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`
`t l" -1
`
`re"n. . .._1~ Jt-,1-,
`
`$2.50
`
`NCE
`
`lr • L.~ ':1
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`•
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`I
`AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE
`
`*********~****t~··
`115 6 SCl 7 ~ 263 6 ~
`NATL LIB GF ~Ep
`1 S INDEX MEDICLS
`€60C ROCKV ILLE ~IKE
`2C 20S
`BET HESDA , DC
`
`PFIZER EX. 1041
`Page 1
`
`

`

`IS SN 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 . Gou gh , J . Toomre . . . .. . . · ·
`
`Intrinsic and Extrinsic Factors in ·Protein Antigenic Structure: J . A. Berz(~f1·ky . . . ·
`
`Sequence and Structure of a Human Glucose Transporter: M . Mu eckler et al. .. . ·
`
`911
`
`914
`
`921
`
`923
`
`932
`
`941
`
`NEWS AND COMMENT Reagan Announces New ASAT Test ... . . . .... . .................... . ..... . .. .
`NIH to Award 2200 New Grants .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Bomb Scandal Highlights French Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`U.S. , Mexico Pledge Smelter Controls
`
`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 .... ... . . ............... . . . ...
`
`" Spy Dust" Irritates Diplomats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`RESEARCH NEWS Continental Drift Nearing Certain Detection ... ...... .... .. . ........ . . . .. . . .. .
`
`An Agenda for Space Physics ............ .... ..... ... . .... .. ............... .
`
`946
`
`947
`
`948
`949
`
`950
`
`952
`
`953
`
`954
`
`WIMP's, Cosmions, and Solar Neutrinos... . .. .. . .. ... . ... . ... . .. . ......... . .
`
`955
`
`Nitrogen Fixation Briefing : Fixing Nitrogen Without Molybdenum '?; Gene
`Rearrangements in a Prokaryote . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`956
`
`BOARD OF DIRECTORS
`
`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
`
`CHAIRMEN AND
`SECRETARIES OF
`AAAS SECTIONS
`
`DIVISIONS
`
`MATHEMATICS (A)
`Daniel Zelinsky
`Lynn Arthur Steen
`
`CHEMISTRY (C)
`Rustum Roy
`Jean'ne M. Shreeve
`
`ASTRONOMY (D)
`David Morrison
`John E. Gaustad
`
`PSYCHOLOGY (J)
`John I. Lacey
`William N. Dember
`
`SOCIAL, ECONOMIC, AND POLITICAL SCIENCES (K)
`David Mechanic
`David L. Sills
`
`HISTORY AND PHILOSOPHY OF SCIENCE (L)
`Edward Grant
`Arthur L. Norberg
`
`ENGINEERING (M)
`Henry McGee
`W. Edward Lear
`
`EDUCATION (Q)
`John F. Scha«
`Joseph D. Novak
`
`DENTISTRY (R)
`Gordon H. Rovelstad
`Harold M. Fullmer
`
`PHARMACEUTICAL SCIENCES (S)
`Edward G. Ripple
`Betty-ann Hoener
`
`INFORMATION, COMPUTING, AND COMMUNICATiON (T)
`Karen B. Levitan
`Elliot R. Siegel
`
`ARCTIC DIVISION
`
`CARIBBEAN DIVISION
`
`Robert White
`President
`
`Gunter E. Weller
`Executive Secretary
`
`Juan A. Bonnet, Jr.
`President
`
`Lucy Gaspar
`Secretary-Treasurer
`
`PACIFIC DIVISION
`Alan E. Le~:~~tor
`Executive
`
`Walter Gardner
`President
`
`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
`
`. I
`
`AAAS NEWS
`
`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
`Confere nce Slated for October in Tucson ... ..... . . .. . .... . .... . .... . .... .
`
`958
`
`BOOK REVIEWS
`
`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 . ... . ....... . ......... .. .... .... . ....... . .. . .... . . .... . .. . ... .
`
`REPORTS
`
`Rare Earths: Atmospheric Signatures for Oil-Fired Power Plants and Refineries:
`I. Olmez and G. E. Gordon .. . . . ...... ....... .. . ........ . ... .. . . . ..... . .
`
`Crassulacea n Acid Metabolism in the Strangler Clusia rosea Jacq. :
`/ . P. Ting, E. M. Lord, L. da S. L. Sternberg, M. 1. DeNiro . . . . . . . . . . . . . . .
`
`A 1500-Year Record of .Tropical Precipitation in lee Cores from the Quelccaya lee
`Cap, Peru: L. G. 1hompson, E. Mosley-Th ompson , J. F. Bo/zan , B. R. Koci
`
`Amplification of a Novel v-erbS-Related Gene in a Human Mammary Carcinoma:
`C. R. King, M. H . Kraus, S. A. Aaronson.. . . ... . .. . .. . . ....... . . . ...... .
`
`The neu Gene: An erbB-Homologous Gene Distinct from and Unlinked to the
`Gene Encoding the EGF Receptor: A. L. Schechter et al.
`. . . . . . . . . . . . . . . . .
`
`Isolation and Propagation of a Human Enteric Coronavirus: S. Resta , J.P. Luby,
`C. R o Rosenfeld, J.D . Siegel. . .... . .... .. . .. .... . .. .. ....... . .. .. .......
`
`Recombinant Vaccinia Virus: Immunization Against Multiple Pathogen s:
`M . E. Perkus, A. Piccini, B. R . Lipinskas, E. Paoletti. .... . ... . ............
`
`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.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Hallucinogenic Amphetamine Selectively Destroys Brain Serotonin Nerve
`Terminals : G. Ricaurte, G. Bryan, L. Strauss , L. Seiden, C. Schuster . .. .. 0 .
`
`Gonadotropin-Releasing Hormone Binding Sites in Human Breast Carcinoma:
`K. A. Eidn e, C. A. Flanagan, R. P. Millar ..... . . ... .. ..... . ...... 0. .. .. ..
`
`Tissue Factor Gene Localized to Human Chromosome I (I pter--7 1 p21 ):
`S. D . Carson , W. M . Henry, T. B. Shows ........................ 0.......
`
`961
`
`966
`
`969
`
`971
`
`974
`
`976
`
`978
`
`981
`
`984
`
`986
`
`989
`
`991
`
`~~01'1-iY
`""'1N E. SA~KEIRN
`<lEOLQ
`r:ieiJi ~\1AND GEOGRAPHY (E)
`1.1 len~.~. ·~.~c::~~~~~u
`EOICAL
`~~ P, ...,SCsh~~CES (N)
`r,
`.. ~,
`--"1111
`an E. Rhoads
`~~:.~TICS (U)
`Ectv,""'" Hunt
`ard J. w 81
`8()
`&gman
`
`SHEILA E. WIDNALL
`LINDA S. WILSON
`
`WILLIAM T. GOLDEN
`Treasurer
`
`BIOLOGICAL SCIENCES (G)
`
`~~~rh ~-1,~=~~e
`
`AGRICULTURE (O)
`~~Yp~J~~~~~acken
`
`ERIC (W) GENERAL (X)
`ATMOSPHERIC AND HYDROSPH
`Harold P. Green
`F. Kenneth Hare
`Bernice Ackerman
`Rodney W. Nichols
`
`liTHWESTERN AND ROCKY MOUNTAIN DIVISION
`Oonatd J
`M. Michelle Balcomb
`Preaid
`· Nash
`'The Am
`Executive Director
`ant
`~~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.
`
`WILLIAM D. CAREY
`Executive Oftlcer
`
`ANTHROPOLOGY (H)
`Albert C. Spaulding
`Priscilla Reining
`
`INDUSTRIAL SCIENCE (P)
`Robert H. Pry
`Robert L. Stern
`
`COVER
`
`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 theoreti(cid:173)
`cally predicted acoustic resonances in
`t~e sun. f\nalysis of observed frequen(cid:173)
`Cies 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
`Arizon~
`P .O . Box 26732, Tucson
`85726]
`'
`
`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.
`
`Publisher: WILLIAM D. CAREY
`
`Editor: DANIEL E. KosHLAND, JR.
`
`Deputy Editors
`PHILIP H. ADELSON (Engineering and Applied Sciences), JOHN
`I. llRAUMAN (/'hysical Sciences), GARDNER LINDZEY (Social
`Sciences)
`
`Editorial Board
`PHILIP W. ANDERSON, DAVID IJALTIMORE, ANSLEY J. COALE,
`JOSEPII L. GOLDSTEIN, LEON KNOPOFF, SEYMOUR LIPSET, WAL(cid:173)
`TER MASSEY, OuvER E. NELSON, ALLEN NEWELL, Rum PAT(cid:173)
`RICK, VERA C. RUDIN, HowARD E. SIMMONS, SoLOMON H.
`SNYDER, RooERT M. SoLOw
`
`lloard of Reviewing Editors
`JAMES P. ALLISON, QAIS AL-AWQATJ, Lu1s w. ALVAREZ, DoN
`L. ANDERSON, KENNETII J. ARROW, C. PAUL lliANCIII, EuzA(cid:173)
`DETII H. IJLACKBURN, FLOYD E. BLOOM, MICIIAEL s. llROWN,
`JAMES H. CLARK, SrANLEY FALKOW, NINA V. FEDOROFF, GARY
`FELSENFELD, DoUGLAS J. FuruniA, TIIEODORE H. GEBALLE,
`STEPIIEN I'. GoH, PATRICIA S. GOLDMAN-RAKIC, RICHARD M.
`HF.I D, GLORIA HEPPNER, JOliN IMBRIE, ERIC F. JOHNSON,
`KoNRAD ll. KRAUSKOPF, PAUL E. LACY, JOSEPII ll. MARTIN,
`JoliN C. McGIFF, MoRTIMER MISIIKIN, JoliN S. PEARSE,
`YESIIAYAU
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`ROTIIMAN, RONAI.lJ H. SCIIWARTZ, OTTO T. SOLBRIG, ROBERT
`T. N. TJJAN, VIRGINIA TRIMBLE, GEERAT J. VERMEJJ, MARTIN
`G. WEIGERT, GEORGE M. WIIITESJDES, WILLIAM ll. WooD,
`HARRIET ZUCKERMAN
`
`Editorial Stalf
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`KERR, GINA KOLATA, JEAN L. MARX, ARfiiUR L. RORINSON, M.
`MITCHELL WALDROP
`AdminiJtrative As.\·i.\·tant, Nt•ws: SCIIERRAINE MAcK; Editorial
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`Associate t'ditors: MARTIIA COI.LJNS, SYLVIA EBERHART,
`CAITILIN GORDON, WILLIAM GREAVES, BARBARA JASNY, STE~
`PIIEN KEI'I'LE, EDJfll MEYERS, LOIS SCIIMITT
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`mation for Contributors" sec page xi, Scintce, 28 June 1985.
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`llroadway (212-730-1050); ScOTCII PLAINS, N.J. 07076: C. Rich(cid:173)
`ard Callis, 12 Unami Lane (201·889-4873); CIIICAGO, ILL 60611:
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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 four(cid:173)
`letter 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 deter(cid:173)
`minations 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 expedi(cid:173)
`tionary 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 person(cid:173)
`alities 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 erythrobla stosis virus . In
`a human mammary carcinoma , a DNA sequence was identified that is related to v(cid:173)
`erbB but amplified in a manner that appeared to distinguish it fi'om the gene for the
`EGF receptor. Mole cular 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, th e
`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 transform(cid:173)
`ing retroviru ses have counterparts, des(cid:173)
`ignated proto-oncogenes, that are con(cid:173)
`served within the human genome (1).
`The human sis proto-oncogene encodes
`one major polypeptide chain of platelet(cid:173)
`derived growth factor (PDGF) (2), and
`the erbB proto-oncogene appears to en(cid:173)
`code the receptor for epidermal growth
`factor (EGF) (3) . A number of other
`proto-oncogenes , like erbB, share nucle(cid:173)
`otide sequence homology with the tyro(cid:173)
`sine kinase-encoding src gene (4). The
`fact that cellular receptors for several
`growth factors or hormones , including
`the EGF receptor, possess this enzymat(cid:173)
`ic activity suggests that other proto-on(cid:173)
`cogenes may encode growth factor re(cid:173)
`ceptors as well.
`Genetic alterations affecting proto-on(cid:173)
`cogenes of the tyrosine kinase family can
`play a role in spontaneous tumor devel(cid:173)
`opment. A specific translocation affect(cid:173)
`ing the c-ab/locus, for example, is asso(cid:173)
`ciated with chronic myelogenou s leuke(cid:173)
`mia (5). Several recent studies have also
`documented amplification or rearrange(cid:173)
`ment 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 tyrosi ne kinase fam(cid:173)
`ily 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 mem(cid:173)
`bers of some proto-oncogene families
`has emerged from findings of related
`sequences amplified sufficiently in a par(cid:173)
`ticular tumor to allow detection (8) . Be(cid:173)
`cause of our interest in genes coding for
`
`974
`
`growth factor receptors, we used the v(cid:173)
`erbB gene to probe for related genes that
`might be candidates for other receptor
`coding sequences . We selected moderate
`stringency hybridization conditions un(cid:173)
`der which different oncogenes of the
`tyrosine family did not cross-hybridize.
`Thus, any gene detected might be ex(cid:173)
`pected to have a closer relation ship to v(cid:173)
`erbB than to other members of the tyro(cid:173)
`sine kinase family.
`DNA prepared from ti ssue of a human
`
`..
`A
`;:
`..
`
`Cl)
`(,)
`
`ii:
`
`,._
`...
`...
`0
`<
`::E
`
`;;;
`
`'<t
`
`< -I
`
`.,
`8
`;:
`(,) .,
`ii:
`
`Cl)
`
`:::
`0
`<
`::E
`
`...
`"' '<t
`<
`
`kbp
`~ 9.4
`
`-~ ~6.6
`
`~4 . 4
`
`..... 2 .3
`~ 2.0
`
`Fig. I. Detection of v-erbB- and pMAC 117-
`specific gene fragment s in normal human pla(cid:173)
`centa, A431 cells, or human mammary carci(cid:173)
`noma 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 so(cid:173)
`dium citrate , at 42°C (19). The v-erbB probe
`(A) was a mixture of the 0.5-kbp Bam HI(cid:173)
`Bam 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 sul(cid:173)
`fate 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) th at
`diffe red both from that observed with
`DN A of normal human pl ace nta and
`from that observed with the A43 1 squ a(cid:173)
`mou s-cell carcinoma line, which con(cid:173)
`tains amplified EGF rece ptor genes (7).
`In A43 1 DNA , four Eco Rl fragment s
`were detected th at had increased signal
`intensities co mpared to those of corre(cid:173)
`sponding 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 (cid:173)
`fied compared to corresponding frag(cid:173)
`ments obse rved in both A43 1 a nd place n(cid:173)
`ta DNA's (Fig . l A). T hese finding s were
`consiste nt with the poss ibility that the
`MAC 11 7 tumor contained a n amplified
`DN A sequence rel a ted to , but di stinct
`from , the cellular erhB proto-oncogene.
`To clone the 6-kbp fragment, we di(cid:173)
`gested 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 infec(cid:173)
`tion. 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 sub(cid:173)
`clone containing a 2-kbp Bam HI frag(cid:173)
`ment of A. MAC 117 that hybridized with
`v-erbB probes . The region of pMAC 117
`to which v-erbB hybridized most in(cid:173)
`tensely 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 single-copy probe homologou s to v(cid:173)
`erbB . 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 ybrid(cid:173)
`ization to Southern blots containing seri(cid:173)
`al dilution s of MACII7 genomic DNA
`indicated an approximate amplification
`of 5- to I 0-fold when compa red to human
`pl acenta DNA .
`The nucleotide seque nce of the por(cid:173)
`tion of pMACI17 located between the
`Nco I and Ace I sites contained two
`region s of nucleotide sequence homolo(cid:173)
`gous to v-erb B separated by 122 nucleo(cid:173)
`tides (Fig. 3) . These region s shared 69
`identity
`perce nt nucleotide seque nce
`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 se(cid:173)
`quence (7). Furthermore, these two pu(cid:173)
`tative coding region s of the MACII7
`sequence were each flanked by the AG
`and GT dinucleotides that border the
`exons of eukaryotic genes (9) . These
`finding s suggest that the seque nce 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 exo ns is homolo(cid:173)
`gous 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 ob(cid:173)
`se rved 42 percent to 52 percent homolo(cid:173)
`gy with the predicted amino acid se(cid:173)
`quences of other tyrosi ne kinase-encod(cid:173)
`ing genes. At 25 percent of the positions
`there was identity among all
`the se(cid:173)
`quences analy zed (Fig. 3). A tyrosine
`re sidue in the ;>..MACI I7 putative codi ng
`sequence , conserved among the tyrosine
`kinases ana lyzed , is the site of autophos(cid:173)
`phorylation of the src protein (10).
`The avai labi lit y of cloned probes of
`the MA C 117 gene made it possible to
`in vestigate its expression in a variety of
`cell type s . The MA C 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 co mpl e(cid:173)
`mentary DNA. Thu s , MACII7 repre(cid:173)
`sents a new fun ctional gene within the
`tyrosine kina se fami ly, closely related
`to, but di stinct from , the gene encoding
`the EG F receptor.
`There is precedent for the identifica(cid:173)
`tion of genes related to known onco(cid:173)
`genes on the basis of their amplification
`in human tumors . For example, the high
`degree of ampli fication of N-myc in cer(cid:173)
`tain malignancies made it detectable by
`mean s 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 carcino(cid:173)
`ma made it possible to identify this se(cid:173)
`quence again st a complex pattern of
`EGF receptor gene fragments . Analysis
`of DNA from ten additional mammary
`carcinomas has not revealed amplifica(cid:173)
`tion of the MAC II7 gene. However,
`extensive studies wi ll be required to de(cid:173)
`termine the frequency of MACII7 ge ne
`amplification in different human malig(cid:173)
`nancies.
`The MAC 117 coding sequence , as de(cid:173)
`termined by nucleotide and predicted
`amino acid seque nce , 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 tran(cid:173)
`script size. Detailed structural analysis
`of the complete coding seq uence should
`give insights into the possible functions
`
`of this v-erhB-related ge ne. Neverthe(cid:173)
`less, because of its close relationship to
`the seque nce of the EGF receptor, it is
`possible to speculate that the MAC 117 _
`coding seque nce may also be derived
`from a gene encoding a growth factor
`receptor. An oncogene in a chemicall y
`
`B BXbR
`B
`BXh
`B
`AMAC117--~----++--~~----~~~-r---
`
`1 kb
`t-----1
`
`pMAC 1 '17
`
`' '
`
`N BgANN
`
`' I
`B,'
`I
`
`v-erbB
`related
`
`0.5 kb
`f---l
`
`zo
`') 0
`8 0
`7 0
`6 0
`5 0
`'•0
`.5 0
`10
`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 o u 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/\GGC l 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 Pr o II n l y~ T r p f>1 nt:ll I "L nuG I u :i 1~ r I I o l n uAr 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 electrophoretic analys is
`of the products of single and double digestion. Regions homologou s 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 seque nce, 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 / ).
`
`*** * *
`
`* ***
`
`(.)
`<(
`~
`c.
`
`pMAC117
`Human EGF Receptor
`v-erbB
`v-src
`v-ill
`v-TiiiS
`Human In sulin
`Receptor
`
`.... 285
`
`.... 185
`
`+
`* ***
`*
`OETEYHA-OG-GK-- VP IKWMALE S I L
`pMAC117
`Human EGF Receptor
`w~
`v-erbB
`EON
`v-src
`IT
`v-ill
`v-fmS
`NO
`Human Insulin Receptor Yl
`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 acid 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 ami no acid sequences of human EGF receptor (7), v-erbB (4), v-src (23), v(cid:173)
`ab/ (24) , v ~fms (4) , and human insulin receptor (25) were aligned by the computer program
`described (26). The homology observed with the predicted amino acid sequences ofv-yes and v(cid:173)
`f 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(cid:173)
`Iabeled probe from pMACI17 (Bgii-Bam HI fragment) or human EGF receptor complementary
`DNA (PE7: 2-k