"D BIOLOGY
`
`try and Drug Addiction
`
`lmes
`
`nberg
`
`dler
`
`PS
`’
`
`rmelmk
`
`I‘ION
`ic G. Brunngraber
`
`her Biologicals
`
`
`
`CELL SUBSTRATES
`Their Use in the Production of
`Vaccines and Other Biologicals
`
`Edited by
`
`John C. Petricciani
`and Hope E. Hopps
`Bureau of Biologics of the Food and Drug Administration
`Bethesda, Maryland
`
`and
`Paul J. Chapplc
`W. Alton Jones Cell Science Center
`Lake Placid, New York
`
`PLENUM PRESS ° NEW YORK AND LONDON
`
`Merck Ex. 1072, pg 1564
`
`
`Merck Ex. 1072, pg 1564
`
`

`
`
`Library of Congress Cataloging in Publication Data
`Symposium on Cell Substrates and Their Use in the Production of Vaccines and
`Other Biologicals, 2d, Lake Placid, N. Y., 1978.
`Cell substrates, their use in the production of vaccines and other biologicals.
`
`(Advances in experimental medicine and biology; V. 118).
`“Proceedings of the Second Annual Symposium on Cell Substrates and Their
`Use in the Production of Vaccines and Other Biologicals, held October 23-26,
`1978.”
`“Symposium sponsored by the W. Alton Jones Cell Science Center."
`Includes index.
`1. Vaccines——Congresses. 2. Human cell culture—Congresses. 3. Biological
`products — Congresses. 4-. Cancer cells — Congresses.
`I. Petricciani, John C. II.
`Hopps, Hope E. III. Chapple, Paul J. IV. Tissue Culture Association. W. Alton
`Jones Cell Science Center. V. Title. V1. Series.
`79~13479
`QR189.S93 1978
`574'.07'24‘
`ISBN 0-306-40189-4
`
`
`..__-1
`
`Proceedings of the Second Annual Symposium, on Cell Substrates and Their Use
`in the Production of Vaccines and Other Biologicals, held at the W. Alton Jones
`Cell Science Center, Lake Placid, New York, October 23—26, 1978.
`
`Symposium sponsored by the Tissue Culture Association, Inc., and its
`operational unit, the W. Alton Jones Cell Science Center, with the support of:
`Bureau of Biologics, FDA
`-
`Fogarty International Center, National Institutes of Health
`National Institute of Allergy and
`Infectious Diseases
`SYNTEX
`
`© 1979 Plenum Press, New York
`A Division of Plenum Publishing Corporation
`227 West 17th Street, New York, N.Y. 10011
`
`All rights reserved
`No part of this book may be reproduced, stored in a retrieval system, or transmitted,
`in any form or by any means, electronic, mechanical, photocopying, microfilming,
`recording, or otherwise, without written permission from the Publisher
`Printed in the United States of America
`
`Merck Ex. 1072, pg 1565
`
`Merck Ex. 1072, pg 1565
`
`

`
`K.DOUGALL
`
`rary, Alberta
`
`‘servation of
`
`‘44 in
`Le culture
`.ag Berlin
`
`»n industry.
`,sue culture
`.ue Culture.
`‘ta, Canada.
`none
`Planta
`
`Lction of
`
`§_blumei.
`
`Weiler,
`ts serpentine
`‘anthus
`I. Zenk,
`
`New York.
`'76.
`
`:hnol. §g;
`
`>lant tissue
`letabolites.
`eds.
`_cations.
`
`Ltion by
`)-
`
`USE OF RECOMBINANT DNA TECHNOLOGY FOR THE PRODUCTION OF POLYPEPTIDES
`
`Walter L. Miller, M.D.
`
`Endocrine Research Division and the Department of
`Pediatrics, University of California, San Francisco
`
`I.
`
`INTRODUCTION
`
`There are many polypeptides of biologic and medical interest
`which have not been fully studied because they cannot be obtained
`in adequate quantity with sufficient purity and economy. Recent ad-
`vances in recombinant DNA technology now give promise of in_vivo
`synthesis in bacteria of a wide variety of polypeptide hormones,
`specific immunoglobins, enzymes and other proteins. This presenta-
`tion will first review the procedures for constructing chimeric micro-
`organisms containing the DNA coding for a eucaryotic protein,
`then
`discuss the problems and experience in obtaining such proteins from
`DNA cloned in procaryotic cells.
`
`Both eucaryotic and procaryotic cells synthesize proteins in
`comparable manners: DNA is transcribed by a complex polymerase into
`.RNA, which may require further processing before it can be used as a
`messenger RNA (mRNA); mRNA is translated into protein on ribosomes,
`using amino-acylated transfer RNAS as the interface between nucleic
`acid and polypeptide;
`the resulting polypeptide may then undergo
`further processing before being utilized by the cell. As these steps
`are common to all organisms, one may hypothesize that it should be
`possible to construct chimeric microorganisms containing DNA for a
`desired eucaryotic polypeptide, and that proper arrangement of this
`DNA in the host cell should result in the production of the coded
`protein.
`
`In order to modify microorganisms so that they may produce a
`desired polypeptide, five steps are necessary. First, DNA sequences
`coding for the protein are prepared.
`Secondly, this gene must be
`
`153
`
`Merck Ex. 1072, pg 1566
`
`Merck Ex. 1072, pg 1566
`
`

`
`
`
`154
`
`W.L.M|LLER
`
`covalently linked to the DNA of a suitable vehicle, such as the
`small, circular, non—chromosomal DNA's termed plasmids, or the DNA of
`a bacteriophage.
`The gene for the protein then travels as a pas-
`senger with the vehicular DNA when the vehicle infects a suitable
`recipient host cell. This infection of the host cell is the third
`step,
`transformation. Fourth,
`the transformed host cell must repli-
`cate the infecting DNA faithfully so that all daughter cells will
`carry the gene for the protein. Finally,
`the transformed cells
`must transcribe the foreign DNA into mRNA, and the cellular ma-
`chinery for protein synthesis must accept and translate this mRNA.
`
`while the outlines of molecular biology are the same for both
`nucleated eucaryotic and lower procaryotic cells, a number of dif-
`ferences exist between these classes of organisms which could inter-
`fere with bacterial production of eucaryotic proteins. Various or-
`ganisms exhibit codon preference, i.e., not all the triplet codons
`for a particular amino acid are utilized in equal numbers.
`For ex-
`ample,
`the amino acid leucine may be specified by six different co-
`dons, but CUG is used for 19 out of 31 residues in rat pre-growth
`hormone U) and in ll out of 21 residues in human chorionic soma-
`tomammotropin (2); however,
`the same codon is used in only 16 of 143
`residues in bacteriophage MS2
`(3). Similarly, promoter sequences
`and ribosome binding sites also differ between eucaryotic and pro-
`caryotic organisms (4-6). Each of these types of differences, and
`possibly others, has the potential for altering the efficiency of
`transcription and translation of eucaryotic gene sequences which
`have been inserted into a procaryotic genome by recombinant DNA
`technology.
`
`A
`
`The strategy which has been most widely employed in recombinant
`DNA technology is outlined in Figure 1.
`DNA, foreign to the host
`microorganism and which codes for the polypeptide of interest, is
`isolated and cut with one of several restriction endonucleases.
`restriction endonuclease is a highly specific enzyme which cuts
`double stranded DNA at a uniquely specified sequence of four to six
`nucleotides. These enzymes recognize mirror image sequences of DNA
`(pallindromes) and cut them along an axis of symmetry, commonly
`generating short segments of single stranded DNA which are comple-
`mentary to each other. Such complementary ends of DNA strands are
`termed cohesive termini, or "sticky ends" because they have the
`ability to recognize and adhere to complementary sticky ends by base
`pairing.
`A cloning vehicle,
`in this example a plasmid,
`is chosen
`with a single restriction site for the enzyme used to cut the foreign
`DNA.
`The plasmid is cut with the enzyme and mixed with the foreign
`DNA.
`The complementary sticky ends recognize each other and align
`by hydrogen bonding,
`inserting the foreign DNA into the plasmid.
`The ends are then cemented by DNA ligase.
`The resulting new chimeric
`plasmid, carrying the gene for the desired protein,
`is then put in-
`to bacteria (usually a derivative of E. coli strain K12). Plasmids
`
`Merck Ex. 1072, pg 1567
`
`
`
`
`
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`
`Merck Ex. 1072, pg 1567
`
`

`
`!.L.M|LLER
`
`the
`.s
`' the DNA of
`; a pas-
`;uitable
`:he third
`nust repli-
`Lls will
`cells
`ar ma-
`his mRNA.
`
`for both
`r of dif-
`ould inter-
`.rious or-
`.et codons
`‘“ For ex-
`fferent co-
`
`:e—growth
`.c soma-
`
`Ly 16 of 143
`;equences
`and pro-
`ances, and
`siency of
`as which
`ant DNA
`
`A
`
`recombinant
`i the host
`.erest,
`is
`leases.
`.ch cuts
`four to six
`snces of DNA
`:ommonly
`are comple-
`trands are
`nave the
`ends by base
`is chosen
`ut the foreign_
`the foreign
`r and align
`plasmid.
`g new chimeric
`then put in—
`i).» Plasmids
`
`USEOFRECOMNNANTDNATECHNOLOGY
`
`155
`
`Eucaryoiic DNA
`
`Hind III Site
`
`Hind Ill Site
`
`:_.‘=:::J_—:.:
`
` rWEaxA
`i ”‘"d '"
`
`Hind Ill
`
`4
`7}
`#0
`CC4
`Linear Plasmid
`
`‘
`CV
`P‘
`
`Restriction Fragment
`
`Mix
`
`14 DNA Ugose
`
`
`
`Chimeric Piasmid
`
`AAGCTT-—--ATGCTT
`T'TC(3AA----“ITCCEAA
`
`Figure 1. Strategy for cloning foreign DNA in a microorganism using
`a plasmid vector. Foreign DNA is cut with a restriction enzyme such
`as Hind III, yielding fragments with self—complementary cohesive
`termini.
`A plasmid cloning vector having a single restriction site
`for Hind III is also cut with the enzyme.
`The cut ("linear") plasmid
`is mixed with the foreign DNA.
`The complementary cohesive Hind III
`termini of plasmid and foreign DNA align by hydrogen—bond base pari-
`ing, and the DNA strands are sealed with DNA ligase. This "chimeric
`plasmid" may be replicated in transformed bacteria.
`
`Merck Ex. 1072, pg 1568
`
`Merck Ex. 1072, pg 1568
`
`

`
`156
`
`W.L.M|LLER
`
`which have been taken up by the bacteria now behave as benign obli-
`gate intracellular parasites, replicating independently of the host
`chromosomal DNA, and have full access to its protein synthetic
`machinery. Successfully transformed bacteria can be selected on
`agar plates if the plasmid cloning vehicle also confers antibiotic
`resistance to the bacteria.
`
`II.MEANS OF OBTAINING DNA SEQUENCES
`
`Progress made in many laboratories in the past five years has
`made available a wide variety of restriction enzymes,
`ligation pro-
`cedures, cloning vehicles, and hosts. This technology has been limit-
`ed in techniques for preparing DNA for cloning,
`in selecting the pro-
`per colonies of chimeric microorganisms, and in obtaining expression
`of the cloned DNA in the microorganisms.
`The principal difficulty
`in obtaining human DNA has been ensuring its purity. Because of
`speculations concerning the safety of recombinant DNA research,
`regulations are delineated in a series of NIH guidelines (7), where-
`in the source and purity of the DNA,
`the cloning vehicles, and the
`recipient microorganisms are specified so as to provide biologic
`containment. Various levels of physical containment in specially
`built and certified laboratories are also specified by these guide-
`lines. However, it must be emphasized that there is no evidence that
`these containment measures are necessary.
`DNA sequences for cloning
`can be generated in three ways:
`they can be created synthetically
`by elaborate but conventional organic chemistry;
`they can be isolated
`from the chromosomal DNA of an organism; or they can be enzymatically
`copied from purified mRNA. Each of these techniques has different
`strengths and weaknesses which would then dictate the best approach
`to cloning the DNA for a specific protein.
`
`a. Chemical Synthesis
`
`Chemically synthesized DNA offers formidable advantages for
`many recombinant DNA experiments. Chemical synthesis can yield a
`product of great purity, without the possibility that the DNA se-
`quences for other genes have been co~isolated with the DNA sequences
`to be cloned.
`Secondly, as the DNA is being chemically synthesized,
`its precise nucleotide sequence must be designed by the experimenter.
`This permits selection of triplet codons favored by the recipient
`host microorganism and permits the creating of designed proteins
`which do not exist in nature.
`Such modified proteins have already
`been created by protein chemistry, resulting in pharmacologically
`useful derivatives of vasopressin, oxytocin, and gonadotropin re-
`leasing hormone.
`However, chemical synthesis is complicated and
`may not be able to produce long segments of DNA efficiently. Further-
`more, as short polypeptides are rapidly degraded by the host bacteria,
`successful production of these peptides has been achieved by covalent
`linkage to large proteins which must be removed without harming the
`
`Merck Ex. 1072, pg 1569
`
`Merck Ex. 1072, pg 1569
`
`

`
`W.L.M|LLER
`
`nign obli-
`f the host
`.hetic
`:cted on
`.ntibiotic
`
`years has
`yation pro-
`[S been limit-
`
`;ing the pro-
`; expression
`lifficulty
`:ause of
`
`search,
`(7), where-
`s, and the
`aiologic
`specially
`iese guide-
`avidence that
`for cloning
`thetically
`1 be isolated
`
`anzymatically
`different
`
`st approach
`
`ages for
`n yield a
`a DNA se-
`
`NA sequences
`synthesized,
`experimenter.
`recipient
`proteins
`ve already
`logically
`ropin re~
`cated and
`
`tly. Further-
`host bacteria,
`d by covalent
`harming the
`
`
`
`USEOFRECOMMNANTDNATECHNOLOGY
`
`157
`
`peptide coded by the cloned DNA. Despite these limitations, this
`approach has been used to produce somatostatin, a 14 amino acid
`peptide,
`in bacteria (8)
`(Figure 2).
`The plasmid pBR322 was modi-
`fied by insertion of the E. coli lag_operon along with the structural
`gene for B-galactosidase.
`A synthetic gene coding for somatostatin
`and containing an extra triplet codon for a amino—terminal methionine
`residue is joined to the B—galactosidase gene at an Eco Rl re-
`striction enzyme site.
`The synthetic somatostatin gene also contains
`two different nonsense or "stop" codons following the codon for the
`carboxy-terminal cystine residue, and is joined to pBR322 at a Bam
`I site.
`Since the lac repressor gene is absent in this plasmid, and
`since the lag repressor of the host E. coli RRl chromosomal DNA can-
`not produce enough repressor molecules to bind to the operator genes
`of all plasmid copies present in the cell,
`the cell essentially be-
`comes constitutive for the production of B—galactosidase.
`Each
`molecule of B—galactosidase coded by the plasmid will then have the
`14 amino acids of somatostatin linked to its carboxy terminus by an
`extra methionine residue. This hybrid protein molecule is then
`cleaved at its methionine residues with cyanogen bromide to yield
`active somatostatin and fragments of B—galactosidase.
`
`b.
`
`Isolation of Native DNA Fragments
`
`The most direct approach to obtaining long sequences of DNA for
`cloning is the isolation of native gene fragments. This is the ap-
`proach used in early experiments cloning bacterial DNA (9) and DNA's
`coding for ribosomal
`(10) and transfer RNAs
`(11). This direct ap-
`proach offers the advantage of permitting the cloning of a whole
`natural gene with its attendant regulatory regions;
`furthermore,
`only the capacity of the cloning vehicle limits the length of DNA
`which can be cloned by this procedure.
`Isolation of a whole natural
`gene makes this a very powerful technique for studying gene structure
`and regulation, but makes it a poor technique for constructing
`chimeric microorganisms which can produce eucaryotic proteins. Pro-
`caryotic host cells might recognize eucaryotic control sequences and
`enzyme binding sites poorly. Furthermore, eucaryotic genes are often
`interrupted by “intervening” sequences (l2,l3).
`Intervening
`sequences are segmentsof DNA,often of considerable length, which
`interrupt the coding regions of certain genes. These sequences ap-
`pear to be transcribed into heterogeneous nuclear RNA, which is then
`processed into mRNA by excision of these sequences (14).
`As pro~
`caryotic organisms do not process their mRNA in this matter,
`they may
`not be able to process the RNA transcribed from a cloned eucaryotic
`gene containing intervening sequences, and hence would not express
`this genetic material as meaningful protein. Another limitation of
`this procedure is the need for a relatively pure probe, a radioactive
`complementary DNA or mRNA which can be used to assay for the presence
`of the desired sequences.
`
`Merck Ex. 1072, pg 1570
`
`Merck Ex. 1072, pg 1570
`
`

`
`158
`
`W. L.M|LLER
`
`.
`
`I’
`
`E can Lac loperon DNA
`
`Lac PO
`
`fi_Ga'
`
`GENETIC CODE ‘
`
`DNA Synthesis
`
`Chemicm
`
`Somatastatin Gene
`GCT oar TGT AAG AAC TTC TTT 1-
`
` cue
`lln Vwo
`
`TGT GCT TCA CTT TCA G‘
`
`
` pBR322 Plasmid DNA
`
`Sam
`15 — Gal
`NH2 Mev » Ala 4 Gly - Cys- Ly:-Asn - PM‘ mm,
`s
`?
`H0 - Cys~Ser~ Thr- Phe - Thr'
`
`Tfp
`Ly:
`
`Inv Vino
`'
`i Cyanogen Bromide
`Cleavage
`
`15- Gal Fragments
`
`+
`
`NH2- Ala - Gly - Cys . Lys -Asn’Phe - Phe,
`;
`S
`H0- C‘ys- S¢r~Thr- Phe » TM’
`Active somatostatin
`
`W
`Lys
`
`Figure 2. Modification of plasmid pBR322 for the production of
`somatostatin.
`A fragment of E. coli DNA containing the lag_operon,
`including the promoter and operator sites, and a portion of the
`structural gene for B—galactosidase had been inserted into the
`plasmid pBR322.
`A chemically synthesized double—stranded segment
`of DNA is attached to the B—galactosidase gene at an Eco RI site
`((G)AATTC) and to the pBR322 at a Bam I site ((C)CTAG(G)).
`The
`sequences coding for somatostatin are linked to the Eco RI site
`by a methionine codon (ATG), and to the Bam I site by two nonsense
`(stop) condons
`(GAT AGT). when inserted into E. coli,the B—galacto—
`sidase and somatostatin sequences are transcribed under control of
`the lag_operon, yielding a large, hybrid protein molecule. Digest—
`tion of this protein with cyanogen bromide cleaves all the methionine
`residues,
`fragmenting the B—galactosidase and liberating active
`somatostatin.
`(Reproduced by permission from Itakura et_§l,, Science
`l§2;lO56, 1977)
`(8). Copyright 1977 by The American Association for
`the Advancement of Science.
`
`6 i
`
`Merck Ex. 1072, pg 1571 1
`
`L
`
`Merck Ex. 1072, pg 1571
`
`

`
`use OF RECOMBINANT DNA TECHNOLOGY
`
`159
`
`
`
`Chromosomal
`DNA
`
`500 Kl
`
`'
`
`Rostrlctlon
`(I'\'°(% Fragments
`~/5§~b
`
`Preparlilvo Agarose
` ' Gel Electrophoresis
`('Gene Machine’)
`
`RPc'5
`c°'"m"
`
`WU
`3 Hybrldlxatlon
`W333? W
`1 Pool rruciions
`
`Gene Machine
`
`FUD
`J Hybridization
`9E_9§jW
`1 Pool Fractions
`
`
`
`Cloning in
`A Phage
`
`Cloning In
`Plasmids
`
`
`
`RPC-5 Column
`
`DNA is prepared from
`Figure 3. Cloning of native gene fragments.
`nuclei and cut with a restriction fragment such as Eco RI.
`The frag-
`ments are roughly separated by size and charge either by agarose gel
`electrophoresis or reverse—phase chromatography. Aliquots of col-
`lected fractions are assayed for the desired sequences by nucleic
`acid hybridization and the appropriate fractions are pooled. This
`partially purified DNA may be cloned directly or purified further.
`
`The general strategy for cloning native DNA sequences is shown
`in Figure 3. Chromosomal DNA is cut with a restriction endonuclease
`such as Eco R1 chosen to give relatively large restriction fragments.
`Digestion of human DNA with this enzyme yields approximately 106
`unique fragments. These may be partially separated according to size
`and charge by preparative agarose gel electrophoresis (15) or
`reverse-phase column chromotography (16). Fractions of the eluate
`are collected and aliquots are assayed for the presence of the sub-
`ject DNA sequences by nucleic acid hybridization (17). This assay
`requires the use of a radioactively labeled probe, usually a strand
`
`Merck Ex. 1072, pg 1572
`
`'.L.M|LLER
`
`l'l
`
`
`
`Phe _
`
`-Th!‘
`
`Trp
`Lfi
`
`-Phe,
`
`Tip
`
`»ThI'
`
`an of
`
`;_Operon,
`f the
`the
`
`segment
`I site
`The
`site
`nonsense
`
`3—galactO—
`ntrol of
`
`Digest-
`methionine
`:tive
`
`L., Science
`iation for
`
`
`
`Merck Ex. 1072, pg 1572
`
`

`
`360
`
`W.L.MlLLER
`
`of DNA synthesized on a template of mRNA by reserve transcriptase(l8),
`and hence complementary to it. Fractions containing the relevant
`DNA sequences are then pooled and the DNA precipitated. This DNA
`is enriched about 100-fold for the desired sequences, but still con-
`tains a vast excess of other DNA.
`The enriched DNA can be purified
`further by either RPC—5 chromatography or preparative agarose gel
`electrophoresis (whichever was not used first) prior to cloning in
`plasmids as described earlier. Alternatively, it may be inserted
`into bacteriophage lambda and cloned when the recombinant phage in-
`fects suitable E. coli,
`taking advantage of the greatly increased
`number of recombinant molecules which may be screened with this
`technique (19). Recently,
`the native gene for ovalbumin (20),and
`a seven kilobase fragment of mouse DNA containing the gene for B
`globin, at least one insert, and extensive regions on both sides of
`the structural gene (21) have been cloned using this approach.
`Figure 4 shows the map of the ovalbumin gene as determined by
`Dugaiczyk et_al,, derived from detailed restriction enzyme analysis
`of several Eco Rl fragments purified and cloned as described above.
`The startling lesson from this map is that a structural gene over
`7,000 bases long is used to code for an mRNA of only 1,859 bases,
`thus the sequences actually coding for protein may constitute a
`small fraction of a structural gene.
`
`c. Reverse Transcription
`
`The third strategy for obtaining DNA sequences for cloning is
`to obtain the RNA for the protein of interest, make a strand of DNA
`complementary to it by reverse transcription,
`then clone this comple-
`mentary DNA (cDNA).
`The advantages of this approach are obvious:
`first, there are no intervening sequences,'as cDNA is copied from
`mRNA, which is free of intervening sequences.
`Second, a nucleic
`acid probe is not crucial to this approach. Third,
`the cDNA can be
`highly labelled with radioactive nucleotides thereby greatly facili-
`tating its handling and anlysis. These advantages may be offset
`somewhat by inefficient mRNA isolation and incomplete reverse trans-
`scription, so that the cloned cDNA may be only a fragment of the
`gene.
`Secondly, although it is possible to obtain mRNA and CDNA
`enriched for a particular sequence, it is almost impossible to ob-
`tain it in an absolutely pure form. As a result, cloning cDNA is
`usually a "shotgun experiment" where bacterial colonies containing
`the desired chimeric plasmids must be selected from colonies con-
`taining extraneous material. This raises a third problem, detecting
`the bacterial clones containing the sequences of interest. Despite
`these difficulties, CDNA cloning has produced chimeras harboring
`genes for growth hormone,
`insulin, ACTH, chorionic somatomammotropin,
`ovalbumin, dihydrofolate reductase,
`immunoglobulin chains, and
`others. Figure 5 illustrates this technique: Tissue which synthe-
`sizes a large quantity of the desired protein, and hence is rich in
`its mRNA,
`is chosen and polyadenylated mRNA is prepared from it by
`standard techniques. Proper choice of tissue or physiologic prepa-
`
`Merck Ex. 1072, pg 1573
`
`Merck Ex. 1072, pg 1573
`
`

`
`I
`
`’.L.M|LLER
`
`riptase(l8),
`elevant
`his DNA
`still con-
`
`purified
`ose gel
`oning in
`nserted
`
`phage in-
`creased
`this
`
`20),and
`for B
`‘ sides of
`ach.
`
`by
`analysis
`led above.
`zne over
`
`> bases,
`.ute a
`
`.oning is
`ind of DNA
`
`:his comple-
`»bViOuS:
`.ed from
`uucleic
`)NA can be
`
`;ly facili-
`offset
`arse trans-
`; of the
`Id CDNA
`.e to ob-
`CDNA is
`
`xntaining
`.es con-
`
`detecting
`Despite
`tboring
`xammotropin,
`and
`
`th synthe-
`.s rich in
`‘om it by
`[ic prepa-
`
`USEOFRECOMMNANTDNATECHNOLOGY
`
`IN
`
`Hinf Mbo 1]
`Taq I
`(E?g;258)
`(308)
`(41)
`[AUG
`
`PS! I
`(477)
`
`Mbo II Hac III
`(603) (818)
`
`[UAA]
`
` Eh7RI
`
`Eco RI
`
`£h9RI
`
`560 RI
`
`20
`L0
`,
`0
`‘——‘+----—-*-——-‘
`kflobaws
`
`I: Structural gene sequence
`
`_ Intervening DNA sequence
`
`Flanking DNA sequence
`
`The ovalbumin-coding
`Figure 4. Structure of the ovalbumin gene.
`sequences totaling 1859 bases, are divided into seven pieces by
`long segments of DNA (intervening sequences or "introns"), so
`that the whole gene is over 7,000 bases long.
`Intervening sequences
`appear to be transcribed into RNA, partially explaining
`the role of HnRNA precursors to mRNA, but are not translated into
`protein.
`(Reproduced by permission from Dugaiczyk 33. al,, Nature
`2135328, 1978)
`(20).
`
`Merck Ex. 1072, pg 1574
`
`Merck Ex. 1072, pg 1574
`
`

`
`
`
`W. L. MILLER
`
`GENERATION OF GENE FRAGMENT ANALOGUES
`BY REVERSE TRANSCRIPTION
`'
`
`GHSCN
`._______..
`
`RNA
`
`Ollgo(dT)-Collulou
`Cliromaiography
`
`AAAA ._.__..
`1 1' 1 1- —__._
`
`‘NOON
`
`“DVD?”
`
`Transcrlpfuso
`
`AAAA ----
`mRNA
`
`Reverse
`
`rrn __..._.._..
`(DNA
`
`Transcrlpfluo
`
`fin
`
`J5 ¢oNA
`
`.____.___.
`__._.__...
`Blunt-Ended
`dz CDNA
`
`Linkers
`
`T4 ligase
`
`Cl__"":_-_EJ
`
`Poll
`<-—-—-—-
`
`________.
`_._.._.._._.
`
`Hind III
`--—-—-> nil-_:.'_’]?
`i
`
`Clone in Plasmid
`
`RNA is
`Figure 5. Preparation of complementary DNA for cloning.
`prepared by guanidine thiocyanate extraction from tissue or cells
`containing a large amount of mRNA coding for a protein of interest.
`Polyadenylated mRNA is selected by affinity chromatography on oligo
`(dT)-cellulose and copied into DNA by avian myeloblastosis virus
`reverse transcriptase. After alkali digestion of the mRNA template,
`the single—stranded CDNA is reverse transcribed into a hairpin-shap-
`ed double stranded CDNA.
`The hairpin is opened and the single
`stranded ends are partially digested with S1 nuclease, and the re-
`maining single stranded ends are filled in by DNA polymerase I to
`yield blunt—ended CDNA. Synthetic decanucleotide "linkers" contain—
`ing the recognition sequence of the restriction enzyme Hind III are
`attached to the blunt—ended CDNA by DNA ligase from bacteriophage
`T4.
`The CDNA is then digested with Hind III to yield cohesive
`termini suitable for ligation into a plasmid as shown in Figure 1.
`
`
`
`
`
`
`
`Merck Ex. 1072, pg 1575
`
`Merck Ex. 1072, pg 1575
`
`

`
`W.L.MlLLER
`
`
`
`-- :m~;:rr-~»-;—‘.g.~—_—_:»..l
`
`
`Iw
`
`RNA is
`or cells
`interest.
`y on oligo
`s virus
`
`A template,
`irpin-shap-
`ingle
`d the re-
`ase I to
`5" contain-
`ud III are
`
`riophage
`asive
`
`?igure 1.
`
`USE OF RECOMBINANT DNA TECHNOLOGY
`
`163
`
`for ex-
`ration can greatly increase the yield of a specific mRNA:
`ample, ovalbumin mRNA constitutes 50% of the mRNA in the oviduct of
`an estrogen—treated chicken, but only 0.01% of the mRNA in the ovi-
`duct before estrogen treatment
`(22). Similarly,
`treatment of the
`cultured rat pituitary tumor cell line GC with thyroxine and dexa-
`methasone resulted in an increased quantity of growth hormone mRNA
`to about 10% of the total
`(23).
`A greater purification can be ob-
`tained by preparing polyribosomes and selecting those carrying the
`desired mRNA by immune precipitation of the attached nascent pro-
`tein, but this yields very little mRNA. Polyadenylated mRNA is then
`copied into CDNA by reverse transcriptase, an enzyme derived from
`certain RNA tumor viruses (24,25). After digesting the mRNA template
`with alkali, a second strand of CDNA is made with the same enzyme.
`This double stranded CDNA must be processed further.
`It can be di-
`gested with a restriction enzyme which will cut its length, but also
`give sticky ends which may be ligated into a plasmid cut with the
`same enzyme. Alternatively,
`the hairpin loop formed by the reverse
`transcriptase may be opened enzymatically with S1 nuclease and the
`unpaired ends filled in by DNA polymerase I to yield "blunt-ended
`cDNA." This full length cDNA can then be cloned using "linkers"
`synthetic double—stranded polynucleotides 8-10 base pairs long, which
`contain the recognition sequence for a restriction enzyme (26).
`The
`linkers are attached to the blunt-ended CDNA with T4 DNA ligase (27)
`and digested with the restriction enzyme to yield sticky ends. This
`cDNA can then be cloned in a plasmid opened with the same enzyme.
`The cDNA may also be cloned by "tailing" it with homopolymeric dC,
`added to its 3' ends with terminal transferase. Opened plasmid
`tailed with dG is mixed with the dC—tailed CDNA under annealing con-
`ditions so that stable dC-dG base pairs form,
`thus recircularizing
`the plasmid with the cDNA insert
`(28,29).
`
`III.HOST-VEHICLE SYSTEMS AVAILABLE
`
`Once we have prepared the DNA, a suitable cloning vehicle must
`be chosen. Three distinct possibilities exist: bacterial plasmids,
`Xphage, and animal viruses, of which only the first two are in gener-
`al use. Bacterial plasmids, also known as R-factors or episomes, are
`small circular pieces of extra-chromosomal DNA found in a wide varie-
`ty of gram-negative bacteria.
`They have long been known to carry
`genes for resistance to various antibiotics and to be transferable
`between bacteria by cell—to—cell contact. Plasmids were the first
`vehicles used for cloning (lO,30), and remain the most widely used
`technique.
`A number of plasmids have been specifically "built" for
`use as vehicles for recombinant DNA research (31,32).
`These are
`derived from the naturally occurring plasmid Col El and created by
`restriction endonuclease excision of unwanted DNA sequences and in-
`sertion of others.
`The plasmid vehicle with its passenger DNA may
`be assimilated into bacteria which have been rendered permeable to
`extracellular DNA by pretreatment with calcium chloride. Because
`
`Merck Ex. 1072, pg 1576
`
`Merck Ex. 1072, pg 1576
`
`

`
`
`
`
`
`~,,§g;~«a:st>r’5.S*rr".v"¢;.:!:.'+'«-<r‘-‘c-,:-—~......,.._.w..-._..:e.~a¢.u..:...-
`
`
`
`A‘-¥f<‘§LV'«:-
`
`4'
`
`2:
`1?,
`
`{I
`
`164
`
`W.L.MlLLER
`
`of present requirements for biological containment, recipient bac-
`teria have been devised by genetic manipulation so that they can
`survive only in uniquely enriched environments which cannot exist
`in nature. For example, E. coli
`1776, which requires a medium con-
`taining both thymidine (for nucleic acid synthesis) and diaminopime-
`lic acid (a constituent of the cell wall) has been derived from
`Such
`strain K12, which itself cannot survive in the human gut (33).
`host—vector systems have been used in hundreds of successful cloning
`experiments, but remain limited by the difficulty of screening large
`numbers of clones.
`To this end, a number of modifications have been
`built into the E. coli phage K (34-38) which permit screening of
`thousands of clones (19).
`In one case, expression of cloned globin
`DNA was obtained in a Xphage system when cloning in a plasmid system
`had failed to produce detectable protein (39). Other cloning vehicle
`systems such as somatic cells infected with simian virus 40 have
`been used (40,41) and may soon be practical for the in vitro pro-
`duction of many biologically useful polypeptides.
`—_
`
`IV.
`
`EXPRESSION OF CLONED DNA
`
`The earliest evidence that recombinant DNA could be expressed in
`transformed E. coli was the expression of genes native to the host E.
`coli itself, such as the lac operator (42,43). Transcribed RNA com-
`plementary to cloned DNA was detected by several investigators,
`(10, 44, 45) but much of this transcription was regarded as "gratui—
`tous" as the protein products of the cloned genes could not be de-
`tected. Using the novel system of "minicells" (46), small fragments
`of E. coli cells containing all normal cellular elements except the
`chromosomal DNA, Megaher et al_demonstrated production of some yeast
`and drosophila proteins from cloned DNA, but could not detect ex-
`pression of cloned mouse mitochrondrial DNA(47). As discussed in
`section IIa, expression of the cloned synthetic somatostatin gene
`has been achieved, but as this entails synthetic DNA, it does not
`answer the question of whether procaryotic organisms can be made to
`transcribe and translate eucaryotic genes to yield useful proteins.
`Production of yeast enzymes has been achieved in both Aphage (48)
`and plasmid (49) systems, but yeasts may not be so dissimilar from
`procaryots as are vertebrates. Recently however, rat proinsulin (28)
`mouse dihydrofolate reductase (29), ovalbumin (50), and rat growth
`hormone (51), have been synthesized in bacteria harboring recombinant
`plasmids. As an example of this technology, we shall now review the
`rat growth hormone work in more detail. Using the strategy of re-
`verse transcription (see section IIC abOVe)
`the entire C0din9
`sequence for pre—RGH,
`the 3' untranslated region of the mRNA, and
`most of the 5' untranslated region was cloned in the plasmid pBR322
`to form the new plasmid pRGHl (l).
`The 800 base—pair rat growth
`hormone gene was then prepared in quantity in pRGHl, excised with
`Hind III, and used to prepare the "expression plasmid" as shown in
`
`Merck Ex. 1072, pg 1577
`
`Merck Ex. 1072, pg 1577
`
`

`
`W.L.M|LLER
`
`ient bac-
`hey can
`ot exist
`medium con-
`
`iaminopime—
`d from
`Such
`(33).
`:ful cloning
`eening large
`s have been
`
`ning of
`ned globin
`smid system
`hing vehicle
`40 have
`
`§£g_pro—
`
`expressed in
`the host E.
`1 RNA com-
`ators,
`as "gratui-
`>t be de-
`
`L fragments
`axcept the
`some yeast
`:ect ex-
`lssed in
`
`;in gene
`loes not
`>e made to
`
`proteins.
`.ge
`(48)
`lar from
`nsulin (28)
`t growth
`recombinant
`eview the
`y of re-
`ling
`NA, and
`id pBR322
`growth
`ed with
`shown in
`
`USEOFRECOMWNANTDNATECHNOLOGY
`
`165
`
`The RGH gene prepared from pRGHl was recloned in the Hind
`Figure 6.
`III site of pMB9. As the sole Hind III site of pMB9 lies a few
`bases into the gene for tetracycline resistance, cloning a segment
`of DNA in this site results in the plasmid conferring resistance
`only to low concentrations of tetracycline,
`thus providing a selec-
`tion technique for the successful recombinants (52).
`A strain of
`recombinants having the poly dA~dT end of the gene (i.e.
`the car-
`boxy—terminus of RGH) oriented toward the Eco Rl site was chosen
`for further treat