D. R. Helinski et al. (eds.), Plasmids in Bacteria
`© Plenum Press, New York 1985
`
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2079
`
`

`
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`
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`
`the stahle ntegration of these proeoter classes (ii, ani it hecane
`olnions that ntense trnscriptlonal activities can notices in
`canine reps nith plrsnil nantenaoce ani strocnal stahilitp.
`to
`foreclose constrnctei i lnilies of plrsnil rectors nhich not onlp
`ailon no to stahlp ntegrate pronoters nlepenientlp of
`their
`strength, int also enabled to to ilentifp none of the prohlens nhich
`iecrilp traoscnihci regions can one to folll-tppe rector spstens
`(ill.
`
`iheprioipleoflnesepiannilspstensisshonn nPig.lani
`their in properties are leccrlhel in the correspnlng legenl.
`
`the stahle ntegration of these proeoter classes (ii, ani it hecane
`olnions that ntense trnscriptlonal activities can interfere in
`canine reps nith plrsnil nantenaoce ani strocnal stahilitp.
`to
`foreclose constrnctei i lnilies of plrsnil rectors nhich not onlp
`ailon no
`to stahlp ntegrate pronoters nlepenientlp of
`their
`strength, int also enabled to to ilentifp none of the prohlens nhich
`iecrilp traoscnihci regions can one to folll-tppe rector spstens
`(fl).
`
`iheprioipleoflnesepiannilspstensisshonn nPig.lani
`their in properties are leccrlhel in the correspnlng legenl.
`
`the stahle ntegration of these proeoter classes (ii, ani it hecane
`olnions that
`intense transcriptional activities can interfere in
`canine reps nith plrsnil nantenaoce ani strocnal stahilitp.
`to
`foreclose constrnctei i lnilies of plrsnil rectors nhich not onlp
`ailon no to stahlp integrate pronoters nlepenientlp of
`their
`strength, int also enabled to to ilentifp none of the prohlens nhich
`iecrilp traoscnihci regions can one to folll-tppe rector spstens
`(fl).
`
`iheprioipleoflnesepiannilspstensisshonn nPig.lani
`their in properties are leccrlhel in the correspnlng legenl.
`
`the stahle ntegration of these proeoter classes (ii, ani it hecane
`olnions that
`intense transcriptional activities can interfere in
`canine reps nith plrsnil nantenaoce ani strocnal stahilitp.
`to
`foreclose constrnctei i lnilies of plrsnil rectors nhich not onlp
`ailon no to stahlp integrate pronoters nlepenientlp of
`their
`strength, int also enabled to to ilentifp none of the prohlens nhich
`iecrilp traoscnihci regions can one to folll-tppe rector spstens
`(fl).
`
`iheprioipleoflnesepiannilspstensisshonn nPig.lani
`their in properties are leccrlhel in the correspnlng legenl.
`
`Mp inner and Plasnil ilcintenance
`
`Mp inner and Plasnil ilcintenance
`
`Mp inner and Plasnil ilcintenance
`
`Mp inner and Plasnil ilcintenance
`
`the cloong of nations
`(Pig. i) pernittel
`Pie pilll rector
`phage if pronoters (ll. honor,
`those font to he not efficient
`nritro cppeacel to be either not clonahio or thep cancel a lrastic
`selection in the copp nnnoer, often accospanici rith the non of the
`tire planil npon prolongei propagation of tie host cells.
`since
`the reason for this phenonenon sold here heeo an oerproinctin of
`M gene prolots as cell as a possible transcriptional roalthtongi
`nto the replication region, as leoelopel
`the pill plasnii fnilp
`(f). on teen shonn (l), ntegration of a M i into on the
`M if site of plliii (nhich destroys the ooing sentence of
`the
`iii-proton (ill ncceasei the piannil copp men i- to l-foll, n-
`licatiog that a possible noerpnolotio of (iris regnlator protenhp
`resltnongh iron npatrean pronoters can relate plaonil copp nnher
`ani nnhsegnent sentence. This is lennstratel hest hp the fact
`that ii pronto P
`I is stahlp nantainel in poll, alheit at a re-
`lncoi coppnonier ((
`. i(. nsertionof an efficient terninatnr at
`site 1 no i of post
`(Pig. 1], hoaener, ncreases the copp nnnler
`again (Pig. 2], sharing that
`transcriptional
`realthrongh into the
`replication region hp itself has a profound effect on plrsnil rep
`licatin.
`in have not onalpnoi nlether this is no to an orerpro
`lnctioo of hill (ii, a possible 'nenranil' nterfereoce rith the
`trnscriptnnal colt for in ii (ii, on hotl.
`
`Strong tranoriptinal rosithrnngh no the replication region
`tonarls the price proeoter (prolong til it, let. i) also niec-
`feres rith plasnil replication.
`listens pronoters no to in lines the
`efliciencp of the l-iactanase prnnoter (PW or P3, nlef. it
`he toieratel, sepnences shin cool this pronoter‘s efliciencp il-
`foll or one cannot stahlp he ntegratel in place of P
`nnlen con-
`ditions niece translation of
`l-nctanase n-iii nnnll
`teen in-
`
`pocsihle (i,il(.
`
`in srnnnanp, for the constnnction of highlp efficient transcrip
`tinal onits nitln pnsnii octorn carrping toiil-tppe replicons,
`it has to he talen nto accont that pronntec effiniencp has to he
`lhaiancel‘
`for
`the on to he stahlp nantainei.
`i'nis can he
`achieoel heat hp nsertng colpstihle transcriptional signals, pre-
`ferahlp in ailition to repressihle proanter elenents.
`
`the cloong of nations
`(Pig. i) pernittel
`Pie pilll rector
`phage if pronoters (ll. honor,
`those font to he not efficient
`nritro cppeacel to be either not clonahio or thep cancel a lrastic
`selection in the copp nnnoer, often accospanici rith the non of the
`tire planil npon prolongei propagation of tie host cells.
`since
`the reason for this phenonenon sold here heeo an oerproinctin of
`M gene prolots as cell as a possible transcriptional roalthtongi
`nto the replication region, as leoelopel
`the pill plasnii fnilp
`(f). on teen shonn (l), ntegration of a M i into on the
`M if site of plliii (nhich destroys the ooing sentence of
`the
`iii-proton (ill ncceasei the piannil copp men i- to l-foll, n-
`licatiog that a possible noerpnolotio of (iris regnlator protenhp
`resltnongh iron npatrean pronoters can relate plaonil copp nnher
`ani nnhsegnent sentence. This is lennstratel hest hp the fact
`that ii pronto P
`I is stahlp nantainel in poll, alheit at a re-
`lncoi coppnonier (fag. i(. nsertionof an efficient terninatnr at
`site 1 no i of post
`(Pig. 1], hoaener,
`increases the copp nnnler
`again (Pig. 2], sharing that
`transcriptional
`realthrongh into the
`replication region hp itself has a profound effect on plrsnil rep
`licatin.
`in have not onalpnoi nlether this is no to an orerpro
`lnctioo of hill (ii, a possible 'nenranil' interference rith the
`transcriptional colt for in ii (ii, on hotl.
`
`Strong tranoriptinal rosithrnngh no the replication region
`tonarls the price proeoter (prolong til it, let. i) also niec-
`feces rith plasnil replicatnn.
`listens pronoters no to in lines the
`efliciencp of the l-iactanase prnnoter (PW or P3, nlef. it
`he toieratel, sepnences shin cool this pronoter‘s efliciencp il-
`foll or one cannot stahlp he ntegratel in place of P
`nnlen con-
`ditions niece translation of
`l-nctanase n-iii nnnll
`teen in-
`
`pocsihle (i,il(.
`
`in srnnnanp, for the constnnction of highlp efficient transcrip
`tinal onits nitln pnsnii octorn carrping toiil-tppe replicons,
`it has to he talen nto accont that pronntec effiniencp has to he
`lhaiancel‘
`for
`the on to he stahlp nantainei.
`i'nis can he
`achieoel heat hp nsertng colpstihle transcriptional signals, pre-
`ferahlp in ailition to repressihle proanter elenents.
`
`the cloong of nations
`(Pig. i) pernittel
`Pie pilll rector
`phage if pronoters (ll. honor,
`those font to he not efficient
`nritro cppeacel to be either not clonahio or thep cancel a lrastic
`selection in the copp nnnoer, often accospanici rith the non of the
`tire planil npon prolongei propagation of tie host cells.
`since
`the reason for this phenonenon sold here heeo an oerproinctin of
`M gene prolots as cell as a possible transcriptional roalthtongi
`nto the replication region, as leoelopel
`the pill plasnii fnilp
`(f). on teen shonn (l), ntegration of a M i into on the
`M if site of plliii (nhich destroys the ooing sentence of
`the
`iii-proton (ill ncceasei the piannil copp men i- to l-foll, n-
`licatiog that a possible noerpnolotio of (iris regnlator protenhp
`resltnongh iron npatrean pronoters can relate plaonil copp nnher
`ani nnhsegnent sentence. This is lennstratel hest hp the fact
`that ii pronto P
`I is stahlp nantainel in poll, alheit at a re-
`lncoi coppnonier (fag. i(. nsertionof an efficient terninatnr at
`site 1 no i of post
`(Pig. 1], hoaener,
`increases the copp nnnler
`again (Pig. 2], sharing that
`transcriptional
`realthrongh into the
`replication region hp itself has a profound effect on plrsnil rep
`licatin.
`in have not onalpnoi nlether this is no to an orerpro
`lnctioo of hill (ii, a possible 'nenranil' nterfereoce rith the
`transcriptional colt for in ii (ii, on hotl.
`
`Strong tranoriptinal rosithrnngh no the replication region
`tonarls the price proeoter (prolong til it, let. i) also niec-
`feres rith plasnil replicatnn.
`listens pronoters no to in lines the
`efliciencp of the l-iactanase prnnoter (PW or P3, nlef. it
`is tolerated, sepnences shin cool this pronoter‘s efliciencp il-
`foll or one cannot stahlp he ntegratel in place of P
`nnlen con-
`ditions niece translation of
`l-nctanase n-iii nnnll
`teen in-
`
`pocsihle (i,il(.
`
`in srnnnanp, for the constnnction of highlp efficient transcrip
`tinal onits nitln pnsnii octorn carrping toiil-tppe replicons,
`it has to he talen nto accont that pronntec effiniencp has to he
`lhaiancel‘
`for
`the on to he stahlp nantainei.
`i'nis can he
`achieoel heat hp inserting colpstihle transcriptional signals, pre-
`ferahlp in ailition to repressihle proanter elenents.
`
`the cloong of nations
`(Pig. i) pernittel
`Pie pilll rector
`phage if pronoters (ll. honor,
`those font to he not efficient
`nritro cppeacel to be either not clonahio or thep cancel a lrastic
`selection in the copp nnnoer, often accospanici rith the non of the
`tire planil npon prolongei propagation of tie host cells.
`since
`the reason for this phenonenon sold here heeo an oerproinctin of
`M gene prolots as cell as a possible transcriptional roalthtongi
`nto the replication region, as leoelopel
`the pill plasnii fnilp
`(f). on teen shonn (l), ntegration of a M i into on the
`M if site of plliii (nhich destroys the ooing sentence of
`the
`iii-proton (ill ncceasei the piannil copp men i- to l-foll, n-
`licatiog that a possible noerpnolotio of (iris regnlator protenhp
`resltnongh iron npatrean pronoters can relate plaonil copp nnher
`ani nnhsegnent sentence. This is lennstratel hest hp the fact
`that ii pronto P
`I is stahlp nantainel in poll, alheit at a re-
`lncoi coppnonier ((
`. i(. nsertionof an efficient terninatnr at
`site 1 no i of post
`(Pig. 1], hoaener,
`increases the copp nnnler
`again (Pig. 2], sharing that
`transcriptional
`realthrongh into the
`replication region hp itself has a profound effect on plrsnil rep
`licatin.
`in have not onalpnoi nlether this is no to an orerpro
`lnctioo of hill (ii, a possible 'nenranil' nterfereoce rith the
`transcriptional colt for in ii (ii, on hotl.
`
`Strong tranoriptinal rosithrnngh no the replication region
`tonarls the price proeoter (prolong til it, let. i) also niec-
`feces rith plasnil replicatnn.
`listens pronoters no to in lines the
`efliciencp of the l-iactanase prnnoter (PW or P3, nlef. it
`is tolerated, sepnences shin cool this pronoter‘s efliciencp il-
`foll or one cannot stahlp he ntegratel in place of P
`nnlen con-
`ditions niece translation of
`l-nctanase n-iii nnnll
`teen in-
`
`pocsihle (i,il(.
`
`in srnnnanp, for the constnnction of highlp efficient transcrip
`tinal onits nitln pnsnii octorn carrping toiil-tppe replicons,
`it has to he talen nto accont that pronntec effiniencp has to he
`lhaiancel‘
`for
`the on to he stahlp nantainei.
`i'nis can he
`achieoel heat hp nsertng colpstihle transcriptional signals, pre-
`ferahlp in ailition to repressihle proanter elenents.
`
`

`
`llhhlllhllhflllhhiihhliihhlhcilfillli
`
`ll
`
`llhillllhllhflllhhiihhliihhlhcilfillli
`
`ll
`
`llhillllhllhflllhhiihhliihhlhcilfillli
`
`ll
`
`llhhlllhllhflllhhiihhliihhlhcilfillli
`
`ll
`
`Fig. l.
`
`
`
`hoth plas-
`ienetio ani phpsical nap oi phhlia ani phil.
`nhls contain 2 easilp assapahle inoctions nhioh can he
`hrooght sales the control oi a single pronotet (ll hnt
`nhich can also he seporatol hp tho insertion oi a terri-
`nator
`ii].
`lhese inoctions are lot
`the phi series:
`h-galactosihase actiritp (oia r-omplenentation at not
`tetracpcline resistance (too); for the pih series:
`tri-
`rethoprhne (oia hlhplrololate rohochase, hM anl chlor-
`mheoisoi
`(chinranphnricoi aoetpl transierase, M ra-
`sistarce.
`hoth plaonils coniet anpioillin resistance [oia
`i-lactnase, M and oarrp the iohil replication region
`of phiiii (M.
`imjor lilierence oi
`the i plnsnhl
`sperm is the presence oi
`the intact nap gene in phhlia
`and a second site for tho integration of a terninatoo
`[site i) in pill.
`l‘oe hireotion of transcription of the
`oohing sepoences is inlicatei.
`iositions and directions
`of trnscription for in ii (prison hill anl iii are
`representel hp cranes.
`lhe phpsioal rape of hoth plasnils
`are not honor to scale.
`integration of cnliphage fl ter-
`ninator into phhlia session in pihlh nhioh oas nsei
`ion
`the cloning of ooliphage hi proosotets ii).
`insertion of
`ternioators lihe t of ooliphage l (in) at site 1, ion at
`hoth sites nalesotlio piasnii an open letter cloning
`vehicle for pitlilti cloning.
`iroonters ash other signals
`conheplooeiinirontoftheM_rgnaeintooarioos
`restriction sites [onlp the Mi site is also here].
`honest the M gene carries its onnn translational initia-
`tion site,
`there is no translation of M-speciiiei
`nessage raises a ihhihtltl hiniing site has preoionslp
`heen fool to the M gene.
`
`Fig. l.
`
`
`
`hoth plas-
`ienetio ani phpsical nap oi phhlia ani phil.
`nhls contain 2 easilp assapahle inoctions nhioh can he
`hrooght sales the control oi a single pronotet (ll hnt
`nhich can also he seporatol hp the insertion oi a terri-
`nator
`ii].
`lhese inoctions are lot
`the phi series:
`h-galactosihase actiritp (oia r-omplenentation at not
`tetracpcline resistance (too); for the pih series:
`tri-
`rethoprhne (oia hlhplrololate rohochase, hM anl chlor-
`mheoisoi
`(chinranphnricoi aoetpl transierase, M ra-
`sistarce.
`hoth plaonils coniet anpioillin resistance [oia
`i-lactnase, M and oarrp the iohil replication region
`of phiiii (M.
`imjor lilierence oi
`the i plnsnhl
`sperm is the presence oi
`the intact nap gene in phhlia
`and a second site too the integration of a terninatoo
`[site i) in pill.
`l‘oe hireotion of transcription of the
`oohing sepoences is inlicatei.
`iositions and directions
`of trnscription for in ii (prison hill anl iii are
`representel hp cranes.
`lhe phpsioal rape of hoth plasnils
`are not honor to scale.
`integration of cnliphage fl ter-
`ninator into phhlia session in pihlh nhioh oas nsei
`ion
`the cloning of ooliphage hi proosotets ii).
`insertion of
`ternioators lihe t of ooliphage l (in) at site 1, ion at
`hoth sites nalesotlio piasnii an open letter cloning
`vehicle for pitlilti cloning.
`iroonters ash other signals
`conheplooeiinirontoftheM_rgnaeintooarioos
`restriction sites [onlp the Mi site is also here].
`honest the M gene carries its onnn translational initia-
`tion site,
`there is no translation of M-speciiiei
`nessage raises a ihhihtltl hiniing site has preoionslp
`heen fool to the M gene.
`
`Fig. l.
`
`
`
`hoth plas-
`ienetio ani phpsical nap oi phhlia ani phil.
`nhls contain 2 easilp assapahle inoctions nhioh can he
`hrooght sales the control oi a single pronotet (ll hnt
`nhich can also he seporatol hp the insertion oi a terri-
`nator
`ii].
`lhese inoctions are lot
`the phi series:
`h-galactosihase actiritp (oia r-omplenentation at not
`tetracpcline resistance (too); for the pih series:
`tri-
`rethoprhne (oia hlhplrololate rohochase, hM anl chlor-
`mheoisoi
`(chinranphnricoi aoetpl transierase, M ra-
`sistarce.
`hoth plaonils coniet anpioillin resistance [oia
`i-lactnase, M and oarrp the iohil replication region
`of phiiii (M.
`imjor lilierence oi
`the i plnsnhl
`sperm is the presence oi
`the intact nap gene in phhlia
`and a second site too the integration of a terninatoo
`[site i) in pill.
`l‘oe hireotion of transcription of the
`oohing sepoences is inlicatei.
`iositions and directions
`of trnscription for in ii (prison hill anl iii are
`representel hp cranes.
`lhe phpsioal rape of hoth plasnils
`are not honor to scale.
`integration of cnliphage fl ter-
`ninator into phhlia session in pihlh nhioh oas nsei
`ion
`the cloning of ooliphage hi proosotets ii).
`insertion of
`ternioators lihe t of ooliphage l (in) at site 1, ion at
`hoth sites nalesotlio piasnii an open letter cloning
`vehicle for pitlilti cloning.
`iroonters ash other signals
`conheplooeiinirontoftheM_rgnaeintooarioos
`restriction sites [onlp the Mi site is also here].
`honest the M gene carries its onnn translational initia-
`tion site,
`there is no translation of M-speciiiei
`nessage raises a ihhihtltl hiniing site has preoionslp
`heen fool to the M gene.
`
`Fig. l.
`
`
`
`hoth plas-
`ienetio ani phpsical nap oi phhlia ani phil.
`nhls contain 2 easilp assapahle inoctions nhioh can he
`hrooght sales the control oi a single pronotet (ll hnt
`nhich can also he seporatol hp the insertion oi a terri-
`nator
`ii].
`lhese inoctions are lot
`the phi series:
`h-galactosihase actiritp (oia r-omplenentation at not
`tetracpcline resistance (too); for the pih series:
`tri-
`rethoprhne (oia hlhplrololate rohochase, hM anl chlor-
`mheoisoi
`(chinranphnricoi aoetpl transierase, M ra-
`sistarce.
`hoth plaonils coniet anpioillin resistance [oia
`i-lactnase, M and oarrp the iohil replication region
`of phiiii (M.
`imjor lilierence oi
`the i plnsnhl
`sperm is the presence oi
`the intact nap gene in phhlia
`and a second site too the integration of a terninatoo
`[site i) in pill.
`l‘oe hireotion of transcription of the
`oohing sepoences is inlicatei.
`iositions and directions
`of transcription for in ii (prison hill anl iii are
`representel hp cranes.
`lhe phpsioal rape of hoth plasnils
`are not honor to scale.
`integration of cnliphage fl ter-
`ninator into phhlia session in pihlh nhioh oas nsei
`ion
`the cloning of ooliphage hi proosotets ii).
`insertion of
`ternioators lihe t of ooliphage l (in) at site 1, ion at
`hoth sites nalesotlio piasnii an open letter cloning
`vehicle for pitlilti cloning.
`iroonters ash other signals
`conheplooeiinirontoftheM_rgnaeintooarioos
`restriction sites [onlp the Mi site is also here].
`honest the M gene carries its onnn translational initia-
`tion site,
`there is no translation of M-speciiiei
`nessage raises a ihhihtltl hiniing site has preoionslp
`heen fool to the M gene.
`
`The inllnonce of ienninator losition on llasnil iinerination in piil
`
`The inllnonce of ienninator losition on llasnil iinerination in piil
`
`The inllnonce of ienninator losition on llasnil iinerination in piil
`
`The inllnonce of ienninator losition on llasnil iinerination in piil
`
`is shonn alone, placing the ternioator t
`in position 1 at h oi
`pihl escaping the phage ii proaoter hm ohestallishes the sopp
`rather to that of the prosnter-free plaanil ilig. ii. homer.
`in
`analpring the ratio of Iononerlc to iinerlc icons of the plasnii, it
`can he clearlp recognised that to inpositlon i increases the hllhti
`
`is shonn alone, placing the ternioator t
`in position 1 or i oi
`pihl escaping the phage ii proaoter hm ohestallishes the sopp
`rather to that of the prosnter-free plaanil ilig. ii. homer.
`in
`analpring the ratio of Iononerlc to iinerlc icons of the plasnii, it
`can he clearlp recognised that to inpositlon i increases the hllhti
`
`is shonn alone, placing the ternioator t
`in position 1 or i oi
`pihl escaping the phage ii proaoter hm ohestallishes the sopp
`rather to that of the prosnter-free plaanil ilig. ii. homer.
`in
`analpring the ratio of Iononerlc to iinerlc icons of the plasnii, it
`can he clearlp recognised that to inpositlon i increases the hllhti
`
`is shonn alone, placing the ternioator t
`in position 1 or i oi
`pihl escaping the phage ii proaoter hm ohestallishes the sopp
`rather to that of the prosnter-free plaanil ilig. ii. homer.
`in
`analpring the ratio of Iononerlc to iinerlc icons of the plasnii, it
`can he clearlp recognised that to inpositlon i increases the hllhti
`
`

`
`48
`
`H. BUJARD ET AL.
`
`
`
`Fig. 2.
`
`Electrophoretic analysis of pDS1 derivatives at various
`cell stages (for details, see Ref. 4).
`The roman numerals
`designate the nucleic acid preparations
`from cells of
`stage I to IV, M are concentration standards.
`The dupli-
`cate probes can be identified by the headings whereby the
`following abbreviations are used:
`P
`, coliphage T5 pro-
`moter P 07 inserted into the EcoRI site of pDS1 (Fig. 1);
`tol
`and tol
`describes the insertion of Ehe coliphage A
`terminator t
`at site 1
`in functional
`(1 ) and nonfunc-
`tional
`(1_) grientation, respectively;
`the same holds for
`:02
`and t 2 .
`The first band on the top of each gel re-
`presents thb dimeric,
`the lowest
`the monomeric form of the
`plasmid.
`In comparing the dimeric forms in stage II and
`stage IV,
`the influence of
`the positioning of
`t
`can be
`seen most clearly.
`
`0
`
`of dimeric structures. This is confirmed by the experiment shown in
`Fig. 3.
`Since it has been shown that multimerization decreases the
`maintenance of Co1E1-derived plasmids (11),
`these observations show
`that
`even such seemingly trivial
`topographic
`rearrangements of
`otherwise identical signals can affect
`the stable propagation of a
`vector. This result
`is puzzling in a special way:
`proposing that
`heavily transcribed regions may have some impact on a plasmid's rep-
`lication, a size reduction of this region by moving t
`from position
`2 to 1 would be expected to have the opposite effect.°
`
`Increase of Recombinational Events in Heavily Transcribed Regions
`
`We have constructed a pDS1 derivative containing 3 identical T5
`promoter sequences tandemly fused around 100 bp apart (12).
`Since a
`
`

`
`INSEHHONOFTRANSCmPWONALfl£MENTS
`
`49
`
`
`
`Fig. 3.
`
`Influence of the positioning of terminators on the dimeri—
`zation of pDS1.
`The different probes of
`the electropho-
`retic analysis in agarose gels can be identified by the
`heading of
`the figure.
`The promoter used is P2
`from
`coliophage T5.
`The
`increase in plasmid dimerizaglon in
`presence of P207 and terminator t
`in position 1
`(Fig. 1)
`is seen in lane 2 (from the left)P
`
`battery of 3 strong promoters was assumed to possibly create prob-
`lems,
`the promoters were previously fused to a
`lac operator se-
`quence.
`In addition,
`terminator t was placed in position 1 and no
`ribosomal binding site was presentoto allow the production of DHFR.
`This construct is perfectly stable if grown in the presence of suf-
`ficient
`amounts of
`lac repressor
`(Fig. 4).
`Upon induction with
`IPTG, colonies appear at high rates wherein the plasmid has lost one
`or 2 promoters by homologous recombination (evidenced by fine map-
`ping of restriction sites and sequence analysis). Transferring the
`plasmid into rec A or rec F strains markedly increased the stability
`of
`the construct
`(Fig. 4).
`There are 2
`interpretations to those
`results:
`
`- Increased intensity of transcription has an impact on the fre-
`quency of
`recombinational
`events whereby
`it
`is not clear
`whether
`the tandemly repeated promoter
`regions
`represent
`a
`special situation, or whether similar results can be obtained
`with tandemly repeated sequences
`further downstream of
`the
`transcriptional initiation sites.
`
`low frequency of elimination of promoters
`- There is a normal
`but, under condition of
`transcription, plasmids with less in-
`tense transcription (i.e.,
`lower promoter number) have a rep-
`licative advantage.
`
`the first interpretation, we have never observed
`In favor of
`differences
`in copy numbers between plasmids
`in which promoters
`differ severalfold in their efficiency-—as long as translation of
`the overproduced m-RNA and readthrough into the replication region
`is prevented.
`
`

`
`50
`
`H. BUJARD ET AL.
`
`
`
`Fig. 4.
`
`tandemly repeated promoter/operator con-
`a.
`Stability of
`struct.
`The upper part of the figure shows the electro-
`phoretic analysis (6% polyacrylamide) of pDSl containing
`t
`in position 1 (Fig. 1) and the triple promoter/operator
`cgnstruct shown in the lower part of the figure.
`The dis-
`tance between the promoter/operator
`regions
`is 100 bp.
`The host system and the conditions of growth (t
`IPTG) of
`the different plasmid isolates can be taken from the head-
`ings. At
`the left side the numbers of promoter/operator
`regions within the different isolates are indicated, which
`are
`600,
`400,
`or
`200 bp
`in length,
`respectively,
`as
`pointed out at the left side of the figure.
`The cultures
`were grown to OD600 9 2.0 in LB medium.
`
`Plasmid Carried Translational Initiation Signals and the Possible
`Limits of the Translational Capacity of the Host System
`
`insertion permits
`system carrying a proper promoter
`The pDSl
`quantification of different translational initiation signals by in-
`serting the unknown signal in front of the dhfr gene.
`The resulting
`polycistronic m—RNA (terminator in position 2) codes for 2 proteins:
`DHFR and CAT.
`Since the amount of CAT depends on its original
`translation initiation signal,
`the ratio of CAT to DHFR can be used
`to compare translation initiation signals of unknown activity (13).
`The products of both indicator genes (dhfr and cat) are tolerated by
`the cell in concentrations of up to 50% of the total soluble protein
`if only one of
`the genes is translated or up to 30% each if both
`gene products are allowed to be synthesized. Using this system, we
`have identified some unusually efficient
`translational
`initiation
`signals.
`Some of these, however, can only be cloned in presence of
`a
`strong promoter
`if an efficient
`transcriptional
`terminator
`is
`placed in position 2 of the plasmid (Fig. 1). Attempts to integrate
`
`

`
`lihillllliilillllliiiiiillliihlflillfllii
`
`hi
`
`lihillllliilillllliiiiiillliihlflillfllii
`
`hi
`
`lihillllliilillllliiiiiillliihlflillfllii
`
`hi
`
`lihillllliilillllliiiiiillliihlflillfllii
`
`hi
`
`terninator in
`on signals into plannils carrping the iieatical
`position i failel.
`hnr interpretation of these experience is that
`the axons of hill proincel onler these coalitions nap hearse tonic
`to the cell.
`lnaeoer.
`loaning stoichloetric accents of
`ill-
`rihosoaal hinling sites (omission in position i) cropeting rith
`the signal in front of the hits gene appears to allon the snrninal
`of the sell. Since nnier these coalitions arornl ill of the scalp
`spnthesinol proteins consist of it nsl hill, these esperisents seen
`to iniicate that, hp one of the proper expression signals inanlti-
`copp plasaila. at least in transiatinai ehinerp oi the host can
`he exhaoatei at certain stages of grnath.
`ionsepoantlp,
`the stable
`propagation of a plasnii can also iepnol on phpsinlogical parareters
`oi this tppe.
`
`terninator in
`on signals into plannils carrping the iieatical
`position i failel.
`hnr interpretation of these experience is that
`the axons of hill proincel onler these coalitions nap hearse tonic
`to the cell.
`lnaeoer.
`loaning stoichloetric accents of
`ill-
`rihosoaal hinling sites (omission in position i) cropeting rith
`the signal in front of the hits gene appears to allon the snrninal
`of the sell. Since nnier these coalitions arornl ill of the scalp
`spnthesinol proteins consist of it nsl hill, these esperisents seen
`to iniicate that, hp one of the proper expression signals inanlti-
`copp plasaila. at least in transiatinai ehinerp oi the host can
`he exhaoatei at certain stages of grnath.
`ionsepoantlp,
`the stable
`propagation of a plasnii can also iepnol on phpsinlogical parareters
`of this tppe.
`
`terninator in
`on signals into plannils carrping the iieatical
`position i failel.
`hnr interpretation of these experience is that
`the axons of hill proincel onler these coalitions nap hearse tonic
`to the cell.
`lnaeoer.
`loaning stoichloetric accents of
`ill-
`rihosoaal hinling sites (omission in position i) cropeting rith
`the signal in front of the hits gene appears to allon the snrninal
`of the sell. Since nnier these coalitions arornl ill of the scalp
`spnthesinol proteins consist of it nsl hill, these esperisents seen
`to iniicate that, hp one of the proper expression signals inanlti-
`copp plasaila. at least in transiatinai ehinerp oi the host can
`he exhaoatei at certain stages of grnath.
`ionsepoantlp,
`the stable
`propagation of a plasnii can also iepnol on phpsinlogical parareters
`of this tppe.
`
`terninator in
`on signals into plannils carrping the iieatical
`position i failel.
`hnr interpretation of these experience is that
`the axons of hill proincel onler these coalitions nap hearse tonic
`to the cell.
`lnaeoer.
`loaning stoichloetric accents of
`ill-
`rihosoaal hinling sites (omission in position i) cropeting rith
`the signal in front of the hits gene appears to allon the snrninal
`of the sell. Since nnier these coalitions arornl ill of the scalp
`spnthesinol proteins consist of it nsl hill, these esperisents seen
`to iniicate that, hp one of the proper expression signals inanlti-
`copp plasaila. at least in transiatinai ehinerp oi the host can
`he exhaoatei at certain stages of grnath.
`ionsepoantlp,
`the stable
`propagation of a plasnii can also iepnol on phpsinlogical parareters
`of this tppe.
`
`hlilllliii Willi
`
`hlilllliii Willi
`
`hlilllliii Willi
`
`hlilllliii Willi
`
`ile resnlts lescrihel here nnere ohtainei as "rile prolnots' in
`a prngra in iaicl plaoils are serelp heing nsel as tools to pro-
`iaae all to strip signals of gene expression in nitrn anl innino.
`teen in scale sisple constructs as lfscnsssl here, share an iron the
`ill sepneneas anl the topograplp of the rain locations, it appears
`that one often cannt preiict
`the antenna of senninglp hnnalogons
`enperlsnnts, anl pnanling pnestions lltiliiiit.
`ihep iniicate that
`can seal straig)ntinrearl artificial plasnil coastrnots cannot he
`hotel at in a static nap.
`lnsteal, they appear as ipnaio entities
`innhicl, for eaanpie,
`the extent of transcription in others re-
`gions nap represent onlp one each nariahie to he tries into accnnt
`shall each a spacer iecone preiictahle.
`
`ile resnlts lescrihel here nnere ohtainei as "rile prolnots' in
`a prngra in iaicl plaoils are serelp heing nsel as tools to pro-
`iaae all to strip signals of gene expression in nitrn anl innino.
`teen in scale sisple constructs as lfscnsssl here, share an iron the
`ill sepneneas anl the topograplp of the rain locations, it appears
`that one often cannt preiict
`the antenna of senninglp hnnalogons
`enperlsnnts, anl pnanling pnestions lltiliiiit.
`ihep iniicate that
`can seal straig)ntinrearl artificial plasnil coastrnots cannot he
`hotel at in a static nap.
`lnsteal, they appear as ipnaio entities
`innhicl, for eaanpie,
`the extent of transcription in others re-
`gions nap represent onlp one each nariahie to he tries into accnnt
`shall each a spacer iecone preiictahle.
`
`ile resnlts lescrihel here nnere ohtainei as "rile prolnots' in
`a prngra in iaicl plaoils are serelp heing nsel as tools to pro-
`iaae all to strip signals of gene expression in nitrn anl innino.
`teen in scale sisple constructs as lfscnsssl here, share an iron the
`ill sepneneas anl the topograplp of the rain locations, it appears
`that one often cannot preiict
`the antenna of senninglp hnnalogons
`enperlsnnts, anl pnanling pnestions lltiliiiit.
`ihep iniicate that
`can seal straig)ntinrearl artificial plasnil coastrnots cannot he
`hotel at in a static nap.
`lnsteal, they appear as ipnaio entities
`innhicl, for eaanpie,
`the extent of transcription in others re-
`gions nap represent onlp one each nariahie to he tries into accnnt
`shall each a spacer iecone preiictahle.
`
`ile resnlts lescrihel here nnere ohtainei as "rile prolnots' in
`a prngra in iaicl plaoils are serelp heing nsel as tools to pro-
`iaae all to strip signals of gene expression in nitrn anl innino.
`teen in scale sisple constructs as lfscnsssl here, share an iron the
`ill sepneneas anl the topograplp of the rain locations, it appears
`that one often cannt preiict
`the antenna of senninglp hnnalogons
`enperlsnnts, anl pnanling pnestions lltiliiiit.
`ihep iniicate that
`can seal straig)ntinrearl artificial plasnil coastrnots cannot he
`hotel at in a static nap.
`lnsteal, they appear as ipnaio entities
`innhicl, for eaanpie,
`the extent of transcription in others re-
`gions nap represent onlp one each nariahie to he tries into accnnt
`shall each a spacer iecone preiictahle.
`
`liiiiiillii
`
`liiiiiillii
`
`liiiiiillii
`
`liiiiiillii
`
`1.
`
`i.
`
`a.
`
`l.
`
`5.
`
`trials, i.l., nah i. lnjanl (iili) interaction of o. ol_i iii
`polpnerase rith pronters:
`the relative rates of conpiex for
`nation anl lecap rith prolnters of seoeral phage anl plannil
`iil's.
`trot. lath. isal. ill, on ihtihi-iii.
`innner, i.
`(hill) lntersnshongen nor
`iechsdnirhng non o.
`oofi iii-iolpserase nit irnnotoren.
`iiplnnarheit, hnineraitit
`leiielierg, hi ieilelherg, iii.
`iohen. anl a. open
`lento, n., a. ianpner, nor. snag. s.n.
`(till)
`iloning anl analpais of
`strong proeoters
`in sale
`possihiehp the loanstrean plaoeaent oi an ill ternination
`signal.
`tron. iati. ical. ici., iii lirlililill.
`itnaher, i., anl l. injarl (lill) transcription fro: efficient
`proeaters can interfere rith plassil replication al lininisi
`expression of plasail specifiel genes.
`filo iciiii-ilil.
`lesarini, t., i.i. loosing. anl i. ioiisip (hill) antral of
`iolii ill replication:
`the top gene prslnot negatinelp affects
`transcription