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`United States Patent
`
`{:9}
`
`Bujard et al.
`
`{11} Patent Number:
`
`4,495,280
`
`[45] Date of Patent:
`
`Jan. 22, 1985
`
`Casadaban et 31., J. Mol. Biol., 138, 179-207, (1980).
`West (:1 8L, Gene, 9, 175493, (1980).
`Gabain as 31., PNAS, 7'60), 189-193, (1979).
`Sliiber et al., Molec. Gen. Genet, 166, 141-149, (1978).
`Stiiber at al., PNAS USA, 78(1), E67471. (K981).
`
`Primary Examinermfisther M. Kepplinger
`Attorney, Agent. or Firm-—Bertram I. Rowland
`
`{S7}
`
`ABSTRACT
`
`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 intzxcz promoters.
`These promoters are inserted into vecrom separated
`from a baianced terminator by a gene ofimerest and me
`terminator is desirabiy followed by a marker aliowing
`for selection of Iransformants. High efficiencics in tran-
`scription of ‘DNA can be achieved with the highly ac~
`rive T5 promoters. The promoters and terminators are
`used in hybrid DNA for efficient expression of struc~
`turai genes and transcription :0 provide RNA sequcm
`(:63.
`
`15 Ciaims, 1 Drawing Figure
`
`
`
`[54] CLONED HIGH SIGNAL STRENGTH
`FROMOTERS
`
`[75]
`
`Inventors: Hermann G. Bujard, Heidefiberg,
`Fed. Rep. of Germany; Annie C. Y.
`Chang, Palo Alto; Stanley N. Cohen,
`Portola Valley, both of Calif.
`The Board of Trustees of the Leland
`Stanford Jr. University, Stanford,
`Calif.
`
`[73] Assignee:
`
`[21] App1.No.: 265,276 .
`
`[22] Filed:
`
`May 20,1981
`
`[SE]
`
`1111. CL3‘
`
`....................... .. C12Q 1/68; C12Q 3/02;
`C121’ 21/00; C12? 19/34; C12N 35/00
`[52] US. Cl. ........................................ .. 435/6; 435/29;
`435/68; 435/172.3; 435/9!; 536/27; 935/6:
`935/36: 935/37; 935/41; 935/76
`Field of Search ..................... .. 435/29, 68, 6, I72.
`435/240. 253, 254, 257, 258. 317, 91', 536/27.
`28, 29
`
`[58]
`
`{S6}
`
`References Cited
`
`PUBLICPCQIONS
`Stuber at 31., PNAS, 78(1), 16747]. (Jan. 1981).
`
`Page 2
`
`
`
`
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`

`
`1
`
`4,495,280
`
`2
`SUMMARY OF THE INVENTION
`
`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 Dentsche Forschungsgcmeinschaft.
`BACKGROUND OF THE INVENTION
`
`l0
`
`Methods for cloning, sequencing and using strong
`promoters and terminators are provided, as well as
`compositions resulting from the methods. By cleaving
`T5 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 by a marker allowing for
`selection of transformants. Upon introducing the result-
`ing plasmid into a microorganism host, efficient tran-
`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 theabove described construct.
`The construct or regulatory portions thereof are used
`for efficient transcription of RNA or gene expression.
`BRIEF DESCRIVFION OF THE DRAWING
`
`FEG. 1 is a flow chart of the preparation of a plasmid
`for cloning strong promoters and terminazors.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`Methods and compositions are provided for prepar~
`ing and cloning strong promoter and terminator regula~
`tory signals 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 appropri-
`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 T5 phage promoter; op~
`tionally a structural gene which may be a marker: a
`balancing terrninalor; and optionally a marker allowing
`for selection of transformants containing the construct,
`which marker has n 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 up
`and at least comparable to and normally greater than
`the combination of lac and up promoters. For the most
`part. the strongest promoters among prokaryotes are
`the T5 phage promoters and these will be employed as
`exemplary of naturally occurring or synthetic strong
`promoters. it is to be understood, that other prokaryotic
`undeultaryotic promoters. either naturally occurring or
`synthetic. could find application in the subject inven-
`non.
`
`The compositions of this invention will include linear
`segments for insertion of DNA having the strong regu»
`latory signal sequences (i.::., 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.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`1. Field of the Invention
`Having established the feasibility of producing 3 wide
`variety of naturally occurring and synthetic polypep- .
`15
`tides by means of hybrid DNA technology, there are
`continuing and extensive efforts to provide for more
`efficient and economic methods for producing the poly-
`peptides. In developing a process for the commercial
`production of polypeptides, many factors will be in-
`volved in optirnizing the economic and efficient pro—
`duciion of the polypeptides. Included among these Fae»
`tors are regulatory signals, which are DNA sequences
`involved with the regulation of replicaton, 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 milled a promoter.
`It has been observed that promoters vary in their activ»
`ity, 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, which may or may not be contigu~
`ous. The more active promoters are referred to as
`strong promoters
`it was found that when introducing a strong pro»
`moter into a vector and employing the resulting plasmid
`for transformation, one could not select rransformants
`based on expression of markers which allowed for selec~
`tiou. Tiierefore, cloning of the strong promoters was
`not feasible. It is therefore desirable that methods be
`provided which would allow for the screening of strong
`promoters and terminstors and their subsequent cloning
`to be used in conjunction with the replication, transcrip—
`tion and translation of the genes for production of 45
`DNA, RNA, and polypeptides.
`2. Description of the Prior Art
`}’romoters from bacterial and viral sources have been
`cloned in E colt‘. and their signal strength in vitro has
`been studied using expression from distal promotcrless
`sequences encoding Bgalsctosidase or other proteins
`(Casadaban and Cohen (I980) J. Mol. Biol 138, 1794.07;
`West and Rodriguez (1980) Gene 9, I75-l93). Attempts
`to clone small DNA fragments‘ carrying the strong
`promoters of bacteriophage T5 have been unsuccessful
`(V. Gabain and Bujard (2979) PNAS USA 76, l39, l93),
`Fragments of T5 DNA having both a strong promoter
`and a strong termination signal have been cloned.
`(Breunig (I979) Dissertation (Universitat Heidelberg,
`Heidelberg, Germany)) Analysis has shown that tran-
`scriptional regions of several E. coil‘ plasmids are orga»
`nized in units where initiation and termination signals.
`are balanced. (Stuber and Bujard (1981) PNAS USA 78:
`167-171) P25 and P26 promoters of the T5 bacteriophage
`are reported as among the most efficient RNA polymer-
`ase binding sequences. (Stuber er al (1978) Mol. gen.
`Genet.
`I66 l4l—l-49; Niemann (I98!) Diplomarbeit
`(Universitat Heidelberg, Heidelberg, Germany».
`
`50
`
`55
`
`bl}
`
`65
`
`Page 4
`
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`

`
`3
`The ternnni may be blunt or staggered ended, having
`the same or different termini to allow for directed posi~
`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-
`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 nonstructural gene or a structural gene providing
`RNA e.g.
`ribosornal or messenger, or providing a
`poly(amino acid).
`.
`Desirably, the gene is followed by one or a plurality
`of translational stop coclons e.g. oop or nonsense co-
`dons, or preferably a plurality, usually up to about six,
`more usually from about two to live, 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 andexpression.
`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 about one-fourth of the times that a gene
`upstream from the terminator is transcribed, where both
`genes are under the control of the same prornoter._ Usu-
`ally, it will be desirable that a sufficient amount of the
`marker is transcribed to allow for selection of transfor—
`tnants In view of the fact that the marker is transcribed
`from a strong promoter, it will usually be sufficient that
`the marker is transcribed in relationto the transcription
`of the gene intermediate the promoter and terminator in
`only about l 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 roarlter 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-
`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~
`nals may be included, such as additional promoters,
`operators, initiators, catabolite activator protein binding
`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
`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
`derived from chromosomal or extrachromosomal
`sources. The sources include plasmids, viruses (phage),
`chromosomes, or the like. In addition, the vector or the
`essential portions thereof may be prepared syntheti-
`cally.
`The plasmids may then be used for transformation of
`an appropriate microorganism hcfst. Methods of intro»
`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-
`fection, and conjugation. Descriptions of these methods
`may be found in Genetic Engineering, ed. Setlaw and
`Hollaender, Vol. 1, Plenum Press, New York and Lon—
`
`I0
`
`15
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`20
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`25
`
`30
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`35
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`55
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`60
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`65
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`4,495,280
`
`4
`l979; Molecular Cloning of Recombinant DNA,
`don,
`ed. Scott and Werner, Vol. 13, Academic Press, Inc.
`New York,
`i973, and references cited therein.
`In order to allow for flexibility in preparing the com
`struct and self-replicating sequence or plasmid contain‘
`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~
`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
`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«
`est
`in transforming another product. which may be
`proteinaceous or non-proteinaceous. The proteins may
`be prepared as 3 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, eg. lipid
`acids, phosphorolation, sulfonation or the like. The
`different classes of proteins which may be prepared
`include protamines, histories, albumins globulins, sclero-
`pfoieins. pilosphoproteins, mucoproteins, chromopro-
`reins, lipoproteins, nuclcoproteins, and the remaining
`proteins which are unclassified.
`The following is a representative list of proteins of
`interest.
`_
`,
`interferon e.g.
`lnsulin; growth hormone;
`leukocyte,
`lurnphoblastoid, 'I‘~immune and fibroblast; Bdipotro»
`pin; B~endorphin; dynorphin; histocompatability prow
`teins; immunoglobulins e.g. IgA, Igl), lg.E, IgG and
`IgM and fragments thereof; hemoglobin, sornatome~
`dins;
`lymphokines; growth factors e.g. epidermal,
`fibroblast, platelet-derived, multiplication stimulating
`and nerve; hemat0poretic~stimulat§ng factors e.g.
`erythropoietin, colony-stimulating, erythroid poten—
`taxing activity or burst-promoting activity and lynx»
`phopoietins; albumin and prealburnin;
`Prealbumin
`Albumin
`<zz~I..ipoprotei.n
`a1~Acid glycoprotein
`at-Antitrypsin
`a 1-Glycoprorein
`Transcortin
`’ 4.65-Postalbumin
`'I‘ryptophan«poor
`ctpglycoprotein
`<1;-x~Glycoprotein
`Thyroztin~binding globulin
`Inter~a~trypsin~inhibitor
`Cvc~globulin;
`(Gc 1-1),
`(Gc 2-1),
`((30 2~2),
`llaptoglobin:
`1'1)!
`2'1)»
`(Hp 2-2).
`Ceruloplasmin
`Cholinesterase
`a2-l..lpoprotein(s)
`<12-Macroglobtzlin
`az~HS~Glycoprotein
`
`Paoe 5
`
`
`
`
`
`

`
`5
`
`Zn-ctz-giycoprotein
`at;-Neuramino-glycoprotein
`Erythropoietin
`8-lipoprotein
`Transferrin
`
`‘Hemopexin
`Fibrinogcn
`Plasminogen
`a2-giycoprotein I
`ag-glycoprotein II
`lmmuncglobulin G
`(IgG) or 7/G-globulin
`Moi. formula:
`wt: or 72?»:
`lmmunoglobuhn A (lgA) or 7A-globulin
`Moi. formula:
`(a:zK2)" Or (a2>\2)"
`lmmunoglobulin M (lgM) or 7M-globulin
`Moi. formula:
`(u2x2)5 or (11»2?~2)5
`Immunoglobuiin D (lgD) or 'yD-Globulin (~/D)
`Moi. formula:
`(32K2) or (32?~2)
`Immunoglobulin E (IgE) or 'yE—Globulin (715)
`Moi. formula:
`(em) 0? (em)
`Free light chains
`Complement factors:
`C’1
`C".lq
`Cir
`013
`C2
`C3
`BIA
`
`a2D
`C4
`C5
`C’6
`C’?
`C8
`C9.
`Important protein hormones include:
`
`Peptide and Protein Plormones
`Parathyroid hormone: (parathorrnone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Rclaxin
`Erythropoietin
`Melanotropin: (Melanocyte~stimulating hormone; inten
`medin)
`Somatotropin: (growth hormone)
`Corticotropin: (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating. hormone
`Leuteinizing hormone: (interstitial cell-simulating hor-
`mone)
`Luteomammotropic hormone: (Luteotropin. prolactin)
`Gonadotropin: (chorionic gonadotropin).
`Tissue Hormones
`
`Secretin
`Gaszrin
`Angiotensin I and II
`Bradykiniu
`Human placental, lactogen
`
`4,495,280
`
`6
`
`Peptide Hormones from the Neurohypophysis
`
`Oxytocin
`Vasoprcssin
`Releasing factors (RF): CRF, LRF, TRF, Somatotro-
`pin-RF, CERF, FSHRF, PIF, MIR
`
`In addition to various non-enzymatic proteins of
`physiological interest, enzymes can also he produced as
`an end product or for intracellular transformatioti of a
`substrate present in the host or substrate introduced
`extracellularly, or for enzymatic transfonrtation in vi-
`tro.
`In accordance with the I.U.Bi classification. the en-
`zymes fall into varying categories such as l. ox§dore~
`ductases; 2. transferases; 3. hydrolases; 4. lyases; 5. isom-
`erases; 6. ligases. Enzymes of particular interest will be
`hydrolases and oxidoreductases for use in commercial
`processing, for example, hydrolases for hydrolysing
`polysaccharides, lipids and polypeptides; oxidoreduo
`tases for oxidation of alcohols and aldehydes, epoxida-
`lion, and the like,
`The microorganism host may be bacteria, such as
`Escherichia, Bacillus, Acrobacter. Klehsiella, Proteus,
`Pseudomonas, Streptococcus, Staphylococcus, Clo-
`stridium, Mycobacterium, Streptomyces and Actino~
`myces; Fungi e.g. Gyrnnomycota, Dimastygornycota,
`Eumycota, Zygomycctcs, Ascomycctes and Basidorny-
`cetes, such as, Candida, Aspergillus, Rhizobus, Micro»
`sporum, and Fonsecaea; Protozoa e.g. Mastigophora,
`Sarcodina, Sporozoa and Celiophora, such as, Trypa.no~
`soma, Codosiga. Protospongra and Entameba, and Alga
`e.g. Dinoflagellates, Euglenoids, and Diatorns.
`Higher cells, c.g., mammalian, may also be employed
`as hosts, where viral, e.g., bovine papilloma virus or
`other DNA sequence is available whiclr has plasmid-
`iike activity-
`Depending upon the nature of the host, a wide vari—
`ety of vectors may be employed. The vector will have
`an intact replicon and be capable of replication in the
`host. In addition, replicons can be developer: which
`may have one or more other regulatory signals. Regui'a~
`tory signals can include temperature sensitive replicons,
`runaway-replication sequences,
`temperature sensitive
`operators, and the like. Various additional DNA se-
`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
`and ease of separation of the construct from the vector
`by molecular weight separation techniques eg. electro~
`phoresis and density gradient centrifugation.
`The subject invention also provides for a technique
`for screening the strength of promoters and terminators.
`thus allowing for the determination of the use of a pm
`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
`or terminator of known activity. A DNA construct is
`made having the sequence described previously, where
`appropriate restriction sites are provided For introduc~
`ing the various elements. The construct provides in the
`direction of transcription the promoter.
`21 first gene
`marker, optionally nomination cocloris, such as non-
`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
`
`l0
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`
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`

`
`7
`terminator are properly balanced, there willbe a sub-
`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
`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 extrachromc»
`sornal element, such. as aplasmid or double minute,
`isolate the promoters by selective binding with RNA
`polymerase and insert the DNA fragments which bind
`to the RNA polymerase into the previously described
`construction. By detennining 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 se-
`quences having terminator sequences, insert the sequen-
`ces into the above described construct at the appropri-
`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~
`niently, markers which allow for selection such as resis~
`tancc 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 growth of the host. Alternatively, one
`can use growth factors e.g. having a gene which com-
`plements a. mutant gene in an auxotrophic host, where
`the gene expresses a product necessary for a biosym
`thetic pathway‘. A third marker provides virus incom~
`patibility, preventing plaque formation. Other markers
`which allow for comparison will come readily to mind.
`The tcrminators may be evaluated in the presence
`and absence of rho, so that one can determine the de-
`pendency of the tenninator on tho, as well as the effec~
`“tiveness of the terminator in relation to the concentra-
`tion of rho.
`The methodsfor 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 endonucleasc cleavage to pro~
`vide fragments having the desired intact sequence or
`gene. The fragments can be further processed employ-
`ing cndo— or cxonucleases to remove nucleotides unre-
`lated to desired regulatory sequences. or structural
`genes. 8y appropriate choice of restriction enzymes,
`cohesive or blunt ended fragments can be generated.
`Furthczmore, chains can be extended with single nucle-
`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
`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 2: T5 promoter and a terminator,
`desirably having at least one stop“ codon on the up-
`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-
`tor, activator. ribosomal start signal sequence, or the
`like,
`to allow for controlled expression of the inserted
`gene(s).
`
`5
`
`l0
`
`l5
`
`20
`
`'25
`
`30
`
`35
`
`-15
`
`50
`
`55
`
`65
`
`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-
`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«
`tion sites or restriction sites limited to the region be»
`tween the promoter and terminator 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 com
`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
`speciiicitics commercially available.
`The following examples are offered by way of illus~
`tration and not by way of limitation.
`'
`EXPERIMENTAL
`Materials and Methods
`Restriction endonucleases Hindlli, Hincll, Sail,
`Bamiil and T4 Ligase were from New England Biolabs
`(Beverly, Mass, USA), Sau3A was from BRL (Neu-
`Isenburg, Germany) and ECORI and Hpall from (Bo-
`ehringer Mannheim, Federal Republic of Germany).
`DNA polymerase I, phage fd DNA (rcplicative form),
`plasmid A1316/30 containing a 28 bp Hfindlll/Bamlril
`adapter sequence and, lac rcpresser were supplied by
`private sources. The isolation of bacteriophage T5
`DNA, plasmid DNA E. colt‘ RNA polymerase and E.
`colt‘ termination factor rho have been described previ~
`ously, (v. Gahian and Bujard, Moi. gen. Genet. (1977)
`157, 30l~3l l; 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 13. colt‘ lac regu»
`latory region (promoter/operator: P/D), an N~tcrminal
`portion of the ,8~galactosid'ase structural gene sufficient
`for intracistrcmic cornplementation. of the M15 deletion,
`as well as a portion of the i-genc(i) was isolated from a
`Hincll digest of a pACYC 214 plasmid (aplasmid re-
`lated to pACYC 184 by insertion at a Bamfll site of a
`BamHI restriction cleavage fragment from F‘~lac carry~
`ing the lac gene) by rcpressor binding and subsequent
`
`Page 7
`
`
`
`
`
`

`
`9
`adsorption to nitrocellose. This fragment was then em-
`ployed in the construction of an exemplary plasmid for
`analyzing strong promoters and strong terrninators as
`depicted in FIG. 1.
`Utilizing the Hpall cleavage site within the ,8-gal
`structural genes the fragment was reduced in size and
`provided with Bamlil and Sell cleavage sites by vari~
`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-
`tural gene coding for 66 amino acids (a). Introduction
`of this fragment by blunt end ligation into the Hindlll
`site of pACYCl84 (Chang and Cohen (1978) J. Bac-
`teriol. 134,
`ll4l»—ll56) yielded pBUlO, a vector suit-
`able for terminator cloning. The major terminator of the
`coliphage fd genome was isolated as a 338bp Sau3A
`fragment {Beck et al.
`(1978) Nucl Acids Res. 5,
`4495-4503)’,
`ligated with a Bamlrll/'1-Iindlll adaptor
`sequence and integrated into pBUl0 to yield pLBUl.
`Cleavage of the lac sequence in pBUl0 by Hpall
`destroys the lac promoter and liberates upon cleavage
`with Hindlll a fragment containing the lac operator and
`a region coding for a functional o.-fragment. Integrating
`this DNA sequence into pBR322 leads to pBU12a. Fi~
`nally,
`replacement of the Iriindlll/Sail portion of
`pBUl2a by a partial digest of the Hindlll/Sail 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
`cliloramphenicol (Cm), ampicillin (Ap) and tetracycline
`(Tc) resistance are indicated as cat, bla and tet respec-
`tively.
`The plasmid, pBU it), had the following properties: (i)
`it contains the a. fragment of B-galactosidase (avprotein)
`and complemented the M15 deletion of the lac operon;
`(ii) the To resistance it specified was under the control
`of the lac promoter, as shown in M15 E4 strains; (iii) the
`Hindlll site between the lac gene fragment and the tet
`gene was restored; the stop codon immediately follow-
`ing the Hindlll site limited the length of the lacZ gene
`product to 68 amino acids.
`insertion of the id terminator upstream to the teat gene
`resulted in a 90% reduction in the level of’l‘c resistance,
`but no detectable change in the levels oi‘B~galactosidase
`activity in M35 deletion strains. The results obtained as
`to the properties imparted to various E. coil‘ strains by
`various plasmids is set forth in the following table:
`Seven independent plasmid isolates from clones con-
`tained the expected 35223;) Hindlll-generated fragment
`containing the fol terminator. Electrophoretic analysis
`of all seven Barnl-[Lcleaved isolates and DNA sequence
`analysis of one of these showed that in all instances the
`fd terminator had been integrated in an orientation op»
`posite the direction of transcription within the fcl phage
`genome. Sequence analysis also revealed a translational
`stop codon in frame with the or-protein less than lObp
`down stream from the HindIII cleavage forming the
`conjunction between the lac—derived segment and the
`fd-derived DNA fragment; is translational stop signal on
`this position would be expected to result in an a-frag~
`merit containing ‘ll amino acids.
`For the cloning of exogenous promoters, the lac pro-
`moter on the lac/tet construct bad to be removed or
`destroyed in such a way that a site for the subsequent
`integration of promoters was retained. To do this, the
`Hpall cleavage site at position «—- l7 of lac was em~
`played. as described above. Colonies that showed both
`a reduced level of To resistance and the presence of :3
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4,495,280
`
`10
`lac operator sequence, which could be detected on the
`rnulticopy plasmid by its ability to bind the lac represser
`and induce chromosomal B-gal synthesis, were identi~
`fled. Endonuclease analysis (liindlll/EcoRI double
`digest) of plasmids recovered from several
`isolates
`yielded two types of vectors: one of these represented
`by pBUl2 harbored the expected 253bp lac fragment;
`the other represented by pBU12a yielded a 420bp frag-
`ment. DNA sequence analysis showed that in pBUl2a,
`a l60bp fragment of unknown origin, ‘containing two to
`three stop codons in each of the possible translational
`reading frames, had been integrated between the Ecokl
`site and position —— l5 of the lac promoter. The presence
`of the stop codons made the fragment an efficient termi-
`nator of any translation that occurred upstream of the
`translational initiation site for the ct-fragment.
`When the Hindlll/Sall segment of p}3Ul2a was re-
`placed with an identically generated fragment of
`pLl3Ul carrying the id terminator. the plasmid pLBU3
`was obtained, which conferred neither Tc resistance
`nor 8~gal activity to E. colt‘ M15 strain. Although this
`plasmid contained a tet region and a DNA sequence
`encoding the <1-fragment of lac, it conferred neither
`rcsistance nor B—gal activity to the E. coli Mlistrain. It
`was therefore chosen as the T5 promoter cloning vchi~
`ole.
`A population of about 200 short fragments of T5
`DNA was obtained by double digestion of the l20kb
`phage genome with Haelll and Alul cndonuclcases’.
`These fragments were ligated with excess synthetic
`EcoRI links and the resulting molecules were cleaved
`with Beam endonuclease and ligated into the EcoRI
`cleavage site of pLBU3. Transforxnation ofE. coir‘ C600
`and selection for fi~gal activity plus high level Tc»resist~
`ance yielded 35 colonies resistant to Tcwconcentzations
`between 8 and 70 pg/ml. Plasmids were isolated from
`13 colonies resistant to 70 gag/ml, which earlier experi~
`merits using multicopy plasmids had suggested was the
`highest level detectable in E. coli K12 (Cabcllo et al.
`(1976) Nature 259, 285-29,0).
`‘
`Digestion of the various isolates with EcoRI endonu~
`clease liberated between one and ten fr