB 82

S I'C

43 abl 42

yes 42

fg r 41

ros 38

fp s 37

mil 28

.fins 27

mos/rl'i < 25

PFIZER EX. 1042 Page 12

Biochemistry : Semba e t a/.

a h

• • F IG. 5. Con ~e rva ti o n of th e c-l'rb/J -2 ge ne in ve rt e bra t e~. Ca)

Conserved C-l'rb/J -2 ~eque nce~ in chicken and mou ~e. N itrocc llul o~e filt ers containing Eco Rl dige~ t s of DNA from human placenta IIane 1), mouse FM3A ce ll ~ (lane 2). and chicken blood <lane 3) wen: probed with th e K X DNA. (b) Clo~c ~ imi l arit y of th e C-l'r!JB -2 gene to th e neu oncogene. Ra t D NA was cleaved with either L coRI (lane l) or Eco Rl plu ~ /JamHI IIane 2). The d i ge~ t ~ we re analyzed by Southern blot hybridi za ti on w ith the K X probe. Arrowhead\ indica te Positions of fragmenh from Hind I II digeq ion of bac teriophage 'II DN A (see Fig. la ).

These findings lead us to speculate that there is a close relation betwee n c-erbB-2 and neu.

Amplifi cation of pro tooncogcnc~ such a~ myc and m s h a~ been reported to be associated w ith variou s human cancers (26-30) . Thi s amplifi ca tion suggests that the protein products of the amplified protooncogenes are in vo lved in some stage oftumorigenesis. Of parti cul ar int eres t i 'i th e role(s) of growth factor receptors in tumori genes is, w hich is inferred from the Observations that EG F receptors arc ex pressed in large numbers on the surface of ce ll s of epidermoid carcinomas (31-33). Since the c-erbB-2 gene could code for a protein ~imil ar to the EG F receptor or at leas t it s intracellular domain and was amplified in an adenocarc inoma of the sa li vary gland , the gene products ex pressed abnormall y in thi s tumor

b

2 4 2 4

\

F1o. 6. A mplification of the e-nbB-2 gene in human primary 1Urnors. (a) Nitroce llulose filt ers containing Eco Rl digests of tum or DNAs were probed with c-erbB-2-~pec ifi c K X DNA. (b) After r·ernoval of the fir st probe, th e fi lt ers were then hyb ridi zed with the t GF receptor eDNA probe. H ybridi za ti on was carried out under l tr·ingent conditi on ~. D NA~ were i ~olat ed fmm a neuroblastoma (J<tne 1), an adenocarcinoma of the ~a li va r y gland (J ane 2), two 1quamous cell carcinomas (lanes 3 and 4). placent a IIane 5). and the A431 cell line (fane 6). M ark ers at left in 11 and h indica te posit ions Of fragments from J-/indlll dige~ tion of >.. DNA.

Proc . N at/. A CliC!. Sci. USA 82 ( 1985 ) 6501

might funciion in es tabli shing and maintaining th e cancerou s state.

Note Added in Proof. King 1'1 a/ . (34 ) have independentl y isolat ed an n bB-2 gene amplified in a human mammary carcinoma .

We thank Y. Miura and H. Kawano for exce llent technical ass istance. M . N ishi zawa and N . Mi yajima for helpful di sc 11 ss ions. and S. Sasaki for typing thi s manuscript. We also wi sh to th ank N. Nomura for DNA from a human primary neuroblastom a and 1. Pa ~ t a n fo r recombinant p l a ~ mid pE7. Thi s work was support ed in part by a Grant -in -A id for Spec ial Project Research Ca nce r-Biosc i­ence from th e Mini stry of Educa tion. Sc ience. and C ulture of .J apan.

1. Land . H .. Parada . L. F. & Weinbe rg . R. /\. il 'iXJ ) Scien ce 222, 77 1- 778. 2. Cohe n. S .. Carpent er. G . & King. 1 .. ( 19801 J . 1/io / . C lt cm. 255,

4834 - 4842 3. Nishimura. J .. Hung. H . S. & Deuel. T . F . 11982 1 Proc. N ut/ . Arm/. s·ci

USA 79, 4303- 4307. 4. Ka , ugil , M .. Zick. Y .. Ulit hc. D. L .. Crcllaz. M. & Kahn . C. R. 11982)

Nu ture ( l.oll(/on ) 298, 667- 61>9 . 5. Ru bin . J . ll .. Shia . M. ,\ . & Pil ch. 1' . F . !19811 N uturc U.oll(/on J 305 ,

438- 440. 6. Yamil lllOio . '1 .. Nishida. T .. Mi yaj ima . N .. Ka wai . S ., Ooi. T . &

Toyoshimil . K I 19831 Cell 35, 71- 78. 7. Dow nwa rd . J .. YanJcn . Y .. Ma yes. E .. Sc race. G .. Toll y. N. ,

Stockwel l. P .. Ullri ch. A .. Sc hless inge r . .1 . & Wat erfi e ld . M. D. (1 9H4 ) Nu ture (Lo ndon) 3117, 52 1-527.

H. Ullri c h. A .. CouS\ens. 1~ .. Haylli c k. J . S .. Dull , T . .1 .. G ray. A .. Ta m. 1\. W .. Lee. J .. Yarden. Y .. Libermann . T . /\ .. Sc hle ss inge r. J . . Down­ward . J .. Ma yes. E. L. V .. Whillle . N .. Wat erll e ld . M D & Sec burg, P. H (19R4) N utun• (/ .oll(/on ) 309,4 18- 425

9. Wat .: rlleld . M. D .. Sc riiCC. G. T .. Whill le. N ., Stroobant . P .. John sson , /\., Wastcson . /\., Wes te rmark . U .. Heluin . C.- H .. Huang. J . S. & Dcud . T . F. (1 983) N ature (/ .oll(/on ) 304, 35-39.

10. Doolill le. R. F .. Hunk<lpi llc r . M. W .. Hood. L. E .. Dcvare. S. G., Robbin ~ . K. C .. Aaro n">n. S. A. & /\ntoniad e~. H. N. i i9Xl) .1i ciencc 22 1, 275-277.

II . Maniati >. ·1 .. Hardi son . R. C .. Lacy. E .. Laue r. J .. O 'Co nnc l ... Quon , D .. S im . G. K. & Ermatia dis. A. (197X l C!'il IS, 687- 701

12. Bento n. W. D . & Davis. R. W. ( 1977 1 Sc ience 196, 1HO- IX2. 13. Mani ati s. T .. Jcllrey. /\. & Klcid . D . G. 11975 ) Proc. Nat / . Acod . Sc i.

USA 72, 11 H4 - 11 HX. 14. Dav is . R. W .. Botste in . D. & Rot h . J. R. 11 9X2) Arln<nc•· d lla c teria/

Ger"' tics (Cold S pring Harbor L.ahorat ury, Cold S pring Harbor . NY) , pp . 109- 111 .

15. Ma xa m./\ . M. & Gil be rt . W. 119X0) M r· riwrls Ln ~ymol. 65, 499- 560 . 16. Sa nge r. F .. Nick lc n. S. & Cuuhon. /\. R. (19771 Proc. Nu t/. i\ r'(((/ . Sci .

us,, 74, 5463 -5467. 17. Mes , ing. J .. Crca . R. & Secburg. P. H !1'18 1) N ucl<' ic ,\ c ids li<'.l' . 9,

30'1- 121. 18. Mess ing. J . & Vi eira . J. (1 982) (ienc 19, 269- 276. 19. Southe rn . E. M. (1975) J . M ul. /Jio l. 98, 503-5 17 20. Chirgwin . J . M .. Przy byl;, ,/\. E .. Mac Don ald . R. J. & RuiTer. W. J.

(197'11 /Jioc hem is tr\' IH, 5294-5299 21. l.ehrach. H .. Dia;no nd . K .. Wozney, J. M. & Uocdtke r. H. 11977 )

/Ji()(' h emistry t6, 4743- 475 1. 22. Xu. Y.-H .. Ishii . S .. Clark . A J . L .. Sulli van , M .. Wil son. R. K . Ma ,

D. P., Roc. U. /\., Merlino. G. T . & Pas tan. I. (J 9X4) N aturr· (l.o ndon ) 309, !:<06- 810 .

23. Merlino. G. T .. Xu. Y. · H .. hhii . S .. Clark . /\. J. l ... Scmba , K., Toyoshima . K., Ya mamoto, '!' & Past<lll , I. (1984) Scienn· 224, 4 17- 41 9.

24. Sc hec ht er. /\. L., Stern . D. F .. Vaid yanathan . 1 ... Dec ke r, S . J. , Drcb in . J. A .. Greene. M. I. & Wein be rg, R. /\ . (19X4 1 N at /11'<' (London) 312 , 513- 516.

25. Dcrbin . J . /\.,S te rn . D. F . Link . V. C .. We in be rg, R. A. & Green . M. I. (1984) N lllfll'<' (Lo udon ) 312, 545 - 54X.

26. Collin s, S. J. & Groudinc. M. !1 982) Na ture (/ .one/o n ) 298, 1>7 '1 - 68 1. 27. Dalla-ho vc ra. R .. Wung-S taal. F. & Ga ll o. R. C. ( 19X2) N ature (/.onc/o nj

299, 61- 63. 28. Alit aro. K .. Sc hwab. M .. Lin . C. C .. Va rmus. H . L . & Bi shop. J . M.

(1983) Proc. N11 tl . ,\cad. Sci . USA 80,1 707- 1711. 29. McCoy. M.S .. Toole . J . .1 .. Cunningham . .1 . M .. C hang, E. H .. I.owy,

D. R. & We in be rg, R. A. (19831 N11 turr· (/ .ondcm J 302 , 7'1 - XI. 30 . Schwab. M .. 1\ litalo , K .. Varmu s. H . E .. Bi shop , .1 . M . & Gc m gc. D.

!1 983) N ature• ( l.onrlorl) 303, 497-501 . 31. ';trpc nt cr . G .. King, 1 .. L. & Cohe n. S. 11'1791 J . Bioi . Ch ern . 254,

48X4 - 4R9 1. 32. Fab ri cant . R .. De larco. J. L. & Tod;11·o. G. J. ( 1'1771 Proc. Nu t/. A n ul .

Sci. USA 7-1, 565-569. 33. Cow ly. J .. Gutason. B . S mi th . .1.. Hendlor. F. & () za nnc. ll. 11984) in

Can en (' ,·1/s. eds. Lev ine. A . J .. Va nde Wuudc. G. F .. T npp . W. C. & Watson. J. IJ . (Co ld Spring Harbor l,ahmatory, Co ld Spring Harbor . NYI. Vo l. I . pp. -~- 10 .

J.t. King. C. R .. Kra '"· M. H. & Aa ron son. S. 1\. i1 '.JH5) S ci"" ' '''· in press.

fh is m.ateri a I w as co~i ed atth.e NLM and may t>e

Subject UCS Co~yright Law s

PFIZER EX. 1042 Page 13

Biochemi <; try : Semba e1 a /.

b

-•

• FtG. 5. Con ~e rva ti o n of the c- t·rh/J -2 gene: in ve rt ebrate\. (a)

Conse rved c-ah/J -2 \ equ c: nce \ in chi cke n and mou ,e . Nitrocellulo' e filt ers containing L'co RI dige'i t\ of DNA from human placenta (lane 1), mo u\ e FM 3A ce ll \ (l ane 2) . and chicke n blood (lane 3) we re Probed with the K X DNA . (IJ) Clo'ie 'i imilarit y of th e c-erh /J -2 ge ne to the neu oncoge ne. Rat DNA wa\ cleaved with either /~·co R 1 (lane 1) or l:.'co Rl plu \ /JIIIn lf l (lane 2) . The dige\ t\ we re analy;:ed by Southe rn blo t hyb ridi za tio n with the KX probe . Arrow head' ind ica te Po~ iti on s of fragme nh from /l ind II I dige'i ti on of bacteriophage ;.. DN A (see Fig. la ).

These findings lead us to speculate that th ere is a clo 'ic re lat ion bet wee n c-erh/J -2 and neu.

Amplifica tion of protooncogcne-; 'iUCh a-; myc and m s ha-; been reported to be a-;soc iated w ith va r ious human ca nce rs (26-30) . This amplifica tion suggests that th e protein rroduct 'i of the amplifi ed protooncoge nes are in vo lved in <;omc qage Of tumorigenesis. Of part icular in terest i -; th e rolch) of growth factor receptors in tumorigene'i is. whi ch is inferred from the observations that EGF rece rtors are ex pressed in large number<; on the 'i llrface of ce ll s of epidermoid ca rcinoma <; (31-33). Since the c-erh/J-2 gene could code for a prot ein sirnil arto the EG F receptor or at least it -; intrace llular domai n and was amplifi ed in an adenocarc inoma of th e -;a li va ry gland , the gene product-; ex pressed abnormall y in thi s tumor

h

2 4

• I

. -

• FtG. 6. Amplifi ca ti on of th e c-eriJJJ -2 ge ne in human primary

tumors . (u) Nitrocellulose fi lt e r~ cont aining t :co R l dige\ IS of tumor DNAs were probed with c-eriJ/J-2-\ pecific KX DNA. (h) Aft er remova l of th e fir~ t probe. th e filt e r~ we re then hybridi zed with the tGF receptor e DNA probe . Hyb ri di za ti on wa \ carri ed out und er \tringent condition\ . DNA 'i were i\o lated from a neuroh la\ toma Oane 1), an adenocarcinoma of the \ a li va ry gland (lane 2). two \qu amou s cell carc inomas rl anes 3 and 4). pl acent a (lane 5). and the 1\431 cell li ne (lane 6). Mark e r~ at le ft in a and h indicate positions 01' fragment s from /l ind II I dige ~ ti o n of>.. DNA.

Proc. Na t/. Acad. Sci. USA 82 (1985) 6501

might !'unct ion in es tablishing and maintaining the cancerous state.

Note Added in Proof. King e / a/. (34) have independentl y isolated an er!J/J -2 gene amplified in a human mammary carcinoma .

We tln nk Y. Miura and H. l<i.awano for excellent tec hnical a> \ i ~ tanc~. M. Ni shi zawa and N. Miyajima for helpful di scussions, and S. S;sak i for typing thi s man uscript. We also wi sh to thank N. No mttr<t for DNA from a human primary neuroblastoma and I. Pa ~ tan l'rr recombi nant plas mid p£7 . Thi s work was supported in pa rt by a Grant -in-Aid for Specia l Project Research Cancer-Biosci­ence !'ron, th e Mini stry of Educa tion, Science, and Culture of Japan.

1. Land. H .. Parada . L . F. & Wei nbe rg, R. A. (1983) Science 222,77 1- 778. ~. Coh c~t . S .. Carpe nt e r. G. & King. L. (19SO) J . Bioi. Chem. 255,

~ X l4 -4S42 . .1. Ni , hinlllra . J .. Hung , H . S. & De ue l, T . F. (1982) Proc. Na t/. Awd. Sci.

L/.)',1 79, 4.103- 4307. 4 . K : "u~a . M .. Zic k . Y .. l:l lithc, D. L .. C re tl az. M. & Kahn, C . R. (1982)

N 11111r<' (Lo ndon) 298, 667- 669 . ~- Rubi n. J . IJ .. Shia . M.A . & Pilc h. P. F. (1983) Na ture (Lon don) 305,

~ l X - ,40.

6. Ya nmnoto . T .. N ishida, T .. Miyajirna. N .. Kawai, S . , Ooi, T . & T oy u: hima . K . (1983) Cell 35, 71- 78.

7. Dt\\v,;ward. J .. Yarden. Y., Ma yes, E .. Scrace, G .. Toll y , N. , S tockwell . P .. Ullri c h . A .. Sc hl ess inge r , J . & Wate rfie ld , M. D. (1984) Nllt lirt• (London ) 307 , 521- 527.

X. Ul lr• ch . A .. Coussc ns . L. . Hay lli c k. J . S .. Dull. T . J .. G ray, A . , Tam, /1. W .. Lee. J ., Yard c n , Y .. Libcrmann. T . A., Sc hl ess inger, J .. Down­ward. J .. Ma yes . E. L . V .. Wh iutc. N .. Wate rfi e ld. M.D. & Seeburg, 1' . II (1984) N alllrt• (London) 309,4 18- 425.

9. Wat e rfi e ld . M. D .. Sc race. G. T .. Whilll e. N., Sl rooban l , P., Johnsson , A . Was te so n , A .. Wcste rmark . B .. He ldin, C.- H .. Hua ng, J . S. & lkue l. T . F. (1983) N 11 111re (London) 304, 35- 39.

10 . Jloo li•tlc. R. F .. Hunkapill e r . M. W. , Hood, L. E .. Devare . S. G., Rohh n ~ . K. C .. Aaronso n . S. A. & Ant oniades. H . N. 0983) Science 22 I. :75-277.

II . Mani:.t i ~ . T .. Hardi ~on . R . C .. Lacy, E .. Laue r. J .. O'Conne l, C .. Quon , ll .. S m . G. K . & Efstra tiacl is . A. (1978) Ce//f5 , 687-701.

\ ~. Be nt t n . W . D. & Da vis . R . W. 0977) Science 196, 180- 182. \ '1. M<u1iat is . T .. Je ffrey. A. & Klcid . D. G. (1975) Proc . Na t/ . Acml. Sci .

lJ.)A 72, 11 84- 11 88. 1·1. \l iiv i,, R. W .. IJot stc in . D. & Rot h . J . R. (1982) Advanced Bacterial

(;<'lf< ' lic .l· <Cold S pring Harbor Laborato ry, Cold Spring Harbo r . NY), pp . 1119- 111.

15. Ma x :~1n . /1. M. & Gi lbe rt . W . (1980) M ethods Enzymol. 65,499- 560. 16. S:u1gcr . F .. N ic klc n , S. & Co ul son . A. R . (1977) Proc. Nat/. Amd. Sci.

U.\A 74, 5463 - 5467. 17. Me"1ng. J.. C rea. R. & Sccburg. P. H . (!981) N ucleic Acids Res . 9,

\0') - )21. IH. Me,\lng. J . & Vie ira . J. 11982) Gene 19,269- 276. 19 . Soutl1: rn . E. M. (1975) J . M ol. Bioi. 91! , 503- 517. ~ II Ch irg.v in . J . M .. Przy byla. A. E .. Mac Dona ld, R. J . & Ruffer, W. J.

(I 'J79 . /Jioclwmistrv 1!!, 5294 - 5299. 2 1. l.c hr;d1. H .. Dia;nond , K .. Wozncy. J . M . & Iiocd tkcr, H. (1977)

!liuc/wmistry 16, 4743 - 475 I. 22. Xu. 'i .- H .. Ishii . S .. C lark . A. J . L .. S ullivan, M .. Wil son. R. K . , Ma ,

D. 1' . Roc. B. A .. Merl ino. G . T . & Pasta n . I. (]9S4) Na ture (London) 309, f0(> - 810 .

~3 Me rl ino. G. T .. Xu . Y .-1-1 .. Ishii . S .. C lark. A. J. L., Scmba, K., T o yo•,h ima. K .. Ya mamoto, T. & Pasla n . I. (1984) Science 224,417- 419.

2~. Sc hechte r . A. L .. S te rn . D. F .. Va id ya nalhan , L. , Decker. S. J . , Drc bi1. J . A .. Gree ne. M. I. & Weinbe rg. R. A. (1984) Nature (London) J t 2, _1 13-5 16 .

25. Dc rbi1. J. A .. S te rn . D. F .. Li nk. V. C .. We inbe rg. R. A. & G reen, M I. (1 984) N 1111tr f ( i.o ndon) 312, 545-548.

26. Co lii il'i , S. J. & G ro udin c. M. ( 1982) Na ture (L ondon) 298, 679-681. 27. Dal la -Favcra. R ., Wo ng-Staa l. F. & Gall o, R . C . (]982) Nat ure (London)

299, (·1- 63. 28 . Alit au . K .. Schwab. M .. L in . C. C .. Varmus, H. E. & Bishop, J . M.

11983 JPmc. Nat/ . A ctld. Sci. USA 80, 1707- 1711 . 29 . McCl1y. M.S .. Toole. J . J .. C unningham. J. M .. C ha ng. E. H., Lowy,

D. R. & Weinbe rg, R . A. (1983) Nature (London) 302 , 79- 81. 30 . Sc hwab, M .. Ali ta lo. K .. Va rmu s . H . E .• Bishop. J. M. & George , D.

( I 9X l i Nat11re I /. o/1(/on) 303, 497- 50 1. 3 1. Ca rpen te r . G .. King. L. E. & Cohe n , S. (1979) J . Bioi . C!t em . 254,

~XX-1 -489 1 .

.1~ Fahri< a nt . R .. Dc la rco. J . E. & Todaro. G. J. (1977) Proc. Nat /. Acad. Sci. USA 74, 565-569

31 Co w \y, J.. Gu lason. B .. S mith . J .. Hc ndlor. F. & Ozann e, B. (1984) in Can n r Ce lls. cd s . Lev ine. A . .1.. Vande Woudc. G. F .. Topp, W. C. & W:~ t "•n . J . D. <Cold S pring Harbo r Laborato ry, Cold Spring Harbor. N Yl . Vol. I . pp . 5- 10.

l·l . Kin g. C. R .. Kra u\, M. H . & Aaronson. S . /1. (1985) Science. in press.

PFIZER EX. 1042 Page 14

Biochemi stry: Semba eta/.

b

2 2

-

• • Ftc 5 Cons ·. · Conserva ti on of the c-erhB-2 ge ne in vert eb rates. (a)

Glt etveu c-erb/3-2 ~equence~ in chi cken and mouse . Nitroce llu lose 1) ers cont aining l:'coR I digests of DNA from human place nt a (lane

, tnouse FM . . , Probed . 3A ce ll ~ (lane 2), and cht cken blood (lane 3) wc te to tl Wllh the KX DN A. (/;) Close similarit y of th e c-erbB-2 ge ne 1) o:e 1~e u oncoge ne. Rat DNA was cleaved with ei ther Eco RI (lane South ELOR I plus /JamH I (lane 2). The diges ts were analyzed by Posit i C tt~ bl?t hybridi za tion with the KX probe. Arrowheads indicate DNA o(ns of fragme nt s fro m Hind! II diges tion of bac teriophage A

~ee Fig. la).

The se f . . rela . tncltngs lead us to speculate that there ts a close tton b ,

A . etween c-er!JB-2 and neu. b tnpltfi cat ' r ·1 • • has een . ton o protooncogcnes such as myc anu l£1 ,\ ·

(26_3 t ep~rted to be associated w ith various human cancers or th eO). 1 hts_ amplificat ion sugges ts that the protein products Oftun ampltft ed protooncogencs are in vo lved in some stage fact

1 ~ngenes i s. Of particular interest is the ro le(s) of growth obs~; r_eceptors in tumorigenes is, w hich is inferred from the nu

111b va~ r o n s that EG F receptors are expressed in large

(31_3~)r~ Son _the surface of ce ll s of epidermoid_ carcm_o mas ~ trn · 1 • . tn ce the c-erbB-2 gene could code for a protem

'"1 <1 r loth E . . . . · and e G F receptor or at leastrt s mtracelluldr domarn glanuwas amplified in an adenocarcinoma of the sali vary

' the gene products expressed abnormall y in thi s tumor

b 6

• I

• Pte ( •

tttn . 1· Am rt· . . · . -D 10 r~. ( ) N· P 1 teat ton of the c-erhB-2 ge ne 111 human pnma1 Y NA.s wa. ttrocellulose fi lters cont a ining EcoR l digests of tumor

~lllova J eft e Pr_?bed with c-erbB-2-specific KX ON. A. · .(b) After o:G•- 0 the I · · · · · I h . -" rec li st Probe th e fi lters were th en hybnd1 zecl wtt 1 l e Str1 Cptor cON ' . . . .. · . d , (I ngent , . . A probe. Hyb ndt zatton was cattt ed out un er . ane l ) ~Ond t t t on s . ON As were isohted from a neuroblastoma squ- ' <1 n 'ld · ' ' 1\

4 '1 1llou~ Cell ~noca rcmoma of th e ~a li vary gland (l ane 2), two

f 31 Ce JI I' ca rcmornas (Janes 3 and 4) , placent a (Jane 5), and the

0 fr· tne (Jan , 6) · .•. · · ·t · ns <~gnle nt s f · e . · Markers at left in a and h tllu!ca te post 10 10 1n Htnd lll digestion of A DNA .

Proc. Nat /. A cad . Sci. USA 82 ( 1985) 6501

might function in establi shing and maintaining the cancerous state.

Note Added in Proof. King eta/ . (34) have independently isolated an erbB-2 gene amplified in a human mammary carcinoma .

We thank Y . Miura and H. ~awano for excellent technical ass istance, M. Ni shizawa and N. Mi yajima for helpfu l di scussions, and S. Sasak i for typing thi s manuscript. We also wish to thank N. Nomura for DNA from a human primary neuroblastoma and I. Pastan for recombinant plasmid pE7. This work was supported in part by a Grant-in:Aid for Special Project Research Cancer-Biosci­ence from the Mmtstry of Educatton, Sctence, and Culture of Japan.

1. Land . H., Parada. L. F. & Weinberg, R. A. (1983) Science 222, 771-778 . 2. Cohen. S .. Carpenter. G. & King, L. (1980) J . Bioi . Chem. 255,

4834- 4842. 3. Nishimura. J .. Hung, H. S. & Deuel, T. F. (1982) Proc. Nat/. A cad . Sci .

USA 79, 4303 - 4307. 4. Kas uga. M .. Zick. Y., Blithe, D. L.. Crettaz, M. & Kahn , C. R. (1982)

Na ture (Londo n) 298, 667-669 . 5. Rubin. J . B .. Shia, M. A. & Pilch . P. F. (1983) Na ture (London) 305,

438- 440. 6. Yamamoto. T .. Nishida. T .. Miyajima, N., Kawai, S .. Ooi, T. &

Toyoshima. K. (]983) Cell 35, 71-78. 7. Downwa rd . J .. Yarden. Y., Mayes, E., Scrace. G., Totty, N.,

Stockwell , P .. Ullnch , A .. Schlessmger, J . & Waterfield , M. D. (1984) Na ture (Lo ndon) 307, 521-527.

8. Ull ric h. A .. Coussens. L., Hayllick. J . s.,. Du ll , T . J .. Gray, A .. Tam, A. w .. Lee. J .. Varden. Y., L1bermann, 1 . A .. Schless inge r, J., Down­ward. J .. Mayes. E. L. V .. Whitt le, N., Waterfi eld, M.D. & Seeburg P. H . ( 1984) Na turt• (London) 309, 418- 425. '

9. Waterfield. M.D .. Scrace. G. T. , Whitt le. N .. Stroobant. P .• Johnsson A .. Wasteson. A .. Westermark . B .. Heldin. C.- H .. Huang, J. s·. i_ Deuel. T. F. (1983) Na ture (London) 304, 35-39.

10. Doolittle . R. F .. Hunkapiller . M. W .. Hood, L. E .. Devare, S. G . . Robbi ns, K. C .. Aaronson . S. A. & Antoniades, H. N. (1983) Science 22 1, 275 - 277.

11. Maniati s, T .. Hardison. R: C., Lacy . E .. Lauer , J .. O'Connel, C .. Quon, D .. Sim , G. K. & Efstratmd1 s, A. (1978) Cell IS, 687-701.

12. Benton. W. D. & Davis. R. W. (1977) Science 196, 180- 182. 13. Maniatis. T . . JcfTrey. A. & Kleid , D. G. (1975) Proc. Na t/. Acad. Sci.

USA 72, 11 84- 11 88. 14. Da vis. R. W. , Bots tein, D. & Roth, J . R. (1982) Advanced Bacteria l

Ge11<•tics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) , pp . 109- 111.

15. Maxam. A. M. & Gilbert, W. (1980) Metlwd>· Enzym ol. 65, 499-560. 16. Sanger, F., Nick len, S. & Coulson. A. R. (1 977) Proc. Nml. Acad. Sci.

USA 74, 5463-5467. 17. Mess ing , J .. Crca. R. & Seeburg, P. H. 0 981) N ucleic Acitls R e>. 9,

309-32 1. 18. Mess ing, J. & Vieira. J . (1982) Gene 19, 269-276. 19 . Southern. E. M. (1975) J . Mol. Bioi. 98, 503- 517 . 20. Chirgwin . J . M .. Przybyla. A. E., MacDonald. R. J. & Ruffer. W. J.

(1979) Biochemistry 18, 5294-5299. 21. Lchrach. H. , Diamond . K., Wozney . J . M. & Eioed tker, H. (1977)

BioC" hemistry 16, 4743-475 1. 22. Xu. Y.- H .. Ishii , S . . Clark. A. J . L .. Sull ivan. M., Wi lson, R. K., Ma,

D. P .. Roc. B. A., Merl1no. G. T. & Pastan , I. (1984) Nature (London) 309, 806- 810

23. Merlino. G. T. , Xu. Y. -H., Ishii , S., Clark , A. J . L.. Semba K Toyoshima , K .. Yamamoto, T . & Pas tan, I. 0 984) Science 224, 417~419:

24. Sc hecht er , A. L.. Stern . D. F .. Vaidyanathan , L .. Decker, S. J. , Drebin . J. A .. Greene. M. I. & Weinberg, R. A. (1984) Nmure (London) 312, 513 -5 16.

25. Dcrbin , J. A .. Stern , D. F .. Link, V. C., Weinberg, R. A. & Green, M. I. (1984) N ature (London) 312, 545-548.

26. Collins: S. J . & Groudin ~, M. (_1982) Na ture (London) 298, 679- 681. 27. Dalla-foavera , R., Wong-S taa l, l·. & Gallo, R. C. (1982) Nature (London)

299, 61- 63. 28. Alitaro, K .. Schwab, M., Lin. C. C., Vannus. H. E. & Bi shop, J. M.

(1983) Proc. Na t/ . Acrui. Sci. USA 80, 1707-1711. 29. McCoy. M. S .. Toole, J . J .. Cunn ingham , J . M .. Chang. E. H .. Lowy,

D. R. & Wconberg, R. A. 0 983) Na ture (London) 302, 79-81. 30. Schwab. M., Alita lo. K .. Vannu s, H. E .. Bishop, J . M. & George D

(1983) Na ture (Lo ndon) 303, 497-501. ' · 31. Carpenter. G .. King. L. E. & Cohen, S. (1979) 1 . Bioi. Chem. 254,

4884 - 4891. 32. Fabricant. R. , Dclarco. J . E. & Todaro, G. J . (1977) Proc. Nat/. Acad.

Sci. USA 74, 565-569 . 33. Cowly. J. , Gu tason. B._. Smith . J ., Hcndlor, F. & Ozanne. B. (1984) in

Cancer Cells, cds . Levmc . A. J .. Vande Woudc , G. F., Topp, w. c. & Wat son. J. D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Vol. I . pp . 5-10.

34. King. C. R .. Kraus. M. H. & Aaronson, S. A. (1985) Science, in press.

PFIZER EX. 1042 Page 15