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`UNITED STATES DEPARTMENT OF COMMERCE
`
`United States Patent and Trademark Office
`
` June: 27, .23 E6‘
`
`THIS IS TO CERTIFY THAT ANNEXED IS A TRUE COPY FROM THE
`
`RECORDS’ OF THIS OFFICE. OF THE FILE WRAPPER AND CONTENTS
`
`
`
`OF;
`
`
`
`
` APPLICATION NUMBER: 06/265,} 76
`
`
`FILING DATE: May 20, 1981
`
`
`
`PATENT NUMBER: 4,495,230
`
`ISSUE DATE: January 22, 1985
`
`
`
` By Authority of the
`Under Secretary of Commerce for §nie§Iectua1Praperty
`Director of the United Stat
`*
`
`
`
`
`|PR2015-01624
`
`
`
`Sanofi v. Genentech
`
`Exhibit 2141
`
`
`
`IPR2015-01624
`Sanofi v. Genentech
`Exhibit 2141
`
`-1-
`
`

`
`(Rev. 8/73‘)
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`RETENTiON LABEL
`
`3 2
`
`”“?
`
`
`
`-2-
`
`-2-
`
`

`
`TOWNSEND and TOWNS
`Sgeuart Street Tower
`'
`Market Flaza
`_.. ancisco. CA 94105
`
`.5
`
`‘
`
`3~960O
`
`
`255275
`
`‘Case Docket Now.
`
`5490-35
`
`Date
`
`May 19, 1981
`
`THE COMMISSIONER OF PATENTS & TRADEMARKS
`Washington. D. C. 2023}
`
`Sir:
`
`Transmitted herewith for filing is the patent application of
`
`JHVBUTOFI Herrman Bujard, Annie C..Y. Chang, Stanley N. Cohen
`
`Fm?
`
`Cloned High Signal strength Promoters
`
`Enciosed are:
`
`._.,.l_____sheetsofdrawing.
`
`The B05955 of Trust"-eeS~0f the Leland
`An assignmemof the invention to
`_Sta.nford Junior University, a California _co2_:_goration
`
`E] A certified copy of a pplication_
`
`[1 Associate power of attorney.
`
`
`
`I
`CLAIMS AVS FELED
`(3)
`NUMBER EXTRA,
`
`(4)
`RATE
`
`15)
`.BAS!C FEE
`$65.00
`
`
`
`
`
`(2)
`NUMBER FELED
`
`m
`FOR’
`TOTAL
`
`4
`
`E NDENT
`
`
`
`'TOTAL_'FlLiNG
`FEE
`
`.
`127 . 00
`
`
`
`Please_ charge my Deposit Account N0( W.o.gu1 the amount of
`S 127 .. 00 A duplicate copy of this-sheet is enclosed.
`
`ix 1 The Commissioner is hereby authorized to charge any additional fees which may
`be required. or credit any overpayment to Account N0._.Z,Q,:;3-W53 30
`A duplicate copy of this sheet is enclosed‘
`
`D A check in the amount of___.__.____.__..,____to cover the fifing fee is enclosed.
`
`
`
`TEzT 25 REV.
`
`-3-
`
`

`
`
`
` 28527§?/
`/gay/e
`Ԥ;2/7/27
`
`CLONED HIGH SIGNAL STRENGTH PRDMOTERS
`
`5490~35/BOTSOZD
`
`BACKGROUND OF THE INVENTION
`
` 5
`
`Field of the Invention
`
`'
`
`Having established the feasibility of producing a
`
`wide variety of naturally occurring and synthetic polypep~
`
`tides by means of hybrid.DNA technology,
`
`there are continuing
`
`»vand extensive efforts to provide for more efficient and
`
`economic methods for producing the polypeptides.
`In develop»
`ing a process for the commercial production of polypeptides,
`
`many factors will be involved in optimizing the economic and
`
`efficient production of the polypeptides.
`
`Included among
`
`these factors are regulatory signals, which are DNA sequences
`involved with the regulation of replication,
`transcription
`and translation.
`
`One area of interest is at the level of transcrip~
`
`tion.
`
`Transcription involves the enzyme RNA polymerase.
`It has
`
`RNA.po1ymerase binds to a site called a promoter.
`
`The
`
`been observed that promoters vary in their activity, as
`
`evidenced by the number of initiations of RNA per unit time
`
`or the strength of binding of the enzyme to the promoter
`
`site.
`
`The promoter may have one or more sequences that bind,
`
`10
`
`15
`
`20
`
`25
`
`The more active promo»
`which may or may not be contiguous.
`ters are referred to as strong promoters.
`
`It was found that when introducing a strong
`
`promoter into a vector and employing the resu1ting’p1asmid‘
`
`for transformation, one conld not select transformants based
`mu gage i 134
`3o,&GEH
`Séflrb Offim/B1 Héhmfé
`'
`”
`' "”~
`'
`on expression of markers which allowed for selection. There~
`
`30
`
`fore, cloning of the strong promoters was not feasible.
`
`It
`
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`
`
`
`
`
`2
`
`is therefore desirable that methods be provided which would
`
`allow for the screening of strong promoters and terminators
`
`and their subsequent cloning to be used in conjunction with
`
`the replication, transcription and translation of the genes
`
`5
`
`for production of DNA, RNA,
`
`and polypeptides.
`
`g.‘ :7‘
`
`H.
`
`Descrigtion of the Prior Art
`A
`Promoters from bacterial and viral sources have
`
`and their signal strength in vitro
`been cloned in E. coli
`has been studied using expression from distal promoterless
`
`sequences encoding figalactos‘dase or other prote%%i;g?
`(Casadaban and Cohen (l980)é;lr%E$§3Biol+ l§§,k,
`'
`;
`
`West
`
`Attempts to clone
`lfiéifififi.
`and Rodriguez (1980) Gene g,
`small DNA fragments carryingfithe strong promoters of
`bacteriophage T5 have been ugggfigessful.
`(V. Gabain and
`Bujard (1979) PNAS USA 2g,”
`, Fragments of T5 DNA
`having both a strong promoter and a strong termination signal
`
`have been cloned.
`
`(Breunig (1979) Dissertation (Universitat
`
`Heidelberg, Heidelberg, Germanyj) Analysis has shown that
`
`transcriptional regions of several E. coli plasmids are
`
`organized in units where initiation and termination sig§als£
`(Stuber and Bujard (1981) pms USA 7f’&’ ‘/7’ )
`IL
`
`are balanced.
`
`and P26 promoters of the T5 bacteriophage are reported as
`among the most efficient RNA polymerase binding sequences.
`
`(Stuber et al
`
`(1978) Mol. gen. Genet.
`
`lgg l4l—l49; Niemann
`
`(l98l) Diplomarbeit (Universitat Heidelberg, Heidelberg,
`
`Germany)).
`
`SUMMARY OF THE INVENTION
`
`Methods for cloning, sequencing and using strong
`
`promoters and terminators are provided, as well as Composi-
`
`tions resulting from the methods.
`
`By cleaving TS phage and
`
`selecting fragments specifically binding to RNA polymerase,
`
`fragments containing promoters are isolated. A vector is
`
`constructed having a strong promoter,
`
`followed by a DNA
`
`sequence of interest, optionally followed by one or more
`
`translational stop codons in one or more reading frames,
`
`followed by a balanced terminator,
`
`followed byva marker
`
`allowing for selection of transformants. Upon introducing
`
`the resulting plasmid into a microorganism host, efficient
`
`25
`
`30
`
`35
`
`-5-
`
`

`
`
`
`
`
`3,
`
`transcription of the gene is obtained with substantially
`
`lesser expression of the marker as Compared to the gene-
`
`The
`
`level of expression of the marker permits selection of
`The
`
`transformants having the above described construct.
`
`construct or regulatory portions thereof are used for effi-
`
`cient transcription of
`
`RNA or gene expression.
`
`BRIEF DESCRIPTION OF THE DRAWING
`l is a flow chart of the preparation of a
`
`Fig.
`
`plasmid for cloning strong promoters and terminators.
`
`or?
`
`10
`
`DESCRIPTION OF THE-SPECIFIC EMBODIMENTS
`
`Methods and compositions are provided for preparing
`
`and cloning strong promoter and terminator regulatory signals
`
`‘fits:
`
`15
`
`and utilization of the strong regulatory sequences in the
`
`transcription and expression of genes of interest.
`
`Particularly, T5 phage promoters are isolated,
`
`cloned in conjunction with a strong terminator, and approw
`
`gpriate Vectors developed for insertion of DNA sequences of
`
`interest, usually structual genes,
`
`to provide for high and
`
`efficient transcription and/or expression of the sequence.
`
`20
`
`.The compositions of this invention are character-
`
`ized as having in the downstream direction of transcription
`
`the following units:
`
`a strong TS phage promoter; optionally
`
`a structural gene which may be a marker; a balancing termina—
`
`25
`
`tor; and optionally a marker allowing for §election.of transw
`formants containing the construct, which marker has a rela-
`
`tively low level of expression in comparison with the amount
`
`of RNA polymerase initiation at the promoter.
`
`In the absence
`
`of a promoter in the construct,
`
`the construct can be used for
`
`the cloning and characterization of promoters of different
`
`30
`
`strengths‘
`
`In referring to strong promoters, it is intended
`
`that the binding affinity of RNA polymerase is stronger than
`
`the commonly employed promoters such as lag and Egg and at
`
`least comparable to and normally greater than the combination
`
`35
`
`%mW
`bV}HH
`
`of lac and try promoters.
`
`For the most part,
`
`the strongest
`
`promoters among prokaryotes are the T5 phage promoters and
`
`these will be employed as exemplary of V
`
`-
`
`occurring or
`
`synthetic strong promoters.
`
`It is to be understood,
`
`that
`
`-6-
`
`

`
`
`
`
`
`4
`
`other prokaryotic and eukaryotic promoters, either naturally
`
`occurring or synthetic, could find application in the subject
`invention.
`
`The compositions of this invention will include
`
`linear segments for insertion of DNA having the strong
`
`regulatory signal sequences (i.e.,
`
`the promoter and termi~
`
`nator) adjacent opposite ends of the linear segment and
`
`plasmids formed by introducing a DNA sequence from a source
`other than the source of the promoter as a bridge between the
`
`The termini may be blunt
`strong regulatory signal.sequences.
`or staggered ended, having the same or different termini to
`
`allow for directed positioning or inserted sequences.
`
`The Compositions which are employed as already
`
`indicated have a promoter, a balanced terminator, and desir-
`
`ably a marker which are in the direction of transcription
`
`when the signal sequences are joined.
`
`The terminator is
`
`balanced, so that a small but useful amount of expression of
`the marker can occur. Normally,
`in most situations, a gene
`
`10
`
`15
`
`20
`
`will bridge the promoter and terminator to provide circular
`DNA.
`
`The gene may be a non-structural gene or a structural
`
`gene providing RNA e.g. ribosomal or messenger, or providing
`
`a poly(amino acid).
`
`Desirably,
`
`the gene is followed by one or a plural-
`
`ity of translational stop codons e.g- oop or nonsense codons,
`
`or preferably a plurality, usually up to about six, more
`
`usually from about two to five, where there is at least one
`
`stop codon in each reading frame-
`
`The stop codons aid in the
`
`efficiency of termination, both at the level of transcription
`
`and expression.
`
`Next in the direction of transcription is the __
`
`terminator sequence,
`
`with the promoter in the s nse
`hat the marker is transcribed
`fl?m£»~'
`wthe times that a gene up-
`not more than aboutf;;¢
`stream from the terminator is transcribed,
`
`The terminator sequence is balanced
`
`
`
`
`where both genes
`
`are under the control of the same promoter. Usually, it will
`be desirable that a sufficient amount of the marker is tranw
`
`scribed to allow for selection of transformants.
`
`In View of
`
`the fact that the marker is transcribed from a strong pro—
`
`25
`
`30
`
`35
`
`-7-
`
`

`
`
`
`
`
`5
`
`moter, it will usually be sufficient that the marker is
`
`transcribed in relation to the transcription of the gene
`
`§.1¢p(H<+p
`
`intermediate the promoter and terminator in only about 1 to
`
`20, usually only 5 to l5 number % of the gene.
`
`The strength
`
`of the promoter is reflected in the level of expression of
`
`the marker which is transcribed from the same promoter as the
`
`gene whose expression is desired.
`
`In addition to the DNA sequences indicated above,
`
`there will normally be other regulatory signals necessary for
`
`expression involved with the DNA sequence, such as transla-
`
`tional start and stop.sites.
`
`In addition to the foregoing
`
`regulatory signals, other regulatory signals may be included,
`
`such as additional promoters, operators,
`
`initiators,
`
`catabolite activator protein binding sites, etc. Further~
`
`more,
`
`the promoter and terminator may be separated by more
`
`than one gene, that is, a plurality of genes,
`
`including
`
`multimers and operons-
`
`The above DNA sequence construct will have a repli~
`
`cating system or be cleaved and be inserted into a vector to
`
`provide a plasmid.
`
`The vector is distinguished by having one
`
`or more DNA sequences which serve to insure stable replica-
`
`tion of the plasmid and may also provide opportunities for
`
`high copy numbers of the plasmid in the microoganism host.
`
`The vectors may be derived from chromosomal or extrachromow
`
`somal sources-
`
`The sources include plasmids, viruses
`
`(phage),
`
`chromosomes,
`
`or the like.
`
`In addition,
`
`the Vector
`
`or the essential portions thereof may be prepared
`
`syntheticallyr
`
`10
`
`15
`
`20
`
`25
`
`30
`
`»The plasmids may then be used for transformation of
`an appropriate microorganism host. Methods of introducing
`
`...rrg
`
`35
`
`DNA into an appropriate host are well known.
`
`Illustrative of
`
`such methods, but not exhaustive of such methods, are trans»
`
`formation e.g. calcium shock,
`
`transfection, and conjugation,
`
`Descriptions of these methods may be found in Genetic
`
`Engineering, ed. Setlaw and Hollaender, Vol. 1, Plenum Press,
`
`New York and London, 1979; Molecular Cloning of Recombinant
`
`DNA, ed. Scott and Werner, Vol.
`
`l3, Academic Press,
`
`Inc. New
`
`York, 1973,
`
`and references cited therein.
`
`-8-
`
`

`
`
`
`
`
`6
`
`In order to allow for flexibility in preparing the
`construct and self—replicating sequence or plasmid containing
`
`the construct restriction sites should be present to allow
`
`for unique insertions and isolation of the various elements.
`
`The restriction sites may be naturally present,
`
`introduced by
`
`linkers, result by partial sequential nucleotide removal from
`
`a chain using an exonuclease, or the like. Desirably,
`
`the
`
`restriction sites will provide for different ends to permit
`
`only the proper orientation of the inserted fragment.
`
`A wifie variety of structural genes are of interest
`
`for production of proteins,
`
`including but not limited to
`
`proteins of physiological interest, proteins as chemicals,
`
`and enzymes which may be of direct interest or of interest in
`
`transforming another product, which may be proteinaoeous or
`
`non—proteinaceous.
`
`The proteins may be prepared as a single
`
`unit or as individual subunits and then joined together in
`
`appropriate ways.
`
`oFurthermore, as appropriate,
`
`the protein
`
`products may be modified by glycosylation, acylation with
`
`aliphatic acids, e.g. lipid acids, phosphorolation, sulfona~
`
`tion or the like.
`
`The different classes of proteins which
`
`may be prepared include protamines, histones, albumins
`
`globulins, scleroproteins, phosphoproteins, mucoproteins,
`
`ohromoproteins,
`
`lipoproteins, nucleoproteins, and the remain~
`
`ing proteins which are unclassified.
`
`The following is a representative list of proteins
`of interest.
`
`Insulin; growth hormone;
`
`interferon e.g.
`
`leukocyte,
`
`lumphoblastoid, T~immune and fibroblast; Bwlipotropin;
`
`fiwendorphin; dynorphin; histocompatability proteins;
`
`immune»
`
`IgG and IgM and fragments
`IgE,
`IgD,
`globulins e.g- IgA,
`thereof; hemoglobin, somatomedins;
`lymphokines; growth
`
`factors e.g. epidermal, fibroblast, platelet—deriVed,
`
`multiplication stimulating and nerve; hematoporeticestimulaw
`
`ting factors e.g. erythropoietin, colonywstimulating,
`
`erythroid potentating activity or burst—promoting activity
`
`and lymphopoietins; albumin and prealbumin;
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`£2
`
`.él
`
`-9-
`
`

`
`
`
`
`
`Prealbumin
`
`Albumin
`
`al—Lipoprotein
`al~Acid glycoprotein
`al—Antitrypsin
`ml-Glycoprotein
`Transcortin
`
`é.6S—Postalbumin
`
`Tryptophan—poor
`
`al-glycoprotein
`aV—x~G1ycoprotein
`1
`Thyroxin—binding globulin
`Inter—a-trypsin—inhibitor
`
`Gc-globulin:
`
`(GC l~l),
`
`(Gc 2~l),
`
`(Gc 2-2),
`
`Haptogldbin:
`
`{HP 1"1},
`
`(HP 2*1),
`
`(HP 2“2).
`
`Ceruloplasmin
`Cholinesterase
`
`a2~Lipoprotein(s)
`a2—Macr0globu1in
`a2~HS~Glycoprotein
`Zn~a2~glycoprotein
`a2—Neuramin0—glycoprotein
`Epythropoietin
`
`Bnlipoprotein
`Transferrin
`
`Hemopexin
`
`Fibrinogen
`
`Plasminogen
`
`B2~glycoprotein I
`B2~glycoprotein II
`Immunoglobulin G
`
`(IgG) or ycmglobulin
`
`-10-
`
`10
`
`15
`
`20
`
`30
`
`35
`
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`
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`formula:
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`Immunoglobulin A (IgA)
`or yA~globulin
`formula:
`{a2K2)n or
`lmmunoglobulin M
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`Mol.
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`(IgM) or yM—globulin
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`Mol.
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`5
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`formula:5
`(PZAZJ
`(HZKZ) Or
`Immunoglobulin D (IgD)a
`or yD~Globulin (yD)
`formula:
`
`Mol.
`
`(62K2) or (GZAZ)
`Immunoglobulin E (IgE)A
`or yE—Globulin (yE)
`formula:
`A
`(a2K2) or (82. 2)
`Free light chains
`Complement factors:
`C‘l
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`C rig
`C’1£
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`C’3
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`.
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`Importain protein hormones include:
`
`Peptide and Protein Hormones
`Parathyroid hormone
`
`(parathormone)
`
`-11-
`
`-11-
`
`

`
`
`
`
`
`Thyrocalcitonin
`
`Insulin
`
`Glucagon
`
`Relaxin
`
`Erythropoietin
`
`Melanotropin H
`
`(Melanocyte«stimulating hormone;
`
`intermedin)
`
`Somatotropin M
`(growth fiormone)
`
`Corticotropin fl
`(adrenocorticotropic hormone)
`
`Thyrotropin
`
`Follicle-stimulating hormone
`
`Leuteinizing hormoneifi
`(interstitial cell~simulating hormone)
`
`Luteomammotropic hormoneflg
`.{Luteotropin, prolactin)
`
`y
`Gonadotropin
`(chorionio gonadotropin)-
`Tissue Hormones
`
`Secretin
`
`Gastrin
`
`Angiotensin I and II
`
`Bradykinin
`
`5
`
`15
`
`F._,.;
`
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`&;b
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`?$
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`L
`
`93%?
`
`WfiamA
`
`93%
`
`at
`
`20
`
`J3
`
`M
`
`Ct
`
`FEE
`
`25
`
`Human placental lactogen
`
`Peptide Hormones from the Neurohypopnysis
`
`Oxytocin
`
`Vasopressin
`
`Pats
`
`if a
`
`‘
`
`%
`
`30
`
`Releasing factors (RF)\
`CRF, LRF, TRF, Sofiatotropin~RF, GRF, FSH«
`
`RF, PIF, MIF.
`
`A
`
`In aédition to various non~enzymatic proteins of
`
`physiological interest, enzymes can also be produced as an
`
`end product or for intracellular transformation of a sub~
`
`35
`
`strate present in the host or substrate introduced extra—
`
`cellularly, or for enzymatic transformation gg vitro.
`
`-12-
`
`-12-
`
`

`
`
`
`
`
`10
`
`In accordance with the I.U.B. classification,
`
`the
`
`enzymes fall into varying categories such as l. oxidoreduc~
`
`I.
`
`tases; 2.
`
`transferases; 3. hydrolases; 4.
`
`lyases; 5.
`
`iso-
`
`merases; 6.
`
`ligases.
`
`Enzymes of particular interest will be
`
`hydrolases and oxidoreductases for use in commercial prom
`
`cessing,
`
`for example, hydrolases for hydrolysing polysac-
`
`charides,
`
`lipids and polypeptides; oxidoreductases for oxida—
`
`tion of alcohols and aldehydes, epoxidation, and the like‘
`
`The microorganism host may be bacteria, such as
`
`Escherichia, Bacillus, Aerobacter, Klebsiella, Proteus,
`
`Pseudomonas, Streptococcus, Staphylococcus, Clostridium,
`
`Mycobacterium, Streptomyces and Actinomyces; Fungi e.g.
`
`Gymnomycota, Dimastygomycota, Eumycota, Zygomycetes, Ascomyw
`
`cetes and Basidomycetes, such as, Candida, Aspergillus,
`
`Rhizobus, Microsporum, and Fonsecaea; Protozoa e.g.
`
`Mastigophora, Sarcodina, Sporozoa and Celiopnora, such as,
`
`Trypanosoma, Codosiga, Protospongra and Entameba, and Alga
`
`e.g. Dinoflagellates, Euglenoids, and Diatoms.
`
`10
`
`15
`
`20
`
`Higher cells, e.g., mammalian, may also be
`employed as hosts, where viral, e.g., bovine papilloma virus
`
`or other DNA sequence is available which has plasmid~like
`
`activity,
`
`Depending upon the nature of the host, a wide
`
`variety of vectors may'be employed“
`
`The vector will have an
`
`25
`
`intact replicon and be capable of replication in the host.
`
`In addition, replicons can be developed which may have one or‘
`
`more other regulatory signals. Regulatory signals can
`
`include temperature sensitive replicons, runawaywreplication
`
`30
`
`temperature sensitive operators, and the like.
`sequences,
`Various additional DNA sequences may be present providing for
`
`restriction sites, markers,
`
`termination sequences, or the
`
`the vector should be of a substantially
`like. Desirably,
`different size from the construct to allow for excision of
`
`the construct and ease of separation of the construct from
`
`35
`
`the vector by molecular weight separation techniques e.g.
`
`electrophoresis and density gradient centrifugation.
`
`The subject invention also provides for a technique
`
`for screening the strength of promoters and terminators,
`
`thus
`
`-13-
`
`-13-
`
`

`
`
`
`
`
`11
`
`allowing for the determination of the use of a promoter or
`
`terminator in a particular application, where it may be V
`
`desirable to have promoters or terminators or combinations
`
`thereof of varying strength.
`
`In.screening promoters and/or
`
`terminators one employs a promoter or terminator of known
`
`activity.
`
`A DNA construct is made having the sequence
`
`described previously, where appropriate restriction sites are
`
`provided for introducing the various elements.
`
`The construct
`
`provides in the direction of transcription the promoter, a
`
`first gene marker, optionally termination codons, such as
`
`nonsense codons and oop terminator,
`
`the terminator, and a
`
`second gene marker. This DNA construct is inserted into an
`
`appropriate vector; Where the promoter and terminator are
`
`properly balanced,
`
`there will be a substantial differentia»
`
`tion between expression of the first gene marker and expresm
`
`sion of the second gene marker.
`
`The ratio of expression
`
`between the first and second marker will provide for a comm
`
`parative evaluation of the activity of the promoter or
`
`terminator, depending upon which is of known value.
`
`Thus,
`
`one can degrade a DNA sequence such as a chromosome, or an
`
`extrachromosomal element, such as a plasmid or double minute,
`
`isolate the promoters by selective binding with RNA poly~
`
`merase and insert the DNA fragments which bind to the RNA
`
`polymerase into the previously described construction.
`
`By
`
`determining the relative proportion of expression of the
`
`first and second gene markers, one can determine the strength
`
`of promoters in relation to a fixed terminator. Similarly,
`
`one can isolate DNA sequences having terminator sequences,
`
`»insert the sequences into the above described Construct at
`
`the appropriate site and then measure the relative expression
`
`of the two gene markers.
`
`Various markers can be chosen for evaluating the
`
`relative activities of promoters and terminators. Conveni-
`
`ently, markers which allow for selection such as resistance
`
`to antibiotics,
`
`toxins or heavy metals can be used.
`
`By
`
`varying the concentration of the selective agents in the
`
`nutrient medium, one can determine the relative proportions
`
`of the enzyme expressed by the genes in relation to the
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`-14-
`
`-14-
`
`

`
`
`
`
`
`12
`
`growth of the host.
`
`Alternatively, one can use growth fac—
`
`tors e.g. having a gene which complements a mutant gene in an
`
`auxotrophic host, where the gene expresses a product necesw
`
`sary for a biosynthetic pathway.
`
`A third marker provides
`Other
`
`virus incompatibility, preventing plaque formation.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`markers which allow for comparison will come readily to mind.»
`
`The terminators may be evaluated in the presence
`
`and absence of rho, so that one can determine the dependency
`
`of the terminator on rho, as well as the effectiveness of the
`
`terminator in relation to the concentration of rho.
`
`The methods for preparing the subject compositions
`
`will be conventional.
`
`’The various DNA fragments and
`
`sequences can be obtained from a variety of sources by
`
`restriction mapping and endonuclease cleavage to provide
`
`fragments having the desired intact sequence or gene.
`
`The
`
`fragments can be further processed employing endo~ or
`
`exonucleases to remove nucleotides unrelated to desired
`
`regulatory Sequences or structural genes.
`
`By appropriate
`
`choice of restriction enzymes, cohesive or blunt ended frag-
`
`ments can be generated.
`
`Furthermore, chains can be extended
`
`with single nucleotides or oligonucleotides,
`
`linkers can be
`
`added, or otherwise processing to provide for termini having
`
`desired properties.
`
`Desirably, a vector is employed having appropriate
`
`restriction sites, a competent replication system for the
`
`intended host, and optionally one or more markers which allow
`for selection.
`For hybrid DNA technology it would be useful
`
`to have a plasmid having a unique restriction site between a
`
`T5 promoter and a terminator, desirably having at least one
`
`stop codon on the upstream side of the terminator.
`
`In this
`
`manner, one or more structural genes may be introduced
`
`between the promoter and terminator.
`
`As appropriate, downstream from the promoter, but
`
`remaining proximal to the promoter, may be an operator,
`
`activator, ribosomal start signal sequence, or the like,
`
`to
`
`allow for controlled expression of the inserted gene(s).
`
`The strategy described above provides a vehicle
`
`which can be used with one or more hosts for gene expression,
`
`“T1
`
`-15-
`
`-15-
`
`

`
`
`
`
`
`13
`
`where the gene after processing in a predetermined way can be
`
`directly inserted into the vehicle to provide a competent
`
`plasmid for expression of the desired gene(s).
`
`Alternatively,
`
`the gene{s) of interest may be
`
`ligated to the appropriate regulatory signal sequences before
`
`insertion into the vehicle.
`
`In this instance, only the
`
`promoter and terminator regulatory signals need be present.
`
`To provide for enhanced flexibility,
`
`the region
`
`between the promoter and terminator may be designed so as to
`
`provide for a_plurality of restriction cleavage sites, allow-
`
`ing for the introduction and removal of,DNA fragments without
`
`interruption of the remainder of the vehicle.
`
`Thus, by
`
`having a plurality of unique restriction sites or restriction
`
`sites limited to the region between the promoter and terminam
`
`tor in the.downstream direction of transcription, regulatory
`
`signals and genes may be readily inserted and removed.
`
`Another strategy is to prepare a Construct having
`
`all of the desired DNA sequences for transcription and
`
`expression in appropriate sequence, with the construct having
`
`predetermined termini and inserting the construct into an
`
`appropriate Vector which has been linearized to provide
`
`complementary termini.
`
`10
`
`15
`
`20
`
`In developing the construct, a vector will normally
`
`be used in order to clone the various sequences.
`
`The conu
`
`25
`
`struct will allow for the insertion of the different
`
`sequences in the correct direction and desirably only in the
`
`proper orientation. Therefore, it will usually be desirable
`
`to have the sequence and insertion site be asymmetric in
`
`30
`
`having different termini with the termini of the sequence and
`insertion site being complementary.
`
`The particular restriction enzymes will vary widely
`
`with the various sequences,
`
`there being a large number of
`
`restriction enzymes of known base or sequence specificities
`
`commercially available.
`
`35
`
`The following examples are offered by way of illus»
`
`tration and not by way of limitation.
`
`......,_.
`
`-15-
`
`-16-
`
`

`
`14
`
`EXPERIMENTAL
`
`Materials and Methods
`
`Restriction endonucleases Eigdlll, gigcll, gall,
`
`gagfil and T4 Ligase were from New England Biolabs (Beverly,
`
`Mass., USA), Sau3A was from BRL (Neu—Isenburg, Germany) and
`§ggRI and ggall
`from (Boehrigfier Mannheim, Federal Republic
`of Germany).
`DNA polymerase I, phage gg DNA (replicative
`
`form), plasmid ADl6/30 containing a 28 hp gindlll/gagfil
`
`adapter sequence and,
`
`lac represser were supplied by private
`
`sources.
`
`The isolation of bacteriophage T5 DNA, plasmid DNA
`
`E; coli RNA polymerase and'§; coli termination factor rho
`have been described previously,
`(V. Gabian and Bujard, Mol.
`
`gen. Genet.
`
`(1977) l§Z, 30l~3ll; Clewel and Helinski (l969)
`
`PNAS USA gg,
`
`ll57~ll66; and Knopf and Bujard (1975) Eur. J.
`
`Biochem. §§, 37l~385).
`A 780bp DNA fragment carrying the E. coli lac
`regulatory region (promoter/operator: P/O), an fi~terminal
`
`portion of the B~galactosidase structural gene sufficient for
`
`intracistronic complementation of the M15 deletion, as well
`
`as a portion of the i—gene(i) was isolated from a gincll
`
`digest of a pACYC 214 plasmid (a plasmid rel te, to pACYC 184
`by insertion at a gagfll site of a §agHI&£;tsustion cleavage
`fragment from F’»lac carrying the lac gene) by repressor
`
`binding and subsequent adsorption to nitrooellose. This
`
`fragment was then employed in the construction of an
`
`exemplary plasmid for analyzing strong promoters and strong
`
`terminators as depicted in the Figure.
`htilizing the ggall cleavage site within the B~gal
`
`structural genes the fragment was reduced in size and pro~
`
`vided with gagfil
`
`and gall cleavage sites by Various subw
`
`cloning.
`
`The resulting fragment (left most part of Figure)
`
`contains the intact control region of the lag operon and an
`
`N~terminal portion of the Bwgal structural gene coding for 66
`
`amino acids (u).
`
`Introduction of this fragment by blunt end
`
`ligation into the gindlll site of pACYCl84 (Chang and Cohen
`(1978) J. Bacteriol.
`;_3_4_L, 1141-4155) .3/ielded pBUlO, a vector
`
`suitable for terminator cloning.
`
`The major terminator of the
`
`coliphage gg genome was isolated as a 338bp
`
`Sau3A fragment
`
`20
`
`30
`
`-17-
`
`
`
`u«»-»-.»:...«,...,.A....AK
`
`‘kb»
`
`-17-
`
`

`
`
`
`
`
`3%
`
`{L (Beck et al.
`
`M5»;/5223
`15
`(1978) Nucl. Acids Res. §, aeesssoa),
`
`ligated
`
`with a gamfil/gindlll adaptor sequence and integrated into
`
`pBUlO to yield pLBUl.
`
`was».an!:N,‘
`
`l0
`
`Cleavage of the lag sequence in pBUlO by gpall
`
`destroys the lag promoter and liberates upon cleavage with
`
`gindlll a fragment containing the lac operator and a region
`
`coding for a functional u«fragment.
`
`Integrating this DNA
`
`sequence into pBR322 leads to pBUl2a- Finally, replacement
`
`of the gindlll/gall-portion of pBUl2a by a partial digest of
`
`the gindlll/gall fragment of pLBUl containing the fd termi-
`
`nator resulted in pLBU3, a vector suitable for integration of
`
`efficient promoters at the EEQRI site.
`
`The regions encoding
`
`chloramphenicol
`
`(Cm), ampicillin (Ap) and tetracycline (Tc)
`
`resistance are indicated as cat, bla and tet respectively.
`
`l5
`
`\"\
`
`The plasmid, pBUlO, had the following properties: Mwg
`(i) it contains the a fragment of B~galactosidase (u—proteim%
`complemented the M15 deletion of the lag operon; (ii) the Tc.
`
`fa}
`
`
`
`.12,:"~f»
`
`ti;
`
`resistance it specified was under the control of the lag
`promoter, as shown in M15 lq strains; (iii) the gindlll site
`
`20
`
`25
`
`30
`
`between the lag gene fragment and the tet gene was restored;
`
`the stop codon immediately following the gindlll site limited
`
`the length of the lagz gene product to 68 amino acids.
`
`Insertion of the fig terminator upstream to the Egg
`gene resulted in a 90% reduction in the level of To resis«
`
`tance, but no detectable change in the levels of
`
`B—galactosidase activity in Ml5 deletion strains.
`
`The
`
`results obtained as to the properties imparted to various E;
`
`coli strains by various plasmids is set forth in the folw
`
`lowing table:
`
`Seven independent plasmid isolates from clones
`
`contained the expected 352bp §indIII—generated fragment
`
`containing the gg terminator. Electrophoretic analysis of
`
`all seven §amHI~cleaVed isolates and DNA sequence analysis of
`
`one of these showed that in all instances the gg terminator
`
`had been integrated in an orientation opposite the direction
`
`Sequence
`of transcription within the gg phage genome.
`analysis also revealed a translational stop codon in frame
`
`with the a~protein less than lObp down stream from the
`
`-18-
`
`,‘\
`
`ayes»
`
`-18-
`
`

`
`
`
`?
`
`gm.
`
`
`
`
`
`l6
`
`gindlll cleavage forming the conjunction between the
`
`lag—derived segment and the gg-derived DNA fragment; a
`
`translational stop signal on this position would be-expected
`
`to result in an a—fragment Containing 71 amino acids.
`
`the lag
`yiogqfihe cloning of exogenous promoters,
`promoter£em—t§e lag/get construct had to be removed or
`destroyed in such a way that a site for the subsequent inte~
`
`gration of promoters was retained.
`
`To do this,
`
`the flggll
`
`10
`
`Cleavage site at position ~17 of leg was employed, as
`described above. Colonies that showed both a reduced level
`
`15
`
`20
`
`25
`
`30
`
`35
`
`of To resistance and the presence of a lag operator sequence,
`
`which could be detected on the multicopy plasmid by its
`
`ability to bind the lag represser and induce chromosomal
`
`B~gal synth