`Bujard et al.
`
`[I I] Patent Number:
`[45] Date of Patent:
`
`4,495,280
`Jan. 22, 1985
`
`[54] CLONED HIGH SIGNAL STRENGTH
`PROMOTERS
`
`[75]
`
`Inventors: Hermann G. Bujard, Heidelberg,
`Fed. Rep. of Germany; Annie C. Y.
`Chang, Palo Alto; Stanley N. Cohen,
`Portola Valley, both of Calif.
`
`[73] Assignee: The Board of Trustees of the Leland
`Stanford Jr. Uni'Yersity, Stanford,
`Calif.
`
`[21] Appl. No.: 265,276
`
`May 20, 1981
`[22] Filed:
`Int. Cl.J ......................... C12Q 1/68; CI2Q 1/02;
`[51]
`Cl2P 21!00; CI2P 19/34; Cl2N 15/00
`[52] u.s. Cl ........................................... 435/6; 435/29;
`435/68; 435/172.3; 435/91; 536/27; 935/6;
`935/36; 935/37; 935/41; 935/76
`[58] Field of Search ....................... 435/29, 68, 6, 172,
`435/240, 253, 254, 257, 258, 317, 91; 536/27,
`28, 29
`
`[56]
`
`References Cited
`PUBLICA ~IONS
`Stuber et al., PNAS, 78(1), 167-171, (Jan. 1981).
`
`Casadaban eta!., J. Mol. Bioi., 138, 179-207, (1980).
`West et a!., Gene, 9, I 75-193, (1980).
`Gabain eta!., PNAS, 76(1), 189-193, (1979).
`StUber eta!., Malec. Gen. Genet., 166, 141-149, (1978).
`StUber era!., PNAS USA, 78(1), 167-171, (1981).
`
`Primary Examiner-Esther M. Kepplinger
`Attorney, Agent, or Firm-Bertram I. Rowland
`
`ABSTRACT
`
`[57]
`Method for preparing high signal strength promoters
`and terminators and DNA compositions employing
`such promoters and terminators. T5 phage is cleaved to
`provide for DNA sequences having intact promoters.
`These promoters are inserted into vectors separated
`from a balanced terminator by a gene of interest and the
`terminator is desirably followed by a marker allowing
`for selection of transformants. High efficiencies in tran(cid:173)
`scription of DNA can be achieved with the highly ac(cid:173)
`tive TS promoters. The promoters and terminators are
`used in hybrid DNA for efficient expression of struc(cid:173)
`tural genes and transcription to provide RNA sequen(cid:173)
`ces.
`
`IS Claims, 1 Drawing Figure
`
`Sanofi/Regeneron Ex. 1 002, pg 44
`
`Merck Ex. 1002, pg 44
`
`
`
`ter.(fd} •
`
`Ligase
`+
`..
`
`/
`Hindill
`~ tetjHindm
`
`Mndm H1ndm
`
`Sail
`
`pLBUI
`
`~artial Hind m
`\Soli
`
`Hindm Hindm
`
`~ •
`t/.)
`•
`~
`
`~ a.
`
`~
`
`~
`
`00
`Ul
`
`~
`~
`\0
`...
`Ul
`N
`00
`0
`
`Hincii
`HincH
`/3 -gal
`PIO
`----t-=:1• } - - - -
`
`tpant
`I
`I
`I
`I
`~ oe Sa/1
`
`(f)
`Q)
`::::J
`
`0 -::::::.:
`
`:::0
`CD
`(()
`CD
`::::J
`
`CD a
`::::J
`m
`;><
`.....>.
`0
`0
`1\.)
`
`"'0
`(()
`.t:>.
`01
`
`BamHI
`~
`DNA Pol.
`pBUIO
`Ligase
`
`+
`pACYCI84
`(Hindm)
`
`cat
`
`Hpa II I DNA Pol.
`~ndm
`EcoRI
`
`~
`+Ligase bla
`pBR322
`(EcoRIIDNA Poi./Hndm)
`
`EcoRI f
`
`Jr Soli
`
`pLBU3
`
`..0.
`
`I
`
`Hindm 1 h
`+
`te ~ Soli Htndm
`
`Sal!
`
`pBU 12a
`
`N
`N
`
`'" -\0
`
`Merck Ex. 1002, pg 45
`
`
`
`1
`
`4,495,280
`
`CLONED HIGH SIGNAL STRENGTH
`PROMOTERS
`
`The government has. rights in the invention pursuant
`to Grant Nos. AI 08619 and GM 27241 awarded by the
`National Institute of Health.
`The research was supported in part by a grant from
`the Deutsche Forschungsgemeinschaft.
`
`2
`SUMMARY OF THE INVENTION
`Methods for cloning, sequencing and using strong
`promoters and terminators are provided, as well as
`5 compositions 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,
`10 optionally followed by one or more translational stop
`codons in one or more reading frames, followed by a
`balanced terminator, followed by a marker allowing for
`selection of transformants. Upon introducing the result-
`ing plasmid into a microorganism host, efficient tran(cid:173)
`scription 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
`transformants having the above described construct.
`The construct or regulatory portions thereof are used
`for efficient transcription of RNA or gene expression.
`
`BACKGROUND OF THE INVENTION
`I. Field of the Invention
`Having established the feasibility of producing a wide
`variety of naturally occurring and synthetic polypep(cid:173)
`tides by means of hybrid DNA technology, there are 15
`continuing and extensive efforts to provide for more
`efficient and economic methods for producing the poly(cid:173)
`peptides. In developing a process for the commercial
`production of polypeptides, many factors will be in(cid:173)
`volved in optimizing the economic and efficient pro- 20
`duction of the polypeptides. Included among these fac(cid:173)
`tors are regulatory signals, which are DNA sequences
`involved with the regulation ofreplicaton, transcription
`and translation.
`One area of interest is at the level of transcription.
`Transcription involves the enzyme RNA polymerase.
`The RNA polymerase binds to a site <elled a promoter.
`It has been observed that promoters vary in their activ(cid:173)
`ity, as evidenced by the number of initiations of RNA 30
`per unit time or the strength ofbinding of the enzyme to
`the promoter site. The promoter may have one or more
`sequences that bind, which may or may not be contigu(cid:173)
`ous. The more active promoters are referred to as
`strong promoters.
`It was found that when introducing a strong pro(cid:173)
`moter into a vector and employing the resulting plasmid
`for transformation, one could not select transformants
`based on expression of markers which allowed for selec(cid:173)
`tion. Therefore, cloning of the strong promoters was 40
`not feasible. It 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, transcrip(cid:173)
`tion and translation of the genes for production of 45
`DNA, RNA, and polypeptides.
`2. Description of the Prior Art
`Promoters from bacterial and viral sources have been
`cloned in E. coli, and their signal strength in vitro has
`been studied using expression from distal promoterless 50
`sequences encoding ,B-galactosidase or other proteins
`(Casadaban and Cohen (1980) J. Mol. Bioll38, 179-207;
`West and Rodriguez (1980) Gene 9, 175-193). Attempts
`to clone small DNA fragments carrying the strong
`promoters of bacteriophage TS have been unsuccessful 55
`(v. Gabain and Bujard (1979) PNAS USA 76, 189, 193),
`Fragments of TS DNA having both a strong promoter
`and a strong termination signal have been cloned.
`(Breunig (1979) Dissertation (Universitat Heidelberg,
`Heidelberg, Germany)) Analysis has shown that tran- 60
`scriptional regions of several E. coli plasmids are orga(cid:173)
`nized in units where initiation and termination signals,
`are balanced. (Stuber and Bujard (1981) PNAS USA 78:
`167-171) P25 and P26 promoters oft he T5 bacteriophage
`are reported as among the most efficient RNA polymer- 65
`ase binding sequences. (Stuber et a! ( 1978) Mol. gen.
`Genet. 166 141-149; Niemann (1981) Diplomarbeit
`(Universitat Heidelberg, Heidelberg, Germany)).
`
`BRIEF DESCRIPTION OF THE DRAWING
`FIG. 1 is a flow chart of the preparation of a plasmid
`for cloning strong promoters and terminators.
`
`25
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`Methods and compositions are provided for prepar(cid:173)
`ing and cloning strong promoter and terminator regula(cid:173)
`tory signals and utilization of the strong regulatory
`sequences in the transcription and expression of genes of
`interest.
`Particularly, TS phage promoters are isolated, cloned
`in conjunction with a strong terminator, and appropri-
`35 ate 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.
`The compositions of this invention are characterized
`as having in the downstream direction of transcription
`the following units: a strong TS phage promoter; op(cid:173)
`tionally a structural gene which may be a marker; a
`balancing terminator; and optionally a marker allowing
`for selection of transformants containing the construct,
`which marker has a relatively low level of expression in
`comparison with the amount of RNA polymerase initia-
`tion 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 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 lac and trp
`and at least comparable to and normally greater than
`the combination of lac and trp promoters. For the most
`part, the strongest promoters among prokaryotes are
`the TS phage promoters and these will be employed as
`exemplary of naturally occurring or synthetic strong
`promoters. It is to be understood, that other prokaryotic
`and eukaryotic promoters. either naturally occurring or
`synthetic, could find application in the subject inven(cid:173)
`tion.
`The compositions of this invention will include linear
`segments for insertion of DNA having the strong regu(cid:173)
`latory signal sequences (i.e., the promoter and termina·
`tor) 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 strong regulatory signal sequences.
`
`Sanofi/Regeneron Ex. 1002, pg 46
`
`Merck Ex. 1002, pg 46
`
`
`
`4,495,280
`
`00
`
`3
`The termini may be blunt or staggered ended, having
`the same or different termini to allow for directed posi(cid:173)
`tioning of inserted sequences.
`The compositions which are employed as already
`indicated have a promoter, a balanced terminator, and
`desirably a marker which are in the direction of tran(cid:173)
`scription 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 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(arnino acid).
`Desirably, the gene is followed by one or a plurality
`of translational stop codons e.g. oop or nonsense co(cid:173)
`dons, 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. The terminator sequence is balanced with the
`promoter in the sense that the marker is transcribed not
`more than abo).lt one-fourth of the times that a gene
`upstream from the terminator is transcribed, where both
`genes are under the control of the same promoter. Usu(cid:173)
`ally, it will be desirable that a sufficient amount of the
`marker is transcribed to allow for selection of transfor(cid:173)
`mants. In view of the fact that the marker is transcribed 30
`from a strong promoter, it will usually be sufficient that
`the marker is transcribed in relation to the transcription
`of the gene intermediate the promoter and terminator in
`only about 1 to 20, usually only 5 to 15 number % of the
`gene. The strength of the promoter is reflected in the 35
`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 neces- 4D
`sary for expression involved with the DNA sequence,
`such as translational start and stop sites. In addition to
`the foregoing regulatory signals, other regulatory sig(cid:173)
`nals may be included, such as additional promoters,
`operators, initiators, catabolite activator protein binding 45
`sites, etc. Furthermore, 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
`replicating system or be cleaved and be inserted into a so
`vector to provide a plasmid. The vector is distinguished
`by having one or more DNA sequences which serve to
`insure stable replication of the plasmid and may also
`provide opportunities for high copy numbers of the
`plasmid in the microorganism host. The vectors may be 55
`derived
`from chromosomal or extrachromosomal
`sources. The sources include plasrnids, viruses (phage),
`chromosomes, or the like. In addition, the vector or the
`essential portions thereof may be prepared syntheti-
`~y.
`The plasmids may then be used for transformation of
`an appropriate microorganism host. Methods of intro(cid:173)
`ducing DNA into an appropriate host are well known.
`Illustrative of such methods, but not exhaustive of such
`methods, are transformation e.g. calcium shock, trans- 65
`fection, and conjugation. Descriptions of these methods
`may be found in Genetic Engineering, ed. Setlaw and
`Hollaender, Vol. l, Plenum Press, New York and Lon-
`
`4
`don, 1979; Molecular Cloning of Recombinant DNA,
`ed. Scott and Werner, Vol. 13, Academic Press, Inc.
`New York, 1973, and references cited therein.
`In order to allow for flexibility in preparing the con-
`s struct and self-replicating sequence or plasmid contain(cid:173)
`ing 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 nucle-
`10 otide 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 wide variety of structural genes are of interest for
`IS 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 inter(cid:173)
`est in transforming another product, which may be
`proteinaceous or non-proteinaceous. The proteins may
`20 be prepared as a single unit or as individual subunits and
`then joined together in appropriate ways. Furthermore,
`as appropriate, the protein products may be modified by
`glycosylation, acylation with aliphatic acids, e.g. lipid
`acids, phosphorolation, sulfonation or the like. The
`25 different classes of proteins which may be prepared
`include prolamines, histones, albumins globulins, sclero(cid:173)
`proteins, phosphoproteins, mucoproteins, chromopro(cid:173)
`teins, lipoproteins, nucleoproteins, and the remaining
`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; ,6-lipotro(cid:173)
`pin; ,6-endorphin; dynorphin; histocompatability pro(cid:173)
`teins; immunoglobulins e.g. IgA, IgD, IgE, IgG and
`IgM and fragments thereof; hemoglobin, somatome-
`dins; lymphokines; growth factors e.g. epidermal,
`fibroblast, platelet-derived, multiplication stimulating
`and nerve; hematoporetic-stimulating factors e.g.
`erythropoietin, colony-stimulating, erythroid poten(cid:173)
`tating activity or burst-promoting activity and lym-
`phopoietins; albumin and prealbumin;
`Prealbumin
`Albumin
`at-Lipoprotein
`at-Acid glycoprotein
`at-Antitrypsin
`at-Glycoprotein
`Transcortin
`' 4.6S-Postalbumin
`Tryptophan-poor
`a 1-glycoprotein
`at-x-Glycoprotein
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin:
`(Gc 1-1),
`(Gc 2-1),
`(Gc 2-2),
`Haptoglobin:
`(Hp 1- 1),
`(Hp 2-1),
`(Hp 2-2),
`Ceruloplasmin
`Cholinesterase
`a2-Lipoprotein(s)
`a2-Macroglobulin
`az-HS-Glycoprotein
`
`Sanofi/Regeneron Ex. 1002, pg 47
`
`Merck Ex. 1002, pg 47
`
`
`
`5
`
`Zn-az-glycoprotein
`az-Neuramino-glycoprotein
`Erythropoietin
`13-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`az-glycoprotein I
`az-glycoprotein II
`Immunoglobulin G
`(lgG) or yG-globulin
`Mol. formula:
`')'zKz or ')'2A2
`Immunoglobulin A (IgA) or yA-globulin
`Mol. formula:
`(azKz)n or (az/..z)n
`Immunoglobulin M (IgM) or yM-globulin
`Mol. formula:
`(}.Lz~<:z)5 or (J.LzAz)5
`Immunoglobulin D (IgD) or yO-Globulin (yD)
`Mol. formula:
`(ozKz) or (oz/..z)
`Immunoglobulin E (IgE) or yE-Globulin (yE)
`Mol. formula:
`( E2Kz) or (ez/..z)
`Free light chains
`Complement factors:
`C'l
`C'lq
`C'lr
`C'ls
`C'2
`C'3
`/t1A
`azD
`C'4
`C'S
`C'6
`C'7
`C'8
`C'9.
`Important protein hormones include:
`
`Peptide and Protein Hormones
`Parathyroid hormone: (parathormone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`Erythropoietin
`Melanotropin: (Melanocyte-stimulating hormone; inter-
`medin)
`Somatotropin: (growth hormone)
`Corticotropin: (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Leuteinizing hormone: (interstitial cell-simulating hor(cid:173)
`mone)
`Luteomammotropic hormone: (Luteotropin, prolactin)
`Gonadotropin: (chorionic gonadotropin).
`
`Tissue Hormones
`
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`
`4,495,280
`
`6
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`5 Releasing factors (RF): CRF, LRF, TRF, Somatotro(cid:173)
`pin-RF, GRF, FSHRF, PIF, MIF.
`
`In addition to various non-enzymatic proteins of
`physiological interest, enzymes can also be produced as
`10 an end product or for intracellular transformation of a
`substrate present in the host or substrate introduced
`extracellularly, or for enzymatic transformation in vi(cid:173)
`tro.
`In accordance with the I.U.B. classification, the en-
`IS zymes fall into varying categories such as 1. oxidore(cid:173)
`ductases; 2. transferases; 3. hydrolases; 4. lyases; 5. isom(cid:173)
`erases; 6. ligases. Enzymes of particular interest will be
`hydrolases and oxidoreductases for use in commercial
`processing, for example, hydrolases for hydrolysing
`20 polysaccharides, lipids and polypeptides; oxidoreduc(cid:173)
`tases for oxidation of alcohols and aldehydes, epoxida(cid:173)
`tion, and the like.
`The microorganism host may be bacteria, such as
`Escherichia, Bacillus, Aerobacter, Klebsiella, Proteus,
`25 Pseudomonas, Streptococcus, Staphylococcus, Clo(cid:173)
`stridium, Mycobacterium, Streptomyces and Actino(cid:173)
`myces; Fungi e.g. Gymnomycota, Dimastygomycota,
`Eumycota, Zygomycetes, Ascomycetes and Basidomy(cid:173)
`cetes, such as, Candida, Aspergillus, Rhizobus, Micro-
`30 sporum, and Fonsecaea; Protozoa e.g. Mastigophora,
`Sarcodina, Sporozoa and Celiophora, such as, Trypano(cid:173)
`soma, Codosiga, Protospongra and Entameba, and Alga
`e.g. Dinoflagellates, Euglenoids, and Diatoms.
`Higher cells, e.g., mammalian, may also be employed
`35 as hosts, where viral, e.g., bovine papilloma virus or
`other DNA sequence is available which- has plasmid(cid:173)
`like activity.
`Depending upon the nature of the host, a wide vari(cid:173)
`ety of vectors may be employed. The vector will have
`40 an 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. Regula(cid:173)
`tory signals can include temperature sensitive replicons,
`runaway-replication sequences, temperature sensitive
`45 operators, and the like. Various additional DNA se(cid:173)
`quences may be present providing for restriction sites,
`markers, termination sequences, or the like. Desirably,
`the vector should be of a substantially different size
`from the construct to allow for excision of the construct
`so and ease of separation of the construct from the vector
`by molecular weight separation techniques e.g. electro(cid:173)
`phoresis and density gradient centrifugation.
`The subject invention also provides for a technique
`for screening the strength of promoters and terminators,
`55 thus allowing for the determination of the use of a pro(cid:173)
`moter or terminator in a particular application, where it
`may be desirable to have promoters or terminators or
`combinations thereof of varying strength. In screening
`promoters and/or terminators one employs a promoter
`60 or terminator of known activity. A DNA construct is
`made having the sequence described previously, where
`appropriate restriction· sites are provided for introduc(cid:173)
`ing the various elements. The construct provides in the
`direction of transcription the promoter, a first gene
`65 marker, optionally termination codons, such as non(cid:173)
`sense codons and oop terminator, the terminator, and a
`second gene marker. This DNA construct is inserted
`into an appropriate vector. Where the promoter and
`
`Sanofi/Regeneron Ex. 1002, pg 48
`
`Merck Ex. 1002, pg 48
`
`
`
`4,495,280
`
`8
`The strategy described above provides a vehicle
`which can be used with one or more hosts for gene
`expression, where the gene after processing in a prede(cid:173)
`termined 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 be-
`tween the promoter and terminator may be designed so
`as to provide for a plurality of restriction cleavage sites,
`allowing for the introduction and removal of DNA
`fragments without interruption of the remainder of the
`vehicle. Thus, by having a plurality of unique restric(cid:173)
`tion sites or restriction sites limited to the region be(cid:173)
`tween the promoter and terminator in the downstream
`20 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 con(cid:173)
`struct into an appropriate vector which has been linear-
`ized to provide complementary termini.
`In developing the construct, a vector will normally
`be used in order to clone the various sequences. The
`construct 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 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.
`The following examples are offered by way of iiius(cid:173)
`tration and not by way of limitation.
`
`7
`terminator are properly balanced, there will be a sub(cid:173)
`stantial differentiation between expression of the first
`gene marker and expression of the second gene marker.
`The ratio of expression between the first and second
`marker will provide for a comparative evaluation of the 5
`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 extrachromo(cid:173)
`somal element, such. as a· plasmid or double minute,
`isolate the promoters by selective binding with RNA 10
`polymerase 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 15
`fixed terminator. Similarly, one can isolate DNA se(cid:173)
`quences having terminator sequences, insert the sequen(cid:173)
`ces into the above described construct at the appropri(cid:173)
`ate 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. Conve(cid:173)
`niently, markers which allow for selection such as resis(cid:173)
`tance to antibiotics, toxins or heavy metals can be used.
`By varying the concentration of the selective agents in 25
`the nutrient medium, one. can determine the relative
`proportions of the enzyme expressed by the genes in
`relation to the growth of the host. Alternatively, one
`can use growth factors e.g. having a gene which com(cid:173)
`plements a mutant gene in an auxotrophic host, where 30
`the gene expresses a product necessary for a biosyn(cid:173)
`thetic pathway. A third marker provides virus incom(cid:173)
`patibility, preventing plaque formation. Other markers
`which allow for comparison wiii come readily to mind.
`The terminators may be evaluated in the presence 35
`and absence of rho, so that one can determine the de(cid:173)
`pendency of the terminator on rho, as well as the effec(cid:173)
`tiveness of the terminator in relation to the concentra(cid:173)
`tion of rho.
`The methods for preparing the subject compositions 40
`will be conventional. The various DNA fragments and
`sequences can be obtained from a variety of sources by
`restriction mapping and endonuclease cleavage to pro(cid:173)
`vide fragments having the desired intact sequence or
`gene. The fragments can be further processed employ- 45
`ing endo- or exonucleases to remove nucleotides unre(cid:173)
`lated to desired regulatory sequences or structural
`genes. By appropriate choice of restriction enzymes,
`cohesive or blunt ended fragments can be generated.
`Furthermore, chains can be extended with single nucle- 50
`otides 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 55
`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 TS promoter and a terminator,
`desirably having at least one stop codon on the up- 60
`stream 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 opera- 65
`tor, activator, ribosomal start signal sequence, or the
`like, to allow for controlled expression of the inserted
`gene(s).
`
`EXPERIMENTAL
`Materials and Methods
`Restriction endonucleases Hindiii, Hincll, Sail,
`BamHI and T4 Ligase were from New England Biolabs
`(Beverly, Mass., USA), Sau3A was from BRL (Neu(cid:173)
`Isenburg, Germany) and EcoRI and Hpall from (Bo(cid:173)
`ehringer Mannheim, Federal Republic of Germany).
`DNA polymerase I, phage fd DNA ~replicative form),
`plasmid AD16/30 containing a 28 bp Hindiii/BamHI
`adapter sequence and, lac represser were supplied by
`private sources. The isolation of bacteriophage TS
`DNA, plasmid DNA E. coli RNA polymerase and E.
`coli termination factor rho have been described previ(cid:173)
`ously, (v. Gabian and Bujard, Mol. gen. Genet. (1977)
`157, 301-311; Clewel and Helinski (1969) PNAS USA
`62, 1157-1166; and Knopf and Bujard (1975) Eur. J.
`Biochem. 53, 371-385).
`A 780bp DNA fragment carrying the E. coli lac regu(cid:173)
`latory region (promoter/operator: P/0), anN-terminal
`portion of the j3-galactosidase structural gene sufficient
`for intracistronic complementation of theM I 5 deletion,
`as well as a portion of the i-gene(i) was isolated from a
`Hincll digest of a pACYC 214 plasmid (a plasmid re(cid:173)
`lated to pACYC 184 by insertion at a BamHI site of a
`BamHI restriction cleavage fragment from F' -lac carry(cid:173)
`ing the lac gene) by repressor binding and subsequent
`
`Sanofi/Regeneron Ex. 1002, pg 49
`
`Merck Ex. 1002, pg 49
`
`
`
`4,495,280
`
`9
`adsorption to nitrocellose. This fragment was then em(cid:173)
`ployed in the construction of an exemplary plasmid for
`analyzing strong promoters and strong terminators as
`depicted in FIG. 1.
`Utilizing the Hpall cleavage site within the ,B-gal
`structural genes the fragment was reduced in size and
`provided with BamHI and Sail cleavage sites by vari(cid:173)
`ous subcloning. The resulting fragment (left most part
`of Figure) contains the intact control region of the lac
`operon and an N-terminal portion of the .B-gal struc- 10
`tural gene coding for 66 amino acids (a). Introduction
`of this fragment by blunt end ligation into the Hindiii
`site of pACYC184 (Chang and Cohen (1978) J. Bac(cid:173)
`teriol. 134, 1141-1156) yielded pBUlO, a vector suit(cid:173)
`able for terminator cloning. The major terminator of the 15
`coliphage fd genome was isolated as a 338bp Sau3A
`fragment (Beck et al. (1978) Nucl. Acids Res. 5,
`4495-4503), ligated with a BamHI/Hindlll adaptor
`sequence and integrated into pBUIO to yield pLBUl.
`Cleavage of the lac sequence in pBUIO by Hpall 20
`destroys the lac promoter and liberates upon cleavage
`with Hindlll a fragment containing the lac operator and
`a region coding for a functional a-fragment. Integrating
`this DNA sequence into pBR322 leads to pBU12a. Fi(cid:173)
`nally, replacement of the Hindlii/Sall portion of 25
`pBU 12a by a partial digest of the Hindlll/Sall fragment
`of pLBUl containing the fd terminator resulted in
`pLBU3, a vector suitable for integration of efficient
`promoters at the EcoRI site. The regions encoding
`chloramphenicol (Cm), ampicillin (Ap) and tetracycline 30
`(Tc) resistance are indicated as cat, bla and tet respec(cid:173)
`tively.
`The plasmid, pBU 10, had the following properties: (i)
`it contains the a fragment of .B-galactosidase (a-protein)
`and complemented the MIS deletion of the lac operon; 35
`(ii) the Tc resistance it specified was under the control
`of the lac promoter, as shown in MIS H strains; (iii) the
`Hindlll site between the lac gene fragment and the tet
`gene was restored; the stop codon immediately follow(cid:173)
`ing the Hind III site limited the length of the JacZ gene 40
`product to 68 amino acids.
`Insertion of the fd terminator upstream to the tet gene
`resulted in a 90% reduction in the level ofTc resistance,
`but no detectable change in the levels of .B-galactosidase
`activity in MIS deletion strains. The results obtained as 45
`to the properties imparted to various E. coli strains by
`various plasmids is set forth in the following table:
`Seven independent plasmid isolates from clones con(cid:173)
`tained the expected 352bp Hindlll-generated fragment
`containing the fd terminator. Electrophoretic analysis 50
`of all seven BamHI-cleaved isolates and DNA sequence
`analysis of one of these showed that in all instances the
`fd terminator had been integrated in an orientation op(cid:173)
`posite the direction of transcription within the fd phage
`genome. Sequence analysis also revealed a translational 55
`stop codon in frame with the a-protein Jess than !Obp
`down stream from the Hindiii cleavage forming the
`conjunction between the lac-derived segment and the
`fd-derived DNA fragment; a translational stop signal on
`this position would be expected to result in an a-frag- 60
`ment containing 71 amino acids.
`For the cloning of exogenous promoters, the lac pro(cid:173)
`moter on the lac/tet construct had to be removed or
`destroyed in such a way that a site for the subsequent
`integration of promoters was retained. To do this, the 65
`Hpall cleavage site at position --17 of lac was em(cid:173)
`ployed, as described above. Colonies that showed both
`a reduced level of Tc resistance and the presence of a
`
`10
`lac operator sequence, which could be detected on the
`multicopy plasmid by its ability to bind the lac represser
`and induce chromosomal ,B-gal synthesis, were identi(cid:173)
`fied. Endonuclease analysis (Hindiii/EcoRI double
`digest) of plasmids .recovered from several isolates
`yielded two types of vectors: one of these represented
`by pBU12 harbored the expected 253bp lac fragment;
`the other represented by pBU12a yielded a 420bp frag(cid:173)
`ment. DNA sequence analysis showed that in pBU12a,
`a 160bp fragment of unknown origin, containing two to
`three stop codons in each of the possible translational
`reading frames, had been integrated between the EcoRI
`site and position -15 of the lac promoter. The presence
`of the stop codons made the fragment an efficient termi(cid:173)
`nator of any translation that occurred upstream of the
`translational initiation site for the a-fragment.
`When the Hindlll/Sall segment of pBU12a was re(cid:173)
`pla