UNITED STA TES p A TENT AND TRADEMARK OFFICE
APPLICATION NO. FILING DATE
12/644,666 12/22/2009
28986 7590 02/18/2016
HARRIET M, STRIMPEL, D, PhiL
New England Biolabs, Inc.
240 COUNTY ROAD
IPSWICH, MA 01938-2723
FIRST NAMED INVENTOR
Yu Zheng
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www .uspto.gov
ATTORNEY DOCKET NO. CONFIRMATION NO.
NEB-316-US 6781
EXAMINER
FRONDA, CHRISTIAN L
ART UNIT PAPER NUMBER
1652
NOTIFICATION DATE DELIVERY MODE
02/18/2016 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address( es):
STRIMPEL@NEB.COM
mcgrane@neb.com
stafford@neb.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
BEFORE THE PATENT TRIAL AND APPEAL BOARD
Ex parte YU ZHENG and RICHARD J. ROBERTS 1
Appeal2013-005800
Application 12/644,666
Technology Center 1600
Before JEFFREY N. FREDMAN, ULRIKE W. JENKS, and
RICHARD J. SMITH, Administrative Patent Judges.
JENKS, Administrative Patent Judge.
uECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 from the Examiner's final
rejection of claims 42, 43, 49, and 50. We have jurisdiction under 35 U.S.C.
§ 6(b ).
We AFFIRM.
1 According to Appellants, the Real Party in Interest is New England
Biolabs, Inc. (Br. 2.)
Appeal2013-005800
Application 12/644,666
STATEMENT OF THE CASE
Claims 42, 43, 49, and 50 and can be found in the Claims Appendix of
the Appeal Brief. 2 Claim 42 is representative of the claims on appeal, and
reads as follows:
42. A preparation comprising an isolated protein having an N-
terminal conserved domain corresponding to SEQ ID NO: 55
and/or a C-terminal conserved domain corresponding to SEQ
ID NO: 57.
Appellants seek review of the Examiner's rejection of claims 42, 43,
49, and 50 under 35 U.S.C. § 103(a) as unpatentable over Database
Accession No. A3PUQ5 3 and Guan. 4
Because the claims are not separately argued, we focus our discussion
on representative claim 42.
Findings of Fact
FFl. The Specification provides that SEQ ID NO: 22, contains the
N-terminal consensus sequence of SEQ ID NO: 55. (Spec. i-f 10.)
FF2. The Examiner finds that "[a]ccession A3PUQ5 teaches a restriction
endonuclease [derived] fromMycobacterium sp. strain JLS that has
100% identity to SEQ ID NO: 22." (Ans. 5; see also Database
Accession No. A3PUQ5.)
2 Claims 1-11, 24--41, 44--48, and 51-55, and SEQ ID NOs: 7-21 are drawn
to a non-elected invention and are thereby withdrawn from consideration.
(Final Act. 2; see also Ans. 3.)
3 Accession No. A3PUQ5_MYCSJ, submitted Feb. 2007 to the
EMBL/GenBank/DDBJ databases.
4 Guan et al., US 5,643,758, issued July 1, 1997.
2
Appeal2013-005800
Application 12/644,666
FF3. Guan discloses "[m]ethods and products ... for producing and/or
purifying virtually any hybrid polypeptide molecule employing
recombinant DNA techniques." (Guan, Abstract.)
FF4. Guan discloses that it was known in the art that the "restriction gene
and the methylase gene are transcribed divergently from the promoter
region between the two genes." (Guan 14, 11. 64---66.)
FF5. The Examiner finds that Guan "teach[ es] expression vectors,
prokaryotic host cells such as E. coli and eukaryotic host cells such as
yeast, and methods for making, expressing, isolating, and purifying
any protein fused to the E.coli maltose binding protein (MBP) using
the said expression vectors, prokaryotic and eukaryotic host cells such
as yeast." (Ans. 6.)
Principle of Law
"The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results."
KSR Int 'l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
Analysis
Claim 42 is directed to a composition comprising an isolated
polypeptide. The Examiner's position is that "[t]he claims simply
encompass any preparation comprising any isolated protein comprising the
elected amino acid sequence of SEQ ID NO: 22, and are not further limited
to posses[s] other properties and features." (Ans. 7.) Furthermore, the
Examiner finds that "the claims as written do not recite any cognate
methylase and [also] do not limit the invention to require the absence of
3
Appeal2013-005800
Application 12/644,666
cognate methylase for making the preparation comprising the isolated
protein." (Ans. 12.) We note that SEQ ID NO: 22 contains the consensus
sequences of SEQ ID NO: 55 (FFl).
We find no error with the Examiner's claim interpretation that
claim 42 is directed to composition comprising a protein comprising SEQ ID
NO: 22. We adopt the Examiner's findings of fact and reasoning regarding
the scope and content of the prior art (Ans. 5-12; see also FF2-FF5) and
agree that the claims are rendered obvious based on the combination of
Database Accession No. A3PUQ and Guan. We address Appellants'
arguments below.
Appellants contend that the database merely describes a hypothetical
protein. "[I]t is incorrect to refer to the 'reference restriction endonuclease'
because there was no such prior art protein-merely an identification of an
open reading frame in a DNA sequence, without an enabled method for
making the protein." (Br. 3.) "All that Accession A3PUQ5 indicated was
that the sequenced Mycobacterial genome included an open reading frame
that could hypothetically encode a restriction endonuclease." (Br. 4.)
We recognize, but are not persuaded by, Appellants contention that
the prior art needed to have produced the claimed protein in order to render
such a protein obvious based on the combination of references. Just because
protein production may require the technical skill of the ordinary artisan
does not make the combination as proposed by the Examiner less obvious.
Here, the Examiner identified Database Accession No. A3PUQ5 as
disclosing a protein sequence that has 100% sequence identity with SEQ ID
NO: 22. Additionally, the reference discloses that the protein sequence
possess endonuclease activity. (FF2.) The Examiner combines this
4
Appeal2013-005800
Application 12/644,666
reference with Guan, a reference that discloses a method of producing and
purifying hybrid polypeptides, including endonucleases. (FF3-FF5.) The
prior art cited by the Examiner adequately provides a reasonable expectation
of success that the combination of references would arrive at a protein that
contains the claimed structure. See 0 'Farrell, 853 F.2d 894, 903---04 (Fed.
Cir. 1988)("[ o ]bviousness does not require absolute predictability of success
... all that is required is a reasonable expectation of success."); see also
Hoffmann-La Roche Inc. v. Apotex Inc., 748 F.3d 1326, 1331 (Fed. Cir.
2014) ("Conclusive proof of efficacy is not necessary to show
obviousness.").
We conclude that the Examiner has met the burden of presenting a
prima facie case based on the combination of Database Accession No.
A3PUQ5 and Guan. After aprimafacie case is made out, the burden shifts
to the Appellants to show a difference between the prior art compounds and
the claimed compound. The PTO lacks the resources to do comparisons. In
re Best, 562 F.2d 1252, 1255 (CCPA 1977). We consider whether
Appellants submit sufficient evidence or argument in rebuttal. In re
Rijckaert, 9 F.3d 1531, 1532 (Fed. Cir. 1993).
Appellants contend that "[t]he Zheng Declaration5 highlights, for
example, that MspJI appears to be inactive in a traditional restriction
endonuclease assay." (Br. 6; see also 9; see also Zheng Dec i-fi-12 and 10)
Specifically, cloning of endonucleases "is complicated by the fact that active
restriction endonucleases are toxic to a foreign host cell such as E. coli. If it
is not toxic, the enzyme is likely to be inactive." (Zheng Dec. i12.)
5 Declaration under 37 C.F.R. i-f l.132 by Yu Zheng signed Nov. 11, 2011.
5
Appeal2013-005800
Application 12/644,666
Claim 42 is directed to a polypeptide having a particular structure but
places no further constraints on the polypeptide. (See Ans. 7 and 12.)
Accordingly, Appellants' argument that the combination of references would
result in a product that has no activity is not persuasive because that
argument is based on limitations not appearing in the claim. See In re Self,
671 F.2d 1344, 1348 (1982).
Appellants contend that
[b ]ecause the prior art did not provide an enabling method that
would have motivated a person of ordinary skill to try to
synthesize the protein of Accession A3PUQ5 in the absence of
a cognate methylase ... [there would have been no] reasonable
expectation of success in making a protein preparation in
accordance with the claimed invention.
(App. Br. 11, see also 5). We are not persuaded.
As recognized by the Examiner
the claims as written do not specifically recite any method steps
for making the preparation and[, fhrthermore,] do not exclude
the use of any cognate methylase for the recombinant
production of the restriction endonuclease of Accession
A3PUQ5, one of ordinary skill in the art would use a cognate
methylase as shown in Example II of the [Guan] patent for the
production of the restriction endonuclease of Accession
A3PUQ5 if needed.
(Ans. 11.) We agree with the Examiner that one of ordinary skill in the art
would have known to include a modification enzyme in order to produce a
restriction enzyme to insure that the restriction enzyme is not lethal to the
cell. (See Guan col. 14, 11. 60-67, incorporating by reference Walder. 6)
6 Walder et al., The Organization and Complete Nucleotide Sequence of the
Pstl Restriction-Modification System, 259 J. Biol. Chem. 8015-8026 (1984).
6
Appeal2013-005800
Application 12/644,666
Appellants contend that the claimed endonuclease has "the
unexpected ability to distinguish modified and unmodified DNA.
Specifically, it cleaves methylated and hydroxymethylated DNA at a fixed
distance from a modified cytosine residue." (Br. 6-7.) It is because of this
unexpected property that allowed the invention be developed into a
commercially successful product. (Br. 8.) We are not persuaded.
As explained by the Examiner,
[ o ]ne of ordinary skill in the art recognizes that the structure of
a protein indicates the function of the protein .... [Here,
a ]ccession A3PUQ5 would inherently have all the properties
and characteristics of the claimed protein comprising SEQ ID
NO: 22 including having the ability to preferentially recognize
and cleave modified DNA as argued by appellants.
(Ans. 10.)
With respect to commercial success "in the obviousness context [this
is relevant] only if there is proof that the sales were a direct result of the
unique characteristics of the claimed invention - as opposed to other
economic and commercial factors unrelated to the quality of the patented
subject matter." In re Huang, 100 F.3d 135, 140 (Fed. Cir. 1996). "In other
words, a nexus is required between the sales and the merits of the claimed
invention." Id. Here, Appellants have not provided sufficient evidence to
establish that it is the enzyme's ability to distinguish DNA in a particular
manner is the reason for the products success. "[T]he inventor's opinion as
to the purchaser's reason for buying the product ... is insufficient. Instead,
the applicant must submit some factual evidence that demonstrates the nexus
between the sales and the claimed invention - for example, an affidavit from
the purchaser explaining that the product was purchased due to the claimed
features." Id.
7
Appeal2013-005800
Application 12/644,666
Having considered all of Appellants' arguments and having found
them to be unpersuasive, we affirm the Examiner's rejection of claim 42 as
obvious for the reasons described above and in conjunction with the reasons
of record. Claims 43, 49, and 50 fall with claim 42.
SUMMARY
We affirm the rejection of claim 42 under 35 U.S.C. § 103(a) as
unpatentable over Database Accession No. A3PUQ5 and Guan. Claims 43,
49, and 50 fall with claim 42.
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).
AFFIRMED
8
Application/Control No. Applicant(s)/Patent Under
Reexamination
Notice of References Cited
Examiner Art Unit
n ..... ,.,. ..... _,
rct!::jt:: UI
U.S. PATENT DOCUMENTS
*
Document Number Date
Country Code-Number-Kind Code MM-YYYY Name Classification
A US-
B US-
c US-
D US-
E US-
F US-
G US-
H US-
I US-
J US-
K US-
L US-
M US-
FOREIGN PATENT DOCUMENTS
*
Document Number Date
Country Code-Number-Kind Code MM-YYYY Country Name Classification
N
0
p
Q
11 ~ I
NON-PATENT DOCUMENTS
* Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages)
Organization and Complete Nucleotide Sequence of the Pstl Reconstruction-Modification System. Roxanne Y. Walder, Joseph
u A. Walder,John E. Donelson, Department of Biochemistrym Uof Iowa, Iowa City52242, Volume 259, No12. The Journal of
Biological Chemistry, 1984 the American Society of Biological Chemist.
v
w
x
*A copy of this reference 1s not being furnished with this Office action. (See MPEP § 707.05(a).)
Dates in MM-YYYY format are publication dates. Classifications may be US or foreign.
U.S. Patent and Trademark Office
PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No.
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tht'! t~!3~<:'~iun~ s~~HJ ... ~l{::~ i_}_.·t~~·~~ ~dd~d t•,~ lU ,u3 Qf x~·wplt: h~jffr:r r:t~r:t<"lin.~ng
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tn t~i;~,nng'{~n:f?.ity b~· th':~ ~:r~f:thnd (:f \V ~~ld~~r ;)_nd Ch:.:1.th~f.~<.:~~ (l~ d~is..-.·{ .. ·~twd
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Jy~ ... :.d r:rK~~r n:d5.-!.;.~~~d ptf:-~~i;:r~:· fi.'lr ~~~ h &t 110 ··c ~·v~~.h (: N tJCL
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:d>~t•.t~:-::-t z~f th~ ('(;.n-e~p:..'lr~d.h-:~; 1·:->yc·gk~h~n ;~.rri:-in(> 2:(.id P~"~$~nt ~t~ ~tw
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t~~<.:~· ...
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;.=t,~:d i~·.:i::; id~:n~-~-fi~d hy tl1::n i~~~~·t t(> sny ~J:l~tX>
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«N3tt: ,<,..i~.hd.n~''tn .. In g;.rept~f::.~tk:n .-.:rf anths.~nun ::~~nin~~t Pstl~ th<~ ~:!&:l°n<~
rabbi=. w~:::. i.n~~-=.ct.f1d ~wl"S..··~J.t<~n~:.Rtsh· at fnt-::r !'.;.~t~:; i;.dth ~n en"tuh;~~n"<
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t~quaJ V\.}l~~nle (>f ... -:.:.n1~>h~~.~: ·Freund\ ~c.ij•Jv({nt .. 'I'h~~ n-::bhil w~~-~ h\JO~~~.~xi
2 ':.>.-'~-::~~ :~~t~r \~:ith 0.2 rn~ l)f th~= s~3.D:lt.' p:N~f-'ara:tion of .P~t.l =in. H.~ l fni
and ~B~ {!·qu.:d ....,.ot..~h~n~~ '.Cf itK~~~~m:~pi~:t~~ Fr~:,,H·Hi'.s >3:~~~u\::.:1nt. <)fH~ w~~~=k t~ft.~~:t
f.h:f~ $B<..~o.nd i.}\f:f:i:t.i<)~., ~Gm; of hl<> .• ·x{ \-:.-~r~ ~~n.:.'.)v&1 hy p:lh"$d.~~H~ ,)f th~~
<.:':H:r Vr ai-,~.ih1:.die~ direct~n <~f ~n ~~q'-J:~l ·"·~:...hrrae i.•J ~:attn1rr.ed ::ann).onh.nn
~ult)~.t{::. pf{ 7. U .... fh~ J}i15t·ipitat~d ra~~t.ed.;!l {':;·-~nt!ih1:in~ (::-;:f~ ~v-.~s t:f..•; ..
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P.N.:t.i:;fn A--s~~ph.at•' . .lWf (:·'...'th~mn n ): ~1 <.~:m} ::~nd ~:h~tt:.ci with :l M J.K~li~~
-~}l()t~f a~ , ... 7i) "C.
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with .t2Sbhit. ::-:..rr~is-B.r:ln."( "'"~:re <:2.rrh~d: (t~·~r Of> ~:n1
::~~ 1 M ~H.;1;.·r-:)~W. H~ ...:. Bi:>~ )ind (t>ntnn;_:;;:~:d ~n ~.t.! t.-"'·oly{~.t:tyfoBnd~~ ~~d~-:-.
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w~~t.::: n·~<~·.PP~X1 ~'~"~1rdi:nf~ ~~) H:~~ rn:t~thi:od clt~\·dp~d by Eh:-rk ~:nd Sh:~r}--.,
~"2~~) usin::=: n~uf:,~ hem.1 r:Hde~j~;~: i:nsb:~~Hl ~rf S.1 nndNm~:.. l~!
t)~~~ f". st.undi.l H~·l ~:r:~sisfi>n11:t:d }v·l~.h pP·~;tl(~5 ~l}: $lt;~in
pr.:·~~-~~~04. hy t.h0 ~-r~}~thr.·:d ·of B·~:tt~';:)nd t't ~sf. (~~7}.
lJtv'tts.::.' ! A}f>f:~t'r~·ra:("ng:-~ ...... rrr.~~f~~"t: j pf1...)t<::<:t?.f}f.: <':).'.p~:rirH<:'.nt~ "';:.m.n:" t:~u-·
) ..~~:d i.•~-~t "'~»-.::.f:n~.\~-:.Uy a~ de~·'..·x~h~·d hy G:3.h~:$ ~nd. s~:·hn:Y!t:..~. ('1R, -~~~:;:
R~;:."tl'l.j~;n~ -..> .. '~n: 'C('nd~l~:~.~~d ~~~ HK~ ~d ~:~:.•.::i;:~n~ st~)p_p~l~ by th~ (l:{~khJ~nn {)f ~!.:"":- ~:i:.l •)f :s~.(;.§.' u~h:
~:<:-titain~t~~ :~; M .1~n::..rrF.~ni~J~n -1i'-"l~t&t>.~ .• 0.2t:-. 'M ELrrA, .~~.nd C:.t:) n~g/:))~
::st?nk:;;~ted s;>.h~v~r: .. ~Pt~r~~'.l D~,·~· 'Th,~ .~t~n~:·~~'"S ..,,.:~~t~ {:~tr<)i;:·t.~d \'lt~tl:
pl'"~<:.f:.(>{ :.=:.t:d thl<)f(itO~".n1~ P~"'{:':-{.~1P~(-.~~.N~ S'\•"H.c~ :ed-v~n(>L ~:.nd tl\ OJ, ff~M l:~IYT"A~ ~} M ·~a·.e'-'~ ~~nd ;\~;~,;~:~: xykn·e ~:.v~~ni_~l.
'Th}:. ~x-nr:p~t:$1 -...-s.•;::.r~: h:: .... :::-:.t~?.d :~t ~~O '\~ f:)!" ·i tnin J:jnd k"}ad~:d t•ni.>:.· :~ ~)t ~}:::~.
fJN /\ ~"1:~t).l~~~S$("h:g g<:.b~ :.::.dj~t~::~:~'"~t ~l~ th:-: ,~h·~"n~~k:~J dt-;;~v;;~gi:~ n~ai::t.k~n~r qf
t.b.s- s.:3.rm: i).N A t<.;..:n-..r~t;slt-,
l) .. :·t.~?trn~·ti..rJ.Lt.'(~n rJi t.l:e i\~~-c-:·d(::oi ide Seq<.:·~~nc~~ · .... 'fh{~ J)!~;-.'\ .8.{~·
qu~~nc.e t.d' the· 4.H·kik~h~~:si:::: 1l~:n ... ~U:U fb)E:tn..c:~nt fro:rr~ r~. ~ilit:..u:u·t~.'.i.
.H34 contuitlu-l.g t.he l..,._StI re~tri~~6t)B ~~nd. rnsxHfk:~tinn g·~r~€s
~-;:·~~-~ d{!t~rnl:~nt:d using" tfH~ (+s<~.~'Ylitul Gh:.·lvag<;: n:t::~tJ:{>d nf
~·IHxiHn and (Hlhf:rt. { i.4~ !SJ~ ·~rhe s~q; .. 1-t:n(:i~1.g $tr;xt~~gy i»
(H.~tht3'\:"d in l•'ig. l. ]~he f1.tt( .. ~..:'.:-t~· indk:~tn~ th~~ d~r~~ctio~l zxnd th.c:~
i:>;.t~:nt of ~~i~qtWnCi3 <)bt{~in~:d fr')X{~ ij~lth fr.;~~~!nt:nt I<.~st.1""k~tiHn
fr.8.gn:tt:nt..:~ fro.tB lfinfl~ 7\1.qJ< SNA
p.c:~J~yn1:t:-r~~s0 ~~ Ji~"~~} [l~ .i:!.~·F~-r1I~ ii..~a.!, ..-Xhf::J ~and x·hol r~:~.tt~t:t1on
fr;xgn):E~nt.s ~~··en~ b" .. :end !abeied \.\;:hh .. f4 p,:)1ynu~~t~~i'>t.id~3 'khHV~<~,
(~v~:dB})ping :~<:"quencc~::; \~"·t~n! .ident~fii:~d. ·hy ~~on~}Hrtn:r analy~~;;
{;}(}~ :)1). :~·for~ thEn :-JO~?~, ··}ftht~ ~~q.~ie:n~{: .-..-c.~{~{:; (:.:.:;:;.:~f~d t:n b<--~th
:~tnuuls. ~rhe (':t)tnph":t~~ n~_3clt~ot~d~~ Si~(lU<;'ntx: o.f gs8-9 b.11~:e P*ir~
i:> :>h<.>wn iH Flg. 2.
~ix .. ~tn~n~tt~.f}·n <~f thf.> &~:qui~nt:E~. i~l ,:~H ~-{~ t<::.~~d.ing fr~~n~~~~. i~:·~~,
LL•••..,.<"-•"•"'''''"'"""" ~:::::~~ ..... -..-,LLL> LLLY"'-<•••'~
-· - ......... ;;~ .. - ~:::-::. ~ ....... _.... . .............. ~ ""~:::::::....
t:-~:<: '
;...;.;.f,•:·t
:>-. ~·--': -
~u '8f
._ •••• -,-,-,-,-,-,-,-,-,-,-,-,-,-LLL~ ...... LLLLLL"""""•••-,,,.....,. ............................ -.-,._._._.~LLLLLLLLLLmdwg ~t.rai.tegy am'! m.av
of thi~ 4.iJ-~.lfobil.S-<1 fr.Qgrn~nt th&! coijt.B.!n.s t.lw .P~d. rest.rk-
th.l-~.nn.:~u.-difi(:~(k~n ·g:~nlt:."i)~ TlH3 ~£g .. ~u\~r <'U't(,~~'-1~ b.v~k~~h~ ~h~: kx:~-:.tk~n,
~§::E~~:y2:~:~i:~;:r~~;~~r~:r;:
p;:)t.entSal ~nitiHtSon ~~nd t~3t1Y~iru~L~nn czd
divi:rg>Jntly· frotn s.~:parai::f~ pr~.):~)1ot~:r?) .. '~Ph~~ opttn S:"ttading fran~~~
~)r~ th~~ top ~trand.~ ~xt.~~nd3ng t-o th;~ d~;~ht: ;~tr~~t-ch~:~ frHn:
potrition 2;3~)t} t{> ;1~37:3 h"'~ fhe tn..tc-1'0·:.)t~d~~ ~-::t~qui?TH~;~ ~nd <:an
pot-n~nti?.~Uy end~~ {~)t ;:t. prHt-t:ir~ of g4{~ ~unino a<.~id~~ ~l'ht:: ~>§)'t::n
re~3-<{~r':K fran1~~ ,)n_ th~ bqttoi-r: .strand e~: th~: an~~ly:?.i~
of ;:t s~rd-€:~} of d~.~~!U'-}r! nr~.s.tants Cr~h{e .i!. rn ~-h~': ~n·i§';·inG.l
pkt~ardd~ pPst20J i tb~ 4·.0--k~h)h~M;t:. frag:n1s~)1t ~:nrodLng th~~ fJ:st~
~~:~tc~rfl i~ ck·H1ed Ht the f{i~~tHrI :-;ite oJ pBH)l2:!~ ~rhe v~1r·iou&
'(}~~ieH~)tl p;.a$:~n~d$. "..\:~?.r~~ construtt~~d utni~.::-irHl t.hi~· ri'~tl·~ctit:>n
~;it.(:~::; i.~1 lthin th~ lrH~~rt shov<.r:. at th~~ to~o of Fig, l tn1d: th5;.
u-ni-q_n_.t~ i-$~~n i~nd. Ran1!-Jr site~ in pBRJZ2, ··rh:e ~xtent of ~~:£tc:·h
{)f th~: dt5]i:~tions· is i:tH.-ik--:&t~d ii:i ··ri~l:4~ I. 'J'h~: syntJu~~is ~)f t.hB
.r~:-::~tdctk~n enzvtne 'v~~· asmrvl:d ~n o~;~:.r~.:)t.h~ ~hock Huid isoh~t~d
frsitiv£'~
H.-~~·'.~t.ti-ct~~>n ~:ni~vr:~H~ ~,u:n~ .. !lty ~~a~ d{:;~t~~cti~bl~"'= o~)ly ~n celh; \>ihk.-h
\~:et~~ phet1otyp~c~~-ny :t~·-~ :I~h€· ~ynthe&i~ of :&,~u\~~~ B~udSfh~ation
~~n::--:\~nH~ V.•'S,S P.;;t~lbHsh~d on thi: h~~Bi~ ()f ~.he n=~-S~StB.fR":~: ~){ th~
d~5]~~~hon ph~stn~d:s ~.o cls~~vage. b:_...- th~ endonu{Ji~.:'l.se~ ··rhe pia$-'
?rtids ~!} h~nrc in ('~)I':.1.fn(~n. in t(~ii:l $1h:e tv·;·i.hln th~ !J~l~~~~tHJ f>~t.J :~lt~~; \vithin. th~~ in~ttlon~ ,>f th~ t·ight ,~,·<.~rd (;,)ding~ Sf~{lU~rH~~~ h&v{::-: 'b~~~n delf~ted~
'T'hf? r~~~uH.h'\g ·ph~~n~>t~·"P1~ i.& t~·tn"· in.t ~:h.ls r~gion
$~TH~Odf?H the re~Htdctk~n enzyrne~ \.V~ h;~s-"B ;;:Jso con:~:trl..~Ch~f} t.~>•/<)
in~;~:rti€n: nu~t<~nt~r Ul thi~ r~,sdi.ng ft·~un~ by- !i~;at~()B <)f ~ ~)~J9 ..
t~~~ .. ~e pair fHi§~ra~~n~. jnto th£~ .li~~t~ri1 :Bites {~t Pt'-~~tions ~~148 and
~-}:34~1 of Lh~ 4-,0---kHo'txs$(:' fragrn.ent ( 4 ). (~.~~u~3 cnrrtab.1i"tBg th.esi~
pla~~.n:d.d~~ \Net{:'! ah.t~:- r'\n·:- (4). h1the1l'.&rd: {)i::>ding ~et.pJe~1J:-£ and th~: hegh,n-h1g (Yf tht:
J~fbN;itd co:d.ing 8<:·qu~~~,,~,:::~ ,~~~":·r~ d~~let{~d ... rn~~~;i?. f:~:ns: ;:~re r··:n. ... j •· ..
~~~tab:U~hin~ th~t thi: $e{:ond ~:od3ng r·~gk)~l i~ th~ g~~n~~· {<)t th~
rn<'tthyl~l»>' .. These re&ult:> i:it1:' folly wnfini.wd b.'" sequ~:ncing
~.i.mfoi,s t,f i:h~ hv;) 1rr<)t.·dm descrih~d bdt>V-'·
8017
·rh~~ p·~~?te{n~; t;f th~~ f>~tl ... '{y$ffnt-~, ... ~-'fh(:· -~-~·, foti n1ini:l~-~=H~
produ.dng >P!li,201 m~d
with e.ad1 of tJrn dehtl.on plMmids descti.bd in T~1hb l in
ln:·ti~~r to hf~nti(y· ~nd ·8tudy tJ1t: synthesi~ nf th(,:;~ restri<'t;.on
and nwdificatiMi em.ymt:,OOl..t Md. <'O·miKr;>.tes with t:trn purifiid {l..-m~~ l'i}, ln a prt:\'i<>m~ Btndy, the ;\{, ·~ .~}B,OOU
poiyp~pd-d~: ~\~(M; not d~tect.~d~ $.nd fh~ nl.od;ficat~lHl eni.y~ne
\~'a .. ~ ~~ .. ~-;igne-d to a Af: ~¥:· 32~(K}0 :polypi:pt.~. th~
r>~Eti·itth)n :r:~nzvs.ne~ t$:Jc..~uk~v~d fron; the di~l1~3totnet:tit~ tr.:lc~: -of
l ·'' , . <> < ,. } ' l l . ' j. ' ·' t ~-~ ;2BJ.tQ.r~u."togt.::a11~ n3 4.(), {"'\_}; nt:-;~~\.'t$i.S.:~~(~ ~)t.~ {.)_(,f.i ~ t. ~rn. is pr,) ... ~an·;y·
rei~rt.}?d ~h diffe.t(;~nces in. th~~ ~"at-t~ of ~ynthr:si~:- r-.-i~ficatinn t~tlzyn);i~&
wil:i ;~l.,n Btiidied in 8 ·~•)Hph;d iti uitro 1.rnnsniptfon/1:rnn$fot.iot1
system programmed wit.Ii pPc::;.t201 (Fig. 4 l. In thi» os;-;e, th•~
n~stri~~tk-:n ~~Jl.~:yn-1i~ ?..\~as idf3.nt1fie.d h·y -hr~.n~tH1(.~p.re:cipit .. atl(ffl
~~ith ~1ndhody .ta.i~f:d agH.inst. tJw· purified p:rotsJin Urui~: 2) ..
:ln!.en~stfogly, the ani..;h<,;dy d)d :w>t l't<>ss·m,~n. ·with i.he m~t.h··
yl.r~~e. ~rhe 1~<:.k ({ honl(~logy ?thich i.hi~ su:~g-e-~t~ ).~ aJs.o .esppar~
~'!·nt. h·()rn th~ tHtn_p~tti$.l)n of the s~{~u~:nc~ <1f the t.w'(S p1oh~irm
~Si~f:'c ~'Tli~eUESi(~~n ~~}.
F'r<>t~~in~ ~'frsth~~~sied in th co-:migrnt;; wlth dm pmdudl'! "3ynHrni;ir.<.~d
in Uw ceU.-fre~' ~y~te:m., l.ndirnting: thai iwith~:r prntifrri !,;
s~1nthe'Siz~'1 as ~$ larg~r :rn~)i~~~u1at \{.>eight pr~fnt:s<>r, ··rhis i~ of
part.ieular Bnt~r€::~t re~~rdin.~rth·~=- te~trictkH1 ~r1zyn~~: ~~ ft&ctinn
,,r which ;~pp-ears to tw tn:nl'!port.ed int<'l t.h<: perip!wimk spa<:e
~: l( :'f1_L
tn. the cndb:ig regio-ns. fol' bnth t"h~ te~trlt~tion e·itiyn:;..e and
methylase, i.hilm in·e i:-.No d~>$tdy ~op{u~1il>id.<.ie~
ne<~r t.:tm NH,~ terminu» ;mdi th;;;J !:hdon
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cy·c~~~ pr~:(:~:-di.ng th":' rn.:\~~·>r pf:ak i::'; };~.:;:~~fi:e~~.ndy ~~}xr:..~~ hi~<.~k·
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7~~~--~ :rrru~.:{'ctipt£un ln.di.~~t~~>n ... ~ite.;:.: .... 'T'h~~ :--frt.~:~ ~~:>r initiat~i•.)~:
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in ~x:•th >~;~s~":s.! .;~:nd are $;h.o\.s··t~ o:rdy ~s··ifh th~' :;rr::.BU~n" ffH§;tn::~nt
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th{:~ ~~X}~n~·:;:;i<>n of th~~ r~~tl .:~y-St.~rB.
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t'::~~--p~;~~~: ~v;:~~ th~: ·~:,~~·h~:$.l' p~ir }(6«:d··Bf:i~l DNA fr::~~;~n<.::nt.~ ~:t··~nd
b.'(h{-~ kd '''"j ~ ~~ ~ ·;· ·' ::·~p 1 A ~y--p :.u~ 1d T-·~ F'-~~~·:·~~J.•.: l.~·-~tid·~· kiJ~,;~.:·:~~. Th« ~-::~~l~-<::.:~j.~~~
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·:~·\~ti.;$~·~t;n $it~- f:>t th~: }"\:.:'] n-~,~~(hy;;:r-~~ ~nR? · ·-~?.r~d k~h~~}~d ~''~th ~ \·.-:·~~p. 1
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th{:n di:~~~·$.t~x~ -;,.~:~th ~-r:.~.H~~ h{'..~~n n~H~~~~~E;.~'.~ ~~ $'.h"1gh: ::;:;fr~~n~J·
~p<::.t~fi}~ :'"~UC~t'~~Si{:· \o;,:·h~ch di~~~~t. both J)~J.A .'.~t~d RN.A ct\ ~~fH.
~rht~ r::-:&uh-in~{ f~---:~tnc:t~nt~ 't"..-:t::r~~ then ele~~tn.::•pb<>{~s;::;~d ~n p~~r~U~1
\'"'~th tJs~' f!"K~r %t:~nd~u·d s~qut:{i:C~;.)g L:i~-~~~:~ ~)f th\'. t~rn.Pt~t~ ·~ln~nd
{f~&- i·-p_ ··rhi: L=~bek~d b:~t~d~;· t>:irr~iN::::'} e~-:tch C(:-rt-~~p~)nd tn ~·ht::
d<~ub~;~·~tn"tnd~~d r~:·gk:-n tetTnin{~f;ng -..)..-ith th~:: ;~·· ... ~~nd (>{th~~
rnR~rA. SiHt~e dlt: ~~hi~-rr~;,:~~} tk~&'-.-i~~~~ r~~({~{sn~: frn' nuc}(::~<~tid~!
~..:::que nc~ng ;:~.r~~ d~~gr:<~~:Ht~~~t.~·· l ~-,.~~ rg~jd~H?:~ \l~rv·~·::~rd~;
frc:rn th~: <:<,;rr::~~-ip.:)nd;ng' po~~t~c·n~~ in rh~ .~<=~q~rc.::.tH:~ng: h5ddet..
'T'he init~.:H:i\>B si: ~~ fo:r tr.:~n~rtr~pl~on ~;>f th~~ r~~~~:rk~t.k~n t~~.-..:-~y~ni~
g~::~~~ :~~r th~~ ~:~d~~lF.>sin.·~~ n~~~du~: ~i:~. F~').~~r~on :t3{-;;\ ~F~ hp Hp$J.t~~;3.rn
~r~~.nj ~:h .. ::~ inJ~:~;~J::.on e~~d~~n {~t~~~ }:\g .. 7}. ~1~r~~n.$ttr~pb<~n (~f thi:
n1~:~:hy~;3.$.{:~ gl?.n~~ ht~g~~t).~ ~~7 hp n.p.~-;~::eHn1 nf thl: ~.n.~~iHti~-:>r: <~<~d~);n
::1:: :.h~~ ::Hit:n(~·~:: ~ne r<::.8~th.~~:· ;;~t nucl~~)Hde p~~x.~d<)t) ~~i91. ..rh~
:.:n~n$,t::·ipb.(.-r~ ~l:H.i~H:i:'::.f~ .8.iti::~s. I~.f~? s.ep~~t::}~ .. ~:d hy nn;;.: 70 bp.
I~-~~~~~ po~_>'!n(°':n1~;e t<': the ·~nt~t~--:;~{.N)n~~~ n::g~{H~ v,:.:*~ sti:~di .. ~d hy
{)N~,~~··wnstn~;-.un frnns ;.h{: ;.tans<:6pt~on in~t.~ ·
~~t.~<'.•n s~t~~· for tht~ n~~~thy3a~~~~ _:~t::n.;,.~~ ln th~~ ~~xp{~°ti~nt~nt ~~h-~n~T~ in
F~g .. S, ~.b·~ ){b-:.:d t~~rn·~i:n1:~.$- :;~:-~::.~~· }~~h<::h~d on t.ht: top s~.r1~.f:d. v,:-:~th
rEh~>::~~~t t<> tb~~ ~}~qu~~f}>~(? a~t v .. ~dttt~n ~n Fi};:"~. 2 and 7. ··rh.e l&b-:::-..fr:d ..
r}r~A }\~2!t~ ~n.cnbHtl~d "'-i¥~th V;:triOU$: -:;,;~ .:;,;:- :: ...... "'5. :_....,. _,,'""'"'"'
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W th~ L'.i'--klkn px~·::~.f~l":t~:·.d. fr-l~~:i:s f)N.:s::~~: { d~~~<"~~-::·:·r~ .. ·p-:>:::jt~(H.~ :t~f;-:· .. ,~:·::LH~ .. ~~
f~tr~.:.."k~,~~:d. ·Th~ ft~~~·fi:-~iSnt .,,,:af.-; D·'·~:"nd .~.~~h~~~d ''=·~th l'~ .. ,.3:~P.l .. ·:\:·rp .;xnd .. f:]
P~.)~Y-~~~--:<:"t-~:{>~-~d~: kin.:-":${;:'. ~t :Jw A'h~~I ~:~tl~ .. f:)u':~'.-'.-':~".·} by (:1(~&·(~~~~~: -,;.;,··~th
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;j~~v~~:c:~ pt<~ch .. :.<~$ cd th~ ~l~:.~~~m--GHh~~tt ::~~~:::~ ... ~~r:.<:h1~ rN~~).~{->~~~;- ~-~f -:."h~~
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h~hd~:·d DN .. :\ t~:·tupl~j:*·~ {~1n r~~o :;.;:',;5:::: i·~·~{~3h&t~:~d ·';.v~th ~·hHi::::-:~r-~*- :::.:·~r:.:f'-:3_n,
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f~~H(:-\'i.··~:.d hy d~g~~stk~~~ '<"t'ith iJ~<~~;:.:{: L Tl~.~~ ft~~::.<~~~t~ts ,~,~~N~ ~;:~:-~~Hn~~·~d
by ~~fo·~:tn:•J",lh(:-~"~:';b; ~n{ ~n S~:~: ~;.f,•~y(~<.~isk~~:·n~d<::·-~H~~~~ S.k~q· ... ~N'::~':iB$~: ~d,
~~y p(~1yat~~~.~nnid~~ g~~l ~~~:c:x:t:r~>phHr~~=)~$ in ~~,;::_n~]}~~~ i;~·:~th th~~ fr~·;~r
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pl~~y~ni:r~m~~ i.8 ::,.,hO\.\'n it~ l.:'i~~· 8, h.1n(" 6. 'r'hf dig<:".8t~{~·n (':f th~
ff~:i.~"'~nent {~ftet ~nrub.;~tion (.'fith 4 ~n~d 12 ~Ng/n:l 1{Ni\ pl)lytn.,
~=--r~~'.$.~ is :~-h::>\~'t~ \n lo.f:~:·s {~- ~~nd ?-.=- r~:~~)ftiiv{;:ty. :.~i th~-s{;: rfi:rH~i~n_ ..
tr~~tk~ns~ bindh~~; ~~ oh~ .. ~~r .. ,:,~d ~n th~~ t"(:gi~)n h~~~-\~l~~~~n n:~si~hw~
2~~C7 ~~nd ~~:\:H). =-rh~~ B.-:~ff~~~ :;~npn:site ~tr.-:~nd {4} ... rhi~:· r~Kinn c<-•rn~~p(>n th~~ ~xp~(:t~d
p·~~)nlot-~~r ~~it~~ k~r ~.h~ xno-ch-fi<:~H-i~Yn ~~n~~~sxn~\ ~:p:n~ning pu~it~<)n$
·----·SO ~:o +:ir; r~~la;-tv~~ to th~~ ~:n~n~triptinn ·tn.~ni~tinn ~·~t-t:?. {~t:t:
Fig~ 7) ... -.fh-t~ tran~c:ription i.nit~atil)H ~it~ :f~n· t.h~ ·~~nd.~)JR1t1eas.e
ge·n~ fft ptj~~ti{:-n ~~:3fi;~ !s <-Je~rrJy <)Ut.~3<{e this region~ E\··~~rt \~:ith
.a &~·f~)!d highBr conci~ntr>:=.tion of f.;. (~oli lil~i~ po~yxrH~·r;tst~~
ttu~re is n~) d~t.ect-ahJt~ bit~ding t~) t}i.e :restriction .en~~-._vxne pro ..
mot;:>t The }'.mw~ l:'<~:>.<:lt. \>m» fomid w;:fo.g tb<:> .Hiti.dH1-Xfwl
fr;~gmn~t- {t1cs!d1:ws l--~H8$) as thto l:empht~; ((i. /\!: i:h~~ high<;r
<:iH);f>~ni.tatiuns ..:.~f .RN .. :\ p()l,yrnera~,e~ a.d~iition~3.1 binding :s.it.e$
V/~t~ ~~~)r~~.;i~t¢n~J~,..: ob~:tf3'."V~"'"d at P<-)$fiN<)fl~ i928u·lHG~? .::1?l:~i;.rt·:{~nc~} of thB ;)BlS:{.}U}tal :P~tihno-~· ;:;eqne-nce ~l'"'
T'"iVfA ...... '\.·r· at r~s-id.\H~~ 24:62-,..~~4.l)·7 :;nH}' H}X~o:c.n1t fo~' the \.Vi:H~~r
b~nd.itlg ~ite {~b~e;rved -~~it:hh1 t.h~~ t·;c;~st.rh.-:tion e:t~~:.yln..c;~ gB~1~-·
"\:V<~ h~}\1('! dete-rnlined th~ fHll$p1.}?.t~3 ~uJc:t~~ntid~-? seq:..HnV~ nf
t.!w 4.0·kik~ha~~' }{indill DNA frnp:m:nt from P. i;tWJrlii 1&4
~::o.t~t..;3:~n\ng both the f-~l{ r~::;t:d.ct.ion iH1_d n.~odifi!;.:~tion ~n~.y11~-e
g~n~~~~ T"h~: tv~o g<:~tH?.'$ ~~~~~}.1~ ux~Bn$b3~~XH}US1y n:~spp~~d and iden· ...
tilfod wii.h tlwir re;;pN:tiw p;'<>tein:s, The g!!n6» at~' vay dt>:;ely
Hnkt~d~ ;~;c;::-pwrated hy· <)n1y 130 tn.~c}~)tid~St~ fftBi ~·~r~~ tr.:~n~.cribi:·d
if~ <~ppo.~ite dh~:·rti<)n:s. ~rhB n2'f-n:ti~::t~.on e.-r~zynH:": i;:; ~~ncoded on
th~;- H~t} ~trand. amin(> ».6ifa \'>'ii:b ;i. Nk~ikr!.lld ;~( ,,, h~ ~n~;difi:eBtk~n. ~~I:;;~-~{Blce i$ Bnc~)df;d nu the
~<)~\:er .$tr~r~d:- <:ont~~ir1~~ $07 re .. ~~dnt~H~ ~~;;d has ;::1 cattulat.eci l}1 ..
"" f',n,itlO, A'S- in ot.hN Tyw U r.,,~,t?id:iim-nwdd:'l1;;i.t.i(m :sysi:t:m$\
~~tn~H~~d {~$.}_. thH funct~~)n~] f~)tn1 of the ·inodifi.t(~Uo:n ~~nz_vxue
is presnn1~hiy ~t rn~)n.~)Hl<~t.
(~.'Jtn_,r.Joris.-Jn .~~,r ~h.0::.-.. 8e:qu~:n.f.:t·}; ul {h.f.' 1~~~i1 Re~:;;t..r-£c.t..i<:u~~ l!)~zy~r~e
ft,nd A:f$~t.l~yl!l$<:·-····Since th~~ .f'"~~t1 rt:si:detion .aad .u1odificah~)n
enz.y-::nes :~et0§;)3~:?~~~i .~nd bind t'"'~ thf: ~a1ntz l)~A {:;t:~rt~t:n::~~\ t~~~
t,)\·'(~ .Pn~3t.f~.~xH; n1ilY :3:.h~3:rf.'. {:{Hl1JXlOTI Et.rU<:t.~.'tl(~~ ~~at:t~res;., \:~'hl(:h B1
th~~ $i~np1~~~{. ~::3..S(: (.).>(H]ld }~e .0v·idt:nt. ft0u1 t~(HnpariBnn ·of t.h~ir
atHi.n~.) .;~c·id *~qUffnce~~ .. ··r,,,,~·o d~ffet~t3t a;~}:::-r;thn~s s\~~r~~ u::3txl tn
CD,_n:~pare tht~ S:~:qu~~nee~~. ))oth ::~n~y .. ~·ing· fht~ 8iiu1gt~ncy of as~
Bignrn.E~nt of h<.-n.i.nlqgous residu~:~ to th:~ varied (;30, ~~.~\t .. ~rh~~
k~ng.{~~~t ::;tr~:h.,_:h ·~>f idt~nt.~caj ~n:frr~o .;3c~d res~du~.s i~ on1y .a
!:<:tt».P<='.Ftiik, Glu-Arg .. Afo-JA~u ilithms l4i.;·MS in thf'
:r~:stri(:ti9n ·t:n~:)'n1~ and 164.~-1$7 in tbt; rru~t.hyl;:=.~~~- E> . .ren a~l.::: ... v--
in:::{ for roru::etv{!~:.Bve .::aninn scid :suhEtit~Jbong and for in~~~r-·
t~~;n:B or d~:h~tion~ v?ithin th~ ~~QU>~ni?'.t~~ th~re d'-)~~~~ nJ&f
to b~ ~u:::y :;ig:~:rificat!t hDn~oio,~:· h€!t~vm::r~ tht:: ts\:~o pfi}teit1$~ 11tP
t\~'<> prntBifi~' {~re al$.G .dJ.sti'n(:t b:n:asunokj.gk~-t~ll:y, itB- :1-h()\\'n. ·~n
F~~g. 4~ antib<)dy ru~s~:d ~g~~inst th .. e p1irH}i~d :t·e~tl'it~tion ffn~yt:-H?.
d<)~:s .not. c~"(~ti~~n~a(:t ·-.vith the :rnethyla~-;u.~ ·~rhe~~ dH'i':t:r~~.nc~s in
8trncb:.ffe m]gg{::st that t:h.i~ t)\.'Q ~Jniyn~-e:s. niay h~1,t~ f':90.lved
diffor~~nt rrn~h~rii:sn<~ ~,f biu.dlni~ to th€ir >:ummon N!<'.:'Ggniti•,ih;>trat;\ m;
:$ho~YH f~)r the t:raR.I end. or th~ ~~klb~:t~·\ltB?:trt
c;it.;j_lytk »tq.)1)). or hnth, h1 rnnt.m"-t .. wh~n i.he '>ame DNA
s(~qui~nee i~ :r~~':'-.)gni~<:J~d ~\Y th~~· rnod~fi~:?~3t{c~n i?.~h .. VTne thi~~ po:;} ..
twn mu~t. lw ;;1.etk~Hy a;>:::<%~i.bk 1\; p-enirit tnnl ,$~-~1d{~1H~$!.yt~nE~thinnin~~ ·t.o the f} .. <:ltniCH)
gr-~}up o.t thi~ adf:t~nsi.nt.~ rt.:~·id~l~!.
Compm·i$<>n ;)!'th~: Bl:!qw:nt the Emf:U r!!~tdd.i'-m l!n--
i_yrfH~ and n1.t{hyl;~:se &h;.Q ·t~V$~<.1fed UtH1~· hor:~o1ngy het.\>/e~~n th~~
t~~·o p:N}te·ins Cl~. B} .. Predictions of th~~r s.0tond.ary st.tul--:ttn:-e
fron) the ~.eq~~~rH~-e& o:r the ei·r~~u1&r di{~ht<>i~n.1 !Sp~~ctr~ like<.\:i~'e
v.:~€:rt of tiw bi~cti:>ri<)pha~'I:' ;\ d and
crH r~pr~s~'>r· pTotetns} ah,\n S·u.g~~st that th~r·~ is n<)t. a )..tniqtH~
i~lM.ion~hip b!!tWi.$~tl t11!! i·~cq;nit!on :seq<.Jence ~~ta1~vtitaHy ~~eiive f{~r~n ~>f th~ ]>{-ifl ~ndn1n.1{~1~ase i.'.~ a
ditner (1.~ 4}. 1'h~ tv· .. (o ~u{Jurdts ~~.r$ pt·Ob$.bly te1~~t~d by a 2.-~
fold $0\1h;;l.rnt('t
<.x>indd.-e~, with the dy~d ml-iii of thi n~cognitkin s.~u*lnrn, <"B
ptopc));3t:d..f:c.n· tf() a.nd cJ {{;}--47}~ and is eyid~~nt frotn th{:: inJtia~
cry~,t.~mpk<:: fl:">f!"l>rt1;d
by C~tHbis~'. ~'.'t. (iL {51 } .. Since in th~ ~rYP~ Jr f(:~t.:ri<;tion-·nl(~~lifi ...
J:~l)tion .sy~t~t?~:~ th~: ~no-diOi.:atiotJ *"'n;~ytlie .is gen:e:~r~~Hy xnnnn·
:rn~:tif~ the p})~Jti?-in rn;2:.y t':~}:ntain ~:. pweud;:i~
h~!t:~~~:e~e11 t·e-~$t~d: ~~QUt'T!C~~s \~:ff.hit~ thi:· IJN._A-ithin the fia.n.k~Bg r~~ior~s furth~:H:~ gttgg~~~t th~~t the~f~ s~-"
qu~:nc•~s may b; rdat.€d tn :f11rKtlon.aHy hnpnrtani. di>mairn\
wit.hi~! tl-a; pmt;~iJi, Tlw "-l>me 1.r-ipeptid!! •Y~~s not fo~-:nd in the
Psti mN.hy.tm1e, ft dM,;; ti:ml:a.in tJ1rN; f*'f>f;~ted pt~ptkfos: A$_p (-11<}--415, 4S1487).
~rh~: J&si: ~~~p~!~lt~:d peptidt! g.{~,_, .sho·\V~ ~). high dt:g~:::?. \~f }1<~nJn1: ·
ogy ·;,\,~JJh~n th~ ~~o:rr~s_ponding !)Nj\ ,se<1uen('.~~:- 8B~;~~~ a:nd n~.;~:y
l;~: $iKnifi.z:~n1t ~11~<:~ t~h~tivi: 1~.)e~~ti·c:n zJf the thte:e PB:P.~·~.desl
h'-.)'-Ve'\:·t~r:- is B:Ot nhvionsb·' re{~ted t:o the BtfU(:tun~.
EviH~ h} sequ0n~_:-~ .. ~J)et::ific l)N._A..-hind·ing proteh1~, ~~ l~rg~~
fr.actk1n of th~~ t.~)tRi binding :tHlznte de~nd€nt int{:rf~t~ti(~n:~, p.riticip;:~Jb.( eh~-ctto~t~~.tic
Ln.tt'!rHcti<)n.S b.t~t-..~e.en th~~ phos;phHt:e b~u::~b~}rB~ and catiuni.l~
t¢';:;id1.~i~:$ of t.ht~ prHti-3in (8~J~ 62_. 1~a} .. r .. ~.fft H~i$ r~a~<).B:- ~~·~
e~.~urdnt-'-i tJH3 ~tqu~nc~8 oft}$~~ _PstI n:S:t.rict.i(Hl eniyn:~~ ~3nd
n1eth.yh~$:e fn
{'ndonndm~st' eontafrm f5,i, and 4l j.){_'>;;it.ivdy ~md nrogaifrefy
ch~~t.ged t-esidn~B> -rt::::tp~~l~'-':~~ly; t..h{J sn.~)difi~:~tk>n .~:.n1:y~T:~ c:tn1·-
h1i.tH~ 58 po·~:~t3vt~ ~~~Hi 5-r-~ negB.t.i'\·'i~· re~;{d.-U~i$. h1 l:K~~h j~nz:rn~es(
i.h}~ fh1itge·d ~z.sidne~ &re dis._~~n~d1 \~·ittK~Ht e,~i&:.nt don"t.~tl:n$
r<:'fot.ed te t.h~ DNA--hlnding $.\!.°t'-
(:od~)n. l}'s~-e~~-.(.odnn ~a~1t:.ction it~ .E..~. ·~(l/i and ;;).{$::0 \tl euk~u· ...
y~Jt~s i£ dl~tint.-:tJy n:..":l'.H':;u::.dnnl} p~u:ti~~n~a:tly fnt highly· ~x·
pri::~-;sed pt<)tein8· {54-----5.B.~~ ~fhi~ pr~~terence an~Qtlg ~(~tl1onyu~cu~
l~O{fOHB in B.t~·ongJy e~})tE':Si:S~d fg~ne~·:- at lt"}::is~~ ix: }!;~ coUt i:.}
d~t.etJnint~d flred~n.1Jinantly by th.t~ r~~lath··e ~lbu.ndance t~f the
iHuai:ct~pting t.R-N.A*.- \:\·:-hen rnt~rt: than. on~ cnd~~n is n~:i~ognixt~d
hy th!? samw t.HNA, \~Gdon l)teforem::~ then "'lW'3ats rebt<::'d tn
{}Ot~t1~h-:ing the ~t$bihtv of the <'-o~:fr!:n'";:~t1tic(uion inten~cd{H~
(56). HoH; foct<)rs sug,;;\.<"st tha!; codon sdpthna{ cO\':h.-:u ·~.;~~g-& f~:::-I th:e t~~·(~ .f·'.$t.r
. l' > • 'I' ~' l'l' <1 ! 'tl n ' ,_. ,·t ge~.).(::s i·:.; '.lSt~t~ ll1 .. ~ ~h)1~:- ' .{·~· ~':"\}}.-..,, :i~:>11lp<~ti3( <,.~H . } ~- .. B~l- }..'l_~~i{. s.
v~rou~d <>ef:B:t by rB.ntion.1 &-::k~(~~.1t~n bet\~··een ~Yl)ony1nou~; ~X»..'Son~
~~nd t(>t ~:roups t)f ... ~.·~~XJy ~H:.d ~;trong~:y expreB.s{~-d t;. coli ptO··
t.eim; (;){)), L~'udne, fm ~;it~, i;; spix diff1irim!.
codnns, one of whkh, CUG, is ~tNdy pr,d'ened in stt-ong'ly
:~ K .. L. ,Ag&n(.(R},. j;-$·;:'${~rs.:~l !:!·Jttn1·:~H~it.":;E{t.i:_).n_ St:!-~d.ies ~)f th~ <..~~n.1Ji~{~l
~it>:~:"j ~,f tht: lip:o2~ <~_ndtiH.Ut":li:~::>;~~ ~n;~ di:':~~:.rih~~d. ~n J~~t .i:t
·"''""""''"' ·- _,,,,,,,,,,,,, ... ~
P!w {}.5 0.':!2
.t..i~tl: !)~17 o_,·;s:
Ht~ o.~~:j \!.'/:">
V111 l\'/'.~f o~~lO
::•ki:- n.& H.8:)
l'tl.:. f},2.5 u .. i :.~
·rhr 0.5 n.M
Afo. {):1r~ i)J3.H
Tyt o.r; H.<~>
Hix (~}~ ll.i$
f}h.l n.:> O.S4
A;m H.f~ ((~h:
Lyt' i};j tt71
(A.$p~ O.f~ ~-~Ji2
G!H i) :) 0 .. 'l5
(Cy:s:) OJ~ ~j)J4
Ar~ U}~~~ o.~r1·
G3y n . .s. ~'J.~:~3
,~l\)l&{ t).4\3 n.~2
...... ,..,,,,, .. ,,, .... ~~~~·
tt2t> O.i'>
0.4g {:>
O~~~~) o.:.>\I
n.7:! 11.1$~
o .. ~~1 o.~->
H.f$~~ f:U-'!
znn t) ... }~1
fl.ti{> n .. 80
0-A.1 o .. ~~n
0.:~~) ~}J)~
ox~7 o ..~~6
~)_ 4i~ 0.lH
(l,\N UJ3$
li.4Z 0.Jl)
1)))9 {J,(}~t
t)f, :.\~)
i:u.n ()A2
0.S<' n.&'\
G.[~:~ 0..4~~
(1!)}
CW)
!2!lj
LH3}
W>)
(;?!.)
H4)
(20)
{lf::}
{Ii)
0.1~
(17)
{~~5~
(20}
{24,~
·!Z}
(t~))
{J~n
f~dl
~ss.<:ti;,,:~l;,;~~
o.1s .;-~~2}
f} !t~D)
o.rn H'tl
{) .. ~~7 .u~~)J
0.44 ~~)~}
i.>.11 (18)
o. ;:;2 c:itn
(~ .. 9~ {3~3)
0.2~ C!1}
li,U\ (>H
tt~1~~ (~~4}
n.2~ (;~9!
i.H)O CW)
O. lH f~?.l}
<.t7··~ {~~z~3
0.2~~ f7}
(t~~~~ ('21 ~
0J;4 {2~~)
•Prft.t: vahl~:' {.)f i>pti~n8~ <.~orkH~ u:~5~0- fof' ~tn·~ng1y ;rnd ..,,v~akh· ~..!.X··
pt:f~S~{:d g~~n~S· ar~ .dt:.th·t~d .frr(f~) -:J·~"i ·~.>:s-1.~:~ ~':.~f_p~·(~~-f::i~):$ ~~·rn3>:~~f th(.: opthnai cndi:)n~ baR·~i un th~~ group f~~.
p:r~·:•:,~in.5:: ~~gr~~$ ):VBH :;~:itf·~ th~l d~t~~rn:~ln~!d pr5::vi(lU~l5 .. · b): n~~~n1HS'.·~~ ~ 54.
:.>:j.\.
r:~::)sf~~SS(:d ptotrb~ f<>t il f:rNjl.l(:n<:y of 0 .. 78) ;;~nd h(:tJ-.~nn, I'h~~ frer
0.17, r;\.•en f~)r \V~Bkiy (~:~yhoff
0~9) ... J'h.~~ <)ve:raB frequt:nc·y of <~ptin.1~~1 tl~don :;~l~ct~or~ fot th~~
g:r<~Up (~f ;stt<)ng!y e)):r~:r~~?3~')°?icl1J,:0:. in t.:l-w i.wn prntt;in;;, Of i:h<:>S~\
tHH1€~ u.tH~,i~~ th.ct:epnng tRNA for GUG ii'>:!-, 58!.
.. rhe )::~~N fr~?.qHi~n~::y of opt~u-~;~1 ~~~x]nn u~.~a~~t~ of th~ ;.~!dl g~~n~~
b;is.~d on j~; .. l;·oh prot~in~~ n~ay he due in l>~Ht to ~~~j~(t·ii;)n
f<~VO:ri.ni~ .~ d.~ff~~tent disttiln.rtio:n, Of ti{t~i\s i·n f~rt~~-~id~?ncia. ~fh~~
m;.ts of opt..itna} H.nd nonopt.:unal c<)dons in oth~~r I>:nh~l:·obw::· ...
t~t~iace&~= in g'fi:ni~J~(~1 ap!-~nr to 1~~;: -s-hx~ilur to th-iX.t :~r~ ~E. e~Jh {55{
67J .. Ho\~·e~{fft'.> lJN'/l h.rbridJ2~ation $itlHii~~~~ lnd.k:-~rt.B th{::.t .f)_~1}-·
1~eu..s· i~nd f.~rtJf..~id~~t?.f:iu ~~piu~ie~~ B.tB' nn}y di?,,_t~rntly re);:,t,~d to th~~
othet n.<~n.1l>t~ts nf thi~ f;:nni!y ~J}O}~ 'The .~0f:d1 g~n.~~ ate ~n {act
vei:-y pN>rly {;.><{;n'ed fot the E<v~;rn}) fr1iqrn~rwy <>f optimal
~~od~!n ~~eiectkn'1 f~)r ._tl1es~?. :prnt~~itm i$ gs and .ttn~~:~:, ~ re~pecth··ely~
fn ~Of!"H~ in~t.an-t.~·~~" the di:$tributi<)B ~u:r;.ong ~Yl~<.>nyn1(HJ~ t:~.) .. ·
dun~ is a}~{.) ~in1il~r to th~t iu th~ f~tl ~yst{;:tn ... rhe npt.frna~
1,'Nhn for tm.1dxm, CUC, w l<~rdy 1.1;,;;d and again UUA is
otedorninarrt Jn both w}~ste.sns, th~ co.din.~ :~,.eq~Jf~uce~ .ar~
highJy ;\ ~r~·.ri{~h. ~fhe .f~f:t.f t~3st.6~)tkrn <~n~~YY~l~ and rnethy~~~$e
ar~ :E_1~~. and fit~~:t b,'T\ ro8peedvet.y .. ~rhe l~\:~of\J: Kent~~ Rf~~ (;5~i·
A ~r ... fh~$ ~$ in par:i re1<~ted tn th~~ !~)~~-· .fr;±ql.3.t~rH-:)/ \)f optirnal
c-o.d.(Bl ~-~~h.=:<~tl(n1 in t:b;s.t th;~. optl:u1~:tl (:od~.)ns h\~V~ ~i h.hiher
<. I-fow;:-v~:r,
in both i;ysi:e1M, t.h!l lfonhn.g '>s RN ;i.!~o highly A'Y
l'"ith, rle~Pit~ fh~?.Be si~nHaritk:~~~ ~JH3:n~ is nn ~~pµsr~~nt seq:~~~nt~
hm:i:m!ogy 1>t:\,'NSen tht> Er1c,Rl and Psd geiw:;,. The s!mi!m-
pRtt~l·T~ of c.odn:n H~~g·~ L~ pi-ob~~bly rel&ted to ~:h<:~ )a.ck of
~:ei.e~~thtt: pres~u~~~ to opt.in::.h:.e tht~ ~~:Kprm~~i(H:t nf n~stdctinu
~n1d n.)od~fii~H.t.ion ~n·;~y:rne g~~ru~s.
'J."'rc~n.-s<:r'·fptiolt und ·frQ::-~s.~;;.1t:ion Sta:rt S.i.t.}~s~-~·Fn:n~1: th't) d~t~J:r-Â
ru~natio:n. nf th(~ 1tH:~:;Jion (lf th~ J):~tl: ~;·t~ne.s v,.-ithJ.n the cJ<)ned
fhndlll frngni,~nt, it W(1~ imrnNli~ii.dy appl'imnt that tlw p_n).·
n~o~-~~r and p{)t~"!ntiaJ f>};&'lJ];cU.:;)l":V ~~'qt~et$Ce:s kn· tK~tli the r:esttk:··
ti~:sn ,~nzyrn~ ,ail:nc,:J in
the inte:r~i~tron-k~ regk':!l i~ ~110\~·n .in gre~:t~:r detaH in J-:'ig_. 7.
B·i)fh of th~~ tt~~:ft$::Cdpts b'~gin ;~··hh ~~d~~n .... )~in{:. ~rh·S~~ t~sith.te ~s:
~.;~th~~ th~~ ~~:q~Jenti: (\:j.A H.nd -'["~\/\ fnr th~~ r-~~~t,t}.ttiun ~~n~yxne
and :· ..:r~t:thyta~fe gt~Hl~S~ n~~p{:-eti,,_:~1Y~ lM .E~ r~;li pron:F)teX'.~( th~~n~
i~ a p-n:f~r~rH~?~ for (~ at p(~~·~6011 ·-, 1 and ~r at +2~ but th~~e
r~s~due~s an~ 1>nly \\'~:ak{y co~J.$;~f~~~?d {b1···H~1}. 'Tl~.€~ $:.equ:~nte~ in
th~ ..... 10 t~~fk~n troJtK~h>gou$ ~«) t.he Pt·ihnol~~ bi;)X ~lr~ ~~rtd~:rfin.~~d.
In hoth ~~;~s.t~$ 4 of (; rm~idnt~~ :rnat<.~h d~e tor~.se.rH~U~ EeqB0t1(:B
6'~TATAAT UH·l.'>'1.L Tho: spac;ng l~tw<:"'11 th~! -·10 t~gion
;~nd i:b~ trans;::ripi:!m1 initiiltimi $lh' i=> g('n<~w!ly he.w*ri 4
and S nu('..i~~ot~dt~s ~~nd B»U<~Hy i-frth~!r 6 Gr 7 nnc[~~ot.id~3s. ~rb~3
~~qnt:·nc~~ idt:ntified tl'.pstn~;3;nl frutr~ lhe ·ineth.yla:~~ ~ene h .. H
h8se p,-xit.s f!"h~ Int~~re~th:!gly~ -~fl b<$.3i.?. pair,~
fon.her iJp:>i:rN,m tb!l N:qwmui TTAATA ;;, found •'>·hich
B.pp~~~.1"'~ rt:l~te.r:tbnO'c·curs 17 'b~3$€" vairs upstrt:~~n.) fro1n the --.·10
.::-~~&inn of th~ .rr~ethJ~lase gt!nt~ s.hare~~ H of{) resid1.M~~; s\,·ith the
,~n)(>log:y ii> simila:t w t;l:.mb~i:- M
know:n .fC. fO!.i. prnrn•:)t~-r:;~ (~}~{),
'J'h.{~ $equen~~i~6 and spacing of the .:.:.(H:.~en.s·us ~~~.gions sug;ge~t.
t.hat the _p.ro~1l;ot~~r:B ~~ . .r~: relativ~ly w(:~~~k. ~r~his plOb:ihly ~~c ...
cnunts ~.n t~trg\:~ p~~rt, ·f<)r th~ po~-n .. expl."t~ssion Qf tJ1i~ f-ls·t:[ ~!nes
in _'{<;. cf.1li: (1 }~ f-l. &-i.H~1rth and i~. l:oli are on~y ~H~t.~~ntly .re1~ti~d
UW}, ;md cn.fi&eqrn~nttr the r1~Ngnii.i\~ ht~X ar~: ut £1 i~t~d l~Z bas~~ )._)~d.t.'.~
up~~tr~~an1 ;torn th~ ~tart ~it~~s for ~,h*:"! It3ethy1a~;~ :3T::d r~~st:rl,~bnn
~:n:.~:~vn:.t~ .. l~:$P~~etiv<::!y. l\~~ _poh~t:!Ni ot.H. ~ilxr .. :·~\ th~ h~tt~:r a1s~)
\:,;/l~uld, pr.t.3'Vid~ the .appropriB.t~ .spaci:1g to B re:g~ol·l horr~<)k"tgn.ns
to th~~ ...... ::~5 <~{~n~~~~nslJ~ sequ~s:t{:~~ .. AJh~·rnahve!~~~. tbe·re ~nay he H
prnt~~~n 01· ~'-f th~~ r~st I
g,.en.t:s .. ~rh~~r{;: ·~~~ h<'·-.~·~, .. tH\ sio ~v:ide.nc{~ ;;:~~ ):.et fc>s· H s~n.~~rate
~- . . . ' ~ .
:reg:-ul~~i.{.or.~· k~us in ,-::~ny 'lthf:':t 1\'Pf~ fl r.~~:.;tric~u'.()n .. ,n:~-xiifk:.atinn
sy~t<:~tn,
~Th~~ initiation {:·odor:. t\)t both tkH~ n~s;.ri~~ih)n ~l1~-:yu3~~ ~).nd.
nl~tJ1y.la;s"~ i.:~ l.he .first .A. lJ{; ft(a11 th~?. 5 ·· :e~nd. of th~ n1e.~:..:3g.tL
~rhe n<.)~rtrB.r:;~laL~~d l~~ad{~r 8.-t~q\.u~nte0 ~rr~ ~)~i and 27 n~J:c}e<)t.ides)
rt:spt~<;:t~·ve.~y~ A.nal;/~$:~ of th~~~-t~ .~equence~ re\.·e~ls tlo app~1rent
ho~nnl9gy v .. •ith th~ f::H:~h1e· .. })&Jg-ar·t3J) s.t:qut~ne~~ {G~\ 66.L ~ThJ~.;
~~g~~h-3 ~~otr.e~Bte~; v,~H-:h the .ht.<:t th::tt th~~ g~xu~~ a!~ not high].y
~:~JH~~~:.;~~d~ hut is proh.ah]y n. • ~· • l' ~· • ,, , • .,, ~ ' ' .. .,
rn~:. tn.gh O:eg~:e to i;.vtnch tbH .J t~nd. <.Yt lb Q :rltN.A ]~ t:~~~:oiidt~ 5" .,,tL.:\[TC/\(} js_ fotrnd Lr: tb~ rH>r~-Â
tran.s·lHt~d J~ader s~~qtH~rices -of both hf the ·~nrfNA~, ~rhe
d.i'St~nc~ ft(~n1 thE ~nitiH.tion C{:.dotl 3:~ 20 t,o 18 tllH~lt~(:tid~~~ i~~r
th~: r<~~~td<~ti{~·n :3.r~d rn.ndificario:n ~niyn1~~~ rnRN/\~~ r~'.'.!.P~t ..
ti~~~{y~ !:n t.b~ l~uek:x~ti~ie ~,~:(tU~n<:~ the;:;l': t·egi()!l~ ·~n·~ $~:par~~t~d
lt.v 80 b~~:~~ s_)~~~r~f suffiei~:nt1.Y f~·n" ~~.p~irt tbt1t ;~_ ~tern·l~:>Dp ~;tn~'>
H~~:r~ \\-'o,~k{ ... he v~~ry ·uniikH1y~ 'l~h.~~ ~}<.)~;Stknl of t.h~:sf. .twLk)\~~~d
' ·- , . . . . ,. . . ' . -. ~ ·· ".; 1· '' '""-'I' ny {i tf;}f'..t: {~i: .l ~ pyrBlBdr~H~~~ Sfl:.~Ur~rn,g rr,it\~t:otg,~:~ ;:_~,'$- \_;;,._~}&-'.:. -'·
}~I(~,v~·vt~r~ thh~ 8€:qU~nc~ is hi:;hh:' (>rich and ther~for~ hs
pr~!h~~h1y not a :r-·b.;;)'·~n.d.~p~:.n-d.~H"Jt tBrn:frnntic·n ~it~ ff}~}. ·~rh~?
~P.~tl 1r~odifi,~ath?:r-! ~nzyna~ is als<~ t~n~)\\:.ed b:~~ i~. s:e~~'i~:}n i;·ht<:h
-:_.,;.1 ul~id fDtxn a s;t{;~?~)_'"iont;• str~wi.nre. ·r~le t~~rn1:n1ut1eo.n <..:(:.Untl 3S
~t po:.;ition '"Z.;·JO- Sp~~nnin~~ thh~ s.H:t~ ~~ an itrv~rt.ed r~_p.tat at
po~ih~>n$• 7;14~~788 ~nd ~rJ.fr.:71tt ;:}~patat~d by ~~ kx)p of 14
·tn~cl~'.'ntidt~~. \.V:h~th~~~· ~~.i:!.b~·r ;pn::.s~}~O!l Q{ th~ l>itt g~~ne::~
n~~n:a.1n;s tl:) b~~ d~~t:ern'lJ:l1~~d.
1:-~...-s~t--tran.s.lo,tio.tj,.aJ f:-rd.Hi<:~rt.i,)·n_
~~r~zyn1e ~y:nth<:::sb~-~~d in h~).t"i.~~si~~ !.nini~<::U8. indicate f~hat the
n1Bthion~:a~ :r~s:~dut' l'$ t~l~'-~ p.1:).rth~Jts ch::s~\·t~d (Fig~ fi}. The$l:·
r~$uh.s p~~r.aUeI ~~<),tfB.n~;J,~tio:r!al _ptoC€'iSsin~- "\'l..··hi~~·h (~t:cu.ts .in
~~uk~~ryotic <.':~n;; in -;,~'hh..;b (:$3~~~~ th~3 rnt~thi<.:nin~~ rt;:.,idn.e- h~ often
{,~h~av.e-d v ..d1e.n th~: s:=:-:t;fn1d ~n:x--i.i-n<> ;:tci.d ~s s~r~~~u {~B·d. un~~hatg·~d~
ff:. i\l::-t (Hy.~ S~:r~ ·'rhr, Of I>:t.1) {€-8~ ~}n). -1~h~ ~;~~t·
t,k)n en<-~3-·Trie ~~~ L.,vn, ..
P r~vi<:l frotn prntt!Q·
~ytk~ pt'fl·C~~J3Hinl~ of tJ'~e· .f~nz.y1YH~. !¥ii}f!~,ov·:t:r,. the Nlirt~ttni·u.a~~
;;i:::qu~1!c~: <)f dH! pT~)teh1 fackt: lhe um' Gf hyd_r<)phohk f':%idux:8,
typic~~.l~y St{; J4 <~n~hso ~~(':ids h1 length( <:h&racter~~t·i~ ;){ '.:;.ign~d
fmptid~~S C-34}. '.f'h~:~{3 ob~:tH"\'.'a.th)nS ~Ugk,':(:St th~t the J>sti' ~ti,.
donuc1t~a:se ~s n.{~t foTth~~r prn.~~BI<3~~~d di.iring m~nl~brane tra:n~~,.
p'."?tt .. ~rn~ ~ignsJ _p<:ptide n:1ay he hx;~~t~!'d lnte:rn.:~.Hs\ ~~s has
h-e~~n pr~1p()~ed f~)t ov~~lbntnin and H. :nun~he::.' <~f h"~t.t!:g1~~~.11xH~l.t~.Â
brant1 prutein$ {'7.1--7,;3.L
·1:h~~f~ct·i:l1:<~t th~~ r~~~~:tittio:n ~:n~~y-nH~ is tnu1~P<~rt<.:x:! i.nto th~;
.P~~ripi<~~:rui<: ~p.act' n:~(.~Y h~~"'~ tat:.iht~:t~:d ~~lonh)g ({ the })stl
sy:Bt~nr~~ In th<~ ~;<:'tl{:ction sch~~n~~~~ th~ t:ndontJt:h~.~t!!lx:, t};.l& •~ppwadl ha<;< ht:<:n nw>tl(c.C!~ssful h1 mme fhan :m
oth<:~t eKa:n. .. }ph?.!) :.;b}<.1..BHtl ··rh~~ }oc:·3don <.lf tht: ·:~stdct.in:n i:nzyntB
i.rs the:~ pndp]a.s:u~d~: ~(pa<~~~ n::.ay fan:~<)r ·restrk:t.ion <~f th~~ J3ha€<:~
h~{ted. th~
end,ontu.-:1~3<:;.s.e. Jn the c~~~se of the .e~~oHJ ~n~d l-fh.aH sy~i.etn.s, ~t
has 1:H.~-~rn ~~hn\:\~n d~:t~~tly that the ~!xpn~~,~~]on <~r t:h~ restr~ction
en.~ryn.~~ aiora~ _i~ i~~tJu'31 t~) the <:·~~n w:si.ng t.en:~:p~tatut·-e .. s:ensitiv~
nlllt{~nt~ -of th(:· rn~thyJ~~stt ~~n(: (t2~ 74). ·~rhis .at:;.o ~~pp~ars. tH
ht~ trne fi)x th·:~ f).-;-;,~r ~~::/S:t~tn.~ \VB have h~!~:n unah1~~ t<) :~~~nlat~
~~~th~~r de1H3:,l·~>J~ <)r ftatne ~hift. nnH:ath>n~ ~~ithjn th~ Ul(~thylas~
g·Ni~' hi th;;: pr~s~:nN': qf ;~ !i.HJ('tfol1;i] .rni>i:detkit> en~yrrw gm1e
{4}. lf', (in~, Biq:>eriim:i:it, the Xbnt-<~ined. ~n
addit~nn of 4 b~~w p.~t~ ~d.· the ~\.t'(d ~:it:t:~ ·~~'l~~ch --\.vo~:lt.!i:'fos h d .. ifferent antinQ ~~esd ins~rt.i(nv; \\.'·~rt·:
oht~:ined frahl~ J\') ~n~d~ in botJ). C~~e~~ .V~~~ld~~d B.U ~1f:th~B
n1~.~th'vs~~~·~ h.1 t:t)nt1a~t to tJB3H~~ rt!~nh:s~ c1on~:s h:s.\'i~ tf;J.f.n
~:~ol~ried ~~ .. hirh t:~~rr_y th~: f-~n~ .. rt7 :re~=:.t.riction ~~n.iyn.i.t~ v .. ~ithnu!.·
tlw: ~?:t:~rr~?.spnnd.ing tnet.h:ila.:-3.Q. H~~). It3 thi~ jngtant~~ ~ ho\.Vf:v~r."
th~) ~n.l0-'~:ne is ;.~cUve ~n eruct~~ t;JH ~~.J;:tr:r-~V~.--t ·.?'i-~7fl!s:i}n'->e of the .Xl~'{'iJ $:·t~·2 r~: •. i:~£r .. tl z~l p)t!.J~Js ,1ri.~.i l-><"'>:.~l-~)-f.~
'Th:I$'. ilu~~H.t1f:~ti:..)n ~~!j:f.'.Y).':.<~s ~Jf pXha.~1 .:jnd J::.){b&6 Jwv~ $.H ~n1:~f.1.~)
-'~f:il.a ;xdd~~d. het,-.:.1»~~~n Lf::t3 27 and A::5;P 2-H (~f th~ f\:;t ! i~:tz"15.n~:ntil1n
'I'CG
AOC
S1,,~
TCG
Af)C
8t~r
::?.;)
'TGi~
ACT
'T'h.t
~')'I.~-~ ·'
;{{)' ... ).
AC'l
"rhr
~'2
GAT'
CTA
l"':~U
GAT
CTA
L~u
.... ,, .. ·--~~,--.
~~~ ~~~
"''''''~-'~'"'·~,-----·.,~---·······
GA.T ('TA err
CTA GAT GAA
1 ... ~u Aiw t~=r~
CAT C1'A GTT
(rfA Gii·T· l'..1AA
Va! As>~1 L~1 .. ~
to h~~ a nt~cl to t~:gul;~J."~ th:<~ ~~~J~re:c;~-;kH1 ~rf the :r~~.st.r~(:tiori
~n'f. .. Ytnt;:· or tneth.Yb3.8~ §;1?.XH~~-- :F.c}]lov;ing r~p.lic~t!on~ r~~~\.·v~y ~~y·n-Â
th~~~·~~~i:~d l")NA i~ h~rnin)i3th:-r.,.:h:tt~~d ~1nd n~r~1.;:r:n~ ~)tot=:~ttt~d.Jrnrn
~.j~;;:~~,~~-~~~ -i~~-' ti,~:- .;~;1for~ th~ ne:~t (":eH d.ivh:;io:rL
Fo:r re~;tri~~n<)t~ -n11-x$.ific~iu~_)J~ g~ne~ ,,,,.Yhh::h "~an b;1 tran~·
fornwd in!<:< lln unrnt~ld a1)pe~~.r t~: h.i!. e~%~rrtint th~1~~$:;; th~~
iwxlific~tkm ~:m.ymi~ \;; Xpte:;1:;;~d f\.r:;;t. to ptc•i.eci tht: DNA of
th~~ h::~~-L svnt.}:H~~~.$ of tht~ :r~:str.k~tion -~t~1.'·lUH: v .. ~onh .. 1 be l-ethnl
f\il th~:- l)r;~g~ t~<1.~>~ncod~~ i~:~trk·tion-n-10tHfie~~t~otl ;:":;y$;t~~n.~~ ·it
h~t~ b·el~r~ ~.b.i;)~~~l{ that tn-o.·dific~3t.J(a::. i.s B0Pr'i?.:3sed 1n <~d-..~Rnt~: (_rf
re$;tricti0n ;n nB-s~t·;v ~nfi~;,.:t>~.d ~~~u:~! rT7). h. is u.ncl-e~:r V·/hV this
n1t:chani~-$~n v ..~~)uld .. ]-~.:iv': ~'/($lY~d f<>r n~stt.~tti~n~.,~nndifi.!.~llti<;·n
:~>-;Stern~ thHt ::1n~ t}n"nt? pom;ihUit.Y .~~~ that thesf: t'..'io 3y&tetns tna)·· h~~ve
arl~~n froni ~ l'tl(~~nt <~hronK-:~;~~)n1H.{ ir-it{!·};~"s3.t:=.on nf ;:t nv~bU.e
~{::nl:tic {'.:k·rn~"t~t (~~tr~r·l ng thg?. n:'SU"ict ~()n ~3~.T~~yrn't: ;~nd tn.~:fb):• ...
~ ~$:{: g'-~ n~s.,
"The ~:nitia1 rnod~:] J)l"O:P'.)Bt-d fc}r !~i~·qne1-rti.;~} e~p·te$is1on of
r.;~~trirti{s.n" rni;>difi.c~it}\..~n ~Y-~ten1~ teqnirt:d ttH~ ~nethy~;lS{;~ b:)
H<:J ~& ~~ positivt~ n!guhitor for th~ ::~xpr~~~~~~h>n <$f th~ te~trk:tion
~~Pnt: !f;, 7,~~, F't:.rtht~r studik!~ ()f th~:-- /:;c.ol~l and ffh{1ir s.ywt:E.~tnS;
(·1:\ 7 .. ~J·> a?:; '..YeH ~i~ 1-=-;stL ;~r.10w· strorig1y ,~g)1in.~t this.; po~s~hB.ity.
For b~)th tlu: l:.~col?J ar~d f-fh.·:dJ s:~·~3t!:~ms, pla~anki§ ),>.;·~bic-b ron-~
tafri tht: r~~~(td.c!.ion ~Hl'l~yrr~e and H t-t:-snpe·r~1tln"i:-.-ser~~itivt:: rrtu~
t~J:r1 t Gf tht~ n1.ethvla~~= w1-nt JH)t tnn--~~furrn Ji . .coli at thf! tH.)n ·
p~~ra:r!,~siV(: t,{;n1p~;t~~tl~l't-1 ~n ... ~p[y~ng that ;:he t~':&t.r.te;~ti syste3:i:-~~ a_~, di&C~~~~sed ab<:r~;..-e~ )VB
h.:-h':t;~>t~ of th<:-~ n~;').tri:etion. f tJB~ m~tJ()·~h~~~~
·~rh::~ VBtied g-~~ne arr~1ngen:~.~3nrs i.n r{~~;tri<~bono°'ni.odd:~c~~ti<~n
_S)•st-t::n1-::-~ ~.ii.~l·~~~~t di:ifer~~nt tne(:hani~In~ of r~Kt~L~t~nn_ Jn th~?.
E'tdlI ;:i;nd LUiaH sy:;:J~rns, Hi<:> !:w;; g:mrn; am ;:u·:rMiged. in
t-a~~d.\3rn (;;on, thf~ ~~~ffl{;'! I.~~~J.A .. ~-str~~nd.: ~~-nd ~u~: vt-~)' ck)~t:"!t~~ Hrd\··~~d
Ci~ 8~ 11}. [n the .lfh.a.JJ ~y~;ti}n)~ the nsethyla-~~~~ gf.::t:~ i.s 5~ to
th:t: R.tu1onuc{eB30. ln thf- odgin~d <~hHH?. i~;nl~~h~di t.be gen~~s
a_pp<;:~u· to b~: tr~~n~~tr~hed ~Hi ~~ pnly~i~tr~)n~c :cn~~sRg~~ fr~)rn the
B--li~ct«!l«JS~~ pnnri<)tf't fri pHH:322 { .U .L Si net: dw ckm~d DNA
fn~g111<3.tEt inch.td..i:$ .tnnn~ Lh~3:fl 700 bas~~ pai:r~; B.J~:s.tn~an1 fro!n
~h~: r.nf~-hyhl$-t~ ~en~~ th~~ ~ndogenou.~ pi"l)xuol~t iB P~'i)b:;~1)t~·
nr~~":~~t~t, ht~t h~ {)niv ~~HakJv a~:th··~ .. if at ~3.U, ~n ;;·, r.c;H. ~T'h~: co~
t.rancd;}~tinn Gf ttH~· g~:n.~:3 i~): th·t~ o.tder of th~.?. tn·t~thylrd"'Y of t.he g~nN; i1"
r·t-v\?.-r~~d~ th~ er~do·n.u<:l€::~~~= p:r<:~c~~dt'!S the ln~~th~'{h~Ee g~~ni~ f'7 ~ 8~
1~~), Fu.$.iOn nf th~3 g{:"n~:-::; to the fr.a~ p.r(Wn~~t.et t~p~tn~an$ ft'tH~·~
th-~~ ~!-nd.<)t3.U~~1~~a:;~ h:ad~~ S..H B.. pB.ta~1:~1 inc re a~~! in thr.. -~xp:r~~~sit}n
of both pnJtf-in~ l~P<$n inducti~)n y .... Sth i~OPl"")py1 .. -S--n-.th.il)g~d~·~>
tn~Edt: ~3.nd. g-.ro·•i~tth -tn gJyct~t(~l (1~i). :rh:i$i su~~ge~t$: that th~ tivo
g~~ne$ a:r~ t.:r~~ns<:ri}:x~xl a~ a po}yt3~t~1Jni<: ?ni~$;s:~g:t' fro~n tht~ 1<.tc
·~}rl'>n::.oter and pt~~lJtn.abiy ar~: H-l~l) C<.) .. tr{n:t~cdhi~d frn:n-~ tht:
~:ndn!i~H(>U1" pr<:mot.~.t~ i.nmscrihHl d3v~:rgimi.ly frNn ~~:p·
~~tat.~ prt~~:r~nt~;:r~. ·~rhe tt·ans<-:ripti.-:)n iniu~~ti<)n :.;H.es {rr~~ m?pH··
t.att~d by <~nty 70 bH~~~ pait.$ {set~ Fig_ TL S~uce th€! -~--;~)nt~~ct.slt~?.s
\.~'.it.h ~$. cal£ ~RN~\. polyrr?~~ra;s(: g~:n~raUy e:~d.f~nd ··~H t.o .SO bB.s·e
pRin1 up~~tte;:an. frQi!l tl~H~ i:nitiati~~bb·· ~.v .. ,erlap. Dt'>~.a~e I pr<:~t.~;.ctit)n .e!)ilJe:rSn-l~~rrt~~ h""'~dir.:~ti:;.
that ltN.l' polynl~:nu~~~ fonns H. n.1ufe ~a.able ~x·nnplex ~,~~th th~~
n).e-t.hyta~,~~ Jn):n.1lot.e.t ~ Sint~~ th-t~r~~ "S\~a.s no det~~t.~ihh~: binding tn
the $.f;:);tl.S<:t:intksn start ~ite fo~r tht~ ru~~tr-ittin:n ~tt~-:.J·:rn~"= gt~n~~
t!ve·u \~..-Sth i·~;trea:-sing cnnct:ntrat.iGns nf pol)·Tt).era.~e~ the o(•(·:u ...
p<~rtt~;:l of f~~{:h (~f th~ tv•..::t) ~.ites. :seet:r:f, tn hB ttn.1tu~~uy r:>~~"'Ju~~jy~1-.
In n:g:t-een~~:n.t. );;.~lth thes.t t!?:;l.~h:$.~ t.ht: xr:inic~n e)$J~~~:ri.u1<~nt~
sug-g~:&t fh;:=.t. th~~ n1~th~~di$$;~;. h; n1()t0 high~y i~>.;J3~:m-;):?.d ~:han th~
rm~tri~~tkH1 ~3Ul·~·nne (Si:~?. Fig;~ ~3.L ~rhe d.iffetentiai r~it.c:~ of sy-n··
-th~~.si~f of th~~ t:~vo _ptntf~ins i~~ appsr~nH~l ~uffic.it~nt tn ~rH-;Hte
th;;t iJg; DN.l\. of t.h>' b<>st is n:1rnid
c~H$ .. i\.~ ~n thB other .r~1stri> ..... tk':ti"°'ro.otiificatitn.<~~Hrne art ~:n~~th:yl;~tJ:-f~Jte tht~ r~~S;t . .ti(~t.ion ~~nr.yn1~?. ~s synth.~~$h:.,?.d~ So.~xa-= d:efa:y in
thP i-!X~pn~&vs~otl of HH: calfil_'.;·ti.:~ ~u~th~~-l~Y ()f th~~ .t~:strictif sit~~·$p~cH1t n~t~o:rnb~natk)n mtxiia$.ed hy th~~
l:~fo.RI ~3XH1onocJt~a~~ ~ug~~e8ts th.a:. 1:i} son~~ e>;t.B:nt r~str~~~t.k~tl
and r-t~Hgation oi thi~ chrf f<:,reign DNA,
.. ::·::;:·~:"-J.v.Jv..d0:_Yi.~~ .. n.::~nt.----~:)>./5:· :.~·j~h t{> t.han.k G~>::.n \~.:'HJ$-~Jr:.~ dir..;i~·r.rrr of th{_)
Pt<>t~in St-.nJctU:re }:\"{<:Hiiy s.t ~bt:: t.) ni··.tef~~~ty i::·f Ji~t.,:a .. f<)t his ~~).$'.:~~tsx~i:.;.~
.in u-w: prd asnino ;3d·d iH~alysis st::.~dies.
.L ·wdd'::'r, rt Y., Hattfoy. ,J. L, r>m:i.;,!ln, ,l, };;; .. 1rnd Wald,';~, ,f. .A.
(1 g:H1.) f~rrr{~ .. .:.\~r>.tl. A<";1:.1d. S{~~-- ~~?. .8 . . 4. 78:- 1f:if}~.~-lfo.Y7
:~-. J\:{~Jdrii2h~ P. l1HS2~ Ch:<:: Cnt fig~.:~ Bi .. J.·~·h.e?~~. 13~ 2S."?~.;$2H
;$, ~1lX1ti(~b. P,~ ~nd ll~~b~:tS--s,, ft .J. ~1~)82} in .~V{.u~l~:f~ .. ~'.::: {Lh~~n:- S, W1,,
and fo1h1:rh, It . .l., 1,(11>} pp H""i--l~i4, C~,1d. Sinin~ ffaI!mr
LshHl'°{)t:ori~~:;;.~ .NY
i. \Valda, H. Y. f.lit84) f'h.D, !.hesi~, Thi; U.ni<.'i't<"ity of fow~
~~- !vian~:~ .. I\:f. B.~ }~K'~ H-- N·.~ &nd $nlith, }:L {), tl~7H~ (J~n<;"'. ~~~ ~7--~
H~
(:, lkU''i1r, f,, l'k•d.rli;.-uez., ft L., Gn:t~n!l, P, S., B~t!aeb, lvt C,
~1:l~:yn..ek~r~ it L.: I-:h.~y~l·., H ... \V.~ C~·ff:$..:X~ .J, }{o ar~d f:a§k<.1,v.: S.
{Uri7} (;~·n'2 ·2.~ -95 .. J 1~~
7. N~:ww;m, i\.. K, foib~i-1, RA., Kim, SAL m~d Mmitid-;, f'. 0 %1l
... r t-?~qL Ch<;rn. 2-5-~l~ :~t::u .. 21 aH
S. G-.~~f:~:rH3~ _p_ :-:i ·~ GuptH, a.1 ., H,~:::-·~~r.- X-L \'$,/ ." fltGwn~ ·\v IS~( :.~nd
Rlw.~~nher§~ .J .. ~·t fJ~)B.l) ~I Bfoi, CiUc?.,~-- 25(~, ~l .:r~-·-215~$
~)~ }(Q~}._~~kh~ V. (L~ B:c.~-~~y».n<'r~·'. ·v. I.~ andI~.ayer~ A ... A ... ;·ir~80} ls.il:l. ()<::'.n.
o'.:~r;:.{:t. 1"'i8:- 7.l 7 ,~ 71$. '
.HJ. G-ir)R~::r:~s.> ~r .. ·R ... and 1h ... {)l)k~ .• ~J, E. (H1&~~) P~·o(: . .tVtd.{ .·4.ct-;.d. }~fr:~:.
u: .9 .. A. 80, 4-!32--4Ul>
tL &~h!J~-~el\ B.~ .l\.~H~:~~\ S.~ a:nd sff~H.h~ fL (}. n.~1'33} GePW ~~,:{ ~ 227.,
2~1$
H~:":'&~~tt~"'-..f .:u~d :Hu .. nrphroy~~ (~. (} {19S:~n G'f:n~~~ 20~ iHJ. ..
1 :t !\iax~n~~ A~ !vt .. and ·GHL---er~~ \V. {lff7'1} .:n.~y~c. ~~hltl ... Atx-i;d. S'{:f.
I_/ .:S. A. ·'U, ~i{ci\.l'";~M
l ~(_. ~·hrK~.n~f A-~ .~nd (~Hh~rt~ \V { l.~~i30} :\f(:tfo·.~-..~~ .Ei:::::;.··r~u.'l, 65~ 49H ..
:).\}\)
:H~. ::\.'fiHiN": D .. L.> Gubh~ns: !E, ~L> r .. ,,~gg~ J~, \V .. lH, :;:'Hld D<)n:i::i$;t)·~~~ .J.
E~" { l ~77) .l-lfrxl:t1:·n~i~-d1':}·' 16". l (i~J1 --10~~8
17. 1.-.::~~=nnn!L 1J. }{_. (1~~7H) ~?(:.h . .:.}'t~ Cl-<)t»iJ i~--2'7) :680----~~35
Hi Bh<>"-"-'H~ A. S.~ i\-·!o1~\ . J .. E_~ }{uni.~r .. :F ... Rw:l lW.un~tL ~t C .. Ll~1&!}
il.'1{~·:l, !_l~~x~n.{;~rn:, 103{ 184 .. 1 ~\)
19. Ya:r~g~ l-LkL, .. Jv<1~hkiv~ L:- Ch~n~ !t-Z.i ZtJh~Y:o (t~ &tH3 CaBh~l~ ~-t
LHt8U) f~n~·f::,. l• ... /;ur .4(:(~-d. s{N-~'. fl, s .. A. 11~- 70~:~)--7fJ;J;J
20. Znh~~Y~ G. { lH72>) _,4.n.n~.: .. J?.~:u. Gs:rwt '(!t: > 2ffl--:2::r;
~! l. 1-h~.rdu~pi:lfa.t~ ~{_. \:\.: ·~ .;.xnd. ilo{·N.\ L. E. { lffi~>;.~~ l-f.~~ .... };_·h.;,.~p~.i-::.tt.·~y 1 7·<
2124··~~1~~~)
21. fh~y~~f~ H.. ·L.: ~Bl:nho.:.fs· }~:·~~zyn·R~t H5, 59fi, .... ~K~H
~!.1. Stad~·,:n; ft {l~)T~n !"·lt:-.-:l{;-:k Ac~ds R~~~:·, •!.:-- o'.i0~}7.40,51
:t~. St~t<:hffe} ·:i~ G. f.1~·)7S~ P"t.nd Lh~).·-;~:·x~ ::L (l97H) . .:.Vl,,~:kk A.:-:~{·,t~
/((~$. 3.~ ~~48·<~0~~
;~4. l\1dJJ.la'tf, f.L~ arid. fJa~V<}f~};·H~: {-J. <) .. 0.~)f:.;:~) -;-}. ;.\f~~t. J:h~1J, 167.:- :~~~~l ··
·~O!~
:~s. 1\{fku~.:;~~i: lt. ~.L: ;.~nd r..~~k·'.,~W::i ... %.i~ "&t, Sr. {1tf7U.~ .~r _!3~< . t t;ht;::.rrL
245 :- .&f}~f.~ -{)G:~ :t
~'6- !)i:-.~-..~~~•.Ht~ J, H.~ (3..nd \~1 ~H~. J~,. 'f). tt~77) l·Jfrx~h·>.:'.rn.h~u:~·· ll}:-· 2.::r;.~ ..
1:i79
;J"l_. Kf~~o:.··~~J~k~~ .D.~ K.rt,~~-ss~r. \~·./~ f}., .-:-:.nd J__,:l~ki\ JC L'.-> afid :Folk~ \V. H:~ fHt77) ~I .a~·<"'· Cf:f.~rn, ~~52"~ ~tlSf.~-~
;3HK~
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