I
`
`'
`
`1 /.. ______ _ _
`
`c . 1
`~ i: ''IJ'' l
`'( l
`'
`
`., ' ' ' )
`
`:
`
`' j } rJ / I I(; ) I)
`
`l
`'
`
`'
`
`•
`
`o.f
`
`I) •'
`
`OCTOBER 1985
`VOLUME 82
`NUMBER 19
`
`Proceedings
`
`C)F THE
`
`National Acaden1y
`of Sciences
`
`This material w as~:u~ iei:l
`at the, NLM ani:! m ay De
`Subje-ct US Copyright Law s
`
`BIOEPIS 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
`
`FRANK PRESS, President
`JAMES D. EBERT, Vice President
`BRYCE CRAWFORD, JR., l/ome Secretary
`WALTER A. RoSENBUTII, Foreign Secretary
`ELKAN R. BLOUT, Treasurer
`
`ROBERT A. ALBERTY
`PAUL BERG
`RONALD BRESLOW
`EDWARD E. DAVID, JR.
`STUART A. KORNFELD
`
`MAXINE F. SINGER, Chairman
`DANIEL E. KOSIILAND, JR.
`PETER D. LAX
`ROBERT E. MARSHAK
`DANIEL NATHANS
`PETER H. RAVEN
`JoHN RoDGERs
`
`HERBERT E. SCARF
`J. EDWIN SEEGMILLER
`ROBERT L. SINSIIEIMER
`SOLOMON H. SNYDER
`FRANK H. WESTIIEIMER
`
`Marzaging Editor: FRANCES R. ZWANZIG
`Senior Associate Editor: GARY T. Coc.Ks
`Associate Editor: CAY BUTLER
`Associate Editor: JOHN M. MALLOY
`Associate Editor: JANET L. MORGAN
`Associate Editor: T. PEARSON
`Associate Editor: DOROTHY P. SMITH
`Assistant Managing Editor: JOANNE D'AMICO
`
`Senior Production Editor: LYNN A. WASSYNG
`Production Editors: BARBARA A. BACON, RUTH E. CROSSGROVE, ScoTT C. IlERMAN,
`PEGGY LEONARD, MICHAEL W. NEFF, JANET L. OVERTON,
`DON C. TIPPMAN, DEBORAH I. WEINER
`Administrative Assistants: DELORES BANKS, BEAULAH EDWARDS
`Manuscript Coordinators: CYNDY MATHEWS, JACQUELINE PERRY
`Circulation: JULIA LITTLE, VIRGINIA TREADWAY
`
`Editorial correspondence: PROCEEDINGS OF THE NATIONAL ACADEMY OF SciENCES, 2101 Constitution Avenue, Wa~hing·
`ton, DC 20418.
`Business correspondence: Circulation Office of the PROCEEDINGS, National Academy of Sciences, 2101 Constitution Ave(cid:173)
`nue, Washington, DC 20418.
`Information for Contributors: See issue Number 1, January 1985.
`Copyright: The National Academy of Sciences has copyrighted this journal as a collective work and docs not own copy(cid:173)
`rights for individual articles. Requests for permission to reproduce parts of individual articles or for reprints of individual
`articles should be addressed to the authors. Microforms of complete volumes are available to regular subscribers only and
`may be obtained from University Microfilms, Xerox Corporation, Ann Arbor, MI 48103.
`Subscriptions: All correspondence concerning subscriptions should be addressed to the Circulation Office of the PROCEED·
`INGS. Subscriptions are entered on a calendar year basis only. For 1985, subscription rates are as follows-in the United
`States: personal, $185; institutional, $215; elsewhere: personal, $220; institutional, $250. Sub~cribcrs are requested to
`notify the Circulation Office of the PROCEEDINGS 6 weeks in advance of any change of address; also the local po\tmaster.
`The Academy is not responsible for nonreceipt of issues because of an improper address unless a change of address is on
`file. The notice of address change should list both the old and new addresses. Claims for replacement copies will not be
`honored more than 60 days after the issue date for domestic subscribers and not more than 90 days after the issue date for
`foreign subscribers.
`Back Issues: Volumes 77-81, January 1980 and thereafter, are available from the Circulation Office of the PRoCEEDINGS.
`The price of a single issue is $21.00 for Volumes 77-78 or $11.00 for Volumes 79-82.
`
`Second cla.rs postage paid at WashinRton. DC, and at additional mailing offices.
`
`PRINTED IN TilE USA
`
`PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (ISSN-0027-8424) is pub(cid:173)
`lished semimonthly by THE NATIONAL AcADEMY OF SciENCES, 2101 Constiflltion Avenue, Washington, DC 20418.
`
`© 1985 by THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA.
`
`POSTMASTER: Send address changes to: PROCEEDINGS, 2101 Constitution Ave., Washington, DC 20418.
`
`This material was copied
`at the NLM and may b<!
`~u bject US Cop'lright Laws
`
`BIOEPIS EX. 1042
`Page 2
`
`

`

`ANNOUNCEMENT
`
`Effective with Volume 83 (1986) the Proceedings
`of the National Academy of Sciences (USA) will
`be available to students and postdoctoral fellows
`at a subscription rate of $65 (United States)/$100
`(elsewhere). Special order forms for this rate will
`be available from the Proceedings Circulation Of(cid:173)
`fice, National Academy of Sciences, 2101 Consti(cid:173)
`tution Ave., Washington, DC 20418, beginning
`Sept. 1, 1985.
`
`//
`
`This material wascopcied
`at th,e N LM and may b,e
`~ubject USCopcyright Laws
`
`BIOEPIS EX. 1042
`Page 3
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`October 1985
`
`Volume 82, Number 19
`
`Table of Contents
`
`AUTHOR INDEX
`
`Physical Sciences
`
`CHEMISTRY
`
`pp. 6367-6722
`
`ix
`
`Peptide mixture sequencing by tandem Fourier-transform mass spectrometry
`
`Robert D. Cody, Jr., I. Jonathan Amster
`and Fred W. McLafferty
`'
`
`6367
`
`Biological Sciences
`
`APPLIED BIOLOGY
`
`Brood care by male bumble bees
`
`Sydney A. Cameron
`
`BIOCHEMISTRY
`
`Apolipoprotein multigene family: Tandem organization of human apolipoprotein
`AI, CIII, and AIV genes
`
`Sotirios K. Karathanasis
`
`Structure of canine pulmonary surfactant apoprotein: eDNA and complete
`amino acid sequence
`
`The "fl-like-globin" gene domain in human erythroid cells
`
`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
`
`Mutagenesis of avian carcinoma virus MH2: Only one of two potential
`transforming genes (8ga!?-myc) transforms fibroblasts
`
`Ren-Ping Zhou, Nancy Kan, Takis
`Papas, and Peter Duesberg
`
`iii
`
`6371
`
`6374
`
`6379
`
`6384
`
`6389
`
`BIOEPIS EX. 1042
`Page 4
`
`

`

`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
`
`J. Evan Sadler, Beverly B. Shelton(cid:173)
`Inloes, 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 Rios(cid:173)
`Candelore, Guillermo Gimenez(cid:173)
`Gallego, Jerry DiSalvo, Carl Bennett,
`John Rodkey, and Susan Fitzpatrick
`
`Matthias A. Hediger, David F. Johnson,
`Donald P. Nierlich, and Irving Zabin
`
`Activation of retinoic acid by coenzyme A for the formation of ethyl retinoate
`
`Duncan A. Miller and Hector F. DeLuca
`
`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 A(cid:173)
`tris(diphenylphenanthroline) cobalt(III)
`
`Sequence of a second human asialoglycoprotein receptor: Conservation of two
`receptor genes during evolution
`
`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
`
`Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo
`chromatin structures
`
`Mark J. Solomon and Alexander
`Varshavsky
`
`Replication timing of the H4 histone genes in Plzysarum polyceplzalum
`
`A developmentally regulated mRNA from 3T3 adipocytes encodes a novel
`serine protease homologue
`
`I
`
`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
`
`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
`
`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
`
`Kentaro Semba, Nobuyuki Kamala,
`Kumao Toyoshima, and Tadashi
`Yamamoto
`
`iv
`
`6394
`
`6399
`
`6404
`
`6409
`
`6414
`
`6419
`
`6423
`
`6427
`
`6431
`
`6435
`
`6440
`
`6445
`
`6450
`
`6455
`
`6460
`
`6465
`
`6470
`
`6475
`
`6480
`
`6485
`
`6490
`
`6492
`
`6497
`
`BIOEPIS EX. 1042
`Page 5
`
`

`

`Contents
`
`Location of cis-acting regulatory sequences in the human T-cellleukemia virus
`type I long terminal repeat
`
`Craig A. Rosen, Joseph G. Sodroski, and
`William A. Haseltine
`
`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
`
`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
`
`Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes
`show homology with Agrobacterium tumefaciens T-DNA
`
`Tetsuji Yamada, Curtis J. Palm, Bob
`Brooks, and Tsune Kosuge
`
`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
`
`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
`
`BOTANY
`
`Active auxin uptake by zucchini membrane vesicles: Quantitation using ESR
`volume and .1.pH determinations
`
`TerriL. Lomax, Rolf J. Mehlhorn, and
`Winslow R. Briggs
`
`Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from
`Anabaena 7120 expressed in Escherichia coli
`
`M. Gurevitz, C. R. Somerville, and L.
`Mcintosh
`
`Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to
`fungal elicitor and infection
`
`Allan M. Showalter, John N. Bell, Carole
`L. Cramer, John A. Bailey, Joseph E.
`Varner, and Chris J. Lamb
`
`CELL BIOLOGY
`
`Peroxisomal organization in normal and cerebrohepatorenal (Zellweger)
`syndrome fibroblasts
`
`Manuel J. Santos, Jose Manuel Ojeda,
`Jorge Garrido, and Federico Leighton
`
`Identification of a nuclear localization signal of a yeast ribosomal protein
`
`Do agonists promote rapid internalization of ,8-adrenergic receptors?
`
`Robert B. Moreland, Hong Gil Nam,
`Lynna M. Hereford, and Howard M.
`Fried
`
`Lawrence C. Mahan, Harvey J.
`Motulsky, and Paul A. Insel
`
`Repeating modular structure of the fibronectin gene: Relationship to protein
`structure and subunit variation
`
`Erich Odermatt, John W. Tamkun, and
`Richard 0. Hynes
`
`Phosphotyrosine-containing proteins are concentrated in focal adhesions and
`intercellular junctions in normal cells
`
`Pamela A. Maher, Elena B. Pasquale,
`Jean Y. J. Wang, and S. J. Singer
`
`Analyzing the components of microtubules: Antibodies against chartins,
`associated proteins from cultured cells
`
`Margaret Magendantz and Frank
`Solomon
`
`6502
`
`6507
`
`6512
`
`6517
`
`6522
`
`6527
`
`6532
`
`6537
`
`6541
`
`6546
`
`6551
`
`6556
`
`6561
`
`6566
`
`6571
`
`6576
`
`6581
`
`DEVELOPMENTAL BIOLOGY
`
`Growing Xenopus oocytes have spare translational capacity
`
`GENETICS
`
`Mark A. Taylor, Andrew D. Johnson,
`and L. Dennis Smith
`
`6586
`
`Ultraviolet light-induced crosslinking reveals a unique region of local tertiary
`structure in potato spindle tuber viroid and HeLa 5S RNA
`
`Andrea D. Branch, Bonnie J. Benenfeld,
`and Hugh D. Robertson
`
`6590
`
`v
`
`BIOEPIS 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
`
`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
`
`Distribution of initial and persistent 2-acetylaminofluorene-induced DNA
`adducts within DNA loops
`
`Ramesh C. Gupta, Nutan R. Dighe, Kurt
`Randerath, and Harold C. Smith
`
`Induction of Rhizobium meliloti nodC expression by plant exudate requires
`nodD
`
`John T. Mulligan and Sharon R. Long
`
`UV-induced mutagenesis of phage Sl3 can occur in the absence of the RecA
`and UmuC proteins of Escherichia coli
`
`Irwin Tessman
`
`Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea
`in mouse spermatogonia
`
`S. Hitotsumachi, D. A. Carpenter, and
`W. L. Russell
`
`One pyrimidine dimer inactivates expression of a transfected gene in xeroderma
`pigmentosum cells
`
`Miroslava Protic-Sabljic and Kenneth H.
`Kraemer
`
`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
`
`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
`
`Monoclonal antibodies against human Ia antigens stimulate monocytes to
`secrete interleukin 1
`
`Ronald Palacios
`
`The human Thy-/ gene: Structure and chromosomal location
`
`MEDICAL SCIENCES
`
`Glial cells metabolically cooperate: A potential requirement for gene
`replacement therapy
`
`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
`
`Visualization of fJ.! opiate receptors in rat brain by using a computerized
`autoradiographic subtraction technique
`
`Robert R. Goodman and Gavril W.
`Pasternak
`
`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
`
`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
`
`vi
`
`6595
`
`6600
`
`6605
`
`6609
`
`6614
`
`6619
`
`6622
`
`6627
`
`6632
`
`6637
`
`6642
`
`6647
`
`6652
`
`6657
`
`6662
`
`6667
`
`6672
`
`6677
`
`BIOEPIS EX. 1042
`Page 7
`
`

`

`Contents
`
`Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B(cid:173)
`and 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
`
`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
`
`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
`
`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
`
`Quantitative in vivo receptor binding Ill: Tracer kinetic modeling of muscarinic
`cholinergic receptor binding
`
`K. A. Frey, R. D. Hichwa, R. L. E.
`Ehrenkaufer, and B. W. Agranoff
`
`Calcium binding in pigmented and albino eyes
`
`Ursula C. Drager
`
`Corrections
`
`BIOCHEMISTRY
`
`In vitro expression of chloroplast genes in Jysates of higher plant chloroplasts
`
`Julie Bard, Don P. Bourque, Mark
`Hildebrand, and David Zaitlin
`
`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
`
`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
`
`BIOEPIS 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
`
`From a human genomic library, we obtained
`ABSTRACT
`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 adenocar(cid:173)
`cinoma of the salivary gland.
`
`At least 19 genes have been identified as rctroviral oncogenes
`that arc responsible for inducing tumors in vivo and trans(cid:173)
`forming 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.
`
`We examined details of the relation between the v-erbB
`gene and the EGF receptor gene and the possible involve(cid:173)
`ment 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 con(cid:173)
`structed from placental DNA as described (11). The library
`was screened for the v-erbS-related sequence by plaque(cid:173)
`hybridization 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% polyvinyl(cid:173)
`pyrrolidone/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 con(cid:173)
`junction with bacteriophage M13 mp19 (18).
`Blot-Hybridization Analysis of DNA and RNA. High mo(cid:173)
`lecular 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) formam(cid:173)
`ide. After hybridization, the filters were washed with 2x
`
`'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.
`
`f\bbrcviati~ns: EGF, epidermal growth factor; kb, kilobasc(s); kbp,
`kilobasc patr(s).
`
`6497
`
`BIOEPIS EX. 1042
`Page 9
`
`

`

`6498
`
`Biochemistry: Semba et a/.
`
`Proc. N at/. A cad. Sci. US A 82 ( 1985)
`
`2
`
`4
`
`6
`
`2
`
`4
`
`c
`
`*
`*
`
`*
`
`*
`
`- -- -- -
`-
`- -
`• -
`
`6
`
`-
`
`a
`
`kbp
`23-
`
`94-
`6.6-
`44-
`
`2.3-
`2.0-
`
`0. 5 -
`
`b
`
`s
`I
`
`S E S
`I I [
`1---i
`1 kbp
`
`X EK
`
`¥
`
`s s s
`I ]I
`
`X
`I
`
`:>. 107
`
`Identification and cloning of the v-erbB-related sequence that is di stinct from the EGF receptor ge ne . (a ) Hybridi zation with the
`FIG. 1.
`v-erbB probe. DNAs prepared from A431 cells (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 n e~ 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-e rb/3 probe under the relaxed
`conditions described in Materials and Methods . Autoradiograph s 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 indi ca te band s 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 a c 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% NaDodS0 4 and then with either 0.2 x
`NaCI/Cit/0.1% NaDodS04 (stringent) 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 Biochem(cid:173)
`icals 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 directly to a nitrocellulose filter and subjected to
`blot hybridization under stringent conditions, a s 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 (de scribed
`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 con(cid:173)
`ditions 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). The se sequences were observed as 8.1- ,
`5.9-, 5.4-, and 3.5-kbp EcoRI fragment s; 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 a s 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 stars in Fig. 1a) . Thi s
`suggested that be sides the EGF receptor gene, there may be
`
`anothe r v-erbB-rela te d ge ne, from w hi c h th e una mplifie d
`restri ction fragments a re gene ra te d .
`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, und e r the re laxed hy(cid:173)
`bridiza tion a nd washin g conditions, a nd iso la te d s ix indepe n(cid:173)
`dent clones . Restri cti o n m a p ana lys is s ho wed th a t all the
`cloned in serts e xce pt th e A- 107 inse rt re pre se nte d one ge ne,
`c-erbB-1 . In these clones, we id e ntifi ed fo ur £co R I fragme nt s
`as e xon-conta ining seque nces b y 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
`
`2 3 4 5
`
`b
`
`2
`
`4
`
`-- 4.8 kb
`
`kb
`-to.o
`-
`5.6
`
`-
`
`2.9
`
`FI G. 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 standard s.
`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).
`
`BIOEPIS EX. 1042
`Page 10
`
`

`

`Bioc hemi stry: Semba eta/.
`
`Proc. Nat/. A cad. Sci. USA 82 ( 1985)
`
`6499
`
`L
`
`I
`I
`5' --------+1--tllll+l-tll---+111-- 3'
`
`E
`
`;>..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·oA 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/\G TGTTG/\GGGA /\IIAC ACC T CCCCCA AA GCCAIICAAA
`I ~ O C
`l ~ O '!' G
`yO
`T
`G 1\1\
`G
`'l'
`,\1.
`II
`,\
`T
`II
`GC 1\
`1\
`,\
`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'GGC T GG 'I'(,;TGGGC TCCCC,\'1' 1\'I'G'l'C 'l'CCC GCC' I'TC T GGGCA T C T GCC 'l'GACI\TCC
`C A 21' 0
`C C d) 0
`CC C G c;
`c; 2 4 0
`C
`C
`C
`1\ 1\A
`Th' Vd l t; 1 n Lt.~ ~, 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 T GC 'I 'CII C II C ,\G C ' ! "I'I\T GCCl' 'l' ~. ·l·c c;c TG CC 'I'C 'I'T IIGI\CC,\ 'l'G 'I' CCGGGAI\A ,\CCGCGGACGCC 'l'GGGC 'l'CCC AGGACCTG
`·n:
`1\ Cl\ l' G
`C G
`T
`C A/\A AC/\A T A T
`T
`C
`!70
`330
`300
`GlyMeLSer'ryrLe ~Gl u A s pva l ArgLeuVal Hi sArgAs pLe ~Al aA l aArg
`Lc u/\ ~ ,,·r r pC y~ M~LLlrl ll cAldLys
`C T GII/\f" I' GG T G'l'IITl ;C IIGN I"I 'GC< . AI\Gg L----- agGGGII T GAGC 'I'IICC'I' GG,\CGA' I'GTGCGGC