`_-""'— CHAPTER#
`
`1
`
`The Plasmid, penazz
`Paulina Balbas
`Xavier Soberon
`Francisco Bolivar
`Raymond L. Rodriguez
`
`
`
`Genetic engineering can be defined as the transfer of DNA between hosts
`{or species} by in vitro enzymatic manipulations. This implies that the DNA
`to be transferred will be duplicated in the new host. Since most DNA
`fragments are incapable of self—replication in E. cofi or any other host cell.
`an additional segment of DNA, capable of autonomous replication, must
`be linked to the fragment to be cloned. This autonomously replicating
`fragment is the molecular cloning vector and, by definition, plays a central
`role in recombinant DNA technology. Most cloning vectors were originally
`derived from naturally occurring extrachromosornal elements such as bac-
`teriophage and plasmids. Bacteriophage vectors like M13 and lambda have
`proven to he very useful as cloning vectors and will be discussed in greater
`detail in later chapters (see Chapters-L5).
`Wild-type plasmids such as pSClOl (Cohen and Chang 1973, 1971';
`Cohen et al. 1973) and ColE] (Clewell 1972; Helinslri and Clewell 19?2)
`served as two of the first cloning vectors. Although both plasmids possessed
`
`We would like to thank AJ. Clark and G. EdJin for their helpful discussion and Kimberly
`Srreuch for her help in preparing the manuscript. We would also like lo acknowledge .l. Mireles,
`P. Saucodo, and A. Diaz for the skillful an work and A, Alvarez for conducting the extensive
`literature review needed to write this chapter.
`
`5
`
`Page 1
`Page 1
`
`Mylan v. Genentech
`Mylan V. Genentech
`IPR2016-00710
`Genentech Exhibit 2085
`
`Genentech Exhibit 2085
`
`IPR2016-00710
`
`

`

`6
`
`The Plaza-kt, 1:33:22
`
`certain features that made them useful as vectors (unique restriction sites,
`autonomous replication. and selectable markers).
`there were no vectors
`available at the time that possessed all of these features in one plasmid.
`Although some of these features may not have been anticipated in the
`early days of plasmid construction, most researchers have adhered to the
`following criteria as a guide to the design of cloning Vectors.
`
`1.1 CRITERIA FOR PLASMID VECTOR DESIGN
`
`1. A plasmid vector should be small, with little or no extraneous genetic
`information. Because the efficiency of transformation of many host cells
`decreases as the size of the plasmid approaches 15 kilobases (lrb) and
`above, the vector should contribute as little as possible to the overall
`size of the recombinant molecule.
`2. Vectors should be well characterized with respect to gene number and
`location, restriction enzyme cleavage sites, and nucleotide sequence.
`3. The vector should be easily propagated in the desired host so that large
`quantities of vector and recombinant DNA molecules can be obtained.
`4. The vector should have a selectable marker (gene) to allow cells har—
`boring the vector to be distinguished from nontransformed cells.
`5. An ideal vector should have an additional genetic marker that can be
`activated or inactivated by the insertion of foreign DNA fragments. The
`marker gene will allow cells harboring recombinant molecules to be
`distinguished from nonrecombinant molecules on the basis of altered
`phenotype.
`6. Finally, the vector should pomss the maximum number of unique
`restriction enzyme cleavage sites located in one or the other genetic
`marker. This provides maximum flexibility for cloning different kinds
`of restriction fragments.
`
`1.2 CONSTRUCTION AND STRUCTUFIE
`
`A good example of how plasmid vectors are constructed is shown in Figure
`1—1, which illustrates the various steps involved in the construction of the
`plasmid pBRSZZ and its derivatives (Bolivar et al. 19??a,b; Bolivar 1978;
`Covarrubias et al. 1981; Covarrubias and Bolivar 1982]. The salient points
`in the construction pathway involve the acquisition of a “relaxed mode“ of
`plasmid DNA replication and the ampicillin and tetracycline resistance genes
`to serve interchangeably as genetic markers for selection and insertional
`inactivation.
`Starting with a clinical isolate harboring the colicin—producing plasmid
`pMBl, the relaxed origin of DNA replication was isolated in the form of
`plasmid pMBS (Betlaclr et a]. 1976}. The tetracycline resistance ETC“) gene
`
`m
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`
`
`FIGURE 1-1 A. Schematic representation showing steps in construction of pBRSZZ
`from pMBl. pSCItli. and pRSF‘2124. Restriction enzyme cleavage sites are repre-
`sean as follows; A, Ami; B, Ball; Brnl. Banal-ll; E, 51:012.]; HIII, Hmdlll; HcII,
`Hincll; HpI, HpeII; P, PrtI; PvI, Pml; PvII, Pvull; 8, Soil; Sm, Small. B. Schematic
`representation showing steps in the construction of pBR329 from pBRSZZ, 13312325.
`and 13312328. Solid bars = Tc“; stippled bars = Ap“; hatched bars = Cm“. (Ree
`produced from Rodriguez, R. L., and Tait, R. C. [1983] Recombimm DNA Tech-
`niques: An lnrradumbn. Benjamin Cummings, Menlo Park, Calif.)
`
`Page 2
`Page 2
`
`

`

`1.2 constructions“ Structure
`
`9
`
`.........._ -P2-_---....--_...
`Clal Hindlll
`
`H
`111:1‘chscrrarmrhenrpiaasmcrrrnrcacacrunnrrotrancscmrcnnccnc
`MGQGTfiChAh TGTCGM'iflBTHfiCTITTCGmTTRCéCC
`"'
`thTTiMICGflTTGCGTCMTCCETI-E‘
`er
`=
`__
`m
`mo
`CGTB‘IRIBRRRTCTAMMRIGEGCTCA‘ICBTCRTCDTDBGCMC5TCHECCT559754:‘IGTflGBCflTABBCTTGGTTH'IG
`NETLysserasnnsnfllaLeul Intel 1 IILDuGImi‘Val‘lm-Leunuphl aUalelyl IDEIyLeuualtE‘l‘
`SCFCAI'flET T1 mu 3' 'I all:6I:6fl5TmhfiTAEGFGCEGTGBCRETGWCCTMME RTCCGTATCCGMECMTM
`Rae I
`Eco RV
`Nhel
`
`700
`TMCGCTRTA
`GENTLE I BCCDEBCETCT 1 GCGGMTLTC GTCCETTCCGRMGEflTCDCmfiTCM TH'I 53:6 IGET
`Prwal LeuPl'uG lyLeume-cyfls p1 lava 1 H1 sserfispScl‘I 117M aSIii-Hi sTyrG ] yval LeuLeu Ii 1 atlu Ty
`GWCHTGRCWCDCGGRGRQCECCCTHT fiscflfifiTflQGGC TGTCGTflGCGGTCQETEBTACCSCRCGACGAT CGCGflTfli’
`
`300
`T5C5TTGlTfiC “FT TTCTRTGCGCRCDCGTTCTCGGHDCRCTGTCCGMCGCT TTGBCCGCCECC CM“: E TGCTC ECTT
`F8 l iLEuHETfiI nPhaLeuCysh J aPI—eva 1 Lei-BI ya 1 aLEuse rnsnnrgfl heG 'l ynrgflrgl’rwal Let-Lena 1 as
`ACGCRRCTMGT TRRHGRTACGCDTWEC HRSMCCTCGYGQCQGGCT GGCGRWCMCWBGE TthCGflBCGflI
`
`32ier
`«co
`1: GCTflCTTmCQCTHTCsHCTMGCEhTCRTGGCGflDCiCRCCCGTCC‘rG—I GR‘I' CCTC'U‘CGCCGGAC acnrc GT8
`2 r' LeuLeuG I y i 1 nThr I lensnlyrn 1 al leHE TA] iTlir ThrP rave 1 Leu‘l n pl leL Eu'ler a6 I. ynrg 1 19081
`EGGITGRHCCT CGGTGRTM‘EC VGHTGCGCTFGTMCDC léBTGTGDBCAGGflEflCCTWmT GCWCC TBCG IflECIK-fi
`O
`B'anll
`
`ECCGBCITC RCCGDCBCCMIDET 5C661 'OC‘OGCGC CYQTRTCEC CBfiCfl 'IC McfifiTGGGGfiM-Il CGGGC‘I’LBCCR
`n 1 a6] y I IQThrG] yfl'l fl hr Bl ,1! I aVl I R I afil 7F 1 ET}? I I9! 1 afls pl l ETIIPRIW l 75 I ukpfll‘gfi I afirgH i
`CGBCCBIRSTGBCCBCBBT 6'[CCQCGCCMCGHICDBCBGAIRTWDGGC‘E fi'lflGTGGCTACCCCTIC TRGCCCGAEESS‘}
`
`FIGURE 1‘2 Nucleotide sequence of me. The symbols and letters appearing
`throughout sequence are used according to the following convention: (a) Restriction
`enzyme cleavage sites that are present no more than three times on 13311322 are
`indicated by vertical arrows at the site of cleavage. (b) Structural genes are indicated
`by corresponding amino acid sequence written above or below the coding strand.
`(c) Promoters are indicated by the designation PM, where N refers to the identity of
`the promoter according to numbering system of Stfiber and Bujard 1981 and Brosius _
`et al. 1982. Boned sequences indicate the location of the —10 and —35 00an
`sequences commonly found in most prokzryotic promoters (Rosenberg and Court
`1979). Horizontal arrows indicate start point and direction of transcription for each
`promoter. Location of traitsu-iption terminators can be found in Figure 1—3. (d)
`Origin of replication is indicated by end—onend arrows, starting at the first base of
`nacent DNA attached to the RNA primer. Horizontal lines are used to highlight
`regions of dyad symmetry thought to be biologically relevant. In the region around
`position scan, the symmetrical sequences involved in the regulation of DNA repli-
`cation are identified with Roman numerals according to standard nomenalature
`{Tomizawa and Itoch 1981). {From Ballads et al. 198?.)
`
`8
`
`The Pier-mid, some:
`
`from 1130101 and the ampicillin resistance (APR) gene from the transposon
`Tn3 (carried on the plasmid pRSFZlZ-i) were incorporated into the plasmid
`pBR312 [Rodriguez et al. 191'6; Boiivar et al. 1970's) This maneuver placed
`the Tc“ and APR genes onto a relaxed replicon capable of amplification with
`inhibitors of protein synthesis (see section 1.4}. All subsequent manipulations
`were designed to maximize the number of unique restriction enzyme cleavage
`sites and to minimize plasmid size. For example, the extra BamHI site near
`the ApR gene in pBR312 was removed by EcoRl‘ digestion (Gardner et al.
`1982). Likewise,
`the two additional Pstl cleavage sites in pBR313 were
`removed by a more complimtcd maneuver to give rise to pBRSZ‘Z. As can
`be seen in Figure 1—2, pBR32‘2 contains twenty-one unique restriction en—
`zyme cleavage sites, eleven of which reside in the Tc“ or nipR genes. Since
`the enzyme Hincll also cleaves the Sail site, it is not included among the
`unique cleavage sites.
`With the exception of pBR32? (Figure 1—13), which lacks 1,089 bp of
`nonessential DNA present in pBR322, subsequent plasmid constructions
`were designed to introduce a unique EcoRJ cleavage site within a plasmid
`gene. As shown in Figure 1—13, both the colicin production gene in pBR324
`(Bolivar 1978) and the chloramphenicol resistance gene in pBR329 (00v-
`arrubias and Bolivar 1982) possess unique EcoRI cleavage sites. With these
`plasmids, EcoRl restriction fragments can be cloned and identified by the
`insertional inactivation of a plasmid encoded gene.
`One feature of pBR322 that has contributed to its wide use as a cloning
`vector and research tool is the availability of a complete nucleotide sequence.
`The original sequence of pBR322 was reported by Sutclirfe (1919) to consist
`of 4,362 bp. This sequence was subsequently revised to 4,363 bp by incor~
`porating the following two corrections (Figure 1—2). First, Heusterspreute
`and Davison (1983) raised the possibility of a mistake in the Tc“ gene
`sequence. This would explain the absence of an open reading frame consis-
`tent with earlier studies of the TcR protein. Mirticell {Meagher et al. 19??)
`and maxicell (Sancar et al. 1919) studies revealed the TcR protein to be
`approximately 40 kDal, whereas the original nucleotide sequence predicted
`a polypeptide of about 20 kDal. This error was later confirmed by Peden
`[1983) and Backman and Boyer (1983), who showed that a GC base pair at
`position 526 in the coding region of the TcR gene had been omitted. The
`revised sequence now shows the TcR gene consisting of an open reading
`frame of 396 amino acids or 1,188 bp (position 86—1133) specifying a 41,500
`kDal (M, 37,000) protein. This protein has been shown to actively exclude
`Tc from the cell (Tait and Boyer 1978) while simultaneously mediating the
`uptake of potassium (Dosch et a1. 1984). There is no evidence to suggest,
`however, that those two functions for the To“ protein are related.
`As shown in Figure l—lA, the Tc“ gene of p‘BR322 was derived as art
`fl:on fragment from the naturally occurring R—factor, pSCIUl (Cohen and
`Chang 1977). The TC“ mechanism of pSC101 belongs to the class C resistance
`group and is closely related to the class A (RPlfl‘nI 221) and class B (T1110)
`
`Page 3
`Page 3
`
`

`

`1D
`
`The Phsmid, palm:
`Banfl
`500
`cTTCGBEC
`RTMBCGCTIGTTIC 566616661 RTEG IEECQGECCCCST aGCCBGGWQCYSTTWGCCITCTCC‘
`tho’J l yLeul‘lETSEr‘ A 1 ac rsF hi5 1 Hal 6] yHE‘Wa l A 1 ME] yPraVl l n 1 a6 1 $5 1 yLIuLIuIT-l ya 1 all ISIH.
`unnecc cans! nucamnéc moms: :acncccn rnccnécaTc casscmcc 56¢ cocérancmcccocearm$
`Sph I
`500
`mn‘lfiCICChYTCC TIGGGGCEGCGG YGGYGIMGBCCTCMCCTM YACTDEGC'IGCTICCTMTflBiDGflUTCBChT
`an“ I. I» ll?!‘ uPhoLeuA 1 III 1 3&1 JV: 1 LnuAIHB 1 yLauflsnL!uLEuLwE I yCySPMd—MIJEYG ] nflluSQI-HI n
`m: 61 act! IfiBTn#6611m:55C6CCGCCMSRGFTGCCSGMTTSEITGQTWCMQRMM? maech ICthG‘Ifl
`noel! Hincfi
`Sail
`700
`RNGBBMBIIB
`cm CWT ECCCT TBRERGCCTT can“:chanc1cc1'rccsarmscassacmmc1nos'r
`Ly 191:8 l uhrgfl rgPrunE‘rProLeunrgAl aPhens nPrnva I sersorfihmrqn pa 1 inrgfi l thI Thr 1 I ova
`1Icccnmscna:‘rrssoYM:econnCTc1c66M511'asa1cMYcemnmcnccmcscccccmmenm
`
`BUD
`CGCCOCflCTIRTBhCT 3111' TCTY1'flTCRTBCMGTCETRBBECRBBTECCMRECECTC IWGTCIITTTTWBCM
`I R 1 ii I aLeuIETT h I‘Vi I P?! BFIIR ] l PHETG i nLluval 6] VS 1 hVaI PI‘DH Iafi l anu'l PFU.‘ I la-PIIQGI ya I uh
`GCGGCBTERA IMTMASHfiGflRflYWGTME‘ITGflGCnTC CTGTCC.“66¢CGTCEEWECCflGTthAGEL‘GC'ICC
`
`ICCDC TTTCGC '[GEMCBCGMERTGATCSGCCTGT WET,“ S9T811mQFITCTTECGCGCCCTCGCTCNGGCCTT:
`wflrgPIn-a rg‘r rnSeral a1 Pu- HETI I 96 1 yLauSerL nun I aV-n l Phifi 1,1 I bLnuH 5 sn 1 new: I as I run I nPhn
`TGSCSRRRECGHC CTE6CEéT6CTflCTflGCDGEiCfiBCB;NICECCATRHGCCI TMRRCETficfififimfiABYTCGfimfiG
`
`Bgu Xmam
`
`909
`GTC‘CTDGYCCCBCCIICCM‘CBI TYCWWRRGCMECGRTTRTCGC DEBC Gangnmncacfic IEGGC “WEST
`Va 1 Th r6 1 yFrnfl 1 aThrLysfl I- gP has I ya] uLysBI rm 1 II In I Ian] 15 1 yHETn 1 an 1 ansnm ll. wG I y 1' yr Ila
`METGflCC RGGBC 891G131 q'TBCMRGCCGCTC 'l TCGT EéGGTflflT 985m CSTMCEECBWTGCWOMCCGHTOCA
`Nwl
`3000
`C Y'HEC‘ BGCGTTC
`BIC DCGRGBC TBGRTGBCC TTECCCHTTHTMTTCT H: TC“ TTWSBCGeCn'chGfl‘I-oc ECG
`1|.»ng I nPl’I an 1 11'?! rflrgfil 1TrpHEI'a l aPthruI 1 NET I l eLeuLanfi l ISIrG 1 ya 1 y l I DB 1 yHE‘IPr on
`an: N12664: II“GI:DI:YOCEEC1WGACC‘IMDCBBAMBBETARTMTIEMMEW BAflGSCCBCCETAmCTMGGGC
`
`1100
`CUTYDCRGGCCJITGCTE 'lCCMMRBETMATMWRCCITCRUEGMSC TTCMGMWTWEGCTCTTRCC‘GE
`1 5Lqu 1 an 1 anETLeuSoMr-gfl 1 n0! 1 nspnlpflspui sBl n5 1 ye i nLeufil nu] ySeI-Leual M I aLeuTIn-Ser
`MMGTCOBGTIICGMMGTCCBTCCRTC WMT661061'n0‘I’CDI:Te1CWETTCCTNGCEMCDCMBEI‘IBBTCE‘
`I
`1
`1200
`CTnncTTcGn‘IchfifimficIMTCETCMWCSNI‘ITATWCGCZjEmmMAmBGT'ImTGG-fl'l .
`LnuTh rSe-v- I l eThrG 1 yPruLeu 1 I oval anm :1 l eT‘yrn 1 an i aSQnfl I as-I-Yh r n pit-M; l yLe-HM n‘n-p 1 I
`GnTTG-nmsc‘mfi'l'6m:CT66:6M1118CWOCCBC'IMIWHCQDCGGIIDCCDCTCBIBTMTTmcnnccfil'flcfi’n
`
`YETRWCGCCGCCCTRTMTI ETCTBCCTCCCDGCB'I'TGCETCBCGBTWflTfififlGCCBGECCflCCTCmeTfiB
`file I 6! ya I aal aLou‘l}I rLqui my sLeuPI-nn I iLlun I-gnl'gol 1A1 aTrySernrgR I aanfier I h r
`ECRYCCGCGGC BEI?fl1RT61;RN:NBRI:mBCBCAheECHDC5I:I:RC8TRCCTCBECCEGBTBGRBCWCTTMC
`
`12 common and Structure
`
`1‘!
`
`35m
`1300
`MfiCCEGCGGCRCCTCBC TMCfiGRTYCACCM?I‘CcanSMTTGGn6CCMTDIIITTCTTECMIICTGTBAR‘IDE
`
`'l'l I:G3CCBCCGTmSCflh;TBECTWERGGTTC;TRIICCTCWTTflGT InMfl-ACOCC'CYTGM “CTTMQGG
`Sli |
`Ava!
`mace-mm:
`'r'rsacmmmrccnIcaca'rccaccn'rc‘rccnacnsccrimcsccscacn'r camcaamnm
`1400
`C10
`C ICCflECMCCGCMGWBEMTCTCWWTTBWYUG TCWCGTECWBCGTWBCCCG TCECMCCC
`Hal I
`1500
`rccmcccncacsmccnmm‘rcsmc ranmmrraneemcceec Iammsccsemmocnncmsrrmcn
`
`a56.nch1Ecccarcaunnamennceneaamsnnnncc TGEGCCMTCCEACCW cccnmacnnmcnmcsr
`
`1600
`amamanTcnccan‘rncscsn6caAm:6'1msccnI:'racToerscmama1cmcenpnmcnncmcn‘rcnm;
`CTTM 'l TIGTBBCTRTECGCTCOCT Tet-“C? 'lcsCTBRCmGhCGTTTTGCIMIRCGCTMTCGTTETTBTflC I'l'ficl:
`
`YE Y TCOG'I' YYCCG’T6I"I'YCGTRWTCTEGIMCEQRAGTCRBCBCCC mtfiCCflTTflTG‘TCCGGflTCTGCl TCDC h
`
`mnmnnmmnméflnmnanccn TnCGCcl’1':M;TC6CBBMCBTEOTflfi’M-IMQCETRMCBTW;
`1700
`WhTGCTfiL‘TaUC‘flCcC ImWI.“CM:I:THCITCTGTETTRMG‘QQCGCTDDCHTYMDCTBWGRTT I' 'n’CYC "3
`
`OCT HCeInCGflCCGflT9ma“CC1'T6m5NTBTHGunC“T;RYTI]:TTCGCMWTRQCTWBQCTCMTRWMGRC
`I
`
`18100
`ErCCCGI:CGCQTCCA‘NL‘CGCCRB'I'TB'I'TTRCCCTCMMCG 'l'm.QTIlCDGDECflTBTTCRTCBmMTflflCCCOTfi
`
`CIGG3I:DECQTflGGTRTEliGGTCMl:flflflT665fl8VeT1GI:flmTCflTTmCCBTREflflGTfiGTflGTCWI TGGGCIT
`
`1900
`YCGTEmTCC1'C1CTm'I'"CA7Cl5G1hTCNI1flCCCI:CB1Wm5MRTT‘CCCCTI’RCfiCmBMflTCflHWHETI
`
`ABflCTCGTQGSMRGCAMWAGGCITRE’TMTWTMT1‘6TCTTTWGGMYGTDCCTCCG’IRBTTCMT
`
`2000
`.
`CCRRHE‘1mflhhflicCBCCC'ITMCATMWCTTTIQTC“RAGCCMGRTTMCGCTICTGGRGRRRC‘FCmflG
`anyse 1 n6 1 uLy5Thrn] aLenmIETM mrthe‘I lam-QSEPGI n‘l'hrL-u‘l luv-Leume 1 uLyILouAsnB 1 u
`551 TTa1CCTTTTTTEOB6&6flRTYfiTflmC5hflflTAEI'CTTCBGTCTETQHTTGCGEMCTCY YTGAGTTGC ‘1:
`
`FIGURE 1-2 Confimd
`
`Page 4
`Page 4
`
`

`

`1.2 Cmsflucflonand Structure
`
`13
`
`CCCET TCullEECflSACCECTDCGCCTTRTCCBNMCTflTCBTCT TENTCCMECEWTWCACGADTTFTCGCEECT
`
`aas:Anarcassnmcmcoennmwcm mamécm: 'rcnsm-mnaccahc'ramc Imnrmccsmi
`
`2700
`sscmcnaccac warnmsm 1mama:enasummsscesmnnc new"@se‘mscmun
`‘
`_____ __.F‘IL ____._;
`CC GT CGYCDBTGHECRTTGTCC IMTCBT CTCBCTCCHTACQTSCUCCM BhTGTCT CEABMTTCNCCRECGGQTTGi
`m
`
`3000
`__..
`
`n:anmammaerasm'rmscactc‘rccwmeccammccncsmmnsam Tsetmnrc‘l
`
`10c :anrs‘rcn‘rmcc‘rficnmm cMAGncgmfimncvcomun-rmngégln T1c1¢fimcnarcsna§
`m —'
`m'
`3:
`31'
`3100
`.
`Tantcmcmmmcmmmsrmcmmfin n r1'r611w:mcmnsn‘rmcacscammmnesmc
`_ _. _.__ __. _— _.__
`nnnwncmnm‘r raméscsancmcsccaccmmnncmm ICE tor s csémc TTTT'I'TYCC‘IE
`'
`_._-_._______Pp--_._'__-_
`1r
`1
`1
`5.200
`TC-fihfilmT DCTT' 5“TCTYTTCTN:GBE5TCTEMGCTCflETGEhMWRnCTC-QCGTTRWGSHT TTT' EGTCfiTGfl
`
`AfiTiiTTCT R56}!fiMTIGgmflTMCCCflEMTGmgfiTCMTTGC TY TTGMTS‘ERAT T CCCTMMCCASTHCT
`Drar
`Dral
`
`GATTQTCMGBFT CTYCRCCTEMTDCTTTTLIETTRHMATMEGTTTT FRITCMTCTIMYQTITATGAGTRR
`
`CTMTnmrm-c :‘rnsnémmm imammnmhn Tncr'tcmmn‘mnhannmm tATnc‘rcan
`'
`
`3360
`“1105111 am:AOTTnCEART8C1TMTGAGTSRGGCNCCTATCTCQGCGATCTGTC TATT‘ICBTTCRTECATRB'I'I’GE
`EWTPFH i .Lynl leLluSl-rfl] 33] VI 1 (8] HM II I25 1 fl“! gfllnfir‘gfi 1 “as pNETT hrh I :l
`1'GAflI:ChEflCT5TCMTGI!TlmIIRTTflGTCRCTCCBT66flTmTCGCTRSRCMflTlflWflflGTMl-ETRTMG
`34m
`:1 canmacmce1nm1mmmnmcsssm r‘rnccaTcchcccmmca‘scnn ran'rnccncanmc
`Gins.rBIyThr‘lha-TyrITWllu'au1QArgS!I'ProLySGlynsnPrnfiIeruhl-afllnl an llfilyflrghrcl
`nucrmsaacnecmnmm rams: Tn1accc1cccmrm1mcssssicmancm Tnc‘rmcscoc rcme
`
`59! I
`5500
`cmcTcno:aaon:t:nsn‘rnmcmcm1mccnscmcccw saccanacecnmswmccmcnncr'r'rn
`
`yhr‘gfi 1 us 1 7h 1 an 1 ysoriysfispn 1 al 1 nPImTr-QGI ynl lFraLrun l asurnquIuLouPI-us 1 ya] nun I LyIA
`GT BCBRBTGEECEREGTCTHMTHBTCGT TNT? EGTEGGTC BREE T T CCCG‘GCTCGCGTCTTCACCRfimGTTGMR T
`3600
`TECSCC TCCITCCAGTCTQ'I‘TNITTBT TGCCGBGflflMT-‘GMTMGTWCGCCHE‘I TRATQBYYTDCGCHMST 1' ST
`snflilfiluHETTrnfl=nl 1ELeusansznaHSormaLé-umuuuuclucAwnriouuuLunrngmmr
`al3GI:m6ETm1Cm1MTTAICAECGGCCC‘ITCBA‘ICTCITTCnTcMECGGICflA TTHTCHWST Tanflflcfl
`
`FIGURE 1—2 Continued
`
`12
`
`The PM [333322
`
`anE
`
`Fvull
`
`CTEGIICGGWATBAACNDDCRGRC fl‘l' CTfi'fSMTJOEm-RDGFCCMGGTMTWCTT Immna‘cwcn CGEBCE
`Lanna all 1 “spa 1 us] n8] nnspl 1 ocyill I usnI-LauHI snsgh’i in 1 mspfi 1 uLeu1yrnr9§arcyleH I. anr
`onccmaccmc‘nc'rccm: 161newan1mnmw3r5cmsmncmcrceaRATmnsmancmnscscsé
`
`2100
`TT'I'CifiaTIIhflII GMmMFCC TCT9“cQCITECHGCTCOCGGAMEGETCECRECTT GTCTETQQGCWTBCCW
`9PM“;lyaspnspBIWlulinLeuEm}
`.
`.
`u
`IMGI:CN:TMCTEECIICTT:fT65fl5M1'GTGTH:5TC8fl38GCCTCTbDCflfiTSTCmeABnEnTTCECT-l TMDDCCCT
`
`2200
`GEflmCIflDOCCGTCm5CflI:BTCIneCmTaTT6ECDOBTBTWCGCQGCCRTMMCACBTABCMTW
`
`
`CBTCWTTC 5BBI:MTDEC8I:5CflETI:DCCCN:MCCGI:ECHCmODEC6TCGBTMTEUBTmSTGCHTCECTflTCé
`
`Accll’Snal
`
`«nun-mi:
`MCI
`
`Wei
`D
`WiGTETflTflL'TGfilCT TflInflTGCGWTCMHGATTGTLCTGWETWOCR “TECGBTGTMIRTHECGCRC
`
`RESP
`
`cc‘rcaca‘l “antenna? ncoc CBTRGTCTCBTnC‘ItTCMBIBBTaTflTfifiCETfi
`——————— ---Pch—————
`
`2‘00
`.
`WER'I'DCGTMGGMWTACCDCRTCMGCGC TCTTCCDCTTCCTCGCTCMT GaCTCBCTGCGCT {2561' CSTTCOQCT
`
`TCTMGE fl'I'TCCTCTT TTJ‘“ BGCGTESTCCWGflIGflC BflflGWGh8T“CTGMESIEEEGflGCC-RGCIRGCCGR
`Afl III
`
`6639:SAGEEGTfl‘lI:RBCTCQC!’Cn3WBIAITm:65TTaflttflEnEMTCflmfinTAnWCFGGWGMLnTfiT
`CGCC DCTCDCCRTMTC5HéTMBTTTCCGDCRTTRTWTflGGTBTCTT RETCCCCTRTT GCGTGET T TCTTETMR
`
`2500
`MECWMGC MBCMIRBUCCRSSRM STAhIflMDCCGCGITDC’TGGCGT TTTTmeMTCCGCCCCCcTW
`
`“mm m” —__
`WIGIIK
`CTCETTT 'I'Gl:BBTCB?TTTCC66TCCTTGGBhTTTTTDCESCGCWFCCGGAflrflméT RTCCGRGSCGGQGWCT;
`mo
`BABEATCRCMflnflTEGMMTCRMTCRGREGTBBEGRMC CCGRCREMCTMTMflEATICCIBGCfi lII TTDCCCCTGG
`
`CTcGTfiBTBTT TTTIQGCTBéWSTTC-NETCTCCICC BCTT'TGGGCTGTCC T GBTRT TTCT‘RTGBYCCGCMREGDBBRCE
`
`ZEOD
`WTCDCTCBTGCGCTCTCC YGTTCCBHICCCTGC CECTTflCCGGflTflDCTGTCCBCCTT TCYCCC TTCBBMDBYW
`
`rtcsnsmcncmmnfimcmsc‘rmsscenfirasc:1aicsncaaccaoaéammsccc‘ncscncé
`2300
`CECTTTCTCRYW TCmYETNEGTflTCTCMST-TCEGTGTMWCWTCGCTCQIECTMGCTBTBT 912ml“:
`
`BI:EhMEhGTRTCGAETWERTCCHTAMGTCRMCEACRTC CI5CflmRGBTTDéHCGCGlCRCmTGCTTGGé
`
`Page 5
`Page 5
`
`

`

`14
`
`The Plasmid. pom
`F's: I
`roccar Tecroc necesrccr 5611:! coco: rco‘rc crrrecuroscr rcnrrEnacrccccrrcccnneen‘lemeec
`
`a 1 aHE‘m 1 an 1 aPr‘oi‘ETTlu‘Th maggot-98! uaspnnniir ol 1 9M ab 1 unsnuuls t uPr r36 1 uT r‘ Mega: pl. eunr
`REGGTRRCGQCE YCCBTQGCMCHCRE 15C mot: RSERQQCCQTMC anneramTcmcccan'r ECTAG'T 73:3
`9qu
`3700
`GAET'I RCRTGR'ICC CCCHTGITB’IGC RWWEG‘GTTAGC T C CT TCGETCCT DEER? CG TTGTCRGMETMBT'GSCC
`91 h I‘Vi I Hi sis DEI WETaInHI ILUIAPHIIII a'l hrLuI’aG luLysPrnG i )5 l y I I9 I'D-I r TI!er OBLIu-flfinfl 1 ill.“
`{I ICMTG'I RC TflfifiGGGTnCfl-‘E RC 371 TI YT: GO CflfiTCGhGGMBCCflEBRGGCT mflflcflfifc TTCWI TChflCEGE
`
`3500
`EEMTB‘I T ETC hCTCflTDG'I TRTGGCflGChCTGCATIRTTCTCTTICTBTCRTDCC ETCCGTHRGRTGCT TTTCTB‘IGM:
`
`i .1 mm: uni risen-IE1 1 I11 1 on I an 1 asercysLeuE urn Noun 1 Th rHETGI ynspl‘ irerqu sLynlS 1 n1}. run]
`CETCflCflflTfiB'I METRE C MI RCCBIC ETGMGTR'I TflflefieflflTG-flcm? 9:061 fiGGCflTTC'InCGMflRmflCT G
`
`Hincll
`RealIScal
`S900
`lfifimflfi'
`cam “RENEE TIC TGREAIITRBTGTRTGCGGCGAC CBQET 'lBCTCTTGCCCGBWIWCHCGSGRTR
`
`PruSeI-l‘yrfiluUalLluasnnsnfil n59 rTy I-Hisl ion rgnr genuine] norms] Mil yer impUalnrgserLe
`accnc tcnronorrsor rcaBTnamcrcrrarcncarncoccocrsecrcnm: ancmaaccccm wrote: for
`Dral
`anI
`
`«can
`a IMCGEGCCACJH‘GCRGRM 'l'l TLRfl-fle'lfifi rchcar IGEMMCGT ICTICBEMCBAfifiM ICTEQRGGH? C TTh
`uUnl a I as l ,1: ynLeuLe-uiala i LysPlto‘l hrserflETNE IPr oPhenI—afi 1 Mn uPrnflrthoSm-B luLoul leLysfi
`TflTDDCECGGTGThTCBTC'FTGflflRTT 1 ICE: SRBTRG'UN‘C CT I ITGCQRGMGCCCCBCTTTT MBRGTTCCTIIGWQT
`
`UCGGTGTTfiAElTCCfiG'I ICE“! GTfiflCC CflDTCGTGCACCC “QC T “I CTTCRSCflTCY TTTMTTTCRCCAGCGT'ITC
`
`lysorflsnLa-IASDLOIIG i I-ll IOTYPE I’Va lira“ I mi yL ELIGI Mine 1 uh] afiIDLyFUI I Lyiil'nl L H: I hrs 1 II
`GMGMARCTCTQGGTCWC TM? fiT‘IGBBl ShBCnCGTISSGTTGM: TQGRMICGIhGnAIfl‘EmnABTDfi Y tee-Inna
`
`4100
`'
`TEEGTGRGCWRRERWCMQQTGCC SWAMEBMHTWBBCW CncmflTGT IMhTflCTCATICTCT
`PruH i aw aP hrua [ProL'IICysPhenl am aPth ha?!‘ a! I eL-un 1 oval nrgPheH 1 [ISI nt 1 nSvri-IET
`.
`flCCCH
`DGTTTTTETCCT'ICfiGT TT I‘M GECGTYT'IT 'l CCCTTRT 1 ECCECTGTGE E TTTIQCMC TTRTBABTHTSMH
`LfldIr mutin-
`Ssp |
`4200
`1,
`TCC‘IT ITTCAflTflTUITTGflRGCflTTTfl'ICflGGG? TATTGTC ‘ cnrmc SEQTACRTR‘I TTBRHTGTNI’T'IBWQT
`
`__________ __P3__ _ _.__ __ .. .. ._
`AEGMRQQGTT fiTMTflfiE+1CGYhh TMTE CCTMTC 5C CTN! 5T“ lTMflTC‘lT I I TA
`Act I!
`4300
`MMARITMBBGT'ICCBCGCMflI TTCCCCGfiflMGTOCC-fi CCTGRcG
`Inmnnnccm TRTTH'I'CWI UflCATTMIC
`
`1T IGTYYI“ CCCCRNBGCGCGTBTRRHGGGGCTITTCMGGT 6GM1i3SfiGhTTCTTTmMTRRTMT-IIC'IGTMI I 6
`
`EcoFII
`CTRTMTRBBC BTRTCMGAEECCC" Y ICGTC TTCMGM
`
`flfl'lfl'i'l 'ITTJITOC BCRTRGiGC chmnneacmnacrrcrr
`
`1.3 Transcriptlonal Signals
`
`15
`
`determinants (Mendez et a1. 1980; Unger et al. 1984). In pSC101, Tc’z is
`induced by subirlhibitor'yI concentrations of Tc (Cohen and Chang 1973; Tait
`er al. 197?; Tait and Boyer 1978) and is mediated by two proteins, mm and
`MR. These proteins are errooded by adjacent genes (although in opposite
`polarity) and controlled by a common regulatory sequence (Unger et a].
`1984). It is now known that the product of retA corresponds to the 40-kDa}
`protein encoded on pBR322, whereas MR is a 27-kDa] repressor protein
`not carried on pBRm. For this reason, expression of Tc“ by [31312322—
`containing celis is constitutive, whereas in cells carrying pSClfll, it is in-
`ducible.
`‘
`The AP“ gene of pBR32‘2 was derived from the transposon, T113 (Mey-
`nell and Dana 1967), by the plasmid intermediates, pRSF2124 (80 et a1.
`19TS) and pBR312 (Rodriguez et a1. 19%; Bolivar et a1. 197m}. The Ap“
`gene on Tn3 produces a TEM-l—B-lactaniase that catalyzes the conversion
`of penicillins to penicilloic acid by hydrolysis of the B-lactam ring (Burman
`et al. 1968). In pBR322, the Ap" gene is encoded by an 858-bp (286 amino
`acid) open reading frame from position 3298 to 4155 (Figure 1—2]. As witl
`be discussed in section 1.3, the Ap" gene is transcribed by two promoters,
`the Ap“ promoter and the Jedi promoter.
`Finally, Heusterspreute and Davison [1933) reported a 2—bp inversion
`at positions 2'13] and 2732, which maps in the region required for DNA
`replication. This difference has been used to distinguish between the ColE]
`and the [3MB] replicons (Selzer et al. 1983}.
`Over the past ten years pBR322 and its derivatives have been used
`extensivelyaas model systems to investigate the various properties of DNA
`molecules in vivo and in vitro. In addition to its intrinsic biological and
`biochemical importance, this information has great practical value for in-
`vestigators wishing to construct more efficient or specialized plasmid vectors.
`Some of the results from these studies will be described in the following six
`sermons.
`
`1.3 TRANSCRIPNONAL SiGNALS
`
`0n the basis of in vitro and in vivo analyses, several transcriptionally active
`regions have been identified and charactelized on pBRBZZ. Perhaps the most
`interesting of these regions is located between coordinates 1 and 80 bp
`(Figure 1—2). Within this short sequence of DNA lie the promoters for the
`TcR gene (mm), the To represent gene (MR), and the operator for the Te
`repressor. Transcription from both MA and tar}? promoters can be blocked
`by the binding of repressor proteins to the Te operator. The Tc operator
`for the cfass C determinant (pSClflllpBRSZZ) consists of two short dyad
`symmetries, centered at positions 2.4 and 53 on the nucleotide sequence map
`of pBR322 (Figure 1—2). It has been demonstrated that this region binds
`four molecules of Tc repressor and bears strong resemblance to other Tc
`operator regions in terms of sequence homologyr and position {Klock et a].
`1985).
`
`Page 6
`Page 6
`
`

`

`1.4 DNA Heplicetlon
`
`17
`
`the end of the TeJR mRNA in vivo. In the case of the Ap“ gene, in vivo
`experiments have demonstrated the presence of three transcriptional ter-
`mination sites (approximate coordinates 3210, 3110, and 3050), close to the
`region where the promoter for the replication primer RNA is located (von
`Gabain et a]. 1983).
`A third actively transcribed region on pBR322-maps between coordi—
`nates 2900 and 3200. Two promoters are located within this region and
`promote transcription of the primer of DNA replication and the inhibitory
`RNA, respectively. The promoter for RNAI (repressor RNA) is the strong—
`est in pBRm, whereas the promoter for the Tc“ gene is die weakest (Stiiber
`and Bujard 1981). The promoters P1 and P3 were found to be equal in
`Strength (von Gabairr et al. 1983}.
`The transcriptional signals responsible for the expression of the rep
`(repressor—of—primer) gene have not been identified. Since a terminator for
`the Tc“ gene has yet to be found, it is likely that rap is cotranscribed, at
`least in pan, with the Te“ gene (Bujard et al. 1983).
`The promoters mentioned previously account for approximately 85% of
`the in vitro transcription of 91312322 {Stfiber and Bujard 1981). The remain-
`ing transcriptional activity is contributed by two promoters located between
`the origin of replication and the rap gene. They appear to serve no purpose
`for the plasmid (Queen and Rosenberg 1931). The transcriptional signals
`mentioned previously are summarized in Figure 1—3.
`
`I
`
`1.4 DNA REPLICA'HON
`
`For gram—negative bacteria, plasmid replication falls into two general modes:
`stringent and relaxed. Stringent replicating plasmids, such as pSC101, require
`protein synthesis; the activity of DNA polymerase [11 (Cabello et al. 1916);
`and the host functions, dnaA, B, C, and G (Hasunuma and Sekiguchi 1917,
`1979). These plasmids are usually present in 1 to 5 copies per cell. On the
`other hand, relaxed replicating plasmids such as ColEI require the activity
`of DNA polymerase I, can replicate in the absence of protein synthesis
`(Kingbury and Helinski 1973; Staudenhauer 1916), and require the host
`functions dual}, dnaC, dnaG, dnoE, and dnaZ (Stauderibauer et a]. 1979;
`Scott, 1984]. These plasmids are usually present
`in 15 to 50 copies
`per cell, and on inhibition of protein synthesis, they will continue to replicate
`while chromosomal replication ceases. This phenomenon is known as am-
`plification and results in approximately a 100-fold increase in cellular plasmid
`DNA. For pBR322, the plasmid copy number increases from approximately
`20 to 1,111] per cell when protein synthesis is blocked by 200 lights] chlor—
`amphenicol (Covarrubias et a]. 1981). Five plasmids belong to the relaxed
`category of replicons: pMBl (ancestor to the pBR-plasmid series), ColEl,
`CloDF13, pRSF1030, and pISA. More information on these plasmids can
`
`15
`
`The Plasmid, pflfiafl
`
`this region comes from the parental plasmid
`As mentioned earlier,
`pSClU] and was first characterized by Rodriguez et a1. (1977, 1919}. These
`studies demonstrated that the unique Hindlfl site, located approximately 30
`bp away from the E‘le site, could be protected from digestion by prein—
`cubation of plasmid DNA with RNA poiymerase holoenzyme. The EcoRl
`site was only partially protected from EcoRI digestions by this treatment.
`Later studies (West and Rodriguez 1930) showed that RNA polymerase
`could form productive initiation complexes at the Hindlll site and that tight
`binding by RNA polymerase owrrred even when the E'le was cleaved.
`(West and Rodriguez 1982). These results suggested the presence of more
`than one promoter in this region. In vitrc transcription studies provided
`convincing evidence for the presence of two active promoters in this SCI—hp
`Sequence. The results of these studies indicate that HindIII site lies 1'? bp
`5’ from the transcriptional start-site for the MA gene promoter (P2) and 9
`bp 3' from the start of transcription for the retii gene promoter (Pl) (Stflher
`and Bujard 1981; Brosius et a]. 1982). It should be noted that it is the
`promoter P1 that drives transcription of the chloramphenicol resistauee gene
`(Coin) in pBR329, not the Cm" gene promoter. The native CmH gene
`promoter was removed during BaBl treatment of pBR328 (Figure 1—1B).
`Additional studies demonstrated that both promoters were active in vivo
`(Close and Rodriguez 1982; West and Rodriguez 1982} and that RNA
`polymerase binding to P2 was strong and salt—sensitive relative to the pro-
`moter for the Ap" gene.
`Nucleotide sequence information (Sutclifle 1979) and RNA polymerase
`filter binding experiments (West and Rodriguez 1980) permitted the pro-
`moter for the Apl“ gene to be mapped to a 189 hp region on pBR322,
`coordinates 4155 to 434-4. However, no obvious consensus promoter se-
`quence (Rosenberg and Court 1979) could be identified in this region. Runofi
`transcription studies (Russell and Bennett 1981) and 81 nuclease mapping
`experiments (Brosius et a]. 1982) were able to locate the site of transcription
`initiation for the ApH gene to position 4190 (35 bp from the ATG codorr
`for Ap“). This promoter, which was designated P3 (Stfider and Bujard 1981;
`Brosius et a1. 1982), was shown to have RNA polymerase binding charac—
`teristics that were rapid (half—time of association and dissociation, 0.5 min
`and 6 h, respectively} (Russell and Bennett 1981) and resistant to 100 mM
`KC] {West and Rodriguez 1930; Russell and Bennett 1981).
`Interestingly, the coding sequence for the Apll gene is also transcribed
`by the MR promoter (P1) in the sense orientation (Strider and Bujard 1981;
`Broeius et a1. 1982; von Gabain et a1. 1983) and by promoter P4 (see section
`1.4) in the antisense direction. The latter transcript (from P4) would explain
`why the mouse dihydrofolate reductase gene was expressed in E. anti when
`cloned into the PM site of pBR322 in both orientations (Chang et a]. 1978).
`A transcription terminator region has been identified in vitro approxi-
`mately 650 bp from the initiation site for the Te“ gene (Stiiber and Bujard
`1981). There is, however, no information about transcription termination at
`
`
`
`
`
`W".gm!wafi_..l..9mm.m:fi.prug.m“flair...
`
`Page 7
`Page 7
`
`

`

`32301l01
`[mlnar'.
`
`
`
`m51HTPERSENSITIVEREGION
`
`
`
`
`
`mm3!HYPERSENSIHVEREOIO'N[Inbminor;3l251l0]
`
`andtranscriptionalfeaturesreviewedintextaremarkedoncircularmap.
`
`
`
`
`
`lindromerecognizedbytheEcoRIendonuclcaSC.Panti-tet=P1;Plet=
`
`
`
`NucleotidesequencenumberingstartswithfirstTinthepa
`
`
`
`FIGURE1—3MapofplasmidpBaniAllthestructural
`
`P2;Pbla=P3;PRNA1=P4.
`
`tfimzml
`(u...________
`thanoazz'fi'hu-..
`
`1.4 DNR Replication
`
`19
`
`he found in a number of recent reviews (Davison 1984; Veltkamp and Stnitje
`1981; Selzer et al. 1983; and Cesareni and Banner 1985).
`DNA replication in pBR322 (and ColEl} initiates from a fixed origin
`located between coordinates 2534 and 2536 (Bolivar et al. 1977c; Tomizawa
`et al. 197?} and proceeds onidircctionally around the plasmid genome (1m
`selburg 1974; Lovett et a]. 1975; Bolivar et al. 1977c). Itoh and Tomizawa
`(1932) showed that the initiation of DNA repIiCatic-n in CoIEl requires DNA
`polymerase I, RNA polymerase. and ribonuclease H (RNAse H). Subse-
`quent semidiscontinuous DNA synthesis is carried out by DNA polymerase
`III and the products of dncB and o‘ncG (Stauhenbauer et al. 1979). Plasmid—
`encoded factors controlling the frequency of initiation include a primer
`precursor RNA (RNALI; itch and Tomizawa 1980); a 108-bp inhibitory and
`incompatibilitymediating RNA CRNAI; Morita and Bite 1979; Tomizawa
`et al. 1981); and