Pl d R li i i Eff fRi b lRNAP

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金沢大学十全医学会雑誌第9 2巻第6号 85 1‑^{8 61} ⁽^{1 9 8 3}⁾ ^{8 5 1}

E f fe cts of R ib o s o m al R N A P r o m ote r s o n

P la s mid R eplic atio n

M ^{a r}iâ dêl P il^{a r} A _gûi^{n a}_gâ

D i vi sio n of B iophysic s, Ca n c e r Re s e a r ch In stitute,K a n a z a wa Univ e r sit y

A bstraet

T he efT tcts of p^{r o} ^m ^{o t}^{e r s} ôfri bosom al R N A opê^rôⁿ^sin the rep li^{c a t}io n orlg^{l n} ^{r e}g¹⁰ⁿ Ôf

the B a cillu s s ubti揖 chrom o s o m e hav e be en s t ud ied on the rep li^{c a t}ion of _pla smids in Esche richia c oli as a ho s t c ell. It ha s be en alre ad y fb und t hat p l^{a s}^mids c a r ry^{l n}g^thes e P^r^O^m ^{O t}^{e r}^S ^{C a n} ⁿ^{O t} be rep li^c^{a t}^ed in B ･ Subt肋^･_,ther efbre the m e chanism of inhibition of

r ep li^{c a t}ion c an no tbe e x a min edin th is ba c te riu m . In th is s tud y ^w êfbu nd tha t eve n in E ^. C Oli the re ar e inhibito ry ê鮎ct s on plasmid r ep li^{c a t}ion dep^{e n}ding ôⁿ ^sit e a nd dire c tio n of inse rtion ofthe p^rô^m ^{o t e}^r^s^. In p B R ^{v e c}^{t o}^r^s(Ê ^. ^cô^li p lâ^{s m}i ds)^,^t^hêp^{r o m} ^{o t}^{e r}^{s c a}ⁿ bein sert ed

eitherin one dire c tion orin bo th dir e ctionsdep^{e n}ding ^{o n} gêⁿê ^côⁿ^{s t}it u tions ar oundthesite Of in sertion . St ab ility ôf the p l^{a s}^mi d is als o afr tc t ed b y ^the site an d dir ec tion of the ins ertio n . In a c o m p^{o s}it e ve c t or betw e e n p B R ^{a n}d _{p U B} (^S^tq pわ^′^わ^{c o}^{c c u s} â ^{u r e u}^s p lâ^{s m}id)^,

theins ertion ofthe p^rô^m ^{o t e}^r^s âlone do e s no ttake p l^{a c}ê bu t o c cu rs with an ac c o m pâⁿy^{l n}g D N A 丘ag^mêⁿ^t^, âlw ay^slo c ated do w ns tre am from the p^{r o} ^m ^{o t e}^r^s^. T h isfiag^m êⁿ^tis de riv ed f

ro m the E ^. coli chrom o s om e and can be rep l^{a c}êd b_y D N A 丘ag ^mêⁿ^{t s} ^côⁿ^{t a}in l ng kno w n te r mina t ors oftrans cr lP^t¹⁰ⁿ ^. Subs eq^{u e n}^tl y ^wê have newl y ^côⁿ^{s t}^r^{u c t e}d a ve c to rtha t m aybe usefu1 a s a te r min at or sear ch ing plâ^{s m}id. T he m e chanis m of inhibition c aused b y ^the s e P^rÔ^m ^{O t}^{e r}^S Ôⁿ P l^{a s}^mid r ep lic ation w as d iscu ssed.

Key ^w ^{o r}ds clo n lng^, ^rR N A _pr o m oters, pla smid r eplic atio n

Introduetiom

D u ring ^the s tud y ^of the str u ctu r e and f

u nc tion ofthe orlg^{l n} ^{r e}g^{1 0 n} ^Of rep li^{c a}^tion of BaciHus subtHね iⁿ ou r labora to ry, a D N A f

rag^m êⁿ^t(^{B 7})^, ^W^{h i}^c^h ^{e x}^{h i}^bⁱ^tê^d ^{s o} ^m êⁱⁿ^{h i b i}^{t o}^‑

ry fu nc tion s on D N A r ep li^{c a}^tio n w as fb u nd With in th is r egi^{o n} (^l)^. B y ^sêq^{u e n c}ing â pâ^r^t Ofthis D N A ftag^m êⁿ^t â ^sê^t ôf tw o t ande m

p^{r o m} ^{o t}^{e r}^s ^w ^{a s} d is c o v e red(²)^. ^{T h}^{e s e} p^r^o^m ^o^t^‑

e r s a r e the m ain loci re spô^{n s}i b le fb^r the S uP p^{r e}^{S S}ion of aut onom ous r ep li^c^{a t}ion of p lâ^{s m}ids c a r ry^{l n}g ^this 丘ag^m êⁿ^tin B ^. s ubtilLs.

T hey ^{e x e r c}is ed str o ng inhibitio n o n p l^a^{s m}id

r ep li^{c a t}ion in Esche rich ia c oliand c aus ed r ap i d

S eg^{r e}g^a^tion ofc ells w hich had alre ad_{y l}o st the

Pl^{a s m}i d(³)^.

It w a s fbund that B 7 c ontains a p^{a r}^t ôf a ri bos om al R N A op^{e r}ôⁿ (^{r r n}Ô) ^{a n}^d ^t^hâ^t ^t^hê

tw o t andem p^rô^m ^{o t e}^r^s in th is 丘ag^mêⁿ^t ^w ^{e r}ê p^{r o} ^m ^{o t}^{e r s} ôfrR N A op^{e r}ôⁿ(^{r r}ⁿp^rô^m ^{o t}^{e r}^s) (⁴^,

5)^. ^W ê ^w âⁿ^tê^d ^{t o} ^k^{n o}^w ^{i f} ^t^hê ⁱⁿ^{hi b i}^{t o}^ry

e晩ct s caused b y ^{r r n}O p^rô^m ^{o t}^{e r s} ^wôûl d be C O m m O n f br oth e r r r n p^rô^m ^{o t e}^{r s} âⁿd t o u nde rs t andthe m echanis m of inh ibition du eto thes e p^{r o} ^m ^{o t}^{e r}^s^･

In g^{e n e r}âl, r r n P^rÔ^m ^{O t}^{e r}^S â^rê ^{C O}ⁿ^Sidered

S trOng P^r^O^m ^{O t e}^{r S} ^Sinc e they ^a^{r e} fieq^{u e}ⁿtl y リボゾ ^ーム R N A プロモ ^ータ ^ーのプラスミド複製^に対す^る作用^: 金沢大学がん研究所生物物理部

マリアデルピラ^ールアギナガ ( 指導教官吉川寛)

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tr a n s cri bed b y th e R N A pôl y ^m ê^{r a}^sê^･ Until now rn ore than l O O d i飴rentp^rô^m ^{o t}^{e r} ^sêqû^{e n}^‑ ces hav e bee n i den tified. Prom o te rs ftom S e V er al bacte rial and vir al g^{e n}ô^m ^{e s} hav e b^{e e n} e a sil_y clo n ed in E^･ C Oli and their s tr e ng^th has been s tud ied (⁶^, ⁷)^. ^H ^{o w e}^{v e r} ^c^lôⁿⁱⁿ^g ô^f

D N A f h g^m ^{e n t s} ^cô^{n t a}in l ng ^S^t^rÔⁿg p^rÔ^m ^{O t}^{e r}^S alon e,SuCh as bacte riophagê T 5 p^rô^m ^{o t}^{e r}^s(⁸)

Or r r n p^rÔ^m Ô^{t e}^r^S Ôf E . coli(⁹),ha v e failed^･ T he m echanism ofth is u nclo nab ility ôf t he S trOng P^{r O} ^{m O t e}^r ha s no t been eluci dat ed _yet･

In th is p^ap^{e r} ^w ^e des cri be the ins e rtion of r r n

p^r^{o m o t e}^r^s in vario us pl^a^{s m}ids and it s e 飴c t s On P l^{a s m}i d r ep li^{c a}tion in E ^･ C Oli^･ Som e ve ctor s c o n s tru c ted in this s tud_y m ay h^{a v}^e ^the

P^Otentialto be u sed a s pl^{a s m}i ds s e arch ing fbr

t^ra n s cri_{p t}io nal ter min a t ors. T he m e chanis m Ofinhibition ca us ed b y ^{s t}^{r o n}g p^r^o^m ^o^t^{e r s} is

also d isc u s s ed.

M ateri als a nd M et hods

Bacte rial s tr ain s. E . c oli C 6 0 0 (^t^hy^, hT u,

血, わ乃｣ , 加, ∫呼ダ) âⁿ^d ^丘^■^･ ^{C O}^〟^{1 0 3 7}(^{r e c}^.⁴ ^, d^{n a}A^‑t岳),W er e uSed as ho st sfb rthe cloning ôf P l^{a s m}i ds contain l ng ^r7^･ⁿ P^{r O} ^m ^{O t e}^r^S Ôf B.

subtihs. W hole ch^rom c som al D N A of B.

Sub^tilLs1 6 8 L T T(^h^{? u}^, ^t⁷P C 2^,^th_y) ^w ^{a s} ^{u s e}^{d fb}^r

SoutheTⁿ h ybri d iza tion e xp^{e r}im ent s･

P lasmi ds. Pla s mi ds used in th is s tud y ^{a r}^e su m m a riz ed in Table l^. T he c ons tr u ctio n of P L A R l l l ^aⁿd p L A R 3 3 1 i^s illu stra ted in F i g^. 5^.

P hagê^s^. Lam bda ch B S O I c ont ain l ng ^{r r n}O Op^{e r}Ôⁿ ^{a n}d la mbda ch B S l l c ontain l ng ^{r r}ⁿA Opê^rÔⁿ(F i g^.1) ^hâ^{v e} ^b^{e e n} p^rê^viou sl y dê^s^{c r}ibed

(^{1 3})^.

Table l^. Pr op^{e r}tie s ofthe pla s mids u s ed in th is study

P la s mid S iz e

(k b) ^C^{o m}^p^{o s}ⁱ^tⁱ^{o n}

Ge n etic

m a rke r s So u r c e

pB R 3 2 2 4^.3 6

pB R 3 22 + pU B l l O

A m p^r, Tc^r (1 0)

pB R 3 2 8 4.9 0 AmpてT^cてC^m ^r 四

pU B l lO 5.0 0 K m ^r (^{1 2})

pM S l O 2^′ 7.7 0 A m ^r,Km ^r (3)

pN O l O l O 5.6 6 pB R 3 2 2 十 E 4^′^[¹^】 A mp^r,Tc^r pr ovi ded by D^r. Oga s a w a r a pM S l O 2^′^･B 6 王3^.6 5 pM S l O 2^′+ B 6^【²] A m p^r,K m ^r p^{r o v}i ded by Dr. Oga s a w a r a

･^p^{L O}^p^･¹ ⁷^.^{1 7} ^pB R 3 2 2 十加Rl/^to【31 Am p^r pr o vi ded by D^r^.M a sa^m ^{u n e} pL A R 9 6.20 pB R 3 2 2十E 1 9(b)^【⁴^】 Am p^r,Tc^r this c o m m u nic atio n pL A R 1 2 7.21 pB R 3 2 2+E 1 4(b) Am p^r, Tc^r this c o m m u nic atio n

pN O l O1 2 6^.7 3 pB R 3 2 8 十E 1 9(a) Am p^r, Tc^r (1 3)

pN O l O1 3 6^.73 pB R 3 2 8+E 1 9(b) Am p^r^, Tc^r (¹3)

pN O 2 0 03 7^.7 5 pB R 3 2 8十E 1 4(^a) Am p^r, Tc^r (13)

pN O 2 0 04 7.7 5 pB R 3 2 8+E 1 4(b) Am p^r, Tc^r ( 13)

pB R 3 2 2^･B 7 9.9 0 pB R 3 2 2+ B 7 A m p^r pr o vided by Dr.Og^{a s a w} ^{a r a} pL A R l O 1 1^.3 6 pM S l O 2^′+E 1 9+ Ⅹ(a) Am p^r, K m ^r th is c o m m u nic atio n pL A R l l 1 3^.4 0 pM S l O 2^′+E 1 4+Y(a) Am _p^r, K m ^r this c o m m u nic atio n pL A R 3 3 13.4 0 pM S l O 2^′+ E 14 + Y^′(b) A mp^r, K m ^r this c o m m u nic atio n pL A R 1 3 1 6.2 5 pM S lO 2^′+2【E14〕⁺ ^Y(^c) ^A ^m p^r^. K m ^r this c o m m u nic atio n pL A R l l l 1 3.4 0 pL A R l l^【⁵^】 A m p^r^, K m ^r this c o m m u nic atio n pL A R 3 3 1 1 3^.4 0 pL A R 3 3^[⁵¹ A m p^r^, K m ^r this c o m m u nic atio n

[¹] ^:pN O l O l O ha s al^.3 k b fr agm e nt(E 4^′) ^{c o n}^t^aining r m O^te r min ato rintr odu c ed intot he m nd IIIsi te of pB R 3 2 2^. [2]^:PM S l O2^′^･B 6 ha s a 5^.9 5 k b fr agm e nt(B 6) (1 3)intr odu c ed intothe Ba mH I site ofpM S l O2^′･

B 6 c o ntai^{n s} E 3^′ in which th e r r nA te r min ato rislo c ated^.[3] ^:pL Op^‑1c o ntai_ps t_{e r m}in ato r s t_{R l} a ndto als o ge n e s Oa nd P of la mb da phagê^. [4】^: ^{T h}êⁱ^{n s e r}^tê^{d f}^{r a}^g^m ^{e n}^t^s ^{a r e} â^{t t}^hê Ê^とÔ^{R I} ^sⁱ^tê ô^f ^t^hê ^{v e c}^tp^r^･ ⁽â⁾ ^･

D ir e ctio n of pr o m ote r sisto wa rds A mp^rge n e:m)^‑^:D ir e ctio n of pr o m ote r sis t_{o w}_{a r}d^s T c^r g^{e n e} ^{l n} pB R 3 2 8

a nd pB R 3 2 2 0 rt_{O W}_{a r}ds pU B^ll O o rigi ⁿⁱⁿ pM S l O 2^′^. (^c)^: T w o E 1 4 fr agm e nts s epa r at_ed byâ D N A fr ag^･ m e nt(Y) with th edir e ctio n ofpr o m ot_{e r s}fa cih_g e a ch othe r. X,Y a nd Y^′ a r e D N A fr ag^m?ⁿ^t^S ^dê^rⁱ^{v e}^d fr o m the E c olichr om o s o m e(S e et_{e X}t)^. [5】^: ^{T h}ê ^励oR Isite up^str e a m fr o m the pr o m ote r s I S m Od i丘ed･

(k b): M ole cula r siz e of D N A in kiloba s e pair s^.