AUGUST 1988
`VOLUME 85
`NUMBER 16
`
`·f.~ Clf TH •
`
`--1 ,.
`0 ..,
`
`J
`
`BIO·•AG RICULTURAL
`LI BRA RY
`
`/.\UG 3 0 1988
`
`JNIVERSITY OF C.A.LIFORNI,'.
`RIVERSIDE
`
`Proceedings
`
`C)F THE
`
`National Acadeiny
`of Sciences
`
`Miltenyi Ex. 1033 Page 1
`
`

`

`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`
`Officers
`of 11,e
`Academ_v
`
`Editorial Board
`of tl,e
`Proceedings
`
`FRANK PRESS, President
`JAMES D. EBERT, Vice President
`PETER H. RAVEN, Home Secretarv
`WILLIAM E. GORDON. Foreign sicretary
`ELKAN R. BLOUT, Treasurer
`
`ROBERT H. ABELES
`GORDON A. BA YM
`WILLIAM f. BRACE
`RONALD BRESLOW
`MICHAEL J. CHAMBERLIN
`
`IGOR B. DAWID, Chairman
`MARY-DELL CHILTON
`EDWARD E. DAVID, JR.
`STUART A. KORNFELD
`DANIEL E. KosHLAND, JR,
`PETER D. LAX
`
`DANIEL NATHANS
`HERBERT E. SCARF
`SOLOMON H. SNYDER
`HAROLD V ARM US
`THOMAS A. WALDMANN
`
`Managing Editor: FRANCES R. ZWANZIG
`Senior Associate Editor. GARY T. COCKS
`Associate Editor: CAY BUTLER
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`
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`
`Miltenyi Ex. 1033 Page 2
`
`

`

`"'
`.. ~~iR!'11
`~
`
`C,
`
`..
`
`"'
`"'
`~
`
`OED
`
`Proceedings
`OF THE
`National Academy
`of Sciences
`OF THE UNITED STATES OF AMERICA
`
`Volume 85, Number 16
`
`Table of Contents
`
`pp. 5759-6226
`
`August 1988
`
`AUTHOR INDEX
`
`Physical Sciences
`
`CHEMISTRY
`
`Conformational analysis of 9J3,19-cyclopropyl sterols: Detection of the
`pseudoplanar conformer by nuclear Overhauser effects and its functional
`implications
`Total chemical synthesis of a 77-nucleotide-long RNA sequence having
`methionine-acceptance activity
`
`W. David Nes, Mabry Benson, Robert
`E. Lundin, and Phu H. Le
`
`K. K. Ogilvie, N. Usman, K.
`Nicoghosian, and R. J. Cedergren
`
`Biological Sciences
`
`BIOCHEMISTRY
`
`Total chemical synthesis of a 77-nucleotide-long RNA sequence having
`methionine-acceptance activity
`Stable expression of rat cytochrome P-45011Bl cDNA in Chinese hamster cells
`(V79) and metabolic activation of aflatoxin B1
`
`p21 ras_induced responsiveness of phosphatidylinositol turnover to bradykinin is
`a receptor number effect
`
`Properties of a copper-containing cytochrome ba 3: A second terminal oxidase
`from the extreme thermophile Thermus thermophilus
`
`K. K. Ogilvie, N. Usman, K.
`Nicoghosian, and R. J. Cedergren
`Johannes Doehmer, Satish Dogra,
`Thomas Friedberg, Solange Monier,
`Milton Adesnik, Hansruedi Glatt, and
`Franz Oesch
`Julian Downward, Jean de Gunzburg,
`Rebecca Riehl, and Robert A.
`Weinberg
`Barbara H. Zimmermann, Carmen I.
`Nitsche, James A. Fee, Frank Rusnak,
`and Eckard Miinck
`
`vii
`
`5759
`
`5764
`
`5764
`
`5769
`
`5774
`
`5779
`
`Miltenyi Ex. 1033 Page 3
`
`

`

`Contents
`
`Structure of the coding sequence and primary amino acid sequence of
`acetyl-coenzyme A carboxylase
`
`Cloning, nucleotide sequence, and potential regulatory elements of the
`glutamine synthetase gene from murine 3T3-Ll adipocytes
`Primary structure of cotranscribed genes encoding the Rieske Fe-S and
`cytochrome f proteins of the cyanobacterium Nostoc PCC 7906
`Effects of ras-encoded proteins and platelet-derived growth factor on inositol
`phospholipid turnover in NRK cells
`Alternative splicing generates messages encoding rat c-erbA proteins that do
`not bind thyroid hormone
`Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase
`mRNA and structures of the 5' and 3' flanking regions of the serine
`dehydratase gene
`
`Specificity of action of guanine nucleotide-binding regulatory protein subunits
`on the cardiac muscarinic K + channel
`
`Formation of stable transcription complexes as assayed by analysis of
`individual templates
`Biosynthesis of catalytically active rat testosterone 5a-reductase in
`microinjected Xenopus oocytes: Evidence for tissue-specific differences in
`translatable mRNA
`Characterization of the gene for the subunit of human factor XIII (plasma
`transglutaminase), a blood coagulation factor
`A repressor heterodimer binds to a chimeric operator
`
`Activation of the polyomavirus enhancer by a murine activator protein 1 (APl)
`homolog and two contiguous proteins
`
`A knotted free minicircle in kinetoplast DNA
`
`Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine
`kinases
`Sensitive homologous recombination strand-transfer assay: Partial purification
`of a Drosophila melanogaster enzyme and detection of sequence effects on
`the strand-transfer activity of RecA protein
`A DNA-binding factor specific for xenobiotic responsive elements of P-450c
`gene exists as a cryptic form in cytoplasm: Its possible translocation to
`nucleus
`Bovine papilloma virus-transformed cells contain multiple E2 proteins
`
`Structure of the carboxyl terminus of the RAS gene-encoded P21 proteins
`
`A milk protein gene promoter directs the expression of human tissue
`plasminogen activator cDNA to the mammary gland in transgenic mice
`
`Protein engineering of antibody binding sites: Recovery of specific activity in
`an anti-digoxin single-chain Fv analogue produced in Escherichia coli
`
`Interferon-induced transcription of a major histocompatibility class I gene
`accompanies binding of inducible nuclear factors to the interferon consensus
`sequence
`
`ii
`
`Fernando L6pez-Casillas, Dong-Hoon
`Bai, Xiaochun Luo, In-Soo Kong,
`Mark A. Hermodson, and Ki-Han Kim
`Basant Bhandari, Karla D. Beckwith,
`and Richard E. Miller
`Toivo Kallas, Susan Spiller, and Richard
`Malkin
`Tohru Kamata and Hsiang-fu Kung
`
`Tomoaki Mitsuhashi, Gregory E.
`Tennyson, and Vera M. Nikodem
`Hirofumi Ogawa, Duncan A. Miller,
`Tracy Dunn, Yeu Su, James M.
`Burcham, Carl Peraino, Motoji
`Fujioka, Kay Babcock, and Henry C.
`Pitot
`Diomedes E. Logothetis, Donghee Kim,
`John K. Northup, Eva J. Neer, and
`David E. Clapham
`Harold Weintraub
`
`Yigal Farkash, Hermona Soreq, and
`Joseph Orly
`
`Akitada Ichinose and Earl W. Davie
`
`Melvyn Hollis, Dario Valenzuela, David
`Pioli, Robin Wharton, and Mark
`Ptashne
`Mark E. Martin, Jacques Piette, Moshe
`Yaniv, Wei-Jen Tang, and William R.
`Folk
`Kathleen A. Ryan, Theresa A. Shapiro,
`Carol A. Rauch, Jack D. Griffith, and
`Paul T. Englund
`H.K. Kole, M. Abdel-Ghany, and E.
`Racker
`James G. McCarthy, Miriam Sander, Ky
`Lowenhaupt, and Alexander Rich
`
`Atsuko Fujisawa-Sehara, Miyuki
`Yamane, and Yoshiaki Fujii-Kuriyama
`
`Nancy L. Hubbert, John T. Schiller,
`Douglas R. Lowy, and Elliot J.
`Androphy
`Paul W. Brandt-Rauf, Robert P. Carty,
`James Chen, Matthew Avitable, Jack
`Lubowsky, and Matthew R. Pincus
`Christoph W. Pittius, Lothar
`Hennighausen, Eric Lee, Heiner
`Westphal, Edward Nicols, Jim Vitale,
`and Katherine Gordon
`James S. Huston, Douglas Levinson,
`Meredith Mudgett-Hunter, Mei-Sheng
`Tai, Jiti Novotny, Michael N.
`Margolies, Richard J. Ridge, Robert E.
`Bruccoleri, Edgar Haber, Roberto
`Crea, and Hermann Oppermann
`Yasuaki Shirayoshi, Peter A. Burke,
`Ettore Appella, and Keiko Ozato
`
`5784
`
`5789
`
`5794
`
`5799
`
`5804
`
`5809
`
`5814
`
`5819
`
`5824
`
`5829
`
`5834
`
`5839
`
`5844
`
`5849
`
`5854
`
`5859
`
`5864
`
`5869
`
`5874
`
`5879
`
`5884
`
`Miltenyi Ex. 1033 Page 4
`
`

`

`Contents
`
`Parathyroid hormone modulates transforming growth factor /3 activity and
`binding in osteoblast-enriched cell cultures from fetal rat parietal bone
`Sequences contained within the promoter of the human thymidine kinase gene
`can direct cell-cycle regulation of heterologous fusion genes
`Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced
`ADP-ribosylation and is not recognized by antisera against the a subunit of
`the stimulatory guanine nucleotide-binding regulatory protein
`Colony-stimulating factor I-mediated regulation of a chimeric c-fms/v-fms
`receptor containing the v-fms-encoded tyrosine kinase domain
`
`Michael Centrella, Thomas L. McCarthy,
`and Ernesto Canalis
`Yong Kyu Kim, Steven Wells, Yun-Fai
`Chris Lau, and Amy S. Lee
`Luis M. Molina y Vedia, Bryan R. Reep,
`and Eduardo G. Lapetina
`
`Martine F. Roussel, James R. Downing,
`Richard A. Ashmun, Carl W.
`Rettenmier, and Charles J. Sherr
`
`BIOPHYSICS
`
`Protein dynamics and reaction rates: Mode-specific chemistry in large
`molecues?
`Statistical geometry in sequence space: A method of quantitative comparative
`sequence analysis
`
`Deprotonation of lipid-depleted bacteriorhodopsin
`Chemical oscillations in closed macromolecular systems
`
`William Bialek and Jose Nelson Onuchic
`
`Manfred Eigen, Ruthild
`Winkler-Oswatitsch, and Andreas
`Dress
`Du-Jeon Jang and M. A. El-Sayed
`Enrico Di Cera, Paul E. Phillipson, and
`Jeffries Wyman
`
`BOTANY
`
`Characterization of a benzyladenine binding-site peptide isolated from a wheat
`cytokinin-binding protein: Sequence analysis and identification of a single
`affinity-labeled histidine residue by mass spectrometry
`
`Binding of the phytotoxin zinniol stimulates the entry of calcium into plant
`protoplasts
`
`A. Chris Brinegar, Geoffrey Cooper,
`Anne Stevens, Charles R. Hauer,
`Jeffrey Shabanowitz, Donald F. Hunt,
`and J. Eugene Fox
`Patrice Thuleau, Annick Graziana,
`Michel Rossignol, Heinrich Kauss,
`Pierre Auriol, and Raoul Ranjeva
`
`CELL BIOLOGY
`
`Autocrine growth induced by the insulin-related factor in the
`insulin-independent teratoma cell line 1246-3A
`Genetic variability between isolates of human immunodeficiency virus (HIV)
`type 2 is comparable to the variability among HIV type 1
`
`Selective stabilization of microtubules oriented toward the direction of cell
`migration
`Characterization of human tracheal epithelial cells transformed by an
`origin-defective simian virus 40
`
`The role of lipids in Plasmodium falciparum invasion of erythrocytes: A
`coordinated biochemical and microscopic analysis
`
`Angiogenin activates endothelial cell phospholipase C
`Cleavage furrow isolated from newt eggs: Contraction, organization of the actin
`filaments, and protein components of the furrow
`Specific binding of murine leukemia inhibitory factor to normal and leukemic
`monocytic cells
`
`Yukio Yamada and Ginette Serrero
`
`J. F. Zagury, G. Franchini, M. Reitz, E.
`Collalti, B. Starcich, L. Hall, K.
`Fargnoli, L. Jagodzinski, H.-G. Guo,
`F. Laure, S. K. Arya, S. Josephs, D.
`Zagury, F. Wong-Staal, and R. C.
`Gallo
`Gregg G. Gundersen and Jeannette Chloe
`Bulinski
`D. C. Gruenert, C. B. Basbaum, M. J.
`Welsh, M. Li, W. E. Finkbeiner, and
`J. A. Nadel
`Ross B. Mikkelsen, Markus Kamber,
`Kalyan S. Wadwa, Peck-Sun Lin, and
`Rupert Schmidt-Ullrich
`Roy Bicknell and Bert L. Vallee
`
`Issei Mabuchi, Shoichiro Tsukita,
`Sachiko Tsukita, and Tsuyoshi Sawai
`
`D. J. Hilton, N. A. Nicola, and D.
`Metcalf
`
`DEVELOPMENTAL BIOLOGY
`
`An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence
`common to all chicken globin genes
`Cell contacts are required for induction by cortisol of glutamine synthetase
`gene transcription in the retina
`
`Todd Evans, Marc Reitman, and Gary
`Felsenfeld
`Lily Vardimon, Lyle L. Fox, Linda
`Degenstein, and A. A. Moscona
`
`iii
`
`5889
`
`5894
`
`5899
`
`5903
`
`5908
`
`5913
`
`5918
`5923
`
`5927
`
`5932
`
`5936
`
`5941
`
`5946
`
`5951
`
`5956
`
`5961
`
`5966
`
`5971
`
`5976
`
`5981
`
`Miltenyi Ex. 1033 Page 5
`
`

`

`Contents
`
`ECOLOGY
`
`Global change: Geographical approaches (A Review)
`
`V. M. Kotlyakov, J. R. Mather, G. V.
`Sdasyuk, and G. F. White
`
`EVOLUTION
`Biparental defensive endowment of eggs with acquired plant alkaloid in the
`moth Utetheisa ornatrix
`
`Western equine encephalitis virus is a recombinant virus
`
`Reconstruction of human evolution: Bringing together genetic, archaeological,
`and linguistic data
`
`GENETICS
`Host function of MAK/6: G1 arrest by a mak/6 mutant of Saccharomyces
`cerevisiae
`Allelic sequence variation of the HLA-DQ loci: Relationship to serology and to
`insulin-dependent diabetes susceptibility
`
`Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell
`line Y79
`
`Recognition and cleavage site of the intron-encoded omega transposase
`
`Physical mapping of large DNA by chromosome fragmentation
`
`Comparative mapping of DNA markers from the familial Alzheimer disease and
`Down syndrome regions of human chromosome 21 to mouse chromosomes
`16 and 17
`
`Tn/0 transposition promotes RecA-dependent induction of a A prophage
`Genetic analysis of indefinite division in human cells: Identification of four
`complementation groups
`Transgenic mice bearing the human c-myc gene activated by an
`immunoglobulin enhancer: A pre-B-cell lymphoma model
`Mobility of P elements in drosophilids and nondrosophilids
`
`RED I: A yeast gene required for the segregation of chromosomes during the
`reductional division of meiosis
`Expression of the human /3-globin gene following retroviral-mediated transfer
`into multipotential hematopoietic progenitors of mice
`
`IMMUNOLOGY
`Rapid induction of lgM-secreting murine plasmacytomas by pristane and an
`immunoglobulin heavy-chain promoter/enhancer-driven c-myc/v-Ha-ras
`retrovirus
`cAMP antagonizes interleukin 2-promoted T-cell cycle progression at a discrete
`point in early G1
`CD3-associated heterodimeric polypeptides on suppressor hybridomas define
`biologically active inhibitory cells
`
`iv
`
`David E. Dussourd, Karel Ubik, Carl
`Harvis, James Resch, Jerrold
`Meinwald, and Thomas Eisner
`Chang S. Hahn, Shlomo Lustig, Ellen G.
`Strauss, and James H. Strauss
`
`Luigi Luca Cavalli-Sforza, Alberto
`Piazza, Paolo Menozzi, and Joanna
`Mountain
`
`Reed B. Wickner
`
`Glenn T. Hom, Teodorica L. Bugawan,
`Christopher M. Long, and Henry A.
`Erlich
`Eva Y.-H. P. Lee, Robert Bookstein,
`Lih-Jiuan Young, Chi-Jen Lin, Michael
`G. Rosenfeld, and Wen-Hwa Lee
`L. Colleaux, L. d'Auriol, F. Galibert,
`and B. Dujon
`Douglas Vollrath, Ronald W. Davis,
`Carla Connelly, and Phillip Hieter
`Shirley V. Cheng, Joseph H. Nadeau,
`Rudolph E. Tanzi, Paul C. Watkins,
`Jayashree Jagadesh, Benjamin A.
`Taylor, Jonathan L. Haines, Nicoletta
`Sacchi, and James F. Gusella
`
`Denise Roberts and Nancy Kleckner
`Olivia M. Pereira-Smith and James R.
`Smith
`Emmett V. Schmidt, Paul K. Pattengale,
`Lawrence Weir, and Philip Leder
`
`David A. O'Brochta and Alfred M.
`Handler
`Beth Rockmill and G. Shirleen Roeder
`
`Stefan Karlsson, David M. Bodine,
`Leland Perry, Thalia
`Papayannopoulou, and Arthur W.
`Nienhuis
`
`Raphael Clynes, Judy Wax, Lawrence
`W. Stanton, Sandra Smith-Gill,
`Michael Potter, and Kenneth B. Marcu
`Kirk W. Johnson, Bruce H. Davis, and
`Kendall A. Smith
`David B. Weiner, Jing Liu, Nabil Hanna,
`Jeffrey A. Bluestone, John E. Coligan,
`William V. Williams, and Mark I.
`Greene
`
`5986
`
`5992
`
`5997
`
`6002
`
`6007
`
`6012
`
`6017
`
`6022
`
`6027
`
`6032
`
`6037
`
`6042
`
`6047
`
`6052
`
`6057
`
`6062
`
`6067
`
`6072
`
`6077
`
`Miltenyi Ex. 1033 Page 6
`
`

`

`Contents
`
`Molecular characterization of T-cell antigen receptor expression by subsets of
`CD4 - CDS - murine thymocytes
`
`Autocrine secretion of tumor necrosis factor under the influence of interferon-;,
`amplifies HLA-DR gene induction in human monocytes
`
`Anti-idiotypic antibodies that react with microtubule-associated proteins are
`present in the sera of rabbits immunized with synthetic peptides from
`tubulin's regulatory domain
`Sequence and organization of the diversity, joining, and constant region genes
`of the human T-cell &-chain locus
`
`Binding site for human immunodeficiency virus coat protein gp120 is located in
`the NH2-terminal region of T4 (CD4) and requires the intact
`variable-region-like domain
`
`Interleukin 4 induces membrane Thy-1 expression on normal murine B cells
`
`MEDICAL SCIENCES
`
`Induction of macrophage antitumor activity by acetylated low density
`lipoprotein containing lipophilic muramyl tripeptide
`LR16, a compound with potent effects on the oxygen affinity of hemoglobin,
`on blood cholesterol, and on low density lipoprotein
`Unusual lipid structures selectively reduce the toxicity of amphotericin B
`
`Adenallene and cytallene: Acyclic nucleoside analogues that inhibit replication
`and cytopathic effect of human immunodeficiency virus in vitro
`
`Mechanism of inhibitory effect of dextran sulfate and heparin on replication of
`human immunodeficiency virus in vitro
`
`Essential fatty acid deficiency prevents multiple low-dose streptozotocin(cid:173)
`induced diabetes in CD-1 mice
`
`Anemia lessens and its prevention with recombinant human erythropoietin
`worsens glomerular injury and hypertension in rats with reduced renal mass
`
`Functional reconstitution of the canalicular bile salt transport system of rat
`liver
`Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis
`of factor VIII fragments expressed in Escherichia coli
`
`Martin Pearse, Pauline Gallagher, Anne
`Wilson, Li Wu, Nella Fisicaro,
`J. F. A. P. Miller, Roland Scollay, and
`Ken Shortman
`Fernando Arenzana-Seisdedos, S9>ren C.
`M9>gensen, Fran~oise Vuillier, Walter
`Fiers, and Jean-Louis Virelizier
`Coralia I. Rivas, Juan C. Vera, and
`Ricardo B. Maccioni
`
`Yoshihiro Takihara, Douglas Tkachuk,
`Effie Michalopoulos, Eric Champagne,
`Jorg Reimann, Mark Minden, and Tak
`W.Mak
`Neil E. Richardson, Nicholas R. Brown,
`Rebecca E. Hussey, Arnita Vaid,
`Thomas J. Matthews, Dani P.
`Bolognesi, and Ellis L. Reinherz
`Clifford M. Snapper, Peter V. Hornbeck,
`Ulus Atasoy, Geraldo M. B. Pereira,
`and William E. Paul
`
`J. Michael Shaw, William S. Futch, Jr.,
`and Lawrence B. Schook
`I. Lalezari, S. Rahbar, P. Lalezari, G.
`Fermi, and M. F. Perutz
`A. S. Janoff, L. T. Boni, M. C. Popescu,
`S. R. Minchey, P. R. Cullis, T. D.
`Madden, T. Taraschi, S. M. Gruner,
`E. Shyamsunder, M. W. Tate, R.
`Mendelsohn, and D. Bonner
`Seiji Hayashi, Shashikant Phadtare, Jiri
`Zemlicka, Makoto Matsukura, Hiroaki
`Mitsuya, and Samuel Broder
`Masanori Baba, Rudi Pauwels, Jan
`Balzarini, Jef Arnout, Jan Desmyter,
`and Erik De Clercq
`James R. Wright, Jr., James B.
`Lefkowith, George Schreiner, and Paul
`E. Lacy
`Diego L. Garcia, Sharon Anderson,
`Helmut G. Rennke, and Barry M.
`Brenner
`Stephan Ruetz, Gabriel Hugentobler, and
`Peter J. Meier
`Dorothea Scandella, Sytske deGraaf
`Mahoney, Mora Mattingly, David
`Roeder, Lisa Timmons, and Carol A.
`Fulcher
`
`MICROBIOLOGY
`
`Many pulmonary pathogenic bacteria bind specifically to the carbohydrate
`sequence GalNAc/31-4Gal found in some glycolipids
`X gene-related sequences in the core gene of duck and heron hepatitis B
`viruses
`
`Howard C. Krivan, David D. Roberts,
`and Victor Ginsburg
`Mark A. Feitelson and Roger H. Miller
`
`NEUROBIOLOGY
`
`Differentiation of bipotential glial precursors into oligodendrocytes is promoted
`by interaction with type-1 astrocytes in cerebellar cultures
`
`Francesca Aloisi, Cristina Agresti,
`Donatella D'Urso, and Giulio Levi
`
`V
`
`6082
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`6087
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`6092
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`6097
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`6102
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`6107
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`6112
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`6117
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`6122
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`6127
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`6132
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`6137
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`6142
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`6147
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`6152
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`6157
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`6162
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`6167
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`Miltenyi Ex. 1033 Page 7
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`

`

`Contents
`
`Importance of monoamine oxidase A in the bioactivation of neurotoxic analogs
`of 1-methyl-4-phenyl-1,2,3 ,6-tetrahydropyridine
`
`Structure and action of buccalin: A modulatory neuropeptide localized to an
`identified small cardioactive peptide-containing cholinergic motor neuron of
`Aplysia californica
`
`Laminar-specific distribution and infrastructural detail of amyloid in the
`Alzheimer disease cortex visualized by computer-enhanced imaging of
`epitopes recognized by monoclonal antibodies
`Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase
`and y-aminobutyrate immunoreactivity
`Activation of protein kinase C differentially modulates neuronal Na+, Ca2 +,
`and y-aminobutyrate type A channels
`A simple "neural induction" model with two interacting cleavage-arrested
`ascidian blastomeres
`Reexpression of a developmentally regulated antigen in Down syndrome and
`Alzheimer disease
`Transsynaptic impulse activity regulates postsynaptic density molecules in
`developing and adult rat superior cervical ganglion
`Early blindness results in a degraded auditory map of space in the optic tectum
`of the barn owl
`Maintenance by glycyl-L-glutamine in vivo of molecular forms of
`acetylcholinesterase in the preganglionically denervated superior cervical
`ganglion of the cat
`Phosphorylation of Alzheimer disease amyloid precursor peptide by protein
`kinase C and Caz+ /calmodulin-dependent protein kinase II
`
`PHYSIOLOGICAL SCIENCES
`
`Atrial-specific granules in situ have high calcium content, are acidic, and
`maintain anion gradients
`
`Richard E. Heikkila, M. Victoria Kindt,
`Patricia K. Sonsalla, Andrew
`Giovanni, Stephen K. Youngster,
`Kathleen A. McKeown, and Thomas
`P. Singer
`Elizabeth C. Cropper, Mark W. Miller,
`Renata Tenenbaum, Mary Ann
`Gawinowicz Kolks, Irving
`Kupfermann, and Klaudiusz R. Weiss
`Ronald E. Majocha, Francine M. Benes,
`Jennifer L. Reifel, Anne M. Rodenrys,
`and Charles A. Marotta
`Nicholas Brecha, Donald Johnson, Leo
`Peichl, and Heinz Wassle
`Erwin Sigel and Roland Baur
`
`Haruo Okado and Kunitaro Takahashi
`
`Benjamin Wolozin, Angela Scicutella,
`and Peter Davies
`Kuo Wu and Ira B. Black
`
`Eric I. Knudsen
`
`George B. Koelle, Kenneth A. Skau,
`Nagendran S. Thampi, Diane M.
`Hymel, and Matthew S. Han
`Samuel Gandy, Andrew J. Czernik, and
`Paul Greengard
`
`A. V. Somlyo, R. Broderick, H.
`Shuman, E. L. Buhle, Jr., and A. P.
`Somlyo
`
`6172
`
`6177
`
`6182
`
`6187
`
`6192
`
`6197
`
`6202
`
`6207
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`6211
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`6215
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`6218
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`6222
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`vi
`
`Miltenyi Ex. 1033 Page 8
`
`

`

`Proc. Natl. Acad. Sci. USA
`Vol. 85, pp. 5879-5883, August 1988
`Biochemistry
`
`Protein engineering of antibody binding sites: Recovery of specific
`activity in an anti-digoxin single-chain Fv analogue produced in
`Escherichia coli
`(biosynthetic analogue/ anti-digoxin antibodies/ variable regions/ gene synthesis/ Escherichia coli expression)
`
`JAMES S. HUSTON*t, DOUGLAS LEVINSON*, MEREDITH MUDGETT-HUNTER+, MEI-SHENG TAI*,
`J11H NOVOTNY+, MICHAEL N. MARGOLIES§, RICHARD J. RIDGE*, ROBERT E. BRUCCOLERI+,
`EDGAR HABER+, ROBERTO CREA*, AND HERMANN OPPERMANN*
`*Creative Biomolecules, 35 South Street, Hopkinton, MA 01748; and *Molecular and Cellular Research Laboratory, and §Department of Surgery,
`Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
`
`Communicated by Michael Sela, May 6, 1988 (received for review March 9, 1988)
`
`ABSTRACT
`A biosynthetic antibody binding site, which
`incorporated the variable domains of anti-digoxin monoclonal
`antibody 26-10 in a single polypeptide chain (Mr = 26,354), was
`produced in Escherichia coli by protein engineering. This
`variable region fragment (Fv) analogue comprised the 26-10
`heavy- and light-chain variable regions (Vu and V L) connected
`by a 15-amino acid linker to form a single-chain Fv (sFv). The
`sFv was designed as a prolyl-Vu•Oinker)-VL sequence of 248
`amino acids. A 744-base-pair DNA sequence corresponding to
`this sFv protein was derived by using an E. coli codon pref er(cid:173)
`ence, and the sFv gene was assembled starting from synthetic
`oligonucleotides. The sFv polypeptide was expressed as a fusion
`protein in E. coli, using a leader derived from the trp LE
`sequence. The sFv protein was obtained by acid cleavage of the
`unique Asp-Pro peptide bond engineered at the junction of
`leader and sFv in the fusion protein [(leader)-Asp-Pro-Vu(cid:173)
`(linker)-V d, After isolation and renaturation, folded sFv dis(cid:173)
`played specificity for digoxin and related cardiac glycosides
`similar to that of natural 26-10 Fab fragments. Binding between
`affinity-purified sFv and digoxin exhibited an association con(cid:173)
`stant [Ka = (3.2 ± 0.9) X 107 M- 1
`] that was about a factor of
`6 smaller than that found for 26-10 Fab fragments [Ka = (1.9 ±
`0.2) x 108 M- 11 under the same buffer conditions, consisting of
`0.01 M sodium acetate, pH 5.5/0.25 M urea.
`
`It is known that antigen binding fragments of antibodies (1, 2)
`can be refolded from denatured states with recovery of their
`specific binding activity (3-6). The smallest such fragment that
`contains a complete binding site is termed Fv, consisting of an
`Mr 25,000 heterodimer of the V Hand V L domains (2, 5-11).
`Givol and coworkers were the first to prepare an Fv by peptic
`digestion of murine lgA myeloma MOPC 315 (2). However,
`subsequent development of general cleavage procedures for
`Fv isolation has met with limited success (7-11). As a result,
`the Mr 50,000 Fab (1) has remained the only monovalent
`binding fragment used routinely in biomedical applications.
`An Fv analogue was constructed in which both heavy- and
`light-chain variable domains (V H and V d were part of a
`single polypeptide chain. Synthetic genes for the 26-10
`anti-digoxin V H and V L regions were designed to permit their
`connection through a linker segment, as well as other
`manipulations (12, 13). The synthetic gene for single-chain Fv
`(sFv) was expressed in Escherichia coli as a fusion protein,
`from which the sFv protein was isolated. ,r The sFv was
`renatured with recovery of binding specificity and affinity
`similar to those of the parent molecule. Thus, variable
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`domains connected artificially to form one polypeptide chain
`can be renatured into properly folded Fv regions.
`
`MATERIALS AND METHODS
`Model Antibody. The digoxin binding site of the lgG2a,K
`monoclonal antibody 26-10 has been analyzed by Mudgett(cid:173)
`Hunter and colleagues (14-16). The 26-10 V region sequences
`were determined from both protein sequencing (17) and DNA
`sequencing of 26-10 H- and L-chain mRNA transcripts (D.
`Panka, J.N., and M.N.M., unpublished data). The 26-10
`antibody exhibits a high digoxin binding affinity (Ka = 5.4 x
`109 M- 1
`) (14) and has a well-defined specificity profile (15),
`providing a baseline for comparison with the biosynthetic sFv.
`Protein Design. X-ray coordinates for Fab fragments (18-
`20) were obtained from the Brookhaven Data Bank (21) and
`analyzed with the programs CHARMM (22), CONGEN (23),
`and FRODO (24). X-ray data indicated that the Euclidean
`distance between the C terminus of the V H domain and the
`N terminus of the V L domain was =3.5 nm. A 15-residue
`linker should bridge this gap, since the peptide unit length is
`=0.38 nm. The linker should not exhibit a propensity for
`ordered secondary structure or any tendency to interfere with
`domain folding. Thus, the 15-residue sequence (Gly-Gly-Gly(cid:173)
`Gly-Serh was selected to connect the V H carboxyl and V L
`amino termini (Fig. 1).
`Gene Synthesis. Design of the 744-base sequence for the
`synthetic sFv gene was derived from the sFv protein se(cid:173)
`quence by choosing codons preferred by E. coli (25). Syn(cid:173)
`thetic genes encoding the trp promoter-operator, the modi(cid:173)
`fied trp LE leader peptide (MLE), and V H were prepared
`largely as described (26). The gene encoding V 8 was assem(cid:173)
`bled from 46 overlapping synthetic 15-base oligonucleotides.
`The V L gene was derived from 12 synthetic polynucleotides
`ranging in size from 33 to 88 base pairs, prepared in
`automated DNA synthesizers (model 6500, Biosearch, San
`Rafael, CA; model 380A, Applied Biosystems, Foster City,
`CA). They spanned major restriction sites (Aat II, BstEII,
`Kpn I, HindIII, Bgl I, and Pst I), and several fragments were
`flanked by Eco RI and BamHI cloning ends. All segments
`were cloned and assembled in pUC vectors. The linker
`between V Hand Vu encoding (Gly-Gly-Gly-Gly-Serh, was
`cloned from two polynucleotides spanning Sac I and Aat II
`sites. The complete sFv gene was assembled from the V H•
`V L, and linker genes to yield a single sFv gene, corresponding
`
`Abbreviations: Fv, variable region fragment (V H V L dimer)~ MLE,
`modified trp LE leader sequence; sFv, single-chain Fv; VH, heavy(cid:173)
`chain variable region; V L• light-chain variable region.
`tTo whom reprint requests should be addressed.
`,iThe sequence reported in this paper is being deposited in the
`EMBL/GenBank data base (lntelliGenetics, Mountain View, CA,
`and Eur. Mol. Biol. Lab., Heidelberg) (accession no. 103850).
`
`5879
`
`Miltenyi Ex. 1033 Page 9
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`

`

`5880
`
`Biochemistry: Huston et al.
`
`Proc. Natl. Acad. Sci. USA 85 (1988)
`
`pBR322 vector, was constructed by a three-part ligation using
`the sites Ssp I, EcoRI, and Pst I (Fig. 2D). Intermediate DNA
`fragments and assembled genes were sequenced by the
`dideoxy chain-termination method (28).
`Fusion Protein Expression. Single-chain Fv was expressed
`as a fusion protein (Fig. 2) with the MLE leader gene (29) and
`under the control of a synthetic trp promoter-operator (29).
`E. coli strain JM83 was transformed with the expression
`plasmid and protein expression was induced in M9 minimal
`medium by addition of indoleacrylic acid (10 µ,g/ ml) at a cell
`density with A 600 = 1. The high expression levels of the
`fusion protein resulted in its accumulation as insoluble
`protein granules, which were harvested from cell paste (Fig.
`3, lane 1).
`Fusion Protein Cleavage. The MLE leader was removed
`from sFv by acid cleavage of the Asp-Pro peptide bond (32-
`34) engineered at the junction of the MLE and sFv sequences.
`The washed protein granules containing the fusion protein
`were cleaved in 6 M guanidine hydrochloride/ 10% acetic
`acid, pH 2.5, incubated at 37°C for 96 hr. The reaction was
`stopped by ethanol precipitation, and the precipitate was
`stored at - 20°C (Fig. 3, lane 2).
`Protein Purification. The acid-cleaved sFv was separated
`from remaining intact MLE-sFv species by chromatography
`on