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PFIZER EX. 1041 Page 1

ISSN 0036-8075

6 September 1985 SCIENCE Volume 229, No. 4717

This Week in Science .... ...... . ........ .. . . .... . .... . ...... . .. . . . . . . . · · · ·

LETTERS The Electricity Industry: A. B . Lovins; M . Crawford; C. Whipple . .. .. . . . . . .. · · ·

EDITORIAL Memorandum to Universal Science Foundation ... . ..... . ..... . ..... . · · · · · · · · ·

ARTICLES Seismology of the Sun: J. Christensen-Dalsgaard , D . Gough , J . Toomre . . . .. . . · ·

Intrinsic and Extrinsic Factors in ·Protein Antigenic Structure: J . A. Berz(~f1· ky . . . ·

Sequence and Structure of a Human Glucose Transporter: M . Mueckler et al. .. . ·

911

914

921

923

932

941

NEWS AND COMMENT Reagan Announces New ASAT Test ... . . . .... . .................... . ..... . .. . 946

NIH to Award 2200 New Grants .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947

Bomb Scandal Highlights French Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948

U.S. , Mexico Pledge Smelter Controls 949

Briefing: Biotechnology' s Movie Debut Worries Industry ; Academy' s Fusion Study Causes a Stir; British Scientists Urge Supercomputer Program ; Ohio State's Telescope Granted 10-Year Reprieve .... ... . . ............... . . . ... 950

" Spy Dust" Irritates Diplomats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 952

RESEARCH NEWS Continental Drift Nearing Certain Detection ... ...... .... .. . ........ . . . .. . . .. . 953

954

BOARD OF DIRECTORS

CHAIRMEN AND SECRETARIES OF AAAS SECTIONS

DIVISIONS

An Agenda for Space Physics ............ .... ..... ... . .... .. ............... .

WIMP's, Cosmions, and Solar Neutrinos... . .. .. . .. ... . ... . ... . .. . ......... . . 955

Nitrogen Fixation Briefing : Fixing Nitrogen Without Molybdenum'?; Gene Rearrangements in a Prokaryote . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 956

DAVID A. HAMBURG Retiring President, Chairman

GERARD PIEL President

LAWRENCE BOGORAD President-Elect

ROBERT McC. ADAMS ROBERT W. BERLINER

MILDRED DRESSGEEL~:~~G DONALD N. LAN

MATHEMATICS (A) Daniel Zelinsky Lynn Arthur Steen

PSYCHOLOGY (J) John I. Lacey William N. Dember

EDUCATION (Q) John F. Scha« Joseph D. Novak

CHEMISTRY (C) ASTRONOMY (D) Rustum Roy David Morrison Jean'ne M. Shreeve John E. Gaustad

SOCIAL, ECONOMIC, AND POLITICAL SCIENCES (K) David Mechanic

HISTORY AND PHILOSOPHY OF SCIENCE (L) ENGINEERING (M) Edward Grant Henry McGee Arthur L. Norberg W. Edward Lear David L. Sills

DENTISTRY (R) Gordon H. Rovelstad Harold M. Fullmer

PHARMACEUTICAL SCIENCES (S) INFORMATION, COMPUTING, AND COMMUNICATiON (T) Edward G. Ripple Karen B. Levitan Betty-ann Hoener Elliot R. Siegel

ARCTIC DIVISION CARIBBEAN DIVISION PACIFIC DIVISION

Robert White Gunter E. Weller Juan A. Bonnet, Jr. Lucy Gaspar Walter Gardner Alan E. Le~:~~tor President Executive Secretary President Secretary-Treasurer President Executive

SCIENCE Is published weekly on Friday, except the last week In December, by the American Asaoclatlon for the Advancement of Science, 1333 H Street, NW, Washington, D.C. 20005. Second· class postage (fublication No. 484460) paid at Washin~ton, D.C., and at an additional entry. Now combined with The Scientific Monthly® Copyright <C> 1985 by the American Association for t~e Advancement o Science. Domestic individual membership and subscription (51 issues) : $60. Domestic institutional subscription (51 Issues): $98. Foreign postage extra: Canada $24, other (surface m~w $27, air-surface via Amsterdam $65. First class, airmail, school-year, and student rates on request. Single copies $2.50 ($3 by mall); back Issues $3 ($3.50 by mail); Biotechnology Issue, $5 ($5.50 by m~ ; classroom rates on request. Change of address: allow 6 weeks, giving old and new addresses and seven·digit account number. Authorization to photocopy material for internal or personal use un e circumstances not falling within the lair use provisions of the Copyright Act is granted by AAAS to libraries and other users registered with the Copyright Clearance Center (CCC) TransactiOnal Reporting Se0r· · vice, provided that the base fee of $1 per copy plus $0.10 per page is paid directly to CCC, 21 Congress Street, Salem, Massachusetts 01970. The Identification code for Science Is 0036·8075183 $1 + ·1 · Poatmaater : Send Form 3579 to Science, 1333 H Street, NW, Washington, D.C. 20005. Science is Indexed In the Reader's Guide to Periodical Literature and In several specialized indexes.

PFIZER EX. 1041 Page 2

AMBRICAN ASSOCIATION FOR THB ADVANCBMBNT OF SCIBNCB

AAAS NEWS

BOOK REVIEWS

REPORTS

Professional Ethics Group Reports on Spring Meeting: S. Painter; Call for Nominations; Summer Fellows Work on Environmental Issues and with Mass Media; Issues on Arms Control Featured in New Publications; AAAS Offers Desk Diary for 1986; Call for Nominations: 1986 General Election; Arid Lands Conference Slated for October in Tucson ... ..... . . .. . .... . .... . .... . .... .

Reproductive Decisi<:ns, reviewed by J . B . Silk; The Neurobiology of Motivation and Reward, F. Tf?ates; Geomorphology, P. C. Patton; The Creation of Quantum Mechamcs and the Bohr-Pauli Dialogue , L. Wessels ; Books Received . ... . ....... . ......... .. .... .... . ....... . .. . .... . . .... . .. . ... .

Rare Earths: Atmospheric Signatures for Oil-Fired Power Plants and Refineries: I . Olmez and G. E. Gordon .. . . . ...... ....... .. . ........ . ... .. . . . ..... . .

Crassulacean Acid Metabolism in the Strangler Clusia rosea Jacq. :

958

961

966

/ . P. Ting, E. M. Lord, L. da S. L. Sternberg, M. 1. DeNiro . . . . . . . . . . . . . . . 969

A 1500-Year Record of .Tropical Precipitation in lee Cores from the Quelccaya lee Cap, Peru: L. G. 1hompson, E. Mosley-Thompson , J. F. Bo/zan , B. R. Koci 971

Amplification of a Novel v-erbS-Related Gene in a Human Mammary Carcinoma: C. R. King, M. H . Kraus, S. A. Aaronson.. . . ... . .. . .. . . ....... . . . ...... . 974

The neu Gene: An erbB-Homologous Gene Distinct from and Unlinked to the Gene Encoding the EGF Receptor: A. L. Schechter et al. . . . . . . . . . . . . . . . . . 976

Isolation and Propagation of a Human Enteric Coronavirus: S. Resta , J.P. Luby, C. R o Rosenfeld, J.D . Siegel. . .... . .... .. . .. .... . .. .. ....... . .. .. ....... 978

Recombinant Vaccinia Virus: Immunization Against Multiple Pathogens: M. E. Perkus, A. Piccini, B . R . Lipinskas, E. Paoletti. .... . ... . ............ 981

Growth Regulation of Human Melanocytes: Mitogenic Factors in Extracts of Melanoma , Astrocytoma, and Fibroblast Cell Lines: M . Eisinger, 0. Marko , S.-1. Ogata, L. J. Old ...... . ..... . 0.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984

Hallucinogenic Amphetamine Selectively Destroys Brain Serotonin Nerve Terminals : G. Ricaurte, G. Bryan, L. Strauss , L. Seiden, C. Schuster . .. .. 0. 986

Gonadotropin-Releasing Hormone Binding Sites in Human Breast Carcinoma: K. A. Eidne, C. A. Flanagan, R. P. Millar ..... . . ... .. ..... . ...... 0. .. .. .. 989

Tissue Factor Gene Localized to Human Chromosome I (I pter--7 1 p21 ): S. D . Carson , W. M. Henry, T. B . Shows ........................ 0....... 991

~~01'1-iY ""'1N E. SA~KEIRN SHEILA E. WIDNALL WILLIAM T. GOLDEN <lEOLQ LINDA S. WILSON Treasurer

WILLIAM D. CAREY Executive Oftlcer

COVER r:ieiJi ~\1AND GEOGRAPHY (E) BIOLOGICAL SCIENCES (G)

1.1 len~.~. ·~.~c::~~~~~u ~~~rh ~- 1,~=~~e ANTHROPOLOGY (H) Albert C. Spaulding Priscilla Reining

EOICAL ~~ P, ...,SCsh~~CES (N) AGRICULTURE (O) INDUSTRIAL SCIENCE (P) --"1111 r, .. ~, Robert H. Pry

an E. Rhoads ~~Yp~J~~~~~acken Robert L. Stern

~~:.~TICS (U) ERIC (W) GENERAL (X) Ectv,""'" Hunt ATMOSPHERIC AND HYDROSPH Harold P. Green ard J. w 81 F. Kenneth Hare

8() &gman Bernice Ackerman Rodney W. Nichols

liTHWESTERN AND ROCKY MOUNTAIN DIVISION Oonatd J Preaid · Nash M. Michelle Balcomb

'The Am ant Executive Director

~~tofu~';:" Association for the Advancement of Science was founded in 1848 and Incorporated in 1874. Its objects .... rnp,ove th the work of scientists to facilitate cooperation among them, to foster scientific freedom and respo~sibllity, .,.,..8Ciatton 8 electiveness of science in the rromotlon of human welfare, and to Increase public understanding and

of the importance and promise o the methods of science in human progress.

Power as observed in solar 5-minute oscillations with frequency v (from about 1.5 mHz at bottom to 5.7 mHz at top and degree I (from about 7 on left to 170 on right) . The narrow ridges of ~oncentrated power (shown by the hghter tones) corresponds to theoreti­cally predicted acoustic resonances in t~e sun. f\nalysis of observed frequen­Cies permits study of the structure and dynamics of the solar interior. See page 923 . [T. L. Duvall , Jr. , and J. W . Harvey , National Solar Observatory P .O . Box 26732, Tucson Arizon~ 85726] '

. I

PFIZER EX. 1041 Page 3

6 September 1985, Volume 229, Number 4717 SCIENCE

AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE

Sdenct.: serves its readers as a forum for the presentation and discussion of important issues related to the advancement of science, including the presentation of minority or conflicting points of view, rather than by publishing only material on which a consensus has been reached. Accordingly, all articles published in Science-including editorials, news and comment, and book reviews-are signed and reflect the individual views of the authors and not ollicial points of view adopted by the AAAS or the institutions with which the authors are atnliated.

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To: Universal Science Foundation Planet Utopia Galaxy 7,073,216

From: Intergalactic Cultural Anthropology Expedition Sections IV and XXI

Re: Anomalous Behavior Patterns

The expedition to examine subcultures and behavioral patterns on Planet Earth has uncovered an anomaly that defies explanation by the rational principles and Cartesian logic of our own planet. Sections IV and XXI traveled independently in separate cruise missiles and randomly selected inhabitants for analysis by our noninvasive probes; that is, acoustical eavesdropping and quantitative three-dimensional gossip.

At 3:00 p.m. Earth Time on 4 August, Section IV located an individual with a red face speaking into a telephone at the 103-decibel level. The subject was using arcane linguistic techniques with multisyllable words such as "nincompoop" and "incompetent" occasionally interspersed with four­letter words not available in captured dictionaries. This species, which Section IV calls "Author," was complaining bitterly to something called The Journal that his manuscript had received no decision in 3 weeks despite (i) it represented better work than had ever appeared in that journal for the last decade and (ii) it was easily the best of his 176 papers, none of which had been treated so shabbily. It was ascertained that this work had taken 2 years to complete, 3 months to write up, and 1.5 months to be criticized by colleagues of the Author before being sent to The Journal. At 3:37 p.m., Section IV moved on to study behavior of one horse, two bullfinches, and a garter snake, all of which behaved in a classical and rational Cartesian manner_

At 4:00 p.m. on 4 August, Section XXI located an individual with a red face speaking into a telephone at the 10-3-decibellevel using multisyllable words such as "impossible" and "inconceivable" occasionally broken by signs, groans, and anguished looks at the ceiling. This species, which Section XXI calls "Referee," was apologizing to something called The Journal that (i) the manuscript that he had received for review had only recently arrived, having been delayed in the mails; (ii) he had in fact been studying the manuscript for weeks; and (iii) it had come during a period when he was out of the country, writing a grant, lecturing to 300 students, and lying flat on his back in the hospital being fed intravenously. He promised that the manuscript would be put in the mail "tomorrow" and complained that it was unreasonable of The Journal to expect a busy Referee such as he was to review a manuscript in less than 3 weeks. Section XXI was unable to obtain a definition of the word "tomorrow" before it moved on to study the viscosity of rush-hour traffic.

The anomaly in the case was not recognized until the two sections received laboratory reports of their remote-sensing DNA-sequencing deter­minations and optical surface imagery. The former indicated identical DNA sequences for the two species and optical photographs revealed identical clothing and facial characteristics. The sections concluded that it was theoretically astounding, but experimentally conclusive, that both expedi­tionary units had observed the same individual. No explanation for the subject's behavior could be suggested until Professor X 173 discovered that there were two hemispheres of the brain of Homo sapiens. We conclude that a single body houses both species, but that the Author species uses the left hemisphere and the Referee species the right hemisphere, and there is no cross-correlative system. Professor X173 predicts that such split person­alities will create wars, famines, and two types of Coca-Cola.

-DANIEL E. KosHLAND, JR.

PFIZER EX. 1041 Page 4

Amplification of a Novel v-erbB-Related Gene in a

Human Mammary Carcinoma

Abstract. The cellular gene encoding the receptor for epidermal growth factor (EGF) has considerable homology to the oncogene of avian ery throblastosis virus . In a human mammary carcinoma , a DNA sequence was identified that is related to v­erbB but amplified in a manner that appeared to distinguish it fi'om the gene for the EGF receptor. Molecular cloning of this DNA segment and nucleotide sequence analysis revealed the presence of two putative exons in a DNA segment whose predicted amino acid sequence was closely related to, but different from, the corresponding sequence of the erbBIEGF receptor. Moreover, this DNA segment identified a 5-ki/obase transcript distinct from th e transcripts of the EGF receptor gene. Thus , a new member of the tyrosine kinase proto-oncogene family has been identified on the basis of its amplification in a human mammary carcinoma .

c. RIC HTER KING

MATTHIAS H. KRAUS

STUART A. AARONSON

Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20205

The oncogenes of the acute transform­ing retroviruses have counterparts, des­ignated proto-oncogenes, that are con­served within the human genome (1). The human sis proto-oncogene encodes one major polypeptide chain of platelet­derived growth factor (PDGF) (2), and the erbB proto-oncogene appears to en­code the receptor for epidermal growth factor (EGF) (3) . A number of other proto-oncogenes , like erbB, share nucle­otide sequence homology with the tyro­sine kinase-encoding src gene (4). The fact that cellular receptors for several growth factors or hormones , including the EGF receptor, possess this enzymat­ic activity suggests that other proto-on­cogenes may encode growth factor re­ceptors as well.

Genetic alterations affecting proto-on­cogenes of the tyrosine kinase family can play a role in spontaneous tumor devel­opment. A specific translocation affect­ing the c-ab/locus, for example, is asso­ciated with chronic myelogenous leuke­mia (5). Several recent studies have also documented amplification or rearrange­ment of the gene for the EGF receptor in certain human tumors (6) or tumor cell lines (7). We now report the detection and partial isolation of a gene that is a new member of the tyrosine kinase fam­ily and is amplified in a human mammary carcinoma. This gene is closely related to , but di stinct from, the EGF rec(!ptor gene.

The identification of additional mem­bers of some proto-oncogene families has emerged from findings of related sequences amplified sufficiently in a par­ticular tumor to allow detection (8) . Be­cause of our interest in genes coding for

974

growth factor receptors, we used the v­erbB gene to probe for related genes that might be candidates for other receptor coding sequences . We selected moderate stringency hybridization conditions un­der which different oncogenes of the tyrosine family did not cross-hybridize. Thus, any gene detected might be ex­pected to have a closer relationship to v­erbB than to other members of the tyro­sine kinase family.

A

DNA prepared from ti ssue of a human

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~ 9.4

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~4 .4

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Fig. I. Detection of v-erbB- and pMAC 117-specific gene fragment s in normal human pla­centa, A431 cells, or human mammary carci­noma MACI17. DNA (15 IJ.g) was cleaved with Eco RI , separated by electrophoresis in agarose gels , and transferred to nitrocellulose paper (18). Hybridization to the 32P-labeled probe (20) was conducted in a solution of 40 percent formamide, 0.75M NaCl , 0.075M so­dium citrate , at 42°C (19). The v-erbB probe (A) was a mixture of the 0.5-kbp Bam HI­Bam HI fragment and 0.5-kbp Bam Hl-Eco RI fragment of avian erythroblastosis proviral DNA . The pMAC117 probe (B) was a 1-kbp Bgl 1- Bam HI fragment. After hybridization, the blots were washed first in 0.3M NaCI plus 0.03M sodium citrate at room temperature, and then in 0.015M NaCI , 0.0015M sodium citrate , and 0.1 percent sodium dodecyl sul­fate at 4rC (A) or at 52°C (B). Hybridization was detected by autoradiography.

This m ate·ri a I w as. copied

mammary carcinoma, MACII7, showed a pattern of hybridization (Fig. I A) that diffe red both from that observed with DN A of normal human placenta and from that observed with the A43 1 squa­mou s-cell carc inoma line, which con­tains amplified EGF rece ptor genes (7). In A43 1 DNA , four Eco Rl fragment s were detected that had increased signal intensities compared to those of corre­sponding fragme nts in placenta DNA (Fig . l A) . In contrast, MAC II 7 DNA contained a single 6-k il obase pair (kbp) fragment , which appeared to be amp li ­fied compared to corresponding frag­ments obse rved in both A43 1 and place n­ta DNA's (Fig . l A). These findings were consistent with the poss ibility that the MAC 11 7 tumor contained an amplified DN A sequence rel a ted to , but di stinct from , the cellular erhB proto-oncogene.

To clone the 6-kbp fragment, we di­gested DNA from MACII7 with Eco Rl , ligated it into bacteriophage A.gtWES , packaged it in vitro , and transferred it to Escherichia coli strain BNN45 by infec­tion. A library of 4 x 105 bacteriophages was screened by plaque hybridi zation with radioact ive v-erbB DN A. Ten of 14 hybridizing phages contai ned a 6-kbp Eco RI fragment. Figure 2 shows the physical map of one of these phages, A.MAC II7 , and pMACI I7 , a pUCI2 sub­clone containing a 2-kbp Bam HI frag­ment of A. MAC 117 that hybridized with v-erbB probes . The region of pMAC 117 to which v-erbB hybridized most in­tensely was flanked by Ace I and Nco I sites . Human repetitive sequences were al so localized (Fig . 2, region demarcated by arrows) .

By digestion of pMAC II7 with Bgl I and Bam HI , it was possible to generate a s ingle-copy probe homologous to v­erbB . This probe detected a 6-kb Eco RI fragment that was amplified in MAC 117 DNA and possibly increased in A431 cellular DNA relative to normal DNA (Fig. I B). The sizes of the fragments corresponded to the amplified 6-kb Eco RI fragment detected in MAC 117 DNA by means of v-erbB (Fig. lA). H ybrid­ization to Southern blots containing seri­al dilutions of MACII7 genomic DNA indicated an approximate amplification of 5- to I 0-fold when compared to human placenta DNA.

The nucleotide sequence of the por­tion of pMACI17 located between the Nco I and Ace I sites contained two regions of nucleotide sequence homolo­gous to v-erb B separated by 122 nucleo­tides (Fig . 3) . These regions shared 69 percent nucleotide sequence identity with both the v-erbB and the human EGF receptor gene . The predicted amino

SCIENCE , VOL. 229

PFIZER EX. 1041 Page 5

acid sequence of these regions was 85 percent homologous to two regions that are contiguous in the EGF receptor se­quence (7). Furthermore, these two pu­tative coding regions of the MACII7 sequence were each flanked by the AG and GT dinucleotides that border the exons of eukaryotic genes (9) . These findings suggest that the sequence shown in Fig. 2 represents two exons, separated by an intron , of a gene related to the erhBIEGF receptor gene .

The predicted amino acid sequence of the ;>..MAC 117 putative exons is homolo­gous to the corresponding sequences of several members of the tyrosine kinase fami ly. The most striking homology was observed with the human EGF receptor or erhB (Fig. 3). In add ition, we ob­se rved 42 percent to 52 percent homolo­gy with the predicted amino acid se­quences of other tyrosi ne kinase-encod­ing genes. At 25 percent of the positions there was identity among all the se­quences analyzed (Fig. 3). A tyrosine residue in the ;>..MACI I7 putative coding sequence , conserved among the tyrosine kinases ana lyzed , is the site of autophos­phorylation of the src protein (10).

The avai labi lit y of cloned probes of the MAC 117 gene made it poss ible to investigate it s express ion in a variety of cell types . The MAC 11 7 probe detected a single 5-kb transcript in A43 1 ce ll s (Fig. 4) . Under the stringent conditions of hybridization utilized , thi s probe did not detect any of the three RNA species recognized by EGF receptor comple­mentary DNA. Thu s , MACII7 repre­sents a new functional gene within the tyrosine kinase fami ly, closely related to, but di stinct from , the gene encoding the EG F receptor.

There is precedent for the identifica­tion of genes related to known onco­genes on the basis of their amplification in human tumors . For example, the high degree of ampli fication of N-myc in cer­tain malignancies made it detectable by means of the myc gene as a molecular probe (8) . In the present study, a five- to tenfold amplification of a v-erbS-related gene in the MACII7 mammary carc ino­ma made it possible to identify this se­quence against a complex pattern of EGF receptor gene fragments . Analysis of DNA from ten additional mammary carcinomas has not revealed amplifica­tion of the MAC II7 gene. However, extensive studies wi ll be required to de­termine the frequency of MACII7 gene amplification in different human malig­nancies.

The MAC 117 coding sequence , as de­termined by nucleotide and predicted amino acid sequence , was most closely

6 SEPTEMB ER 1985

related to the erhBIEGF receptor among known members of the tyrosine kinase family. The two genes are distinct, on the basis of sequence diversity and tran­script size. Detailed structural analysis of the complete coding sequence should give insights into the possible functions

B BXh B

of this v-erhB-related gene. Neverthe­less, because of its close relationship to the sequence of the EGF receptor, it is possible to speculate that the MAC 117 _ coding sequence may also be derived from a gene encoding a growth factor receptor. An oncogene in a chemicall y

B BXbR AMAC117--~----++--~~----~~~-r---

1 kb t-----1

pMAC 1 '17

B,' I

' I

N BgANN

' '

v-erbB related

0.5 kb f---l

10 zo .5 0 '•0 5 0 6 0 7 0 8 0 ') 0 G I C T 1\ CI\ 1 GGG 'I GC TT CCC:\ I I CCAGGGG.'1 ! GI\GC T 1\ CC TGG AGG I\ TG I GCGGC TCG T 1\ Cfi CAGGGfiC TT GGCCGCl CGGI\ 1\ CG TGCTGG I CI\1\Gl\G TCC

-G l yl·1 ~>t Jc r I y rl c u Gl ufls pV .ol Ar QLcu Va 1 It i sl\r q fl~. pl eufll ;1 /1. I ;11\ r n l\ s nV ;t ll c u V.1 l L y s::ic r P r

100 CI\IICC /\ TG I C fll\fl l\ 1 TACI\G AC f I CGGGCTGGC TCGGC TGC TGGI\ C/\ TT GI\ CGAGI\ CAGAGT I\ CC I\ TGCI\GI\ TGGGGGC At1Gq} I 1\ GG TGI\ 1\ GGI\CCI\1\G oA •,nH; 'o>V.;Il y!> ll o r hr/\ -J p P h eli l y l cu /\ 1.1 1\ r q l c ul ou A!> pl I oAs p GJ u l h r G I ul y rlt i .._,/\ }.J /\~ pG l yG I yl y s

20 0 G/\GC.'IG/\GG/\GGCl GGG I GG II G TGG TG I CT II GCCC II 1 GGG/\ G/1/\ CTCTG /\ G l GGCC /\ CC l CCCCIIC /\1\ C/\C /\ C/\G fTGG/\GG /\ CTT CC TCTT C TGCCC TC

~UO CCIIGG TGCCC II TC/\1\G TGGI\ l GGCGC l GGIIGTCC A fT CTCCGCCGGCGG T TCI\ CCC I\ CC AGI\C TG/1 1 G TGTGG/\G IT II TGG T g_:r c TG/1 TGGGGGG I G TTG - V,ll Pro II n l y~ T r p f>1 nt:ll I "L nuG I u :i 1~ r I I o l nuAr qll r q Ar qP h GT hrfli ~G J n :i 1! r A~p V.J l T rp :io rTyr G I y

r,o o GG/\GGGG TGGG l G/\GGAGCC /1 TGG

Fig. 2. Restriction-site map of A.MAC 117 and plasmid pMACII7. A, Ace I ; B, Bam HI ; Bg, Bgl I; N, Nco I; R, Eco Rl ; X, Xba I; Xh, Xho I. The sites were located by electrophoret ic analys is of the products of single and double digestion. Regions homologous to v-erbB o r human repetitive sequences (region flanked by arrows) were located by Southern blot hybridization (18) with the v-erbB probe or total human DNA made radioactive by nick translation (20). Hybridization cond itions were as described in Fig. lA. T he nucleotide sequence of pMACII7 between the Ace 1 site and the Nco I sites and regions of encoded am ino ac id sequence homologous to the EGF receptor are shown . T he AG or GT d inucleotides flanking the putative coding regions are underlined. To determine the sequence, Nco I , Hinf I, and Sau 96 I fragments were labeled at the 3' termini by means of the large frag ment of E. coli DNA polymerase , separated into single strands by gel electrophoresis , and chemicall y degraded (2 / ).

pMAC117 Human EGF Receptor v-erbB v-src v-ill v-TiiiS Human Insulin

Receptor

*** * *

pMAC117 + * * ***

OETEYHA-OG-GK-- VP IKWMALE S I L Human EGF Receptor v-erbB w~ v-src EON v-ill IT v-fmS NO Human Insulin Receptor Yl

* *** (.) <(

~ c.

.... 285

.... 185

Fig. 3 (left). Comparison of the putative encoded amino acid sequence in pMAC117 with known tyrosine kinase sequences . Black regions represent homologous amino acids. Differing amino acid residues are shown in one-letter code (22). Amino ac id positions conserved in all sequences are denoted by*· The tyrosine homologous to that autophosphorylated by the v-src protein (10) is shown by an arrow. The v-abl sequence contains a tyrosine residue in this region displaced by two positions . The amino ac id sequences of human EGF receptor (7), v-erbB (4), v-src (23), v­ab/ (24) , v ~fms (4) , and human insulin receptor (25) were aligned by the computer program described (26). The homology observed with the predicted amino ac id sequences ofv-yes and v­f es was 51 percent and 48 percent, respectively. Fig. 4 (right) . Detection of distinct messenger RNA species derived from the A.MAC117 gene and the human EGF receptor gene. Polyadenylated messenger RNA of A431 cells was separated by denaturing gel electrophoresis in formaldehyde (23), transferred to nitrocellulose (/8), and hybridized under stringent conditions (50 percent formamide , 0.075M NaCI, 0.75M sodium citrate, at 42°C) with

32P­

Iabeled probe from pMACI17 (Bgii-Bam HI fragment) or human EGF receptor complementary DNA (PE7: 2-kb Cia r inserted fragment). Fi lters were washed under conditions of high stringency (0 .015M NaCI plus 0.0015M sodium citrate at 5SOC). Hybridization was detected by autoradiography with exposure times of 4 hours for the pMAC 117 probe and I hour for the human EGF receptor probe.

975

PFIZER EX. 1041 Page 6

induced rat neuroblastoma has been de­tected by DNA transfection analysis (11). This oncogene , designated neu, ap­pears to encode a protein immunologi­cally related to the EG F receptor (12). Whether the MAC 117 coding sequence and neu represent the same or different cellular genes awaits further character­ization.

Overexpression of proto-oncogenes can cause cell transformation in culture and may function in the development of human tumors . Amplification of a nor­mal ras gene or its increased expression under the control of a retroviral long terminal repeat (L TR) induces transfor­mation of NIH 3T3 cells (13) . Expression of the normal human sis/PDGF-2 coding sequence in NIH 3T3 cells, which do not normally express their endogenous sis proto-oncogene, also leads to transfor­mation (14). In Burkitt lymphoma, a chromosomal translocation involving myc places its normal coding sequence under the control of an immunoglobulin gene regulatory sequence (15) . The re­sulting alteration in myc expression is likely to be causally related to tumor development (16) . The observation of amplification of myc or N-myc in more malignant phenotypes of certain tumors has supported the idea that overexpres­sion of these genes can contribute to the progression of such tumors (8, / 7) . The erbBIEGF receptor gene is amplified or overexpressed in certain tumors or tu­mor cell lines (6). The five- to tenfold amplification of our v-erbB-related gene in a mammary carcinoma suggests that increased expression of thi s gene may have provided a selective advantage to this tumor. The isolation of a new mem­ber of the tyrosine kinase gene family amplified in a human mammary carcino­ma provides an opportunity to investi­gate the potential role of thi s gene in human malignancy.

Note added in proof: Recently , Semba et a/. (28) independently detected a v­erbB-related gene that was amplified in a human salivary gland adenocarcinoma. Nucleotide sequence analysis of thi s gene indicates its identity to the MAC 117 gene in the regions compared .

References and Notes

I. J . M. Bishop , Annu . Rev. Biochem . 52, 301 ( 1983); P. H . Duesberg , Nature (London) 304, 219 (1983).

2. R. F . Doolittle eta/ . , Science 221, 275 ( 1983) ; M . D. Wate rfield eta/. , Na wre (Lo ndon) 304, 35 (1983) .

3 . J . Downward eta/ ., Nature (London) 307, 521 (1984).

4. A. Hampe, I. Laprevotle, F. Ga li bert, L. A . Fede le , C. J. She rr, Cell 30, 775 (1 982); N. Kitamura, A. Kitamura, K. Toyoshima, Y . Hir­aya ma , M. Yoshida, Nature (London) 297 , 205 (1982) ; M. Shibuya and H . Ha nafusa, Cell 30, 787 ( 1982) ; T . Yama moto et a/., ibid. 35, 71 ( 1983) .

976

5. A . de Klein et a/. , Nature (London) 300, 765 (1982); S. J. Collins and M . T. Gro udin e. Proc. Na t/ . Acad. Sci . U.S.A. 80 , 48 13 ( 1983).

20 . P. W. J. Rigby. M . D ieck man n. C. Rhodes . P. Berg, J. Mol. Bioi. 113 , 237 ( 1977).

21. A. M. Ma xam and W. G il bert , Proc. Na t/. 6. T . A. Liberma nn et a/., Na ture (London) 313.

144 (1985). 7. A. U llri c h eta/. , ibid. 309 ,4 18 (1984) ; Y. Xu et

a/. , ibid. , p. 809; C. R. Lin et a/., Science 224 , 843 ( 1984).

8 . M . Schwab Nawre (Lon don) 305. 245 (1983) ; N. E. Ko hl eta/. , Cell 35, 349 (1983).

9. R. Breathnac h a nd P. C ha mbon , Anntt. Rev. Bioclt<•m. 50, 349 ( 198 1).

10. J . E. Smart et a/. , Proc. Nat /. A cad. Sci. U.S.A . 78 , 60 13 (198 1).

I I. A la n L. Schechte r et u/. , Na ture (Lo ndon) 312, 5 13 ( 1984) .

12. J. A. Drebin , D. F. S te rn . Y. C. Lin k, R . A. Weinberg, M . I. Greene , ibid . , p . 545.

13. E. H. Chang . M . E. Furth. E. M . Scoln ick . D . R. Lowy, ibid. 297 , 479 ( 1982).

14. A. Gazit eta{ , Cell 39 ,89 <1984) ; M . F . C la rke eta/ ., Nature (Lonclorl) 308 , 464 ( 1984).

15. R. Taub eta/ . , Proc. Nat/ . A cad. Sci. U.S.A. 79 , 7837 ( 1982).

16. K. Ni shi ku ra et a/., Science 224. 399 ( 1984). 17. M. Schwab eta/., Proc. Na t{ . A cad. Sci. U.S.A.

81 , 4940 ( 1984). 18 . E. M. Sou the rn , 1 . Mol. Bioi. 98, 503 ( 1975). 19. G. M. Wahl , M . Ste rn , G. R . Stark , Proc . Na t/ .

Acad. Sci. U.S.A . 76, 3683 (1979).

A cud. Sci. U.S.A. 74. 560 < 1977). 22. T he fo llowing abbre viation ' we re used fo r a mi ­

no ac ids: A , a lan ine; C, cyste ine . D . aspa rtic ac id ; E. g lutam ic ac id ; F. phe nyla lan in e ; G, glyc ine; H. hi stid ine; I. iso leuc ine; K . lys ine; L. leuc ine ; M , methionine ; N . a ' paraginc; P . pro­line; Q, g lutamine; R. arginine; S, serine; T. threonine; V. va line; W, tryptop han; Y, ty ro­sine.

23. H . D. Lehrac h , D. Diamond, J. M. Woz nc y. H . Bocdtker , Biochemistry 16 , 4743 ( 1977) .

24. A. P. Cze rnil ofsky <' t ul .. Nat ure (London) 287 . 193 (1980).

25. E . P . Redd y , M. J . S mith . A. Sri ni va s:~ n . Proc. Na t/ . A cue/ . S ci. U.S.A. 80 . 3623 < 1983).

26. A. U ll ri c h et a/., Na ture (Lo ndon) 313. 756 (1985).

27. D . J . Lipman a nd W. R. Pearson. Science 227 , 1435 ( 1985).

28. K. Semba . N. Kamala, K. Toyshima , T . Yama­moto, Proc. Nu t/ . Au1d. Sci. U.S.A., in pre ss.

29. We th a nk I. Pastan and G. Me rlin o fo r providing the hum an EGF compkmcntary ON/\ c io n.; PE7 a nd P. di Fio re for th e polyade nyla tcd A43 I RNA .

22 April I 985 ; accepted 3 J unc I 985

The neu Gene: An erbB-Homologous Gene Distinct from and

Unlinked to the Gene Encoding the EGF Receptor

Abstract. The neu oncogene , identified in ethylnitrosourea-induced ratneuroglio­blastomas, had strong homology with th e erbB gene that encodes th e epidermal growthfactor receptor. This homology was limited to th e region oferbB encoding th e tyrosine kinase domain. It was concluded that the neu gene is a distinct novel gene, as it is not coamplified with sequences encoding the EGF receptor in th e genome of the A43/ tumor line and it maps to human chrom osome 17.

ALAN L. SCHECHTER

MIEN-CHIE HUNG

LALITHA V AIDYANATHAN

ROBERT A. WEINBERG

Whit ehead In stitute for Biomedical R esearch and Department of Biology, Ma ssachusetts In stitute of Technology, Cambridge 02142 TERESA L. Y ANG-FENG

UTA FRANCKE

Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 AXEL ULLRIC H

LISA COUSSENS

Department of Molecular Biology, Genentech , Inc., San Francisco, California 94080

Rat neuroglioblastomas induced by exposure in utero to ethylnitrosourea frequently carry an oncogene detectable upon transfection into NIH 3T3 mouse cell s (/) . This oncogene (which we have termed neu) was found to be related to c­erbB (2), a gene that encodes the recep­tor for epidermal growth factor (EGF-r) (3) . The neu oncogene induces the syn­thesis of a tumor antigen, pl85 , which is serologically related to the EGF-r (2) .

Southern blot analysis of rat DNA after Eco RI digestion revealed at least two erbB-homologous segments, one of

which contained the neu oncogene in a biologically acti ve form (2). It remained unclear whether the same or other DNA segments encode the EGF-r. Other anal­ysis uncovered differences between the products of the two genes. While poly­clonal sera to the EG F-r recognized pl 85, monoclonal antibod ies to pl85 did not react with the EGF-r. Moreover, there was an apparent molecular weight difference of 15,000 daltons between the two proteins (2).

These data rai sed several possibilities regarding the relationship between the neu a nd c-erbB genes . The neu oncogene might be a mutated a lle le of the normal c­erbB gene, or it might be derived from a normal gene, the sequences of which overlap with those of c-erbB. Alte rna­tively, the neu oncogene might have aris­en from a gene that is totally separate and distinct from erbB .

We used three subclones of huma n c­erbB complementary DNA (cON A) (4) and a 0.7-kilobase (kb) subclone of the v­erbB oncogene that had been transduced by the genome of av ian ery throblastosis virus (5) for these studies. All of the neu oncogene li es within a 34-kb Eco Rl segment that is present in the genomes of normal and tumor rat cell s as we ll as in mouse NIH 3T3 cell s that have acquired a neu oncogene via transfection (2) . We

SCIENCE, VOL. 229

PFIZER EX. 1041 Page 7

Breaking of Bodies and Minds Torture, Psychiatric Abuse, and the Health Professions

A documentation of systematic use and effects of physical and mental torture throughout the world

Edited by Eric Stover and Elena 0. Nightingale

With a Foreword by David A. Hamburg ............... __ ~ --..... - .. _ _., ____ 0 .................. - .. -,._._,.,...,_

Contents Part I Torture

Torture and the Ethics of Medicine Albert R. Jonsen and Leonard Sagan

Victims of Torture: Two Testimonies

Compil ed by Corneli us A. Kolff and Roscius N. Doan

Physical and Psychiatric Effects of Torture: Two Medical Studies

Federico Allodi, Glenn R. Randa ll , and others

Torture on Trial: The Case of Joelito Filartiga and the Clinic of Hope

Richard Pierre Claude

Medical Action Against Torture Eric Stover and Michael Nelson

Part II Psychiatric Abuse

Psych iatrists and Dissenters in the Soviet Union

Sidn ey Bloch and Peter l~eddaway

A Question of Conscience The Cases of Alexe i Nikitin

and Anatolyi Koryagin Kevin Close

Unwi lling Patients Anatolyi Koryagin

The Case of General Grigorenko: A Second Opi nion

Wa lter He ich

The World of Soviet Psychiatry Walter Reich

A Response to Psychiatric Abuse Pau l Chodoff and Ellen Mercer

Toward the Prevention of Torture and Psychiatric Abuse

Elena 0. Nightingale and Eric Stover

This. mat eri al w as. copie d

atthe·NLM and m ay !>!!'

This eye-open ing book brings togeth er for th e first time writ ings on the role of medical personnel in cases of torture and psych iatr ic abuse . Through ana l­yses and case histori es, psychiatrists and other health care profess ionals, po li tical scientists, ethi cists , and other writers discuss the systematic use and effects of physica l and mental torture in the Soviet Union. Lat in America, and other parts of the world.

The book also details the comp li city of an alarm ing number of med ica l per­sonn el in torture and psychi atric abuse and examines th e ways in wh ich governments use a medica l rat ionale to seek legitimacy for human des truc­tion. Finall y, it describes efforts by medical and other associat ions both to combat offensive practices and treat vict ims.

T he Breaking of' Bodies and Minds is important reading for anyo ne concerned w1th the

preservation of basic human rights.

1985 352 pages

Paperbound $1 1.95; tv\/\S members $9.50 l lardcover $2 1.95; flAilS members $17.50

Order from MAS Marketing. lJept. ES. 1333 II Street, NW, 8th Ploor. Washington. DC 20005.

Please add $1.50 postage and hand li nf.l per order. Allow 6-8 weeks for delivery. To charge your order to VISA or Mastercard. please ca ll the Sales Department at (202) 326·6405.

PFIZER EX. 1041 Page 8