United States Patent (19J
`Taylor
`
`IP'atent NWl!llber:
`[11]
`[ 45] Date of Patent:
`
`4,476,227
`Oct. 9, 1984
`
`[54] COSMm CLONING VECfORS
`
`[75]
`
`Inventor: Dean P. Taylor, King of Prussia, Pa.
`
`[73] Assignee: Smitbkline Bec!rnuan Corporation,
`Philadelphia, Pa.
`
`[21] Appl. No.: 433,062
`
`[22] Filed:
`
`Oct. 6, 1982
`
`[51]
`
`Int. CI,3 ....................... C12N 1!00; C12N 15/00;
`C12N 1/20; Cl2P 21/00; Cl2P 19/34
`[52] U.S. CI .................................. 435/317; 435/172.3;
`435/68;435/91; 435/253
`[58] Field of Seareb ..................... ,435/68, 70, 91, 172,
`435/253,317, 172.3
`
`(56]
`
`IRererences Cited
`U.S. PATENT DOCUMENTS
`4,304,863 12/1981 Collins .
`
`FOREIGN PATENT DOCUMENTS
`2462476 3/1981 France .
`
`OTHER PUBLICATIONS
`Hohn, Methods in Enzymology 68:299-309 (1979).
`Collins, Methods in Enzymology 68:309-326 (1979).
`
`Collins and Hohn, Proc. Nat!. Acad. Sci. U.S.A.
`75(9):4242-4246 (1978).
`Meyerowitz eta!., Gene !1:271-282 (1980).
`Hohn and Collins, Gene 11:291-298 (1980).
`Plasmid 8:100 (1982).
`Taylor et al., J. Bacteriol. 137(1):92-104 (1979).
`Hashimoto-Gotoh eta!., Gene 16:227-235 (1981).
`Baldacci et al., Nucl. Acids. Res. 9:3575-3588 (1981).
`Marcoli eta!., FEBS Letters 110(1):11-14 (1980).
`Alton eta!., Nature 282:864-869 (1979).
`Nichols et a!., J. Virol. 26(2):429-434 (1978).
`Sutcliffe, Cold Spring Harbor Symposium 43:77-90
`(1979).
`Primary Examiner-Thomas Wiseman
`Assistant Examiner-James Martinell
`Attorney, Agent, or Firm-Edward! T. Lentz; Janice E.
`Williams; Alan D. Lourie
`[57]
`ABSTIRAICT
`Small cosmid cloning vectors having two antibiotic
`resistance markers, selected from chloramphemicol
`resistance, streptomycin and spectinomycin resistance
`and tetracycline resistance markers, at least one having
`a unique restriction site.
`
`6 Claims, 7 Drawing Figures
`
`.::::--
`
`;:;:::
`0
`
`..... s
`~f
`
`Bell
`
`8
`I
`'\
`
`pDPT5SP
`
`SmRfSpR
`\ ...
`
`....__
`cos
`
`Sanofi/Regeneron Ex. 1036, pg 971
`
`Merck Ex. 1036, pg 997
`
`

`
`U.S. Patelllt Oct. 9, 1984
`
`Sheet 1 of 4
`
`4,476,227
`
`pDPT272
`MUA-5
`EcoRI (partial)
`EcoRI
`~stl ""·'-"''""m••••~II!IIIIIIIIIIIIIIIBIIMNIIIIIIIII _____ . / Pstl
`
`pDPT5Cm
`(TcRspscmR)
`
`pDPT5Sp
`(TcRspRcms)
`
`pDPT270
`MUA-5
`EcoRI
`Bell
`'-....,s_1_ .. ,_~-~~~~-'"'•..,.•--/ DNApoll
`
`t Pstl
`
`pDPT6
`(CmRTcR)
`
`pDPT270
`Pstl
`Pvull
`
`'-...., __ ,.._;'Ill·---_..../ Pstl
`
`MUA-5
`EcoRI
`DNApoll
`
`pDPT7
`(SpRTcRt. cos)
`
`pDPT7
`Hindlll
`
`pDPT5Sp
`Hind Ill
`
`pDPTB
`~ Hindlll
`
`pDPT9
`(SpRTcR)
`
`Figure 1
`
`Sanofi/Regeneron Ex. 1 036, pg 972
`
`Merck Ex. 1036, pg 998
`
`

`
`U.S. Patent Oct. 9, 1984
`
`Sheet 2 of 4
`
`4,476,227
`
`Hi nell
`
`pDPT270
`
`Pstl
`
`Figure 2
`
`Figure 3
`
`Sanofi/Regeneron Ex. 1 036, pg 973
`
`Merck Ex. 1036, pg 999
`
`

`
`U.S. Patent Oct. 9, 1984
`
`Sheet 3 of 4
`
`4.,476,227
`
`pDPT5Cm
`
`cos --
`
`Figure 4
`
`Bell
`
`I
`I
`1
`
`\
`
`pDPT5SP
`
`SmRfSpR
`
`\
`
`\
`
`cos --
`
`Figure 5
`
`Sanofi/Regeneron Ex. 1 036, pg 97 4
`
`Merck Ex. 1036, pg 1000
`
`

`
`U.S. Patent Oct. 9, 1984
`
`Sheet 4 of 4
`
`4,476,227
`
`Pvu11
`Pstt
`
`Figure 6
`
`Figure 7
`
`Sanofi/Regeneron Ex. 1036, pg 975
`
`Merck Ex. 1036, pg 1001
`
`

`
`4,476,227
`
`COSMID CJLONING VECTORS
`
`FIELD OF THE INVENTION
`This invention relates to cosmid cloning vectors and 5
`their use in cloning large fragments of foreign DNA in
`bacteria.
`
`15
`
`BACKGROUND OF THE INVENTION
`A cosmid cloning vector is a plasmid which contains 10
`a lambda or lambdoid phage cos site, that is, the recog(cid:173)
`nition site essential for packaging oflinear concatenated
`DNA into lambda or lambdoid phage heads; a replicon,
`at least one selectable genetic marker; and at least one
`unique restriction endonuclease site in a nonessential
`region. These DNA sequences are herein referred to as
`"cosmid DNA".
`Cosmid cloning vectors can be used to clone foreign
`DNA by inserting the foreign DNA into a restriction · 20
`site in the cosmid DNA, packaging the recombinant
`DNA in infectious phage particles, preferably in vitro,
`and transfecting E. coli host cells. Once introduced into
`the host cells, the cosmids replicate in the manner of
`plasmids, due to the presence of the replicon. Trans- 25
`fected cells are selected based on a phenotypic trait
`contributed by the genetic marker, usually an antibiotic
`resistance marker.
`Packaging is most efficient when the linear DNA to
`be packaged inside the phage head is about 23 to 31 30
`Megadaltons (Md), that is, about 38 to 52 kilobase pairs
`(kb). Cosmid DNA can be considerably smaller than 52
`kb. Thus, cosmid cloning vectors can be suitable for
`cloning large DNA fragments such as eukaryotic genes,
`which are typically interspersed in large sequences, and 35
`gene banks or libraries.
`Hohn, Methods in Enzymology 68:299-309 (1979), and
`Collins, Methods in Enzymology 68:309-326 (1979), de(cid:173)
`scribe the morphogenesis oflambda phage particles and
`in vitro packaging Of cosmid vectors. Several known 40
`cosmid vectors are described.
`Collins and Hohn, Proc. Natl. Acad. Sci. U.S.A.
`75{9):4242-4246 {1978), and Collins and Hohn, U.S. Pat.
`No. 4,304,863, disclose preparation of cosmids pJC703
`and pJC720, 17.3 Md and 16 Md, respectively, each 45
`having a rifampicin resistance (RifR) marker, and a
`cosmid of about 27.5 Md having RifR and ampicillin
`resistance (ApR) markers. The authors also disclose
`methods for in vitro packaging and transfection using
`cosmid cloning vectors containing foreign DNA.
`Meyerowitz et al., Gene 11:271-282 (1980), describe
`construction ofcosmids MUA-3, MUA-5 and MUA-10
`and of eukaryotic gene libraries using MUA-3. The
`cosmids are about 3 Md and have tetracycline resistance
`(TcR) markers.
`Hohn and Collins, Gene 11:291-298 (1980), describe
`construction of cosmid pHC79, about 6.4 kb, having the
`two markers of plasmid pBR322, ApR and TcR.
`At the Fifth Annual Mid-Atlantic Regional Confer(cid:173)
`ence on Extrachromosomal Genetic Elements, Oct. 60
`9-ll, 1981, Ocean City, Md., U.S.A., Taylor, the inven(cid:173)
`tor of the cosmid cloning vector disclosed and claimed
`herein, reported cosmids pDPT5Cm and pDPT5Sp,
`each having two genetic markers: chloramphenicol
`resistance (CmR) and TcR in the case ofpDPTSCm, and 65
`streptomycin and spectinomycin resistance (SmRJSpR)
`and TcR in the case of pDPTSSp. An abstract of this
`presentation is published at Plasmid 8:100 (1982).
`
`50
`
`55
`
`2
`Hashimoto-Gotch et al., Gene 16:227-235 (1981),
`describe construction of cosmid pHSG422, about 8.8
`kb, having ApR, CmR and kanamycin resistance (KmR)
`markers.
`Baldacci et al., Nuc/. Acids. Res. 9:3575-3588 (1981),
`describe cosmids pFF1 and pFF2, having a KmR
`marker. The cosmid, pFFI, is claimed in French Patent
`No. 2,462,476 (Derwent Accession Number 33928D).
`As stated previously, there is an upper limit of about
`52 kb on the size of cosmid cloning vectors which can
`be packaged efficiently. Therefore, in order to clone
`large foreign DNA fragments, it is desirable to have
`cosmid cloning vectors in which the cosmid DNA, that
`is, the DNA carrying the cos site,, a replicon, a marker
`and a unique restriction site, is small. Further, to facili(cid:173)
`tate selection of transfected cells containing the desired
`cosmid vector and for subsequent subcloning, it is desir(cid:173)
`able to have cosmid vectors containing two genetic
`markers with at least one of the markers having a unique
`restriction site such that insertion of foreign DNA into
`that site detectabl y affects the phenotype of transfected
`cells by insertional inactivation of the marker.
`
`SUMMARY OF THE INVENTION
`The invention provides a cosmid cloning vector
`wherein the cosmid DNA, in addition to a cos site and
`a replicon, contains two selectable genetic markers
`selected from the group consisting of CmR, TcR and
`SmR!SpR. Particular embodiments of the invention
`include the vector wherein the cosmid DNA is no more
`than about 7.5 kb, the markers are CmR and TcR or
`SmR!SpR and TcR and at least one of the markers has a
`unique restriction site for insertional inactivation. The
`invention also includes a plasmid cloning vector having
`SmRJSpR and TcR markers. The invention includes
`equivalents of the vectors claimed herein, such as, for
`example, RNA and single strand DNA.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`Following is a brief description of the drawings
`which are not drawn to scale but are illustrative of the
`invention or materials which may ibe used in practicing
`the invention.
`FIG. 1 is a flow sheet illustrating preparation of cos(cid:173)
`mid cloning vectors of the invention.
`FIG. 2 is a restriction endonuclease cleavage map of
`pDPT270 showing a deletion for construction of
`pDPT272.
`FIG. 3 is a restriction endonuclease cleavage map of
`MUA-5.
`.
`FIG. 4 is a restriction endonuclease cleavage map of
`pDPT5Cm.
`FIG. 5 is a restriction endonuclease cleavage map of
`pDPT5Sp.
`FIG. 6 is a restriction endonuclease cleavage map of
`pDPT6.
`FIG. 7 is a restriction endonuclease cleavage map of
`pDPT9.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`In the cosmid cloning vector of the invention, the
`cosmid DNA contains two selectable genetic markers
`selected from the group consisting of CmR, SmRJSpR
`and TcR. The vector of the invention includes vectors
`substantially containing only cosmid DNA as well as
`recombinant molecules comprising cosmid DNA and
`foreign DNA. By "foreign DNA" is meant DNA other
`
`Sanofi/Regeneron Ex. 1 036, pg 976
`
`Merck Ex. 1036, pg 1002
`
`

`
`4,476,227
`
`10
`
`3
`than cosmid DNA. Foreign DNA can be of eukaryotic
`or prokaryotic origin and might include, for example,
`one or more genes for expression and production of
`commercially useful products; one or more replicons
`recognized by other species, making the cosmid useful
`as a shuttle vector; and a deoxynucleotide sequence
`comprising all or part of an organism's genome, for
`banking or experimentation. The invention includes all
`forms of the vector, such as, for example, linear, con(cid:173)
`catarneric and circular.
`The preferred embodiment of the invention is about
`5.4 kb and has a CmR marker and a TcR marker. Such
`cosmid is exemplified by pDPT6, characterized sub(cid:173)
`stantially as shown in FIG. 6. The cosmid DNA of
`pDPT6 has substantially the following unique sites: 15
`EcoRI in the CmR gene; Hindiii, BamHI, Sphl, Sail,
`HincH, Xmaiii and Nrul in the TcR gene; and Clal,
`A val and Pstl. All of the unique sites are suitable for
`'cloning. The cosmid DNA lacks restriction sites for
`substantially the following restriction endonucleases: 20
`Kpnl, Bgiii, Hpal, Xhol, Stu!, Smal, 'BstEII, Sacii and
`Pvul. The replicon is derived from pBR322 and is posi(cid:173)
`tioned between the downstream end of the TcR gene
`and the cos site.
`In this context, by "substantially" is meant that the 25
`relative positions of the restriction sites and cosmid
`DNA sequences as illustrated and described are sub(cid:173)
`stantially accurate, that one or more restriction sites can
`be lost or gained by mutations not otherwise affecting
`the vector, and that additional sites for endonucleases 30
`which were not used in mapping procedures probably
`exist.
`In pDPT6, the TcR and CmR genes are promoted
`independently, that is, each has its own promoter; tran(cid:173)
`scription or inactivation of one marker does not affect 35
`the other. The TcR promoter can be inactivated by
`insertion into the Hindlll site immediately upstream of
`the TcR structural gene. Both markers are transcribed in
`the same direction.
`A second embodiment of the invention is exemplified 40
`by pDPT9, which is characterized substantially as
`shown in FIG. 7. It is about 6.3 kb and has a SmRfSpR
`marker and a TcR marker. The cosmid DNA has sub(cid:173)
`stantially the following unique restriction sites: BstEII
`and Bell in the SmRfSpR gene; BamHI, Sail, Hincii 45
`and Nrul, in the TcR gene; and EcoRI, Cia!, Ball,
`Pvull, Pstl, Stu! and Sacll. All of the unique sites are
`suitable for cloning, although cloning in sites in the
`replicon region, such as the Pvuii site, may adversely
`affect the replicon. The cosmid DNA lacks restriction 50
`sites for substantially the following restriction endonu(cid:173)
`cleases: Kpnl, Xhol, Bgiii and Hpal. The replicon,
`which is positioned between the downstream end of the
`TcR gene and the cos site, is (lerived from pBR322. In
`pDPT9, the markers are promoted independently, but 55
`in opposite directions.
`A third embodiment of the invention is exemplified
`by pDPT8. This cosmid is similar to pDPT9, differing
`in the orientation of the Hindiii sequence carrying the
`SmR!SpR structural gene and promoter and the TcR 60
`promoter. It is believed that both markers are tran(cid:173)
`scribed in the same direction from the SmR!SpR pro(cid:173)
`moter and that insertional inactivation of the SmRfSpR
`marker will, therefore, also inactivate the TcR gene.
`Other cosmid vectors of the invention are exempli- 65
`tied by pDPT5Cm and pDPT5Sp which are character(cid:173)
`ized substantially as shown in FIGS. 4 and 5, respec(cid:173)
`tively. pDPT5Cm is about 7.4 kb and has TcR and CmR
`
`4
`markers, the former having unique Hindlll, BamHI,
`Sphl and Sail sites. Additionally, pDPT5Cm has unique
`sites for Pstl and Bell. pDPT5Sp is 7.1 kb and has TcR
`and SmR!SpR markers, the former having unique Sphl
`and Sail sites and the latter having unique Bell and
`BstEII sites. Other unique sites in pDPTSSp are for
`EcoRI and Pstl. The replicons in both vectors are de(cid:173)
`rived from pBR322 and lie between the TcR markers
`and the cos sites as in pDPT6 and pDPT9.
`The cosmid cloning vectors of the invention can be
`prepared by ligating DNA fragments containing the
`lambda cos site, an E. coli replicon and two antibiotic
`resistance markers selected from CmR, SmRfSpR and
`TcR. A DNA fragment containing the cos site and a
`replicon can be prepared by releasing the fragment
`from another cosmid such as, for example, MUA-3,
`MUA-5, MUA-10, pDPT5Sp, pDPT5Cm, pDPT6,
`pDPT8 and pDPT9. MUA-3, -5 and -10, are described
`by Meyerowitz et al, Gene 11:271-282 (1980). Both can
`be constructed from pBR322 and
`lambda DNA.
`MUA-3 and pBR322 are on deposit in the American
`Type Culture Collection, Rockville, Md., U.S.A., under
`accession numbers 37069 and 37017, respectively.
`Insertion of foreign DNA into cosmid DNA to pre-
`pare a cosmid cloning vector of the invention, packag(cid:173)
`ing of the vector and transfection of host cells can be
`carried out by known techniques. Useful techniques are
`disclosed in the references cited above, in the back-
`ground section. There is preferably sufficient foreign
`DNA to form a recombinant molecule in which the
`cosmid DNA and the foreign DNA total about 38 to 52
`kb.
`The above-described cosmid cloning vectors of the
`invention are desirable because their cosmid DNA is
`small, has many restriction sites suitable for cloning and
`subcloning, is comprised of largely defmed DNA se(cid:173)
`quences, contains a multicopy replicon, can be ampli(cid:173)
`fied, is mobilizable only in the presence of a conjugal
`plasmid if mobilization functions are provided in trans,
`is reasonably stable and contains new combinations of
`antibiotic resistance markers, offering valuable alterna(cid:173)
`tive genetic engineering tools. The last-noted fact
`makes the vectors especially useful tools when used, for
`example, in transfection or transformation of cells with
`one or more additional vectors having complementary
`resistance markers, such as, for example pACYC177,
`pSC101 and pHSG422. The vectors also provide alter(cid:173)
`native unique restrictions sites, simplifying cloning of
`restriction fragments using unique restriction sites not
`present in other vectors.
`CmR and SmRfSpR markers permit more efficient
`selection than many other antibiotic resistance markers
`because their products do not interfere with the action
`of the respective antibiotic on non-transfected or non(cid:173)
`producing cells. By contrast, .8-Iactamase, the product
`of the ApR gene, for example, is excreted from produc(cid:173)
`ing cells and acts on ampicillin in the medium. CmR and
`SmRfSpR markers have additional advantages. For
`e/(ample, their products are small, singular proteins
`produced in relatively small amounts. The products of
`certain other resistance markers, including ApR, are
`large, often fragmented into various size pieces, and are
`produced in relatively large amounts and, therefore,
`may interfere with analysis of gene products. By way of
`further example, chloramphenicol and spectinomycin
`are stable in stock solutions making their use more con(cid:173)
`venient than use of several other antibiotics, including
`
`Sanofi/Regeneron Ex. 1 036, pg 977
`
`Merck Ex. 1036, pg 1003
`
`

`
`4,476,227
`
`6
`23, 1982 under accession numbers NRRL B-15159 and
`B-15160, respectively.
`
`5
`rifampicin, tetracycline and ampicillin, which tend to be
`relatively unstable or light sensitive.
`Particular advantages offered by vectors of the in(cid:173)
`vention having SmR!SpR include that the marker can be
`used to select transfectants among cells which are SmR
`but not SpR, such as E. coli HBIOI. Particular advan(cid:173)
`tages offered by vectors of the invention having CmR
`include that the marker is expressed in Streptomyces.
`The plasmid cloning vector of the invention is exem(cid:173)
`plified by pDPT7 which is about 6.1 kb. It is similar to 10
`pDPT9, except that, as explained in Example 3, below,
`it has an additional Hindiii site between the Hindiii
`and Sacll sites downstream of the SmR!SpR gene and
`lacks the cos site and Pstl site. With the exception of the
`Pstl site, pDPT7 has the same unique restriction sites as 15
`pDPT9.
`
`EXAMPLE 2. Construction of pDPT6
`The object of this example was to prepare a smaller,
`and hence more useful, TcR CmR cosmid cloning vec(cid:173)
`tor. The procedure followed is outlined in FIG. 1.
`MUA-5 DNA was digested with EcoRI and the sin(cid:173)
`gle strand ends were filled in using DNA polymerase I
`to produce a linear, blunt-ended fragment. pDPT270
`DNA was digested with Bell. A 0.9 Md Bell fragment
`carrying the CmR marker was JPUrified by agarose gel
`electrophoresis and electroelution. The purified CmR
`fragment, 3X 10-6 g, was treated with 0.1 unit of Sl
`nuclease for 10 minutes at 37', extracted with phenol
`and ether, and precipitated with ethanol.
`The CmR fragment, 3X to-6 g, and 2X to-6 g of the
`EcoRI-cut and repaired MUA-5 were mixed and di(cid:173)
`gested with 10 units of Pstl for 2 hours at 37'. The
`mixture was incubated at 65' for 10 minutes and then
`diluted to 0.1 ml and ligated for more than 12 hours at
`15' using T4 DNA ligase.
`E. coli K 12 strain MM294 was treated with CaCb and
`transformed with the ligated fragments. TcR CmR trans-
`25 formants were selected and found to contain cosmid
`pDPT6, illustrated in FIG. 6, which consists of a Bell
`(nuclease-blunted)-Pstl fragment of pDPT270 carrying
`the CmR marker and a EcoRI(polymerase-repaired)-
`Pstl fragment of MUA-5 carrying the TcR marker, the
`cos site and the replicon. This cosmid has been depos(cid:173)
`ited within E. coli Kl2 strain MM294(pDPT6) in accor-
`dance with the Budapest Treaty and USPTO and EPO
`regulations in the Agricultural Research Culture Col(cid:173)
`lection, Peoria, Ill., U.S.A., on Sept. 23, 1982 under
`accession number NRRL-B-15161.
`
`EXAMPLES
`In the following examples, which are illustrative and
`not limiting, all temperatures are in degress Celsius. 20
`Enzymes and other reagents used in the Examples are
`commercially available. Buffers used in the Examples
`were prepared substantially in accordance with ven(cid:173)
`dors' recommendations.
`
`40
`
`EXAMPLE 1. Construction of pDPT5Cm and
`pDPT5Sp
`The object of this example was to insert an additional
`antibiotic resistance marker into MUA-5, illustrated in 30
`FIG. 3. MUA-5 was provided by Dr. Gregory M.
`Guild, University of Pennsylvania, Philadelphia, Pa.,
`U.S.A. The additional resistance markers were derived
`from pDPT272 which was prepared by in vitro EcoRI
`deletion of pDPT270, the latter being described by 35
`J. Bacterial.
`Taylor et al.,
`137(1):92-104(1979).
`pDPT270 is iilustrated in FIG. 2. The bar in FIG. 2,
`marked delta, illustrates the EcoRI fragments deleted
`from pDPT270 to construct pSPT272. The procedure
`followed is outlined in FIG. 1.
`Purified DNA of MUA-5 and pDPT272, was sepa(cid:173)
`rately digested with Pstl and EcoRI. Varying concen(cid:173)
`trations of EcoRI were used to assure that partial diges(cid:173)
`tions of pDPT272 would produce· EcoRI-Pstl frag(cid:173)
`ments carrying the SmR!SpR marker and the CmR 45
`marker. The fragments from MUA-5 and pDPT272
`were mixed without further purification and ligated at a
`concentration of 10-5 g/mL at 15' for over 12 hours
`using 20-40 units ofT 4 DNA ligase in a volume of 0.1
`mL. The ligated fragments were then transformed into 50
`cells of E. coli K12 strain MM294 made competent by
`treatment with CaCI2.
`TcR CmR and TcR SpR transformants were selected
`on selective medium. Candidate recombinant plasmids
`were purified by cesium chloride density gradient cen- 55
`trifugation and analyzed by restriction endonuclease
`digestion. A TcR CmR candidate having a restriction
`pattern corresponding to the map illustrated in FIG. 4
`was designated pDPT5Cm. A TcR SpR candidate hav(cid:173)
`ing a restriction pattern corresponding to the map ill us- 60
`trated in FIG. 5 was designated pDPT5Sp. Cultures of
`coli Kl2
`strains MM294(pDPT5Cm)
`and
`E.
`MM294(pDPT5Sp),
`carrying
`pDPT5Cm
`and
`pDPT5Sp, respectively, have been desposited in accor(cid:173)
`dance with the Budapest Treaty and U.S. Patent and 65
`Trademark Office ("USPTO") and European Patent
`Office ("EPO") regulations in the Agricultural Re(cid:173)
`search Culture Collection, Peoria, Ill., U.S.A., on Sept.
`
`EXAMPLE 3. Construction of pDPT7 and pDPT8
`The object of this example was to prepare a smaller,
`and hence more useful, TcR SmR;SpR cosmid cloning
`vector. The procedure followed is outlined in FIG. 1.
`MUA-5 DNA was treated as described in Example 2
`to produce an EcoRI(polymerase-repaired) blunt-ended
`linear DNA and then digested with Pstl to excise a
`Psti-EcoRI fragment carrying nonessential DNA.
`pDPT270 was digested with Pstl, Pvuii and EcoRI to
`prepare a 1.5 Md Psti-Pvuii fragment carrying the
`SmR!SpR marker which fragment was readily purified
`from the numerous smaller fragments produced by Pstl,
`EcoRI and Pvull by electrophoresis and electroelution.
`Treated-MUA-5 DNA, 2X Jl)-6 g, and 1.6X J0-6 g
`of the SmR/SpR fragment were ligated in 0.1 mL with
`T4 DNA ligase. The ligated fragments were used to
`transform CaCh-treated E. coli K12 strain MM294.
`TcR SpR transformants were selected and found to con(cid:173)
`tain plasmid pDPT7. This vector has been deposited
`within E. coli Kl2 strain MM294 (pDPT7) in accor(cid:173)
`dance with the Budapest Treaty and USPTO and EPO
`regulations in the Agricultural Research Culture Col(cid:173)
`lection, Peoria, Ill., U.S.A., on Sept. 23, 1982 under
`accession number NRRL B-15162. Analysis by restric(cid:173)
`tion endonuclease digestion revealed that the cos site
`and the Pstl site bordering the cos site in MUA-5 had
`been deleted. Other cosmids having the Pstl site and cos
`site intact can be prepared by the above procedure. A
`Hindlll site near the deletion was intact. The EcoRI((cid:173)
`polymerase-repaired)blunt-ended terminus on MUA-5
`had joined to the Pvull site on the pDPT270 fragment
`regenerating an EcoRI site as desired.
`
`Sanofi/Regeneron Ex. 1 036, pg 978
`
`Merck Ex. 1036, pg 1004
`
`

`
`4,476,227
`
`7
`To prepare pDPT8, a Hindlll fragment of pDPT7,
`carrying the SmR/SpR marker, including the regener(cid:173)
`ated EcoRI site between the SmR/SpR promoter and
`the TcR promoter, was ligated with the largest Hindlll
`fragment of pDPT5Sp, carrying the cos site, the repli(cid:173)
`con and the TcR marker. By this procedure, a cosmid
`containing only two, instead of three, Hindlll sites and
`a Pstl site was prepared, deleting a small (about 150 base
`pairs) Hindiii fragment. The procedure, illustrated in
`FIG. 1, was performed by digesting pDPT7 DNA with to
`Hindiii, BamHI, and Pvuii and pDPTSSp DNA with
`Hindi!!, Pvui and BstEII. The use of the other endonu(cid:173)
`cleases in addition to Hindi II was to reduce the proba(cid:173)
`bility that the undesired fragments would ligate to(cid:173)
`gether to form plasmids that could be selected out of the 15
`ligation mixture upon transformation into E. coli. Each
`digest, O.Sx lQ-6 g, was ligated together in 0.1 mL
`using T4 DNA ligase and the mixture was transformed
`into E. coli Kl2 strain MM294. TcR SpR transformants
`were selected and screened for cosmids of the appropri- 20
`ate size. The cosmid chosen for further study was desig(cid:173)
`nated pDPT8 and mapped with restriction endonu(cid:173)
`cleases. The mapping indicated that the SpR segment
`was in the opposite orientation relative to pDPTSSp
`and pDPT7. In pDPTS, the expression of the TcR gene 25
`is presumed to be driven by transcription initiated at the
`SpR gene promoter reading through the SpR gene into
`the TcR operon. Thus, if a fragment were inserted into
`the SpR gene, inactivating SpR, the expression of TcR
`might also be prevented.
`pDPT8 has been deposited within E. coli Kl2 strain
`MM294(pDPT8) in accordance with the Budapest
`Treaty and USPTO and EPO regulations in the Agri(cid:173)
`cultural Research Culture Collection, Peoria, Ill.,
`U.S.A., on Sept. 23, 1982 under accession number 35
`NRRL B-15163.
`
`8
`ogy 68, 299-309. Samples of packaged DNA were used
`to transfect E. coli Kl2 strain MM294 and CmR colonies
`were selected. The efficiency of recovery of CmR trans(cid:173)
`fectants was estimated at 2.3 X 108/g. Thirty-six percent
`of the CmR transfectants were TcS, indicating that a
`BamHI fragment had been inserted into the cosmid
`vector, inactivating TcR. This frequency of TcS trans(cid:173)
`fectants is not a true indication of the frequency of
`recombinant formation but rather underestimates it.
`When the frequency of KmR transfectants was esti(cid:173)
`mated, almost 30% were TcR. If similar results were
`obtained for all fragments, then the frequency of recom(cid:173)
`binant formation should approach 50%. The TcR re(cid:173)
`combinants probably arose by joining of two vectors in
`a head-to-tail arrangement. The low efficiency given in
`this example is probably due to the unfavorable size
`distribution of the BamHI fragments generated by di(cid:173)
`gestion of the A'::Tn5 DNA and because no efforts
`were made to obtain BamHI fragments of the size
`needed for efficient packaging. Twenty-six clones in(cid:173)
`cluding TcS and four KmR candidates were examined
`by further characterization of plasmid DNA and dem(cid:173)
`onstrated to contain the desired DNA inserts.
`B. The cosmid cloning vector, pDPTSSp, was used
`to clone foreign DNA, namely, EcoRI fragments, in(cid:173)
`cluding one carrying a gene for KmR, of the plasmid
`A'::Tn5, which contains nine EcoRI sites. pDPT5Sp
`DNA was digested completely with EcoRI and A'::Tn5
`30 DNA was partially digested with EcoRI but not size(cid:173)
`fractionated. The DNAs were ligated and packaged as
`described in A, above. SpR transfectants were obtained
`at an efficiency estimated at 2.8X JOII/g. The EcoRI
`site on pDPTSSp is not within either of the antibiotic
`resistance structural genes so insertional inactivation of
`a marker cannot be followed. The frequency of KmR
`transfectants was determined to be 4% and by examin(cid:173)
`ing the DNA of 36 SpR transfectants, 36% were found
`to possess inserts of significant size. As in Example 4,
`the efficiency of packaging and the frequency of inserts
`would have been improved had efforts had been made
`to obtain a more random distribution of fragment sizes
`and to fractionate the DNA to generate DNA most
`suitable for packaging.
`These results demonstrate that the cosmids of the
`invention can be utilized as cosmid cloning vectors. The
`preparation of foreign DNA need not be limited to the
`DNA or the procedures illustrated. For example, a
`structural gene for other protein products can be used
`as the foreign DNA; partial digestion of foreign DNA
`with other endonucleases, such as endonucleases recog-
`nizing a tetrarneric sequence, or by shearing DNA to a
`desired size and adding oligonucleotide linkers can also
`be employed.
`All of the above referenced deposits will be available
`to the public upon issuance of a U.S. Patent on this or a
`related application or upon publication of a European
`Patent Application on this or a related application,
`whichever is earlier.
`I claim:
`· 1. The cosmid cloning vector, pDPT6.
`2. The cosmid cloning vector, pDPT8.
`3. The cosmid cloning vector, pDPT9.
`4. The cosmid cloning vector, pDPTSCm.
`5. The cosmid cloning vector, pDPTSSp.
`6. The plasmid cloning vector, pDPT7.
`* • * • *
`
`EXAMPLE 4. Construction of pDPT9
`The orientation of the SpR segment in pDPT8 was
`reversed by scrambling with Hindiii. pDPT8 DNA, 40
`2X lQ-6 g, was digested with Hindiii, ligated with T4
`DNA ligase and used to transform competent cells of E.
`coli Kl2 strain MM294. TcR SpR transformants were
`selected. Cosmids with the desired orientation were
`obtained by digesting DNA with Sphi and a candidate 45
`with the appropriate size fragments was chosen and
`designated pDPT9, a map of which is illustrated in FIG.
`7. This cosmid has been deposited within E. coli Kl2
`strain MM294(pDPT9) in accordance with the Buda(cid:173)
`pest Treaty and USPTO and EPO regulations in the 50
`Agricultural Research Culture Collection, Peoria, Ill.,
`U.S.A., on Sept. 23, 1982 under accession number
`NRRL-B-1 5164.
`
`55
`
`EXAMPLE 5. Cosmid Cloning of Foreign DNA
`A. The cosmid cloning vector, pDPTSCm, was used
`to clone foreign DNA, namely, BamHI fragments, in(cid:173)
`cluding one carrying a gene for kanamycin resistance
`(KmR), of a plasmid designated A'::Tn5. A'::Tn5 is a
`plasmid with a molecular weight of 6.5 X J07 Md, which 60
`has six BamHI sites. A'::Tn5 DNA was partially di(cid:173)
`gested with BamHI and pDPT5Cm was completely
`digested with BamHI. Digested pDPTSCm, 7.2X lQ-6
`g, and 22X lQ-6 g of digested A'::Tn5 DNA were Ji.
`gated, without fractionation, in 0.02 mL using T4 DNA 65
`ligase. Ligated DNA, !.SX lQ-6 g, was packaged ac(cid:173)
`cording to the procedure ofHohn, Methods in Enzymol-
`
`Sanofi/Regeneron Ex. 1 036, pg 979
`
`Merck Ex. 1036, pg 1005