United States Patent [191
`Bujard et a1.
`
`[11] 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.
`The Board of Trustees of the Leland
`Stanford Jr. University, Stanford,
`Calif.
`
`[73] Assignee:
`
`[21] Appl. No.: 265,276 .
`[22] Filed:
`May 20, 1981
`[51]
`1111.01.3 ....................... .. C12Q l/68; C12Q 1/02;
`CIZP Zl/OO;C1ZP l9/34;C12N 15/00
`[52] US. Cl. ........................................ .. 435/6; 435/29;
`435/68; 435/1723; 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'RIONS
`Stuber et al., PNAS, 78(1), 167-171, (Jan. 1981).
`
`Casadaban et al., J. Mol. Biol, 138, 179-207, (1980).
`West et 211., Gene, 9, 175-193, (1980).
`Gabain et al., PNAS, 76(1), 189-193, (1979).
`Stiiber et a1., Molec. Gen. Genet, 166, 141-149, (1978).
`Stiiber et al., PNAS USA, 78(1), 167-171, (1981).
`Primary Examiner-Esther M. Kepplinger
`Attorney, Agent, or Firm-Bertram 1. Rowland
`[57]
`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 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
`scription of DNA can be achieved with the highly ac
`tive T5 promoters. The promoters and terminators are
`used in hybrid DNA for efficient expression of struc
`tural genes and transcription to provide RNA sequen
`ces.
`
`15 Claims, 1 Drawing Figure
`
`Genzyme Ex. 1002, pg 44
`
`

`
`U.S. Patent
`
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`Genzyme.Ex. 1002, pg 45
`
`Genzyme 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.
`
`l0
`
`is
`
`20
`
`BACKGROUND OF THE INVENTION
`1. 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 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 optimizing the economic and efficient pro
`duction of the polypeptides. Included among these fac
`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 qlled a promoter.
`It has been observed that promoters vary in their activ
`ity, as evidenced by the number of initiations of RNA
`30
`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 transformants
`based on expression of markers which allowed for selec
`tion. Therefore, 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 terminators 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
`Promoters from bacterial and viral sources have been
`cloned in E. 0011', and their signal strength in vitro has
`been studied using expression from distal promoterless
`sequences encoding ,B-galactosidase or other proteins
`(Casadaban and Cohen (1980) J. Mol. Biol 138, 179-207;
`West and Rodriguez (1980) Gene 9, l75—l93). Attempts
`to clone small DNA fragments carrying the strong
`promoters of bacteriophage T5 have been unsuccessful
`(v. Gabain and Bujard (1979) PNAS USA 76, 189, 193),
`Fragments of T5 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
`scriptional regions of several E. 0011' 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 et al (1978) Mol. gen.
`Genet. 166 141-149; Niemann (1981) Diplomarbeit
`(Universitat Heidelberg, Heidelberg, Germany)).
`
`2
`SUMMARY OF THE INVENTION
`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 speci?cally 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 the above described construct.
`The construct or regulatory portions thereof are used
`for efficient transcription of RNA or gene expression.
`
`BRIEF DESCRIPTION OF THE DRAWING
`FIG. 1 is a flow chart of the preparation of a plasmid
`for cloning strong promoters and terminators.
`
`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 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 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
`and eukaryotic promoters. either naturally occurring or
`synthetic, could find application in the subject inven
`
`25
`
`35
`
`55
`
`00
`
`tion.
`_
`The compositions of this invention will include linear
`segments for insertion of DNA having the strong regu~
`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.
`
`65
`
`Genzyme Ex. 1002, pg 46
`
`

`
`3
`The termini 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 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 plurality
`of translational stop codons e.g. oop or nonsense co~
`dons, or preferably a plurality, usually up to about six,
`more usually from about two to ?ve, 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 promoter.’ Usu
`ally, it will be desirable that a suf?cient amount of the
`marker is transcribed to allow for selection of transfor
`mants. 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 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 re?ected 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 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 host. 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
`
`45
`
`55
`
`65
`
`4,495,280
`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 ?exibility in preparing the con
`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 a single unit or as individual subunits and
`then joined together in appropriate ways. Furthermore,
`as appropriate, the protein products may be modi?ed by
`glycosylation, acylation with aliphatic acids, e.g. lipid
`acids, phosphorolation, sulfonation or the like. The
`different classes of proteins which may be prepared
`include protamines, histories, albumins globulins, sclero
`proteins, phosphoproteins, mucoproteins, chromopro
`teins, lipoproteins, nucleoproteins, and the remaining
`proteins which are unclassi?ed.
`The following is a representative list of proteins of
`interest.
`_
`
`Insulin; growth hormone; interferon e.g. leukocyte,
`lumphoblastoid, T-immune and ?broblast; ,B-lipotro
`pin; B-endorphin; dynorphin; histocompatability pro
`teins; immunoglobulins e.g. IgA, IgD, IgE, IgG and
`IgM and fragments thereof; hemoglobin, somatome
`dins; lymphokines; growth factors e.g. epidermal,
`?broblast, platelet-derived, multiplication stimulating
`and nerve; hematoporetic-stimulating factors e.g.
`erythropoietin, colony-stimulating, erythroid poten
`tating activity or burst-promoting activity and lym
`phopoietins; albumin and prealbumin;
`Prealbumin
`Albumin
`al-Lipoprotein
`al-Acid glycoprotein
`ai-Antitrypsin
`a i-Glycoprotein
`Transcortin
`' 4.6S-Postalbumin
`
`Tryptophan-poor
`cal-glycoprotein
`m-x-Glycoprotein
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin:
`(Gc 1-1),
`(Gc 2-1),
`(Gc 2-2),
`Haptoglobin:
`(HP 1'1),
`2_1)v
`(HP 2-2),
`Ceruloplasmin
`Cholinesterase
`a2-Lipoprotein(s)
`ag-Macroglobulin
`az-Hs-Glycoprotein
`
`Genzyme Ex. 1002, pg 47
`
`

`
`4,495,280
`
`6
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF): CRF, LRF, TRF, Somatotro
`pin-RF, GRF, FSHRF, PIF, MIF.
`
`5
`
`Zn-ag-glycoprotein
`az-Neuramino-glycoprotein
`Erythropoietin
`B-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`az-glycoprotein I
`al-glycoprotein II
`Immunoglobulin G
`(IgG) or 'yG-globulin
`Mol. formula:
`
`In addition to various non'enzymatic proteins of
`physiological interest, enzymes can also be produced as
`an end product or for intracellular transformation of a
`substrate present in the host or substrate introduced
`extracellularly, or for enzymatic transformation in vi
`tro.
`In accordance with the I.U.B. classi?cation, the en
`zymes fall into varying categories such as l. oxidore
`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; oxidoreduc
`tases for oxidation of alcohols and aldehydes, epoxida
`tion, and the like.
`The microorganism host may be bacteria, such as
`Escherichia, Bacillus, Aerobacter, Klebsiella, Proteus,
`Pseudomonas, Streptococcus, Staphylococcus, Clo
`stridium, Mycobacterium, Streptomyces and Actino
`myces; Fungi e.g. Gymnomycota, Dimastygomycota,
`Eumycota, Zygomycetes, Ascomycetes and Basidomy
`cetes, such as, Candida, Aspergillus, Rhizobus, Micro
`sporum, and Fonsecaea; Protozoa e.g. Mastigophora,
`Sarcodina, Sporozoa and Celiophora, such as, Trypano
`soma, Codosiga, Protospongra and Entameba, and Alga
`e.g. Dinoflagellates, Euglenoids, and Diatoms.
`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 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 developed which
`may have one or more other regulatory signals. Regula
`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 e. g. 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 pro
`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, a ?rst gene
`marker, optionally termination codons, 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
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Immunoglobulin A (IgA) or yA-globulin
`Mol. formula:
`(022m)" Or (a27t2)"
`Immunoglobulin M (IgM) or yM-globulin
`M01. formula:
`(1L2K2)5 0r (P2>\2)5
`Immunoglobulin D (IgD) or 'yD-Globulin (yD)
`Mol. formula:
`(52K2) 0r (32M)
`lmmunoglobulin E (IgE) or yE-Globulin (7E)
`Mol. formula:
`(62K2) 0r (62M)
`Free light chains
`Complement factors:
`C’l
`
`3 1A
`112D
`C4
`C5
`C6
`G7
`G8
`G9.
`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
`mone)
`Luteomammotropic hormone: (Luteotropin, prolactin)
`Gonadotropin: (chorionic gonadotropin).
`Tissue Hormones
`
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`
`Genzyme 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
`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 ?exibility, 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 con
`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
`speci?cities commercially available.
`The following examples are offered by way of illus
`tration and not by way of limitation.
`'
`
`20
`
`15
`
`25
`
`30
`
`7
`terminator are properly balanced, there will be a sub
`stantial differentiation between expression of the ?rst
`gene marker and expression of the second gene marker.
`The ratio of expression between the ?rst 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 extrachromo
`somal element, such. as a~plasmid 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 determining the relative proportion of
`expression of the ?rst and second gene markers, one can
`determine the strength of promoters in relation to a
`?xed 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
`tance 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 biosyn
`thetic pathway. A third marker provides virus incom
`patibility, preventing plaque formation. Other 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 de
`pendency of the terminator on rho, as well as the effec
`tiveness of the terminator in relation to the concentra~
`tion 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 pro
`vide fragments having the desired intact sequence or
`gene. The fragments can be further processed employ
`ing endo- 0r exonucleases to remove nucleotides unre
`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
`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 a 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).
`
`4-0
`
`45
`
`55
`
`65
`
`EXPERIMENTAL
`Materials and Methods
`Restriction endonucleases HindIII, HincII, SalI,
`BamHI and T4 Ligase were from New England Biolabs
`(Beverly, Mass., USA), Sau3A was from BRL (Neu
`Isenburg, Germany) and EcoRI and HpaII from (Bo
`ehringer Mannheim, Federal Republic of Germany).
`DNA polymerase I, phage fd DNA ‘(replicative form),
`plasmid ADl6/3O containing a 28 bp HindIII/BamHI
`adapter sequence and, lac represser were supplied by
`private sources. The isolation of bacteriophage T5
`DNA, plasmid DNA E. coli RNA polymerase and E.
`coli termination factor rho have been described previ
`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
`latory region (promoter/operator: P/O), an N-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
`HincII digest of a pACYC 214 plasmid (a plasmid re
`lated to pACYC 184 by insertion at a BamHI site of a
`BamHI restriction cleavage fragment from F’-lac carry
`ing the lac gene) by repressor binding and subsequent
`
`Genzyme Ex. 1002, pg 49
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`10
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`30
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`35
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`40
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`adsorption to nitrocellose. This fragment was then em
`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 SalI 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 ((1). Introduction
`of this fragment by blunt end ligation into the HindIII
`site of pACYCl84 (Chang and Cohen (1978) J. Bac
`teriol. 134, 1141-1156) 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 BamHI/HindIII adaptor
`sequence and integrated into pBUlO to yield pLBUl.
`Cleavage of the lac sequence in pBUlO by HpaII
`destroys the lac promoter and liberates upon cleavage
`with HindIII a fragment containing the lac operator and
`a region coding for a functional a-fragment. Integrating
`this DNA sequence into pBR322 leads to pBUl2a. Fi
`nally, replacement of the HindIIl/Sall portion of 25
`pBUl2a by a partial digest of the HindIII/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
`(Tc) resistance are indicated as cat, bla and tet respec
`tively.
`The plasmid, pBUlO, had the following properties: (i)
`it contains the a fragment of B-galactosidase (a-protein)
`and complemented the M15 deletion of the lac operon;
`(ii) the Te resistance it speci?ed was under the control
`of the lac promoter, as shown in M15 I‘! strains; (iii) the
`Hindlll site between the lac gene fragment and the tet
`gene was restored; the stop codon immediately follow
`ing the HindIII site limited the length of the lacZ gene
`product to 68 amino acids.
`Insertion of the fd terminator upstream to the tet gene
`resulted in a 90% reduction in the level of Tc resistance,
`but no detectable change in the levels of B-galactosidase
`activity in M15 deletion strains. The results obtained as
`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
`tained the expected 352bp HindIII-generated fragment
`containing the fd terminator. Electrophoretic analysis
`of all seven BamHI-cleaved isolates and DNA sequence
`analysis of one of these showed that in all instances the
`id terminator had been integrated in an orientation op
`posite the direction of transcription within the fd phage
`genome. Sequence analysis also revealed a translational
`stop codon in frame with the a-protein less than lObp
`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
`ment containing 71 amino acids.
`For the cloning of exogenous promoters, the lac pro
`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
`HpaII cleavage site at position —- 17 of lac was em
`ployed, as described above. Colonies that showed both
`a reduced level of To resistance and the presence of a
`
`4,495,280
`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
`?ed. 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 pBUl2a 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 EcoRI
`site and position — 15 of the lac promoter. The presence
`of the stop codons made the fragment an ef?cient termi
`nator of any translation that occurred upstream of the
`translational initiation site for the a-fragment.
`When the HindIII/SalI segment of pBUl2a was re
`placed with an identically generated fragment of
`pLBUl carrying the fd terminator, the plasmid pLBU3
`was obtained, which conferred neither Tc resistance
`nor B-gal activity to E. coli M15 strain. Although this
`plasmid contained a tet region and a DNA sequence
`encoding the a-fragment of lac, it conferred neither
`resistance nor B—gal activity to the E. coli M15 strain. It
`was therefore chosen as the T5 promoter cloning vehi
`cle.
`A population of about 200 short fragments of T5
`DNA was obtained by double digestion of the l20kb
`phage genome with HaeIII and AluI endonucleases.
`These fragments were ligated with excess synthetic
`EcoRI links and the resulting molecules were cleaved
`with EcoRI endonuclease and ligated into the EcoRI
`cleavage site of pLBU3. Transformation of E. coli C600
`and selection for B~gal activity plus high level Tc-resist
`ance yielded 35 colonies resistant to Tc-concentrations
`between 8 and 70 ug/ml. Plasmids were isolated from
`13 colonies resistant to 70 ug/ml, which earlier experi
`ments using multicopy plasmids had suggested was the
`highest level detectable in E. coli K12 (Cabello et a1.
`(1976) Nature 259, 285-290).
`Digestion of the various isolates with EcoRI endonu
`clease liberated between one and ten fragments of vari
`ous sizes from each constructed plasmid. Complexing of