CA2088379A1 - Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology - Google Patents
Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicologyInfo
- Publication number
- CA2088379A1 CA2088379A1 CA 2088379 CA2088379A CA2088379A1 CA 2088379 A1 CA2088379 A1 CA 2088379A1 CA 2088379 CA2088379 CA 2088379 CA 2088379 A CA2088379 A CA 2088379A CA 2088379 A1 CA2088379 A1 CA 2088379A1
- Authority
- CA
- Canada
- Prior art keywords
- organism
- detectable protein
- control region
- tho
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
- C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
Abstract A method and kit for detecting toxins and pollutants is disclosed. The kit includes a transgenic organism having a stress-inducible control region linked to a gene encoding a detectable protein wherein said control region regulates the expression of said detectable protein; exposing said organism to said sample; and determining the amount of detectable protein produces. Exposure of this organism to a toxin or pollutant induces the production of the detectable protein which can be easily measured. This invention provides a rapid and reliable system for testing samples for the presence of toxins or pollutants.
Description
J~N.29 '9~ 17:35 613-Z32-8440 P.~5 do ot ~1.
TtTL13: Biological ~gLenn~ IncorpDr~ rtre~ln~ueible eenos and r~porltr ee~lrucl- fer onvi~onn~onl~l blon~o~ ln~ ~n~ ~ol~lcolo~r.
YENTO~S:
rt Po~or ~l~r~ C~dido 31~3 ~r. 281h ~v-nue V~
V6L IX~
C~n~-To~:~6o~ 6 og9l Evo t:obriel1e ~l~in~b~m 2-97~ Rln,~woot AY~UO
Y~uveJr, 1 C~n~t-Tol:(604~-8~6-8034 Don~ld Jonor S-6630 SUtlo~ Al~enuo ~by, ~.C.
C~n~
T~ 6~4~-437 4S93 FI~LD; rho effecl~ o~ onvlrol~mo~ ondiliorl~ or toxieolo~lc~ ent~ on C~llu~r or~n~ a . ~ y ~O-l~ b~ O~.pO~IA~ 5t orSIln~m ~
bto~oni~or~ O nvlronm n~ ot eon~ on or in~uC~ Ihen ~nrlyrln~ ro~
btolo~lccl p~temet-r uch ~r vl~bill~y, roptodueliYe ~uccon. Ot ~he level of oo00 moubolllo o~ en~y~o. ~ltorn-tlYoly, îho run~ p~ mour~ y bo nlo~r~re~ OD
nclun1 o~ mC olro~ ptWCoL In ~o onv1lro~enl. ~ucb s~o~monl~ typ/C~
cro CO~PIOA~ bn~t~t~ ~nd Irborlo~; tho cuJlurl~e, oxpo-~ro cod o~Jc~em o~ tho ~cr~ or~anl~m rCquiro~ m~ny ~nlpllblion~ tvon~lo~ deacrlbos~ h~ U~C Or U~onic o~ rm~ to ~vnlto- r~viro~n~l ~oou or îw~;ci~y: 2) ltcn~pnlc rlnirl~ of 1~0 ~ctn~od~. C~elw~ tc~a~J. ~vhlch ro~pond to oetvironmcntcl contllloor ~ prodllcln~ ~n cerily ~ooruro~ protol~ produ~l; 3) ~ ho~ ~nd compo~ o~ rOr o~po~n~ th~ O~rJ~i~m ~o ~c~l Juhrl~ncor; ~) mothod- ~nt coo~od~ionr lor roctout ~ o ~onl-m' ---pon~.
. B~CKOROUND;
T~c n~d ~or In4tho~ of ~ dn~ tho impltct of enviro~nenl~I poIlu~^~nt~ on ocolo~o~I ~g~lom~ h~ Ic~ LO ~C ~C~Iopmcn~ or ptOCOtUIOJ ~hlch utlll~e 11 vin~
or~nr~a~ co bJolo~lc~l mon~ton. r~o ~Implc~t ~nd mo~ convenlel~t o~ îho-e ~y~t~
".n,~ o~ leroofp~ s. ~ir o- lllcy uo mo~ e~ y n~-lntomod ~nd monl~ulolod, Urlic~ cr or~nl~m~, ho~vovcr, uo ~dcqu~ m~d~l~ fos o~i~otin~ Ih~
pote~licl ~b o~ polly~ntJ OD complu~ lo-11ul~ ~nlD~o. cl Ihcy do ~
Ih~ oblllly ~o curr ou~ hlolr~nJform-l103~ lot~n~tormo~lon~ or cben~1c~1 coal~ound~ hy ~ullloolJulu osp~ ltlcont ~clo~ In dotcrrnlnl~ Ih~
o~ot~ ici~y of ~pnu ~o wb)oh ~hcy ~ onpo-~d. Thi~ o~ J~im~l4~d Ih-Jc~rcll ~Or ~dol or~ol~ ]1~ t~o lotto~ o~lopry w~lch co~lId ~orvo l~ bJomonllors-Th ~o~to~lc. Cac~ o~d~ t~OJo~ J ~PCcomc ~ wi~ uKd om~d yJtom hr lc ~nd ~noloo~Jlur blolo~lcol r~ud~c~, ~r~d Ib~ e ~ cu~ nd h~n~ing ~ lh-iC
or~o~rm h~ ~4 lo ~ propwodi we Or ~hl~ ~n~i rol~oa ~po~o~ ~u blD nllon in ~elou~ opplo~do~, ~o~o ~lo~ ~rum Ih~ rc~ oroluro oro:
.
: . ~ ....
JRN.29 '93 17:36 613-232-8440 P.06 2 C'and~d~ ot d.
1. Wllli~cl ~,L, ~enbery, D.B., "Vsi~ ~h~ nom-~do C~norh~dltl,~
~c~a~lJ to prodlcl man~1c~ y to rnot~llic J~l1S." Tol~lcol. Illt, Heallh 4~4)~ 469~8. ~489.
2. WilliJm~, P.L. ~nt I~io~bcrry, ~ ptom1Jln~ ic~,lor o~
Dol~robo~lor~ cl~y ~in~ the nomuodo Cotnorh~ anJ a~ computor u~¢~in~''. Toxlcol. ~;u. Ho~ f3-4), 42S~0, 1990.
TtTL13: Biological ~gLenn~ IncorpDr~ rtre~ln~ueible eenos and r~porltr ee~lrucl- fer onvi~onn~onl~l blon~o~ ln~ ~n~ ~ol~lcolo~r.
YENTO~S:
rt Po~or ~l~r~ C~dido 31~3 ~r. 281h ~v-nue V~
V6L IX~
C~n~-To~:~6o~ 6 og9l Evo t:obriel1e ~l~in~b~m 2-97~ Rln,~woot AY~UO
Y~uveJr, 1 C~n~t-Tol:(604~-8~6-8034 Don~ld Jonor S-6630 SUtlo~ Al~enuo ~by, ~.C.
C~n~
T~ 6~4~-437 4S93 FI~LD; rho effecl~ o~ onvlrol~mo~ ondiliorl~ or toxieolo~lc~ ent~ on C~llu~r or~n~ a . ~ y ~O-l~ b~ O~.pO~IA~ 5t orSIln~m ~
bto~oni~or~ O nvlronm n~ ot eon~ on or in~uC~ Ihen ~nrlyrln~ ro~
btolo~lccl p~temet-r uch ~r vl~bill~y, roptodueliYe ~uccon. Ot ~he level of oo00 moubolllo o~ en~y~o. ~ltorn-tlYoly, îho run~ p~ mour~ y bo nlo~r~re~ OD
nclun1 o~ mC olro~ ptWCoL In ~o onv1lro~enl. ~ucb s~o~monl~ typ/C~
cro CO~PIOA~ bn~t~t~ ~nd Irborlo~; tho cuJlurl~e, oxpo-~ro cod o~Jc~em o~ tho ~cr~ or~anl~m rCquiro~ m~ny ~nlpllblion~ tvon~lo~ deacrlbos~ h~ U~C Or U~onic o~ rm~ to ~vnlto- r~viro~n~l ~oou or îw~;ci~y: 2) ltcn~pnlc rlnirl~ of 1~0 ~ctn~od~. C~elw~ tc~a~J. ~vhlch ro~pond to oetvironmcntcl contllloor ~ prodllcln~ ~n cerily ~ooruro~ protol~ produ~l; 3) ~ ho~ ~nd compo~ o~ rOr o~po~n~ th~ O~rJ~i~m ~o ~c~l Juhrl~ncor; ~) mothod- ~nt coo~od~ionr lor roctout ~ o ~onl-m' ---pon~.
. B~CKOROUND;
T~c n~d ~or In4tho~ of ~ dn~ tho impltct of enviro~nenl~I poIlu~^~nt~ on ocolo~o~I ~g~lom~ h~ Ic~ LO ~C ~C~Iopmcn~ or ptOCOtUIOJ ~hlch utlll~e 11 vin~
or~nr~a~ co bJolo~lc~l mon~ton. r~o ~Implc~t ~nd mo~ convenlel~t o~ îho-e ~y~t~
".n,~ o~ leroofp~ s. ~ir o- lllcy uo mo~ e~ y n~-lntomod ~nd monl~ulolod, Urlic~ cr or~nl~m~, ho~vovcr, uo ~dcqu~ m~d~l~ fos o~i~otin~ Ih~
pote~licl ~b o~ polly~ntJ OD complu~ lo-11ul~ ~nlD~o. cl Ihcy do ~
Ih~ oblllly ~o curr ou~ hlolr~nJform-l103~ lot~n~tormo~lon~ or cben~1c~1 coal~ound~ hy ~ullloolJulu osp~ ltlcont ~clo~ In dotcrrnlnl~ Ih~
o~ot~ ici~y of ~pnu ~o wb)oh ~hcy ~ onpo-~d. Thi~ o~ J~im~l4~d Ih-Jc~rcll ~Or ~dol or~ol~ ]1~ t~o lotto~ o~lopry w~lch co~lId ~orvo l~ bJomonllors-Th ~o~to~lc. Cac~ o~d~ t~OJo~ J ~PCcomc ~ wi~ uKd om~d yJtom hr lc ~nd ~noloo~Jlur blolo~lcol r~ud~c~, ~r~d Ib~ e ~ cu~ nd h~n~ing ~ lh-iC
or~o~rm h~ ~4 lo ~ propwodi we Or ~hl~ ~n~i rol~oa ~po~o~ ~u blD nllon in ~elou~ opplo~do~, ~o~o ~lo~ ~rum Ih~ rc~ oroluro oro:
.
: . ~ ....
JRN.29 '93 17:36 613-232-8440 P.06 2 C'and~d~ ot d.
1. Wllli~cl ~,L, ~enbery, D.B., "Vsi~ ~h~ nom-~do C~norh~dltl,~
~c~a~lJ to prodlcl man~1c~ y to rnot~llic J~l1S." Tol~lcol. Illt, Heallh 4~4)~ 469~8. ~489.
2. WilliJm~, P.L. ~nt I~io~bcrry, ~ ptom1Jln~ ic~,lor o~
Dol~robo~lor~ cl~y ~in~ the nomuodo Cotnorh~ anJ a~ computor u~¢~in~''. Toxlcol. ~;u. Ho~ f3-4), 42S~0, 1990.
3. W111ioms, r.L. u~ Du~on~ry, D.B., "~q~ ~tlc tos~oity ~cs~in~ u~lng Ibo ncm~tod~. Ca~l~or~abdl~l~ el~e~. En~ . Tcxlco1. Chc~ SL- 128S-12gO, 19~0.
. 9~ 0ilo~, ~i,, ''Tho nen~to~io tOJUC]~ J~o~ ucln~ ~anoJr~ r~dl~ w", ~tlXiC. ~-Ct-. 2l.3~- 309~ . 1990.
~ . ~'~ Ko~Jel~ W.~ rocctoc Z~ibor~ R.W, ~r,a ~olnon~ W. "~ S13 o~vlrotu~cnt~l pDllu~n~ on ~ol1 o~pni~nu; ~ dmpl~ c~ o in~ o tollloity o~ cnvlronn~t~ul pollul~n~ oll org~nl~m~, U~De o~dA~lum ohloti~e ~d non~ co~ itot. an~i~n. S-~. ~2), 1~1-190, 1989.
C~r ~ n 15 yc~n ot ~v, It h~ bo-n U b~iJ~d ~t 411 or~ u ro~n4 to hcU ~treU un~ t~ ~ v~rlo~y or chon~c~1 UCJ~C~ b~ 5s~o~cln~ rp~clnD pro~in~ ~blch ~ro n~d- dthor ~ n~h lo~cr lc~ol- or ~ot t c11 uod-t norm~t cn~tltlonr. Thl3 ro~l~clioo bnr lo~ t~ propo~cl~ ~ ~onllorln~ ~hn onvllonmcn~ by ~c~-url~ th lov~1~ o~ Ihe~c prOIcih~ In v~rio~r orpolcm~ (1,2.3). ThC~C ~ppro~c~or irlvobo c~ on oS ~ pto~elnc ~nt ~cc~"-en~nl o~ rhelr bvolr, l~c~clly u~ln~ ~nLibodbr.
'rb~ in~r~Pdoo to~crlt~t ~ordn lltlJIcu ~ ~iiftcton~ ~pprouh, I,e. ~o hvo Itnl~od tno conuol ~o~ion~ whlch re~ul~ pr~t*~n pto~ lon lo ~ hloh produc~c c rodily mcc~ur~lc . c~.yn-c ~C '~tcpo~ no ~ repor~cr ~ene h~
~o~ ul ~i ~to ~he chromo o~o~ or C. ~ a~ o pro~uGo ~-anr~onic c~lr~
~hlch ror~oDt to clns~ ropotl~r ~ nc. TO cnmploto the biomoll~tor tyctor~, mothodo heYo boon de~l~no~ for ~cpitily ~ oonvenlently ~euin~ ~hn nu~ ~ ot the op~r~r ~vytlu ~cu-~lt~, u ~oll ~1~ it~ tilluc 10catlon k~
nomc~od~ c to~om~inm~ lUcinc. ~c~hCt wi~ ~ho o~s~y mc~ho~olo~y, con eon~itut- ~ ~lo~onllori~ 1t" wnlch c~ bo uud ~u dctcol Ihe IcYel ot c~re~r l~o~c4 on ~t otpn4m b~ e~o~-C lo ~ator ~mp1O~ t~ or ~o~h ~cr~ Udp~, oll~"dl a~rocu, pe~icldu, o~c 1~. S~b1M ~RY OF T~E INVENTIO~
1, Tbe r-porter ~e~t--, Th~ nrc~ lo n Jorlo~ of rtre-~ ~cnc-nporl~r co~Jtrutn~ currontly In uu con~l~t ot ~ oll ~one~ JacZ, couPlod to ~ oc~-ln~uclblo prP~Ier dt.tl~ed from ~c p1~ pno of ~ n~nuuo~ C~orh~bdt~f~ J~t. L~CZ onCC~C~ ~e enrym~, p-g~c~dtr~, W ~ ~ hbl~ ~n nen~t~o~c ccll~. cn~ rO~ whleh ~onJluYo h~lo1o~
p~clrophotomolrlc U~ ol~dy oX~
~ n odvon~p of tbo-o TO~W~rli 1~ ~h~ît ~ romolcTl ItO tl~htl~ u1elod, i.t.
lluio o~ ~o ~ooo ectlolly lo praenl unl~n Ihc ~1 1~ b~a ~l~ucd In ~or~ ~cy, ~ tl~o ncmolo~o pOJ~cu~ o p~ ocl~o ~tllvl~r or ll- o~n. lhi~ 0e~ h~l b~cl~o~ld nc~ r in ~ ~y~ rornoly low, ~ e ~ y 1~ 1~0tr ro~ v~ ~ .oSol~.tlo al.~-~ o~ ~peclflc ropo~ur ~co wh~ch h~le ~oo~
con~r~ n h P~,uro 1. NLS, o rucbor loc Ibotlon ~l~ul, br~ o ~
o~oc~ to ~ho nuoleu~ in~ iaOn~irio~l~on Or ~e cu~ c~ n~ uo ~poc ~ouib~o, H~ e he~l dlock or ~ISol- ~uolblo ropbtory olomon~. l~o vcclon b~ve ~n deocribod in ~Irin pl~m U~ ). 1~ oomphl4 ~oquonce- o~ ~he h~ oror ro tor~lbo~ ~ ~4uuu~ C~L (S) od JoneJ 9 C~bcr typc~ ot ~or~r~ ~ic~ ~Ol~ty lho ~bo~o etlletlu cr~ ~Irio eon~ Onl ~i~
~hl~ motho~olo~y. ~o~ In~t~noo, ~ec~er~l ot tlrot~y lucltoruo ~i~hl ~e u-otprovl~e c ~oo~lllvo SCI~y b-x~ at~ pholo~ r~ ny ~er~e prad~ t rot which l~e s~b~nc~o~ ~D di~o IUo ~e rre~ e soll~ ~ ~e pr~ccdure oo~crlh~d bolo~
~OUI~ bo CoNnp~ wlth ~1~ blome~lor cyJ~m~ Othu pmnuNel~. whieh re~pond . ~ .
.:
.'~ ` .
, J~N.29 '9:3 17:37 613-~2-E3440 P.07 0 8 ~3 3 7 9 ~ on~ c~e~ Or o~re~sors or contl~lon~, conld ~Is~ bo u-cd in con,iunc~lon ~vi~h ~
vu~o~l~ ropo~or pno~l c ~ mol~llo~ion~n o~ cy~ochroms P450 prcm3t~r~, or o~har ho~t bl~OCl~ ptomolor-, Det~lled delcrlption o~ streu~r-portor l~e~e ~onftroct~lon~ (Al~o soc ~4~
Thc voctor pPC~I. Illuo~aecd in Pi~utC I bO IhC hc~16~48/1 ts~u~lallonal fuolon co~n~lclo, w~ aor~No~ by inicrllnlt 3~00 bp Hindllr~ fra~g~cat ancom~sslDg ~o loc~ ~c~4 ~nucleolldo~ 18 lo 3~J~ of ~hC oJ~pre~lor~ vcce~r pPDI~.~3 ~8~ In~ O~e J~a I
~lu ~nuclooll~lo 356S of Iho j~ cqu~) o~ Ih~ hlp~ pnc. pPC~1 con~in co~ bt~ pl~U4~/J scno ~lr e~teodlo~ ~n n Bo~ t nuclooeldo 2280 ~ ~ 4a ~ ~It nucleotldo 418~ irl t~o pUb11~1lca iellueDce ~S). Pl~mi~ p~X16.~1 Npre~en~- Ihc eo~p~o~4 ~r~n~l~tionsl ru~lon bo~wco~ Z cnd tho Jl~p~.4tl2 eo~e p/llr, w~Job u~
con~ cted by 1n~orl;ng tacZ 05 ~ 3200 bp XD~ WI fr~l~m~a~ lnto ~ ~p~ 0~ htP-~-2 (n~ lto 169G, t6)). ~h~ ter wu oo~aine~ in 4~ co ~l t~uclooli~e 5~0~ to M~o I
(mlclouLi~ 2~701 ~rugrn~t ~ncomp~ ~g Ih~ ~spt4-~J2 IP p~is, Pl08~ni~ pPCI6.48 1. ~kl~b corrupondo ~ u~nJl~tion o~lon 1 Swlon o~ thc hJp~
~rJ yo~ lr lo ~acZ, wa~ cons~llctot by ln~u~ln~ ~ S~u 3~ ~ro~mone ~ endln~ from r~ucl~DllJo~ 9111 tD 1440 ~ O 010 JaJA Nl ~110 or ~ cm4lodo olcplffsi~n ~cwr ~P~16.~1 u&h Ihu Ih~ isp~6~ promo~cJ wu ptoxlrn~1 to l~cZ, p~C16.1~48 cont~ the ~a~ 3A
rto~tho In~e~e orl~to~lon ~uch ~n~ pJ~ t promo~et i~ clo~o~ ~o JaoZ . A
S~u 3A fill~70nt o~c~inu ~ clootlde~ 1l21 ~o ~S61 ur ~ho hspl ~ locu~ w~
cb~od In~o ~ ~onl H1 6it~ ot ~PD16.~ ~R~) ruch IhCt tho ~Jp 15~J1 promotor w~
a lacZ, ~,onor~tln~ pl3~Aljd pHS1~.2S ~illUtltCICt 0~ ~ho tnn~ loml1 c~.on I ~urlon of 1he pt6~411a ~e~o p~lr).
Plumlt pPC16,48~ o ~sc~ tlon~ lon COJ~ y oJ an ll~nl ~ rt~cnt ~n~ colidc~ 3a1~S lo 3~62) of ;ho h,rp~ ptomotcr clo~ed InLo Ibo Jl~ncr~ ~ito ot Ihe pPD16.51 p~ . pPcto~ s~ o uon~crlptloD~I h~ n consi~o~ oS ~ T~ ~mos cx~en~iAS rrom nucle~ 6g ~o ~409 in thc ~o~ SCne In~or~d ~ ~ Acc I ~ilc o~
~I'DI~.51, All ol t~ onc cunr~ction~ uo do~crlbcd In ~tringhm or ~ 4~.
2. ThC c~rl~lnr Inltl~lly, tr~n~cniC sttlin~ c~rr~lln~ o~trachromo~onul arr~ys o~ the l~sp.l 5-~cZ tranr~ono~ ~c~c~ibcd ~ Ibo proccdi- 1~ eoc~on 9~ oro con~ruc~cd ~n~ entc~l~
oharhc~rlzc~ wllh ro~pCC~ 10 ~hclr lntuc~io3 by hoa~ ~ocl~ ~4), In ~hese ~rain~.
thc ~lrod~eod U~ cl~ic DN~ w~ no~ in~o~to~C~ In~o ~he ho~ rlom~ talb.Or wC~ oosti~ c.~;~tllchrotnoiom-l a~oy~ ob ~Ot- n~t ~u~O~ tc h~ ne~
pnu~tlon wl~ 100 ~ nd~ y ~4). Whi1c Ihcw ~ralm~ prod~lcod l~r~p amo~ o~ ~
~oclWI~U in r~rpo~o IC hoo~ ~tre~r, no on~ymll~io ~o~ivi~y ~ dctoc~od ~ftcr lhc e~n~e1~ h~ cn c~po~c~ tO c~dmlun~ ~4). T~u-. wo co~lndc~ o tlm~ t~l ~e ~pt~ p~- ~oto ~ol mot~l induci~o Ileno- ~4).
rhe ~ enlc no~lo~o rtr~lns cunoD.~ly in usc wcrc produc~d by Inte~r~
bovo reporter ~ o l~o ptlO1116 ~ oxpoctodly. ~he~o ~ullnl prod~cc bo~o~ o In ro~po~N to ~ Yrt~ y or Aucnor~ Includin~ e~tn~lurn, ~nd he~
~uo~ (10,11), Th~lc ~tt~lne ~ o~o~lcol1y ~Ih~le. ~lld wct~ derl~od t o~ ~ho c~ nn~b~c ~tnli~U dc1c~l~ed ~ tlD~h~m el ~ 4), b~ m- Irr~dlulon uld ~otlo ~clec~loD ~'or llnlm~ w1~ich p~l~ed o~ Iho u~eno ~n IOOS ot t~Ir ony ~ cno~ blli~ v~rils~oou~ ~o~tUro C( tb~ nv4nlion~ h~
~llion ~o ~o s~os~cr ~o~ ol Intetort, ~-- p~dcul~.r ~Lre~nJ ~lo cur~ ot ~-ru whlC~ ro~ul~ in ~ c~ orn o~ moYcr~nl of ~ho3c ~ nn~
ai~Lins~ bo~ lrom a~e ~lld-typo orpr~i~r.~. s~ind P~71 ~nt PC:72 c~rr,y ~hc comp1eto ul~nl~ iom~ ~u3ton Jito~vn o~ ~h4 ~o~ ot Pl~.l. S~rllin Pt::73 c~trlos thc hc~ 4~1 ~r~ îtor~ on I tu~lon bo~n ~n ~ho m~d~l- o~ Pig. 1. The c~py J~N.29 '93 17:38 ~13-232-8440 P.0B
Candido e~ ~ 2 0 8 8 ~
~ ber of ~hcJc r~orler tt~Ju~l~nel i~ el~lm~lod ~t ~5~50 per haploid ~enom~
(Y~u~ 2~.
3. E~po~ure o~ thc l~ns~nic oro~nl~m~ to tcit n~t~rl~ (Rehr ~o Fi~ur- S ~o~ Rcrer~nc~ 1I tor ~etatl~d mo~ho0~.
1. U~o ~o~ too~ lub~l or mulli~ell dirht~.
rhou6~nal Or ~n4p~ie L2 or L3 ~t~8od l4rveo c~n bo culturoa ~nd oxpo~et to ~pl~r In mul~i~vcll ~luu4 culture dirho~ h ~ p ~d~um conlainl~
~uA~n ot ~, co~l u ~ rood ~ Jt~ 3). ~ b~bn. numcrou~ mplu oan ~c~tc0 ~imull~noo~ly, ~ tho conccntr~tlon o- llmo o~ o~po~un ~o lhc ~e~ subr~nc~v~od. Tblc ptooo~uro ~eo ~1lDW~ ~onlto,rlD~ or Ibo pnonl he~l~h ot ~o ~nl~l~
turln~ ~ courJo ot r prlm ~ ~ obrav~do~ o til~uctln~; miclo~Copo. .~t ~he onmplotbn of ~o oxporuro lo ~he Icr~ rub~t~ th~ alo ~oo IraNl~o~rod u) J, Ie-t luho, ~ollotod by eo3trl~u~LlOn. ~hot brlo~ly ~ h ~ lllo~ ~t~, po~mo~bill~ wl~h .cetaae ~ld e~yo~i ht p-$~ oJlduo ~etivlt~l by one Or Ihe ~wo n~lhodr dc-edh-d brlow ~PI~uro 3). Al~onn~ivOly. ~ ue culturc di~ho~ col~lolnln~
poruu~ romov~blo In~or~o whieh oro leotono lo~ t~ln~ wo~rl~ ollm~ ny roqulrem~ h~ lo~ tubo~ on~fi Ip~lo~, olublo uuy. ~IAelo~idu~ eloo~o- ~o coloorl4-- ~ub~l~r~o ONPG to ~clc~C rolu~loyollow i~To~luel, orlho~ to~hcnol, whle4 h~ or~ r~ne~ mA~ t ~20 rrn~ undor b~io contltlon~. Uri.~4 ~hlr oDprol~oh ~ho m~,~nitu~e or rh~ on~o o~n ho qu~ntjlhd ~po~l~opho~ lc 11~. ~ItorTI~ti;oly, ~ ion Or ~hc hllltoc~tlc~l ~ub~r~le ~cl ro~ull~
in Lho otn~tioll oi ~ l~o1~h blue prcclpit-~ in ~Itl~, lhu~ provldln~ qu~ o Worm~llo~ ~ Io tl~o t~ e ti~ltib~lion ot ~è tollpn~o (~ e 3).
. ~oii t-~Lir~.
The obovè ~rocc~ure ~ st o~ly i~e modlJ~ol ~lly~tly ~o tUI ~oil ~ sodimont samplc~. A ~ioncc ~h~rty cont~ini~ c ncm~ o~ lac- ~rdrl ~r E. cofi fb ooidod to 12 ~ ot ~oll or ~ed)mont ~vhloh lo con~a~t w~hin ~ ~oll o~ ~ t~R~Uo oul~uro di~h 0~ clnllll^llon ~d~l Ui~ co~pl~tion ot thc e~ ule, thllllCd wolor 1~ ~dti~d to ~oU ~ he l~nimcl~ tloot ~o IhC lOp h- IO~DV~L Tho ro~ri~vc~ ~nl~lo ero ~n~ d. no~l~d on ~0 ~6 ~ucto~t to rcmo~c rom~lnkt~ roll ~no bE c~orl~ ~l 2).
Y~ ~0~ i3~ ololid~ or to~ ;bed ~ovo.
co~1n~d ~ApO~ n~ t~ c~b~ ~sre)~
~ ch~bor ~ ~4eA ~rolopod Jn ~vhlc~ ~ enlm~l- can bo ~tolcd, &~a50d to o~t ~u~l~ct, ~ uoyed ~llhou~ ~ no~ tot pl~o~Ln~ w wnlrifu~ ion. Thl~
~to~lly um~iine~ ~bc ~ro40duro, ~nt ~y m~o i~ ~oulblo lo ule th~ i~lomonl~o- ~n uun o~ ono pol~lbio rd~etrdon of t~e loYt chl~ml~ct 1~ ~own ln R~uto ~. Thl~ dl~un lo ptovi~e~ u ~ r~hct ~uJto u~ tho prec~i~ionet o~
ordlo~ry ic~ n tko r~. ~DC'i ~o ~ot ~o bo ~o~tluod ~ ventbo In u~
Mol-tiel ~oifio~tior~l:
Tho bu~rol ~O ~houl~ ~o Iran~p~ronl ~olcrr p~ c or ~JctJ)~ ll~i Ito~lly ~rr~ c~d In mlIIIl;~o~i ~ rll~-r o~ ~ 04d o~ ~o barrt~ h~uld ~ ~in nemo~Ddo~ fro~ e-rl~ Iorv~l ~lC~O~ onv1or~ bU~ ~IIow Fesc~o o~ ~Iq~ldr ond ~
p~rUclc~ ch ~r b~ctorl~ tO m~cron muh i~ ~u~ blo~ nu~ri~l~ chould bc ro~l~t-nc ~o oceto~o u~ r rdvon~.
Punctionlnc of lhc te~ ch~mb~r~
J~N.29 '93 1~:39 613-~32-R440 P.03 S C~ ct~l. 2~379 ~ rhe ,~c~4~bly ~ o oyrl~gP - ~h- pl~tor~ o~n be drc~n upw~rds or d~wn~ut-, lo ~, In ot C~ l llquld~, ~ospoc~iv61y. To ~ up ~o ~stom, ncn~todc-3te edded ~ tbo b~rol llnd ~o pl~ton 1~ ~d pu~h~d do~ to ~h~ lowl of ~ho gtOp ~4). Tho ~ni~nolJ CUI bo hrt~ in ~hb ~ocombl~ in ~A~'ioU5 ~orm~ bcto~
umll u~od. ThC ch~nbc-~ ~o lo-~ uprl~ lull~bb ~lt t~J l~ Uch ~
~mctGtc~ uub cn tho nulh ~n~ ~hc~ob~l u~ In clo~ con~c1 Wilh oxy~en in ~o ~Ir, ublch 1~ cuc~ o m~nlcln Iholr vl~
T~ oxpooo 1~ n~6~dc~ liquld b~l t~mplc. IbO clllturo Ill~Lid iY c~sollod, ud IhO ~a~pio l~ dr~nvn in, pC-b~p~ rn;~ ol~ t~vlco wllh th~ ~plc. Th~
dc~lrcd ~ol~lm~ Or Ihc b~ o~nplo 4 ~ n kl, n t tllc Acm81Ddo~ bu~
lh4 d~lro~ ~U14~ D- tlmo,.
Rr~t~out of IhC lo~ol ol we~ ducod onzy~o ~CIiVity ~ c~ried ollt in tho ~om~
cpplr3t~s, b~ ox~ t~llt ulDlple CDd t-owi~ 1~ ~h~ ~ro~Jl-tc ~S~Jy solul~ Ln ~ ~r ~ co~orl~olrl6 ~ y, colDr In~n~l~y C~n b~ u~ tcd ~hroullh ~o tr~n-p~rorlt b~rroJ. or tho ~uy iolutlon crll bo oxpollod rO. mo~ure0~nl i~
~peclro~ho~o~e~c-, Mclbod o~ O~er~tt~n~
T~o t~U c41~nbe~ .4~ cont~ir~ ~ pro-toto~ n~mber D~ r~em~lodc3 al 9 ~pc~iflc I-p or dovolopmen~ ~.~B. Ll, L~l, L3, L~" douorlc~ or ~d~t) in ~, nu~ri~nl mo~lum ~S~or-go ~uiiu~, SM).
~ ?lc~, C~D,ds~in~ ot w~r ~mp~u o~ ucou~ extrsct~ o~ w11~. clc. gro i2~ lxo~ lh o c~no~r~lod 5~ oluliD~I ot Sl~ lC ~ d~r~
fiu~l ~onccnk~llOn ~ ~hc ~cdlum.
2. Tho u~mplo i~ ~ jr~ ~ao chcmb~r, u~ the ch~mbor is incub~le~ ~or tbo te~iro4 2imo el ~ ad~rd to~poroluro, u~u~lly be~ n lS ~h~ 2aoc.
3. I'ho ~unplo 1~ ollod, th- ~hmbor 1~ rln~od wilh ~ lli~pb Jcl~ modll~m w tor. ~nd tho rlncc co~ n i~ ox~e~lcd, ~'ho nom~todc~ ~-¢ tc~inod ln Iho ch~mbo~ by tb~ potoo~ filttr or mcrh.
. 9~ 0ilo~, ~i,, ''Tho nen~to~io tOJUC]~ J~o~ ucln~ ~anoJr~ r~dl~ w", ~tlXiC. ~-Ct-. 2l.3~- 309~ . 1990.
~ . ~'~ Ko~Jel~ W.~ rocctoc Z~ibor~ R.W, ~r,a ~olnon~ W. "~ S13 o~vlrotu~cnt~l pDllu~n~ on ~ol1 o~pni~nu; ~ dmpl~ c~ o in~ o tollloity o~ cnvlronn~t~ul pollul~n~ oll org~nl~m~, U~De o~dA~lum ohloti~e ~d non~ co~ itot. an~i~n. S-~. ~2), 1~1-190, 1989.
C~r ~ n 15 yc~n ot ~v, It h~ bo-n U b~iJ~d ~t 411 or~ u ro~n4 to hcU ~treU un~ t~ ~ v~rlo~y or chon~c~1 UCJ~C~ b~ 5s~o~cln~ rp~clnD pro~in~ ~blch ~ro n~d- dthor ~ n~h lo~cr lc~ol- or ~ot t c11 uod-t norm~t cn~tltlonr. Thl3 ro~l~clioo bnr lo~ t~ propo~cl~ ~ ~onllorln~ ~hn onvllonmcn~ by ~c~-url~ th lov~1~ o~ Ihe~c prOIcih~ In v~rio~r orpolcm~ (1,2.3). ThC~C ~ppro~c~or irlvobo c~ on oS ~ pto~elnc ~nt ~cc~"-en~nl o~ rhelr bvolr, l~c~clly u~ln~ ~nLibodbr.
'rb~ in~r~Pdoo to~crlt~t ~ordn lltlJIcu ~ ~iiftcton~ ~pprouh, I,e. ~o hvo Itnl~od tno conuol ~o~ion~ whlch re~ul~ pr~t*~n pto~ lon lo ~ hloh produc~c c rodily mcc~ur~lc . c~.yn-c ~C '~tcpo~ no ~ repor~cr ~ene h~
~o~ ul ~i ~to ~he chromo o~o~ or C. ~ a~ o pro~uGo ~-anr~onic c~lr~
~hlch ror~oDt to clns~ ropotl~r ~ nc. TO cnmploto the biomoll~tor tyctor~, mothodo heYo boon de~l~no~ for ~cpitily ~ oonvenlently ~euin~ ~hn nu~ ~ ot the op~r~r ~vytlu ~cu-~lt~, u ~oll ~1~ it~ tilluc 10catlon k~
nomc~od~ c to~om~inm~ lUcinc. ~c~hCt wi~ ~ho o~s~y mc~ho~olo~y, con eon~itut- ~ ~lo~onllori~ 1t" wnlch c~ bo uud ~u dctcol Ihe IcYel ot c~re~r l~o~c4 on ~t otpn4m b~ e~o~-C lo ~ator ~mp1O~ t~ or ~o~h ~cr~ Udp~, oll~"dl a~rocu, pe~icldu, o~c 1~. S~b1M ~RY OF T~E INVENTIO~
1, Tbe r-porter ~e~t--, Th~ nrc~ lo n Jorlo~ of rtre-~ ~cnc-nporl~r co~Jtrutn~ currontly In uu con~l~t ot ~ oll ~one~ JacZ, couPlod to ~ oc~-ln~uclblo prP~Ier dt.tl~ed from ~c p1~ pno of ~ n~nuuo~ C~orh~bdt~f~ J~t. L~CZ onCC~C~ ~e enrym~, p-g~c~dtr~, W ~ ~ hbl~ ~n nen~t~o~c ccll~. cn~ rO~ whleh ~onJluYo h~lo1o~
p~clrophotomolrlc U~ ol~dy oX~
~ n odvon~p of tbo-o TO~W~rli 1~ ~h~ît ~ romolcTl ItO tl~htl~ u1elod, i.t.
lluio o~ ~o ~ooo ectlolly lo praenl unl~n Ihc ~1 1~ b~a ~l~ucd In ~or~ ~cy, ~ tl~o ncmolo~o pOJ~cu~ o p~ ocl~o ~tllvl~r or ll- o~n. lhi~ 0e~ h~l b~cl~o~ld nc~ r in ~ ~y~ rornoly low, ~ e ~ y 1~ 1~0tr ro~ v~ ~ .oSol~.tlo al.~-~ o~ ~peclflc ropo~ur ~co wh~ch h~le ~oo~
con~r~ n h P~,uro 1. NLS, o rucbor loc Ibotlon ~l~ul, br~ o ~
o~oc~ to ~ho nuoleu~ in~ iaOn~irio~l~on Or ~e cu~ c~ n~ uo ~poc ~ouib~o, H~ e he~l dlock or ~ISol- ~uolblo ropbtory olomon~. l~o vcclon b~ve ~n deocribod in ~Irin pl~m U~ ). 1~ oomphl4 ~oquonce- o~ ~he h~ oror ro tor~lbo~ ~ ~4uuu~ C~L (S) od JoneJ 9 C~bcr typc~ ot ~or~r~ ~ic~ ~Ol~ty lho ~bo~o etlletlu cr~ ~Irio eon~ Onl ~i~
~hl~ motho~olo~y. ~o~ In~t~noo, ~ec~er~l ot tlrot~y lucltoruo ~i~hl ~e u-otprovl~e c ~oo~lllvo SCI~y b-x~ at~ pholo~ r~ ny ~er~e prad~ t rot which l~e s~b~nc~o~ ~D di~o IUo ~e rre~ e soll~ ~ ~e pr~ccdure oo~crlh~d bolo~
~OUI~ bo CoNnp~ wlth ~1~ blome~lor cyJ~m~ Othu pmnuNel~. whieh re~pond . ~ .
.:
.'~ ` .
, J~N.29 '9:3 17:37 613-~2-E3440 P.07 0 8 ~3 3 7 9 ~ on~ c~e~ Or o~re~sors or contl~lon~, conld ~Is~ bo u-cd in con,iunc~lon ~vi~h ~
vu~o~l~ ropo~or pno~l c ~ mol~llo~ion~n o~ cy~ochroms P450 prcm3t~r~, or o~har ho~t bl~OCl~ ptomolor-, Det~lled delcrlption o~ streu~r-portor l~e~e ~onftroct~lon~ (Al~o soc ~4~
Thc voctor pPC~I. Illuo~aecd in Pi~utC I bO IhC hc~16~48/1 ts~u~lallonal fuolon co~n~lclo, w~ aor~No~ by inicrllnlt 3~00 bp Hindllr~ fra~g~cat ancom~sslDg ~o loc~ ~c~4 ~nucleolldo~ 18 lo 3~J~ of ~hC oJ~pre~lor~ vcce~r pPDI~.~3 ~8~ In~ O~e J~a I
~lu ~nuclooll~lo 356S of Iho j~ cqu~) o~ Ih~ hlp~ pnc. pPC~1 con~in co~ bt~ pl~U4~/J scno ~lr e~teodlo~ ~n n Bo~ t nuclooeldo 2280 ~ ~ 4a ~ ~It nucleotldo 418~ irl t~o pUb11~1lca iellueDce ~S). Pl~mi~ p~X16.~1 Npre~en~- Ihc eo~p~o~4 ~r~n~l~tionsl ru~lon bo~wco~ Z cnd tho Jl~p~.4tl2 eo~e p/llr, w~Job u~
con~ cted by 1n~orl;ng tacZ 05 ~ 3200 bp XD~ WI fr~l~m~a~ lnto ~ ~p~ 0~ htP-~-2 (n~ lto 169G, t6)). ~h~ ter wu oo~aine~ in 4~ co ~l t~uclooli~e 5~0~ to M~o I
(mlclouLi~ 2~701 ~rugrn~t ~ncomp~ ~g Ih~ ~spt4-~J2 IP p~is, Pl08~ni~ pPCI6.48 1. ~kl~b corrupondo ~ u~nJl~tion o~lon 1 Swlon o~ thc hJp~
~rJ yo~ lr lo ~acZ, wa~ cons~llctot by ln~u~ln~ ~ S~u 3~ ~ro~mone ~ endln~ from r~ucl~DllJo~ 9111 tD 1440 ~ O 010 JaJA Nl ~110 or ~ cm4lodo olcplffsi~n ~cwr ~P~16.~1 u&h Ihu Ih~ isp~6~ promo~cJ wu ptoxlrn~1 to l~cZ, p~C16.1~48 cont~ the ~a~ 3A
rto~tho In~e~e orl~to~lon ~uch ~n~ pJ~ t promo~et i~ clo~o~ ~o JaoZ . A
S~u 3A fill~70nt o~c~inu ~ clootlde~ 1l21 ~o ~S61 ur ~ho hspl ~ locu~ w~
cb~od In~o ~ ~onl H1 6it~ ot ~PD16.~ ~R~) ruch IhCt tho ~Jp 15~J1 promotor w~
a lacZ, ~,onor~tln~ pl3~Aljd pHS1~.2S ~illUtltCICt 0~ ~ho tnn~ loml1 c~.on I ~urlon of 1he pt6~411a ~e~o p~lr).
Plumlt pPC16,48~ o ~sc~ tlon~ lon COJ~ y oJ an ll~nl ~ rt~cnt ~n~ colidc~ 3a1~S lo 3~62) of ;ho h,rp~ ptomotcr clo~ed InLo Ibo Jl~ncr~ ~ito ot Ihe pPD16.51 p~ . pPcto~ s~ o uon~crlptloD~I h~ n consi~o~ oS ~ T~ ~mos cx~en~iAS rrom nucle~ 6g ~o ~409 in thc ~o~ SCne In~or~d ~ ~ Acc I ~ilc o~
~I'DI~.51, All ol t~ onc cunr~ction~ uo do~crlbcd In ~tringhm or ~ 4~.
2. ThC c~rl~lnr Inltl~lly, tr~n~cniC sttlin~ c~rr~lln~ o~trachromo~onul arr~ys o~ the l~sp.l 5-~cZ tranr~ono~ ~c~c~ibcd ~ Ibo proccdi- 1~ eoc~on 9~ oro con~ruc~cd ~n~ entc~l~
oharhc~rlzc~ wllh ro~pCC~ 10 ~hclr lntuc~io3 by hoa~ ~ocl~ ~4), In ~hese ~rain~.
thc ~lrod~eod U~ cl~ic DN~ w~ no~ in~o~to~C~ In~o ~he ho~ rlom~ talb.Or wC~ oosti~ c.~;~tllchrotnoiom-l a~oy~ ob ~Ot- n~t ~u~O~ tc h~ ne~
pnu~tlon wl~ 100 ~ nd~ y ~4). Whi1c Ihcw ~ralm~ prod~lcod l~r~p amo~ o~ ~
~oclWI~U in r~rpo~o IC hoo~ ~tre~r, no on~ymll~io ~o~ivi~y ~ dctoc~od ~ftcr lhc e~n~e1~ h~ cn c~po~c~ tO c~dmlun~ ~4). T~u-. wo co~lndc~ o tlm~ t~l ~e ~pt~ p~- ~oto ~ol mot~l induci~o Ileno- ~4).
rhe ~ enlc no~lo~o rtr~lns cunoD.~ly in usc wcrc produc~d by Inte~r~
bovo reporter ~ o l~o ptlO1116 ~ oxpoctodly. ~he~o ~ullnl prod~cc bo~o~ o In ro~po~N to ~ Yrt~ y or Aucnor~ Includin~ e~tn~lurn, ~nd he~
~uo~ (10,11), Th~lc ~tt~lne ~ o~o~lcol1y ~Ih~le. ~lld wct~ derl~od t o~ ~ho c~ nn~b~c ~tnli~U dc1c~l~ed ~ tlD~h~m el ~ 4), b~ m- Irr~dlulon uld ~otlo ~clec~loD ~'or llnlm~ w1~ich p~l~ed o~ Iho u~eno ~n IOOS ot t~Ir ony ~ cno~ blli~ v~rils~oou~ ~o~tUro C( tb~ nv4nlion~ h~
~llion ~o ~o s~os~cr ~o~ ol Intetort, ~-- p~dcul~.r ~Lre~nJ ~lo cur~ ot ~-ru whlC~ ro~ul~ in ~ c~ orn o~ moYcr~nl of ~ho3c ~ nn~
ai~Lins~ bo~ lrom a~e ~lld-typo orpr~i~r.~. s~ind P~71 ~nt PC:72 c~rr,y ~hc comp1eto ul~nl~ iom~ ~u3ton Jito~vn o~ ~h4 ~o~ ot Pl~.l. S~rllin Pt::73 c~trlos thc hc~ 4~1 ~r~ îtor~ on I tu~lon bo~n ~n ~ho m~d~l- o~ Pig. 1. The c~py J~N.29 '93 17:38 ~13-232-8440 P.0B
Candido e~ ~ 2 0 8 8 ~
~ ber of ~hcJc r~orler tt~Ju~l~nel i~ el~lm~lod ~t ~5~50 per haploid ~enom~
(Y~u~ 2~.
3. E~po~ure o~ thc l~ns~nic oro~nl~m~ to tcit n~t~rl~ (Rehr ~o Fi~ur- S ~o~ Rcrer~nc~ 1I tor ~etatl~d mo~ho0~.
1. U~o ~o~ too~ lub~l or mulli~ell dirht~.
rhou6~nal Or ~n4p~ie L2 or L3 ~t~8od l4rveo c~n bo culturoa ~nd oxpo~et to ~pl~r In mul~i~vcll ~luu4 culture dirho~ h ~ p ~d~um conlainl~
~uA~n ot ~, co~l u ~ rood ~ Jt~ 3). ~ b~bn. numcrou~ mplu oan ~c~tc0 ~imull~noo~ly, ~ tho conccntr~tlon o- llmo o~ o~po~un ~o lhc ~e~ subr~nc~v~od. Tblc ptooo~uro ~eo ~1lDW~ ~onlto,rlD~ or Ibo pnonl he~l~h ot ~o ~nl~l~
turln~ ~ courJo ot r prlm ~ ~ obrav~do~ o til~uctln~; miclo~Copo. .~t ~he onmplotbn of ~o oxporuro lo ~he Icr~ rub~t~ th~ alo ~oo IraNl~o~rod u) J, Ie-t luho, ~ollotod by eo3trl~u~LlOn. ~hot brlo~ly ~ h ~ lllo~ ~t~, po~mo~bill~ wl~h .cetaae ~ld e~yo~i ht p-$~ oJlduo ~etivlt~l by one Or Ihe ~wo n~lhodr dc-edh-d brlow ~PI~uro 3). Al~onn~ivOly. ~ ue culturc di~ho~ col~lolnln~
poruu~ romov~blo In~or~o whieh oro leotono lo~ t~ln~ wo~rl~ ollm~ ny roqulrem~ h~ lo~ tubo~ on~fi Ip~lo~, olublo uuy. ~IAelo~idu~ eloo~o- ~o coloorl4-- ~ub~l~r~o ONPG to ~clc~C rolu~loyollow i~To~luel, orlho~ to~hcnol, whle4 h~ or~ r~ne~ mA~ t ~20 rrn~ undor b~io contltlon~. Uri.~4 ~hlr oDprol~oh ~ho m~,~nitu~e or rh~ on~o o~n ho qu~ntjlhd ~po~l~opho~ lc 11~. ~ItorTI~ti;oly, ~ ion Or ~hc hllltoc~tlc~l ~ub~r~le ~cl ro~ull~
in Lho otn~tioll oi ~ l~o1~h blue prcclpit-~ in ~Itl~, lhu~ provldln~ qu~ o Worm~llo~ ~ Io tl~o t~ e ti~ltib~lion ot ~è tollpn~o (~ e 3).
. ~oii t-~Lir~.
The obovè ~rocc~ure ~ st o~ly i~e modlJ~ol ~lly~tly ~o tUI ~oil ~ sodimont samplc~. A ~ioncc ~h~rty cont~ini~ c ncm~ o~ lac- ~rdrl ~r E. cofi fb ooidod to 12 ~ ot ~oll or ~ed)mont ~vhloh lo con~a~t w~hin ~ ~oll o~ ~ t~R~Uo oul~uro di~h 0~ clnllll^llon ~d~l Ui~ co~pl~tion ot thc e~ ule, thllllCd wolor 1~ ~dti~d to ~oU ~ he l~nimcl~ tloot ~o IhC lOp h- IO~DV~L Tho ro~ri~vc~ ~nl~lo ero ~n~ d. no~l~d on ~0 ~6 ~ucto~t to rcmo~c rom~lnkt~ roll ~no bE c~orl~ ~l 2).
Y~ ~0~ i3~ ololid~ or to~ ;bed ~ovo.
co~1n~d ~ApO~ n~ t~ c~b~ ~sre)~
~ ch~bor ~ ~4eA ~rolopod Jn ~vhlc~ ~ enlm~l- can bo ~tolcd, &~a50d to o~t ~u~l~ct, ~ uoyed ~llhou~ ~ no~ tot pl~o~Ln~ w wnlrifu~ ion. Thl~
~to~lly um~iine~ ~bc ~ro40duro, ~nt ~y m~o i~ ~oulblo lo ule th~ i~lomonl~o- ~n uun o~ ono pol~lbio rd~etrdon of t~e loYt chl~ml~ct 1~ ~own ln R~uto ~. Thl~ dl~un lo ptovi~e~ u ~ r~hct ~uJto u~ tho prec~i~ionet o~
ordlo~ry ic~ n tko r~. ~DC'i ~o ~ot ~o bo ~o~tluod ~ ventbo In u~
Mol-tiel ~oifio~tior~l:
Tho bu~rol ~O ~houl~ ~o Iran~p~ronl ~olcrr p~ c or ~JctJ)~ ll~i Ito~lly ~rr~ c~d In mlIIIl;~o~i ~ rll~-r o~ ~ 04d o~ ~o barrt~ h~uld ~ ~in nemo~Ddo~ fro~ e-rl~ Iorv~l ~lC~O~ onv1or~ bU~ ~IIow Fesc~o o~ ~Iq~ldr ond ~
p~rUclc~ ch ~r b~ctorl~ tO m~cron muh i~ ~u~ blo~ nu~ri~l~ chould bc ro~l~t-nc ~o oceto~o u~ r rdvon~.
Punctionlnc of lhc te~ ch~mb~r~
J~N.29 '93 1~:39 613-~32-R440 P.03 S C~ ct~l. 2~379 ~ rhe ,~c~4~bly ~ o oyrl~gP - ~h- pl~tor~ o~n be drc~n upw~rds or d~wn~ut-, lo ~, In ot C~ l llquld~, ~ospoc~iv61y. To ~ up ~o ~stom, ncn~todc-3te edded ~ tbo b~rol llnd ~o pl~ton 1~ ~d pu~h~d do~ to ~h~ lowl of ~ho gtOp ~4). Tho ~ni~nolJ CUI bo hrt~ in ~hb ~ocombl~ in ~A~'ioU5 ~orm~ bcto~
umll u~od. ThC ch~nbc-~ ~o lo-~ uprl~ lull~bb ~lt t~J l~ Uch ~
~mctGtc~ uub cn tho nulh ~n~ ~hc~ob~l u~ In clo~ con~c1 Wilh oxy~en in ~o ~Ir, ublch 1~ cuc~ o m~nlcln Iholr vl~
T~ oxpooo 1~ n~6~dc~ liquld b~l t~mplc. IbO clllturo Ill~Lid iY c~sollod, ud IhO ~a~pio l~ dr~nvn in, pC-b~p~ rn;~ ol~ t~vlco wllh th~ ~plc. Th~
dc~lrcd ~ol~lm~ Or Ihc b~ o~nplo 4 ~ n kl, n t tllc Acm81Ddo~ bu~
lh4 d~lro~ ~U14~ D- tlmo,.
Rr~t~out of IhC lo~ol ol we~ ducod onzy~o ~CIiVity ~ c~ried ollt in tho ~om~
cpplr3t~s, b~ ox~ t~llt ulDlple CDd t-owi~ 1~ ~h~ ~ro~Jl-tc ~S~Jy solul~ Ln ~ ~r ~ co~orl~olrl6 ~ y, colDr In~n~l~y C~n b~ u~ tcd ~hroullh ~o tr~n-p~rorlt b~rroJ. or tho ~uy iolutlon crll bo oxpollod rO. mo~ure0~nl i~
~peclro~ho~o~e~c-, Mclbod o~ O~er~tt~n~
T~o t~U c41~nbe~ .4~ cont~ir~ ~ pro-toto~ n~mber D~ r~em~lodc3 al 9 ~pc~iflc I-p or dovolopmen~ ~.~B. Ll, L~l, L3, L~" douorlc~ or ~d~t) in ~, nu~ri~nl mo~lum ~S~or-go ~uiiu~, SM).
~ ?lc~, C~D,ds~in~ ot w~r ~mp~u o~ ucou~ extrsct~ o~ w11~. clc. gro i2~ lxo~ lh o c~no~r~lod 5~ oluliD~I ot Sl~ lC ~ d~r~
fiu~l ~onccnk~llOn ~ ~hc ~cdlum.
2. Tho u~mplo i~ ~ jr~ ~ao chcmb~r, u~ the ch~mbor is incub~le~ ~or tbo te~iro4 2imo el ~ ad~rd to~poroluro, u~u~lly be~ n lS ~h~ 2aoc.
3. I'ho ~unplo 1~ ollod, th- ~hmbor 1~ rln~od wilh ~ lli~pb Jcl~ modll~m w tor. ~nd tho rlncc co~ n i~ ox~e~lcd, ~'ho nom~todc~ ~-¢ tc~inod ln Iho ch~mbo~ by tb~ potoo~ filttr or mcrh.
4. The non~-~o~c~ ~so moto perme~blo ~o thc ~JI~y com~o~onts. A tul~blo rc~unoDl Ir ~O dro~ ~n ~cetono ~d Ir.cubu~o ~t llrnb~cl~l lcmpet~turo tor 1~ fc~v 5Ut~.
~ . T110 cotono 1~ pol~od and tho C~,SUy ro1utloD i~ d~ inlo Ih~ c~mbor.
b. 1~ ulr~y ro~ulu ~c ro~ ~n~r ~ ~uJ~blo In~ lon llmo, t~nein~ trom -~ow mlnule~ to overrdgh~ C ~ o~ture t~nJL~ J~am unblorl ~D 37~. Two lypC-oi~ unlly hovo beon ~mployGd: u 4uulit~liv- hi-~oob~nlic~ s~y ~4hich Indicoto~
~hi~h ~le~uo~ t~vo unter~onc ~hc ~Irou r~pon-o, und o qu~nlil~ c 301ublo ~r~y ~h~h provWu ~ colo~r chlmp 70~10atln~ ~hc lCvCl Or ~ItOU~ uC~d eo2ymc in Ih~
whol- ull~l.
111. DlST~ Q D~SCItlrT101~1 OF SPU:I~IC EM~ODIME~TS OF THE
~IOMONI'rORINC ~YSTISi~
Provlou~ blcm~nllo-in~ ~y~tom~ ~o~/o ton~i~lod i~ n~o~i~oring so~o phy~lolo~lc~l ~orc~o~ot o~ vtld-~ypo o~s~nIun cuch u e microot~ni-m, en Invorlebr~l4 (m~ ol~, cl-m-, Depbnle) or ~ vortobr~to (~ y ~ h ~Ip~c~iO-~durin~ ot ~ftcr o~ ur~ to ~omo ~ cotdillon. The ent-~olnt ot tihe ~r~y i~ ohc~
loth11ly, .~. th~ L~S0 o~ ~ tut Nb~ noo I~ dolenn5n-d ~13,14). O~ CJ~ b~v~
olvo~ tom nl- o~ bth~ ueh ~ nwv-mont o~ 11 cw~,~ rrom lc~t ~mplo~
~15), or tr~~ y of pura~l~ In b~,n~ ucl~ ~- ml~nol~ ); lu Ih~-e cc~, c~mp1~ nd oxpon~livo cloclro~Ie oquIpmon~ ~orm~ p~rt ot ~hc ~y~
~Itor~tlv-ly, ~Dd maro rocon~ly, ~c 1~v~ of ~pocitIc biomoiGculoJ, ucull11y pro~ol~, io ti~o ~o~ pcclo~ h~vo boe~ mul-ur~d, ~ t ~bc curten~ int~c~t In mcP~ o~ o~ pro~oi~ (17), Thc Icll CU~yJ, whilc uslD~ ro~ iwl~ y1c C~ip~oJlt~ novor~h-lo-~ ar~ vory ~,ime~col~amin~. Involve m~ny ~nlpul~lloul, md ~tq~lre ox~onclYo u~i~ in~ ~d oxporl~nce. They uo nol sulted co ~9C in IhO
~lt.
.
- .
. ~ . -:
J~N.29 '93 17:40 613-232-8440 P.10 C~didoetaL 2088379 ~ ho pr~on~ ~p ~o~oh u~ zoJ ~ecl~lly desi,~nod ~r~n~l~cni~ or~ ms vrb~ch re-pon~ lo ur~s~or~ gy protucln~ ~n c~ qu~tlnct en~ymo ~vhlc~ i~ nol norm~lly ~rDdu~od by ~hor4 or~ni~m~ r~hormoro, ~y ~Itili~in~ IhO Im~ll, oaslly n~ni~ul~ed n~ t~d~, C ,rle~ , r~out ~f ~ tos~tr con bo c~rrie~ o~t ra~ldly ond limply in ~ho In~Ac~ org~n~, wltbout ~ho ~occ~ o~ di~-uptl~ o~ Juu or oxuac~lo~ ~ tho nry~u ~ bo lncul~ot r~ ~ray U D be pcrformcd e~rll~ la th~
l~buLl~ry or In ~o Held, by ~or~o~nol with ~1~ nlnin~
~ dvonto~t~ of Ibe ~stom:
~ . Q~an~tlv~ or q~udlbliv~ ~B~U~9 Of ~res~ in ~ul~lcollul~r a. Ie ro~onJivO ~o boO~ nlc ~Ad Inor~cDlc stns~ors G Cell bo uod on ~11 Iy~U ot ~mples: rr~h or ~Cll wa~t ~mplc~, mill omues~ C-C~IOr. slud~o~, soll OIU~IC~, wll~ OJ ~otimon~ poclrlc chomlc~lls or ~I)IXIut~l ot oh4n~io-1~
1~, Moy ~oc corrlcd out In t~ bor~tol~r or i~ ~ fiel~, ~ . ~o~dout requitec ~Inlmcl qulpmoM snt cons~ of ~n o~olly ob~on~blc.
s~blo colour ch~n~e.
F, ~ollow~n~ oxpo~u~o ot tho otpniunr to Iho tost ~mplc, ~c~ulla of tho ~tro~
~J~oy ~se e~oiI~blc wilhin ~Jr~uIo~ îo hou--. dopondln~ on ~hc m~nilude o~ lho im~o~ot Jt-O-J-O. ~- ~o~ c~ bo cu lod o~t ~ Ie Yol~ncJ~ l,e, lo~ ~n 2 mL.
H. Tho ~ort or,pnl-m ~r cho~p ~o ~row l~nd r~im~in, 1. ~ylllom c~ bo YUt fo~ ~no~iloria~ ~olb cluol~lc ~n~ ~cuto ~oxici~y.
~ . Sy~ ~oro ~ondtivo ~b4n currcn~ Iclb~llty îats usln~ ~ild~ c no~ oto~.
K. ~y~l~ provlee~ Inform~tio~ on ti~c ~eclticit~ of Ihe s~ros~ ro3pon3e.
~ t~racc ot th~ n~metoto~:
Thtoo mcl~odr o~ storop of Ihc r~oo~todo- dlr~olly 1~ ~hc les~ cbomb~r ~ro po~d~lo~ ub ~y ~o Itop~ In ~ re~r~t~r ~ncllr 4'C) ht Je~4r~1 d~y~
bchr~ u~, u th~ ~t~pt~d~tO Clutly ~IOWJ ~lr do~olopnx~l. Ii~ C. ~ J i~
st~ tto ~tucrb~v~, whiCh tons~ whcn cu1mt~- ~re Jt rvod, Tb tNor~
tocs no~ ~e~, cnd wn ~ur~lY for uP ~o Ibreo mon~h~ o~t tOtD~ tUto~ t~ea roru~o 1~9 d4vcl~pm4n~ wllcn ~ cnlc~[ wi~h rodl ThU~ ~hc biomonioor otr~ coldd bo olorod ~ tibo ~oet c~mbaJ u d~ul~Yeo for ~p ~o tiuo~ monlh~ un~ll urot; Iho onlm~t~ colld b~ ~cd ~or 8 ~ o ~o con~,crl Ihcm ~o ~on~l L4 luv-o prior to uu b ~UI~ ) C. ~ nr Ll ~ ~ lorv~o 0UI b no~:Cn ~ In~c~inllol~ ~I dry loo oo 11quJd nJl~o~on lomporol~o~. Tboy ooult Ihorohro bo ~torod tro~on ~n~
~hlppod ~Ircolly i~ IhC lo~ eh~oer~: 11 woul~ ~n only ~o nocou-ry t~ cb~n medll~m ~ tood tho nomocoder ~or o to~ ~o~n p~loJ 10 th~lT u~ in ~cr~
Ir. 5XJ~MtL11 6:
'rhe Imr~tlon e~ hstllCt ~0 u~ldou~ood by rero~e~c~ l~ l.hs ~ollowin~
oY~plc~, Thc e~-~mplo- ~-c~¢n~ be1ow crc provj~cd r- ~ futlhor ~uid- ~o ~h~
pr~to~ o~ o~tin~sy ~kl rt, cnd o~ not ~ ~o conJ~r~d o~ ho Iru~0~DD ~ ~ny w-y. ~bo ox~mplo~ u~ tlvl~ qu~lit~vo ~ y; ~) ~C
qu~tl~dv~ U~Cy; o~ Wo ot Ple ~olrcou~ chuob~: t) mhor u~y-.
~ ~u~ll~dvo L~ y-. T~ X~ tc~ny ptocod~e 1~ d4-c~b-t ~n Pirc e~
1. ~0. ~omn,to~cl ~etC ~DO~ 10 tho r~tCuin~ ohcmio~l in ~ ubc~ ~r In multl~oll ~i~poJ~bl~ pl~tb di~)lo~ rlnod wllh Iho bi~lolo~ n, X-pl, ollo~
p~r~ tl~ wllh Jcolono, ~o ~rd reroronce ~ues~ wAditloll. e ho~t thocl~
~ 33~, wC~ cu~bd o~ wi~b ~h~ ~imeJ~ OD ~ Y~ki pl~U conl-lnln~ nUt~io~
c~r. ~ho dnuo ~L~ioln$ p~l~orn ~ Corvo¢i l~nt~ ~ Iow powo~ bl~oc~
mlo~o~co~o. .~ ot ~ ~ho~ ~ ~imC ~ 15 ~ 1 33~C o~Jo~ cl~olo~Jd~o ' :
.
, , J~N.29 '93 17:41 613-232-~440 P.ll 7 Cl~ndido et ~- 2 0 ~ 8 3 7 9 ~ e n~Qd~ tn mo~ uo~ of ~c ~lmsh t4). In ~ddi~io~ r6rioU~ chomlc~ rcJIior~.
lncludin~ ho~y n~ ]o ~uch ~ ~r~cnllo, ~dmlum, cop~or, lo~d. noroury ~n~ sln~.
s1~d Ibo hcrblcldc. purAqu~t. ~oro toun~ ~o oc~lvu~ ~ho roport~ ~pn~ .ner cxposu~
of JO~t~l ~ou~ td ~ dJIyo ~ll. Pi~ur~ S). Iho~ ont~ yhlt dll~Ycrcnl tl~uo plltlO~
or t~ro~ Jn~uc~loD.: ~orouYy ~m~ p~,~q~ Induoo only ~n~ntln~ rer~lon, lo~t clr~tr ph~rn~o~l mwcle, up~cl~lly ~ IhO ba~o o~ thl~ tertnlD~I ~ulb. coppu h~ucc~ in nowonr ~nt muJc1o ~t tho cn~ori~ ont Dl th~ p~uyn~ n~ c~dmlum ~hrou~houl tho ph~ry~x or OCCE~IOA-lly ln th~ ImcJ~ioc, Arscnlt~ O~pO3u~0 ~roducco ou tlrru4 p~ I tlt~ro~lo~ nAor rominbcon~ ot ClBsriC h~trhcek iD~oc~oe\. Tho~ to~cI~J, ~llu~ o~ In ~lotro S, rui3gerl ~ut ~luolno~tlon Of U ~onto ~ complo~ t~r n~y bo ~ u~efl~ ~orJtwo o~ ~hls gio~ool~orln ~y~tel~L It b i~onan~ lo no~ ln~ e~n ill no~~ whlch ~ro no~
o~oud U~ c~ ucr~fi culturo modlum (1~.
i~3 Q~-r~ iVo ~)Oy~ o~o u~porlm~tY, r~ ode~ ~vCr~ oxpo~ed to ~c ~tto~ot C~ i~ 0~, the~ per~lk~d ~i o~oyod u-in~ ONPCi to~ r~Ph-~y~
S~lw~opr~noJ~t~ d~c~ldo~o ub~tretr whloh yloldl ~ ol~blc y-llow prod~ct ~potl re~cLion wIth ~hc onzyme. Ono v~rCIon Or ~ nt~r~ obotoJi c UlOy 5-~o~¢rlbod i'~y ~DUnth~] (t8~, ~nd i~ p~viow u~o wilh c~ tt~ lJ by P1ro (19). ~aeh ~euly l~So~ ~ppso-xîm~ly 200.1~ to~o~, dop~n~ln~ on ~hc OXpO~I~llODt.
~ o dct~rmln~ ir ~h~ onYymo ~cti~ y produco~ ~c~ proporljol~l 10 Iho ~u~nlludo ot ho ~uo~, oll4uot~ of n~m~todu ~voro ho~ hocllot ~t 33~C ~ t10u~ tin~. Thc ptol ~ In Pl~uro 6 I~D~10W~n~S thCt tho on~y-no ~CliYil3' Inducd w~ line~r wi~
t~po¢~ 00 ~o bn~ of ~bo ho~ c~ to~ 30 ml~ o the o~ of IhO oxpcrlmo~ t 90 min. Tl~l~ o~pclt~n~n~ tnllic~cl ~hu ~b,o ~o1uWc O~ Aully i~ g~ ioLo~
~eoJItlv~ end p~Yldc~ qum~tlt ~ mu~ o of ho~t tlrctC.
Su~oquon~ ox~crir~n~ u~d thl~ a~a~Y ~o mea~uo tho o~cc~ o~ chcmic~l r~rn~or~ . Tho rc~lt of u~ oxporlm~n~ in ~ich nc~otodcs ~ore e~tporoa ~0 o~ lum cltlo-l~c rO~ v~1n~ pcrlod- o~ tlme 10 ~hown In E~uro 7.
c) Tbo ~c-f~con~ineC tc~t ch-n~b-r ~9TC). rrototypo STC~ woro cor~uc~ f~om t~oJ~I~ plO~liC l~ngc~, or r.O~ a q~ se pl~er sad t tocllon Or ~ tU~i~S
c tho bàrsel. So~ l hundrod ncmclo~c~ ~c-5 pl~oot ~Ite, Ibo plun~er repl~cc~.
he wholo ~nbly w~o ~ub~cc~ ~o o hccl ~hocl~ a coal-ol SSC w~ ept llt room ~ompon~uso. ~r~o~ tho IncubaslDrl, ell ~pr o~ ~ho Ol~JrO ~c~ were curied ou~
dlroc~y Ic thl~ IISC. Ths; sor,~lt ~U ~1~ to ~ oon ~hon thc uJay- woro tono In 14b~ or woll~: room tompe~hro conlrolc showc~ no co~our dcvolopme~h whilo lltC
boot rboclcod -~p1~ t~volopul ~ Inte~ec ~ollow col~ur wi~hin ~ O n~ln, ~o re~elc hour~ ndln~ upon t~ xbnt ot tb~ hc~ ~hool~ ho number ot o~lr u-~.
d) O~ o~y~. S~cc ~ho nen~todc~ C~a be ~d~ nccblo lo llm~ll enoloculcs In ~ ucl by t~a~ wllh co.t~lln llolvon~r ~uch ~ occ~onc. Ibo~r~tlc~lly rny o~y~o pro~o~l In Ihe olmolr, u~ ~hlch c~ nuuJed url~ lo-~ molocul~r w~l~ht ~u~trll~e~, could bc ucot h ~ho ~bovo upplic~on~. Por ox~mplo.
dohy~ros~-o ~uch ~ o~rophollph-t- dohydro~en~o, notn~ ,ororc~e In wlld-~ypo C. ~ cY~ ~oo~ m~urod ud~ o hblwhemi~ t~in ~ccl~lo far ccoh on~y~o, u ~bown In ~i~urc ~. rhorororo IhO oppu~n~c ond c y In~tho d~crlbed hcro co~ld bo uJod to dclCCt o~c~r potonll~l rcpo~ot onzy~ UI~Cbly o~lnH;rod tran~p~lc ~tr~ln~, or ovon o~r~lly o;ccurrlo~ no~rtodo on~ytno~. Ihe oc~tvl~ ot w~loh o~hl b~ IrltkJtw~ o~ onvl ocrnont~ condilion-.
~ olocubJ s~ch - b~C~t~l ot tlrc~y luclror~ oo~lt bo U~d ~ rcpor~rr cornbltullo~ wl~h tho ~pl~ prDmotor~ (7). O~r pot~n~a~ ro~ tory oq~encol ~l~h~ jnol~do ~romo~cr~ l~duclblo by hc~y mo~ ucb u tho~o of the m~ hlo~olrt pno~, tho cylochJu~ P4SO pro ~ ~or, or pro~ot~rs rcspon~lve w o~ vo ~tto~ ~ to ~ c ~u~n~.
. :
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J~N.29 '93 17:41 613-232-13440 P. lZ
C~nditO et al~ 2 0 ~ g 3 7 9 CONCLUglONI
~ ir pplic~tlon do~crib~ ~ ~ystem ror moslilOti1lg Yl~e~ livin~ or~hl~m.
Thr, 0y~l~ c~lcl- of:
1. ~r~nr~nio ~in~ ol the ne~todol Cacleo-haO~ o~o,~a/~ hi~h ~o~po~d to cor~ onvlro~mo~al ~ond~llon~ by ~yn~ l~ ro~dily ~c~oc~blo enzymo~l;
2. t~Ielho~ ond compo~ition~ tor okpocl~l ~ht ~ni~ Ib ~rl~llr ~IqueOul wlollon4. ~I~.rin, ~oib ~n~ ~lud~
3. ~tu~ nt comporitlooo r"- mo~riD~ thc enzymu ~Ir~lly in ~ nlm~l.
v~nu~es of Ih4 ~yr~om o~or o~er C~d~dr o ~ blc bio~nonlsor- ~ro it~
i~plicily. r~iPl~y, rtl~llvoly low CD~ of qucn1it~llo~ t~ uo ~ oity or ~ho rol~ponco, il~ ro~ nso lo ~ublcth~ ion~ J cclc~tlYI~ ~or c~r~in llreuor~ ~d 11~ po~l~blll~y.
A wmbu ol lo~clll o~odo~- ol ~hic Iy~lem c~ b- nviu~d:
O~r rep~l~ n ~ bo~ he ~ lKIo~ldo~c u~od to doto. co~ld ~e urod ~o ~tv~ to in ~o c~mc way, t~d tho~o co~ld bo oolrollc~ by ~y ~ulre~ pto~no~c-.
. ~noy~c~ olre~ty ~r~ont In tho ~olu~ d typc ot~nl~ eould b~ mo~uro~, iS ~o~ r~ round ~ lr u~ tod or prodllcot in ~on~o Lo c~ng or ir~
~n~lronmo~vl conti~on~.
Ott~:t ~rcc~ rln~ to~c s~c~lo~, wGh a~ C~norho~d~lis b~ sac cr ~na~ t~lVlVI~ coul~l~oo u~e~; t'. r~ft~fvu~ Ir/. F~r~lc~ r r~f~h~ pr~r~rA wrt~itl cdvont~ uc~ ~ lo~vor o~y~on roq~tomcm~ tn~ J~owlb or ourvl~l. or ~re~
o~ o~
I:lre~lio~ of ~e l~tlnel Itonogtnlc or~D~ to~ othet ph~ oing slre---uci~lo plomo~cr~ ~nc re~otlot p~e ~oquoncor.
Vuri~tiot~ o d~ D ol ~ t~l~¢otltoinca ~p~ot~l~ mlllht IDCI~de ~ ~4t;0nbulb ~or oo~ tln~ ~plos ~lo ~be chsm~or, ~nol-~d Or ~ ; l10w-~-ou~h cell~
wi~b ~ tCU~ o~h Or porolU ~ l ~oh ond. ~r pl~ccm-~ in llowln~ nt~r;
mul~ Or In ~hlch ~h~ Du~oo~odos re tot-l~od in ~ uo~ by an ovcrlyln~
potoue ~ r or n~c~h.
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J~N.29 '93 17:4Z 613-Z3Z-8440 P.13 9 C~ndido ~t ~1.
2~379 rerenc~
1. S~n~cr~, B.M. "~hc rolo of ~osr p~atoln r~rpo~e in phy~lologlo~l dd~pullon of~Ino mollu-c ". ~u. ~3n~ e~. 24, 207-~lO ~1988), . S9ndot-, B.~. "SU~ pro~ poten~bl ~ ;t~ biomarl~ors. I~ ~iolo~lc~l m~rl~orll o~ onvironmonl~l conu~L~tion", ~. J. McC~tby ~nd L. 3hu~rt. CRC
Plu~ ur, lloc~ RJcon, Florld~l, 135~ lg90~.
3, S~n~err, ~.iM., M~lr~, L.~" Nel~on, W.o., Pholp~, 4.E~. ~4 ~oh, W. ~R41~lion~hip~
botwoen ~ccunwl~tlon or ~ 6~ r~te~ prototr~ uld ~opc~tor~grow~h n ~yll~
J~ e~ ot lo ~ e of oo,~por concor~r~lor~ ". M~r. Elnvirun. Ro~, 31,111-97 ~99~).
4. ghin~h~m, ~.a.. Di~o~, D.K., loner, D. aut C~n~ito, ~.~.M. ~om~r-l ~n~ Sp~t ~prorl~lor~ P~crn~ Or ~he gm~ll Hoet S~cc~ 6) G~n~r In Tr-n~cnic Cw~orh~bdlll~ tl~a~lJ''. MDIOO. ~1. C~ 233 ~191~2).
S. ~u-~r~ t.H. ~nd ~entldo, ~.P.M. "Locur cncodin~ y of sm~ll hc~t Jbocl~
~nol in Cae~lor~d~ n~: IWlI ~0~ dupllc~ ror~ 3.~ lDbu~ Invetlc~
r, ~SOI. C~ lol. ~6). ~8-1278 ~l9as).
6. Jo~e~, D., Rus~n~, R.H., ~, 2.1. r~nt C~lndido, ~.P.M. "SltUCtUre, oxpr~ on, ~nd 4volutlo~ or ~ ho~t ~hoc~ SC-~O IOC~ In C~ o~labt~r~ c~a~ nl~ke~ by ~Jtlvo ob~". J. ~iol. Chcm. ~(2~), 1200C-120~5 ~lg8~).
. Boylun, !l., ~ellelicr. J. ~ Sei~hen, ~.A. "~u~d ~t4~l~l tu&ife~so subunlts CeUlb~ li~ht Olll~ Orl in el~kuyol~l ~t pro~eryote~ J. Biol. t~b~m. ~, 1915-l!~t8 (lg89).
. F~ ., YVhib H~ o~ 5. o~t ch~n~ mod~ lo~ ot ~-~olaclo~ld~o fu~ion vecto~ fo- J~udrlr~ ~on- r~l,pro~ion ~n C~nor/~Ddl~t cl-RanJ. C;u~ i89 ( 1990).
rin~m, ElØ u,a Cand ~0, ~.P.~q, "T~ elo~ sln~le ocl~ in~cli~n ot p~o producl- 1~ Ca~or~bdl~lJ ~ an~ ~ nc~ ~ot rOt ae~lap~ UU~ie~", J, exp.
Zool., 1~ pro~.
10. gl~in~h~, e.o. ~nd ctodldO, ~.P.M. "~ of U~ ~C ~ o~ ~e ~on~o arh~ cl-~o~J ~ ~lolo~lc~l ~ot~ton Or onv~u~ont-l J~cn"~ ~b~CI- lo ~T~C .~ p,10 (1~g2~, C~1n~, O~, 11. S~i~8h~, E.~. ~ad ~l~di~o. ~.P.~ T~U~enjC A~o~6 IccZ Jlrctns of ~C rDil ~o~tota C~norllabdi~ a~ blolo~lo~l m~ni~or~ ~t ~nvironn cnl~l ~rrou"~
~U~mlllCa.
I~. SallloD, ~F, ~n~ l~rc~t, S, Tht DN~ o~ Ca~n~orh~d~lw ~ a~ erlclic~
10~ (197~)-13. Hclel~-y-mc, ~., Shir~l~hl, H. cn~ Su~ , Y. "~onltorl~ ol Ihc ov-r-ll pc~iold~ IOaiOi-y ar rivor ~lcl lo ~9L~lic or~obrn~ n~ ~toJhw~tol ~hrtn~, Pu4tye co~ o imp~Ylu", Cbo~oa~erc 2~. 229-23~ (~991~.
14. ~lo~y, ~.P., ~o~lu, C.J., O~ , Rl. o~ ~leluy, M.CI, "~enthic InvortcO
bi~eu~y~ ~ilh toxi~ sedin~ nd pre ~IOr~. ~nviron. T~xicol. Chon- ~ 233~24 (~ 990~.
. . . :
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J~N.29 '93 17:43 613-Z32-~440 P.14 ~ C) C~do et ~1, 2 0 8 ~ 3 ~ 9 15 B~borowsSi, M Rac~ho~v61~i, U ~ ~wch-~, H "Molbot ~Ind app~r~ for ~o~rminlnB ~JIar ~oxlolly ~y lhe tc~ ion Jn tho Ien~lh or ~ h ll~y 1~ tho ~ut Ocr~un p~tonI DD 2~85 ycz~nov. ~ Y Kr~l~yui~ov~, ~ N a~t V~ze~O, A C; "D~vloo tor b~1~ic-1 e~tiln~tion o~ c to~icity" So~b~ p~tom 5U ~46027, 17. "S~ble~l blw~sa,Y for ~uYi~oamcnul g~ ", World Pa~o~t No wO ~013023 1~, R~onthd, N L~ "Method~ ~D E~ymology", S~. ~or~a ~nd AR KirNne1. et~, Ac~t~mic Pro -. L~ 704 7~0 (19~7~
19. FIJO, ~, ~'lnte~ti~ mn~orm~tlon or C~lorh~dl~lJ ~l~g~ns" ~l~O ~ ~, 2673-26~0 (l9~6) . . :. :
;
~' . ' :'' : .
- : . ... , . -- - . - . . .. .. . -
~ . T110 cotono 1~ pol~od and tho C~,SUy ro1utloD i~ d~ inlo Ih~ c~mbor.
b. 1~ ulr~y ro~ulu ~c ro~ ~n~r ~ ~uJ~blo In~ lon llmo, t~nein~ trom -~ow mlnule~ to overrdgh~ C ~ o~ture t~nJL~ J~am unblorl ~D 37~. Two lypC-oi~ unlly hovo beon ~mployGd: u 4uulit~liv- hi-~oob~nlic~ s~y ~4hich Indicoto~
~hi~h ~le~uo~ t~vo unter~onc ~hc ~Irou r~pon-o, und o qu~nlil~ c 301ublo ~r~y ~h~h provWu ~ colo~r chlmp 70~10atln~ ~hc lCvCl Or ~ItOU~ uC~d eo2ymc in Ih~
whol- ull~l.
111. DlST~ Q D~SCItlrT101~1 OF SPU:I~IC EM~ODIME~TS OF THE
~IOMONI'rORINC ~YSTISi~
Provlou~ blcm~nllo-in~ ~y~tom~ ~o~/o ton~i~lod i~ n~o~i~oring so~o phy~lolo~lc~l ~orc~o~ot o~ vtld-~ypo o~s~nIun cuch u e microot~ni-m, en Invorlebr~l4 (m~ ol~, cl-m-, Depbnle) or ~ vortobr~to (~ y ~ h ~Ip~c~iO-~durin~ ot ~ftcr o~ ur~ to ~omo ~ cotdillon. The ent-~olnt ot tihe ~r~y i~ ohc~
loth11ly, .~. th~ L~S0 o~ ~ tut Nb~ noo I~ dolenn5n-d ~13,14). O~ CJ~ b~v~
olvo~ tom nl- o~ bth~ ueh ~ nwv-mont o~ 11 cw~,~ rrom lc~t ~mplo~
~15), or tr~~ y of pura~l~ In b~,n~ ucl~ ~- ml~nol~ ); lu Ih~-e cc~, c~mp1~ nd oxpon~livo cloclro~Ie oquIpmon~ ~orm~ p~rt ot ~hc ~y~
~Itor~tlv-ly, ~Dd maro rocon~ly, ~c 1~v~ of ~pocitIc biomoiGculoJ, ucull11y pro~ol~, io ti~o ~o~ pcclo~ h~vo boe~ mul-ur~d, ~ t ~bc curten~ int~c~t In mcP~ o~ o~ pro~oi~ (17), Thc Icll CU~yJ, whilc uslD~ ro~ iwl~ y1c C~ip~oJlt~ novor~h-lo-~ ar~ vory ~,ime~col~amin~. Involve m~ny ~nlpul~lloul, md ~tq~lre ox~onclYo u~i~ in~ ~d oxporl~nce. They uo nol sulted co ~9C in IhO
~lt.
.
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J~N.29 '93 17:40 613-232-8440 P.10 C~didoetaL 2088379 ~ ho pr~on~ ~p ~o~oh u~ zoJ ~ecl~lly desi,~nod ~r~n~l~cni~ or~ ms vrb~ch re-pon~ lo ur~s~or~ gy protucln~ ~n c~ qu~tlnct en~ymo ~vhlc~ i~ nol norm~lly ~rDdu~od by ~hor4 or~ni~m~ r~hormoro, ~y ~Itili~in~ IhO Im~ll, oaslly n~ni~ul~ed n~ t~d~, C ,rle~ , r~out ~f ~ tos~tr con bo c~rrie~ o~t ra~ldly ond limply in ~ho In~Ac~ org~n~, wltbout ~ho ~occ~ o~ di~-uptl~ o~ Juu or oxuac~lo~ ~ tho nry~u ~ bo lncul~ot r~ ~ray U D be pcrformcd e~rll~ la th~
l~buLl~ry or In ~o Held, by ~or~o~nol with ~1~ nlnin~
~ dvonto~t~ of Ibe ~stom:
~ . Q~an~tlv~ or q~udlbliv~ ~B~U~9 Of ~res~ in ~ul~lcollul~r a. Ie ro~onJivO ~o boO~ nlc ~Ad Inor~cDlc stns~ors G Cell bo uod on ~11 Iy~U ot ~mples: rr~h or ~Cll wa~t ~mplc~, mill omues~ C-C~IOr. slud~o~, soll OIU~IC~, wll~ OJ ~otimon~ poclrlc chomlc~lls or ~I)IXIut~l ot oh4n~io-1~
1~, Moy ~oc corrlcd out In t~ bor~tol~r or i~ ~ fiel~, ~ . ~o~dout requitec ~Inlmcl qulpmoM snt cons~ of ~n o~olly ob~on~blc.
s~blo colour ch~n~e.
F, ~ollow~n~ oxpo~u~o ot tho otpniunr to Iho tost ~mplc, ~c~ulla of tho ~tro~
~J~oy ~se e~oiI~blc wilhin ~Jr~uIo~ îo hou--. dopondln~ on ~hc m~nilude o~ lho im~o~ot Jt-O-J-O. ~- ~o~ c~ bo cu lod o~t ~ Ie Yol~ncJ~ l,e, lo~ ~n 2 mL.
H. Tho ~ort or,pnl-m ~r cho~p ~o ~row l~nd r~im~in, 1. ~ylllom c~ bo YUt fo~ ~no~iloria~ ~olb cluol~lc ~n~ ~cuto ~oxici~y.
~ . Sy~ ~oro ~ondtivo ~b4n currcn~ Iclb~llty îats usln~ ~ild~ c no~ oto~.
K. ~y~l~ provlee~ Inform~tio~ on ti~c ~eclticit~ of Ihe s~ros~ ro3pon3e.
~ t~racc ot th~ n~metoto~:
Thtoo mcl~odr o~ storop of Ihc r~oo~todo- dlr~olly 1~ ~hc les~ cbomb~r ~ro po~d~lo~ ub ~y ~o Itop~ In ~ re~r~t~r ~ncllr 4'C) ht Je~4r~1 d~y~
bchr~ u~, u th~ ~t~pt~d~tO Clutly ~IOWJ ~lr do~olopnx~l. Ii~ C. ~ J i~
st~ tto ~tucrb~v~, whiCh tons~ whcn cu1mt~- ~re Jt rvod, Tb tNor~
tocs no~ ~e~, cnd wn ~ur~lY for uP ~o Ibreo mon~h~ o~t tOtD~ tUto~ t~ea roru~o 1~9 d4vcl~pm4n~ wllcn ~ cnlc~[ wi~h rodl ThU~ ~hc biomonioor otr~ coldd bo olorod ~ tibo ~oet c~mbaJ u d~ul~Yeo for ~p ~o tiuo~ monlh~ un~ll urot; Iho onlm~t~ colld b~ ~cd ~or 8 ~ o ~o con~,crl Ihcm ~o ~on~l L4 luv-o prior to uu b ~UI~ ) C. ~ nr Ll ~ ~ lorv~o 0UI b no~:Cn ~ In~c~inllol~ ~I dry loo oo 11quJd nJl~o~on lomporol~o~. Tboy ooult Ihorohro bo ~torod tro~on ~n~
~hlppod ~Ircolly i~ IhC lo~ eh~oer~: 11 woul~ ~n only ~o nocou-ry t~ cb~n medll~m ~ tood tho nomocoder ~or o to~ ~o~n p~loJ 10 th~lT u~ in ~cr~
Ir. 5XJ~MtL11 6:
'rhe Imr~tlon e~ hstllCt ~0 u~ldou~ood by rero~e~c~ l~ l.hs ~ollowin~
oY~plc~, Thc e~-~mplo- ~-c~¢n~ be1ow crc provj~cd r- ~ futlhor ~uid- ~o ~h~
pr~to~ o~ o~tin~sy ~kl rt, cnd o~ not ~ ~o conJ~r~d o~ ho Iru~0~DD ~ ~ny w-y. ~bo ox~mplo~ u~ tlvl~ qu~lit~vo ~ y; ~) ~C
qu~tl~dv~ U~Cy; o~ Wo ot Ple ~olrcou~ chuob~: t) mhor u~y-.
~ ~u~ll~dvo L~ y-. T~ X~ tc~ny ptocod~e 1~ d4-c~b-t ~n Pirc e~
1. ~0. ~omn,to~cl ~etC ~DO~ 10 tho r~tCuin~ ohcmio~l in ~ ubc~ ~r In multl~oll ~i~poJ~bl~ pl~tb di~)lo~ rlnod wllh Iho bi~lolo~ n, X-pl, ollo~
p~r~ tl~ wllh Jcolono, ~o ~rd reroronce ~ues~ wAditloll. e ho~t thocl~
~ 33~, wC~ cu~bd o~ wi~b ~h~ ~imeJ~ OD ~ Y~ki pl~U conl-lnln~ nUt~io~
c~r. ~ho dnuo ~L~ioln$ p~l~orn ~ Corvo¢i l~nt~ ~ Iow powo~ bl~oc~
mlo~o~co~o. .~ ot ~ ~ho~ ~ ~imC ~ 15 ~ 1 33~C o~Jo~ cl~olo~Jd~o ' :
.
, , J~N.29 '93 17:41 613-232-~440 P.ll 7 Cl~ndido et ~- 2 0 ~ 8 3 7 9 ~ e n~Qd~ tn mo~ uo~ of ~c ~lmsh t4). In ~ddi~io~ r6rioU~ chomlc~ rcJIior~.
lncludin~ ho~y n~ ]o ~uch ~ ~r~cnllo, ~dmlum, cop~or, lo~d. noroury ~n~ sln~.
s1~d Ibo hcrblcldc. purAqu~t. ~oro toun~ ~o oc~lvu~ ~ho roport~ ~pn~ .ner cxposu~
of JO~t~l ~ou~ td ~ dJIyo ~ll. Pi~ur~ S). Iho~ ont~ yhlt dll~Ycrcnl tl~uo plltlO~
or t~ro~ Jn~uc~loD.: ~orouYy ~m~ p~,~q~ Induoo only ~n~ntln~ rer~lon, lo~t clr~tr ph~rn~o~l mwcle, up~cl~lly ~ IhO ba~o o~ thl~ tertnlD~I ~ulb. coppu h~ucc~ in nowonr ~nt muJc1o ~t tho cn~ori~ ont Dl th~ p~uyn~ n~ c~dmlum ~hrou~houl tho ph~ry~x or OCCE~IOA-lly ln th~ ImcJ~ioc, Arscnlt~ O~pO3u~0 ~roducco ou tlrru4 p~ I tlt~ro~lo~ nAor rominbcon~ ot ClBsriC h~trhcek iD~oc~oe\. Tho~ to~cI~J, ~llu~ o~ In ~lotro S, rui3gerl ~ut ~luolno~tlon Of U ~onto ~ complo~ t~r n~y bo ~ u~efl~ ~orJtwo o~ ~hls gio~ool~orln ~y~tel~L It b i~onan~ lo no~ ln~ e~n ill no~~ whlch ~ro no~
o~oud U~ c~ ucr~fi culturo modlum (1~.
i~3 Q~-r~ iVo ~)Oy~ o~o u~porlm~tY, r~ ode~ ~vCr~ oxpo~ed to ~c ~tto~ot C~ i~ 0~, the~ per~lk~d ~i o~oyod u-in~ ONPCi to~ r~Ph-~y~
S~lw~opr~noJ~t~ d~c~ldo~o ub~tretr whloh yloldl ~ ol~blc y-llow prod~ct ~potl re~cLion wIth ~hc onzyme. Ono v~rCIon Or ~ nt~r~ obotoJi c UlOy 5-~o~¢rlbod i'~y ~DUnth~] (t8~, ~nd i~ p~viow u~o wilh c~ tt~ lJ by P1ro (19). ~aeh ~euly l~So~ ~ppso-xîm~ly 200.1~ to~o~, dop~n~ln~ on ~hc OXpO~I~llODt.
~ o dct~rmln~ ir ~h~ onYymo ~cti~ y produco~ ~c~ proporljol~l 10 Iho ~u~nlludo ot ho ~uo~, oll4uot~ of n~m~todu ~voro ho~ hocllot ~t 33~C ~ t10u~ tin~. Thc ptol ~ In Pl~uro 6 I~D~10W~n~S thCt tho on~y-no ~CliYil3' Inducd w~ line~r wi~
t~po¢~ 00 ~o bn~ of ~bo ho~ c~ to~ 30 ml~ o the o~ of IhO oxpcrlmo~ t 90 min. Tl~l~ o~pclt~n~n~ tnllic~cl ~hu ~b,o ~o1uWc O~ Aully i~ g~ ioLo~
~eoJItlv~ end p~Yldc~ qum~tlt ~ mu~ o of ho~t tlrctC.
Su~oquon~ ox~crir~n~ u~d thl~ a~a~Y ~o mea~uo tho o~cc~ o~ chcmic~l r~rn~or~ . Tho rc~lt of u~ oxporlm~n~ in ~ich nc~otodcs ~ore e~tporoa ~0 o~ lum cltlo-l~c rO~ v~1n~ pcrlod- o~ tlme 10 ~hown In E~uro 7.
c) Tbo ~c-f~con~ineC tc~t ch-n~b-r ~9TC). rrototypo STC~ woro cor~uc~ f~om t~oJ~I~ plO~liC l~ngc~, or r.O~ a q~ se pl~er sad t tocllon Or ~ tU~i~S
c tho bàrsel. So~ l hundrod ncmclo~c~ ~c-5 pl~oot ~Ite, Ibo plun~er repl~cc~.
he wholo ~nbly w~o ~ub~cc~ ~o o hccl ~hocl~ a coal-ol SSC w~ ept llt room ~ompon~uso. ~r~o~ tho IncubaslDrl, ell ~pr o~ ~ho Ol~JrO ~c~ were curied ou~
dlroc~y Ic thl~ IISC. Ths; sor,~lt ~U ~1~ to ~ oon ~hon thc uJay- woro tono In 14b~ or woll~: room tompe~hro conlrolc showc~ no co~our dcvolopme~h whilo lltC
boot rboclcod -~p1~ t~volopul ~ Inte~ec ~ollow col~ur wi~hin ~ O n~ln, ~o re~elc hour~ ndln~ upon t~ xbnt ot tb~ hc~ ~hool~ ho number ot o~lr u-~.
d) O~ o~y~. S~cc ~ho nen~todc~ C~a be ~d~ nccblo lo llm~ll enoloculcs In ~ ucl by t~a~ wllh co.t~lln llolvon~r ~uch ~ occ~onc. Ibo~r~tlc~lly rny o~y~o pro~o~l In Ihe olmolr, u~ ~hlch c~ nuuJed url~ lo-~ molocul~r w~l~ht ~u~trll~e~, could bc ucot h ~ho ~bovo upplic~on~. Por ox~mplo.
dohy~ros~-o ~uch ~ o~rophollph-t- dohydro~en~o, notn~ ,ororc~e In wlld-~ypo C. ~ cY~ ~oo~ m~urod ud~ o hblwhemi~ t~in ~ccl~lo far ccoh on~y~o, u ~bown In ~i~urc ~. rhorororo IhO oppu~n~c ond c y In~tho d~crlbed hcro co~ld bo uJod to dclCCt o~c~r potonll~l rcpo~ot onzy~ UI~Cbly o~lnH;rod tran~p~lc ~tr~ln~, or ovon o~r~lly o;ccurrlo~ no~rtodo on~ytno~. Ihe oc~tvl~ ot w~loh o~hl b~ IrltkJtw~ o~ onvl ocrnont~ condilion-.
~ olocubJ s~ch - b~C~t~l ot tlrc~y luclror~ oo~lt bo U~d ~ rcpor~rr cornbltullo~ wl~h tho ~pl~ prDmotor~ (7). O~r pot~n~a~ ro~ tory oq~encol ~l~h~ jnol~do ~romo~cr~ l~duclblo by hc~y mo~ ucb u tho~o of the m~ hlo~olrt pno~, tho cylochJu~ P4SO pro ~ ~or, or pro~ot~rs rcspon~lve w o~ vo ~tto~ ~ to ~ c ~u~n~.
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J~N.29 '93 17:41 613-232-13440 P. lZ
C~nditO et al~ 2 0 ~ g 3 7 9 CONCLUglONI
~ ir pplic~tlon do~crib~ ~ ~ystem ror moslilOti1lg Yl~e~ livin~ or~hl~m.
Thr, 0y~l~ c~lcl- of:
1. ~r~nr~nio ~in~ ol the ne~todol Cacleo-haO~ o~o,~a/~ hi~h ~o~po~d to cor~ onvlro~mo~al ~ond~llon~ by ~yn~ l~ ro~dily ~c~oc~blo enzymo~l;
2. t~Ielho~ ond compo~ition~ tor okpocl~l ~ht ~ni~ Ib ~rl~llr ~IqueOul wlollon4. ~I~.rin, ~oib ~n~ ~lud~
3. ~tu~ nt comporitlooo r"- mo~riD~ thc enzymu ~Ir~lly in ~ nlm~l.
v~nu~es of Ih4 ~yr~om o~or o~er C~d~dr o ~ blc bio~nonlsor- ~ro it~
i~plicily. r~iPl~y, rtl~llvoly low CD~ of qucn1it~llo~ t~ uo ~ oity or ~ho rol~ponco, il~ ro~ nso lo ~ublcth~ ion~ J cclc~tlYI~ ~or c~r~in llreuor~ ~d 11~ po~l~blll~y.
A wmbu ol lo~clll o~odo~- ol ~hic Iy~lem c~ b- nviu~d:
O~r rep~l~ n ~ bo~ he ~ lKIo~ldo~c u~od to doto. co~ld ~e urod ~o ~tv~ to in ~o c~mc way, t~d tho~o co~ld bo oolrollc~ by ~y ~ulre~ pto~no~c-.
. ~noy~c~ olre~ty ~r~ont In tho ~olu~ d typc ot~nl~ eould b~ mo~uro~, iS ~o~ r~ round ~ lr u~ tod or prodllcot in ~on~o Lo c~ng or ir~
~n~lronmo~vl conti~on~.
Ott~:t ~rcc~ rln~ to~c s~c~lo~, wGh a~ C~norho~d~lis b~ sac cr ~na~ t~lVlVI~ coul~l~oo u~e~; t'. r~ft~fvu~ Ir/. F~r~lc~ r r~f~h~ pr~r~rA wrt~itl cdvont~ uc~ ~ lo~vor o~y~on roq~tomcm~ tn~ J~owlb or ourvl~l. or ~re~
o~ o~
I:lre~lio~ of ~e l~tlnel Itonogtnlc or~D~ to~ othet ph~ oing slre---uci~lo plomo~cr~ ~nc re~otlot p~e ~oquoncor.
Vuri~tiot~ o d~ D ol ~ t~l~¢otltoinca ~p~ot~l~ mlllht IDCI~de ~ ~4t;0nbulb ~or oo~ tln~ ~plos ~lo ~be chsm~or, ~nol-~d Or ~ ; l10w-~-ou~h cell~
wi~b ~ tCU~ o~h Or porolU ~ l ~oh ond. ~r pl~ccm-~ in llowln~ nt~r;
mul~ Or In ~hlch ~h~ Du~oo~odos re tot-l~od in ~ uo~ by an ovcrlyln~
potoue ~ r or n~c~h.
. .,~ . . , .
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J~N.29 '93 17:4Z 613-Z3Z-8440 P.13 9 C~ndido ~t ~1.
2~379 rerenc~
1. S~n~cr~, B.M. "~hc rolo of ~osr p~atoln r~rpo~e in phy~lologlo~l dd~pullon of~Ino mollu-c ". ~u. ~3n~ e~. 24, 207-~lO ~1988), . S9ndot-, B.~. "SU~ pro~ poten~bl ~ ;t~ biomarl~ors. I~ ~iolo~lc~l m~rl~orll o~ onvironmonl~l conu~L~tion", ~. J. McC~tby ~nd L. 3hu~rt. CRC
Plu~ ur, lloc~ RJcon, Florld~l, 135~ lg90~.
3, S~n~err, ~.iM., M~lr~, L.~" Nel~on, W.o., Pholp~, 4.E~. ~4 ~oh, W. ~R41~lion~hip~
botwoen ~ccunwl~tlon or ~ 6~ r~te~ prototr~ uld ~opc~tor~grow~h n ~yll~
J~ e~ ot lo ~ e of oo,~por concor~r~lor~ ". M~r. Elnvirun. Ro~, 31,111-97 ~99~).
4. ghin~h~m, ~.a.. Di~o~, D.K., loner, D. aut C~n~ito, ~.~.M. ~om~r-l ~n~ Sp~t ~prorl~lor~ P~crn~ Or ~he gm~ll Hoet S~cc~ 6) G~n~r In Tr-n~cnic Cw~orh~bdlll~ tl~a~lJ''. MDIOO. ~1. C~ 233 ~191~2).
S. ~u-~r~ t.H. ~nd ~entldo, ~.P.M. "Locur cncodin~ y of sm~ll hc~t Jbocl~
~nol in Cae~lor~d~ n~: IWlI ~0~ dupllc~ ror~ 3.~ lDbu~ Invetlc~
r, ~SOI. C~ lol. ~6). ~8-1278 ~l9as).
6. Jo~e~, D., Rus~n~, R.H., ~, 2.1. r~nt C~lndido, ~.P.M. "SltUCtUre, oxpr~ on, ~nd 4volutlo~ or ~ ho~t ~hoc~ SC-~O IOC~ In C~ o~labt~r~ c~a~ nl~ke~ by ~Jtlvo ob~". J. ~iol. Chcm. ~(2~), 1200C-120~5 ~lg8~).
. Boylun, !l., ~ellelicr. J. ~ Sei~hen, ~.A. "~u~d ~t4~l~l tu&ife~so subunlts CeUlb~ li~ht Olll~ Orl in el~kuyol~l ~t pro~eryote~ J. Biol. t~b~m. ~, 1915-l!~t8 (lg89).
. F~ ., YVhib H~ o~ 5. o~t ch~n~ mod~ lo~ ot ~-~olaclo~ld~o fu~ion vecto~ fo- J~udrlr~ ~on- r~l,pro~ion ~n C~nor/~Ddl~t cl-RanJ. C;u~ i89 ( 1990).
rin~m, ElØ u,a Cand ~0, ~.P.~q, "T~ elo~ sln~le ocl~ in~cli~n ot p~o producl- 1~ Ca~or~bdl~lJ ~ an~ ~ nc~ ~ot rOt ae~lap~ UU~ie~", J, exp.
Zool., 1~ pro~.
10. gl~in~h~, e.o. ~nd ctodldO, ~.P.M. "~ of U~ ~C ~ o~ ~e ~on~o arh~ cl-~o~J ~ ~lolo~lc~l ~ot~ton Or onv~u~ont-l J~cn"~ ~b~CI- lo ~T~C .~ p,10 (1~g2~, C~1n~, O~, 11. S~i~8h~, E.~. ~ad ~l~di~o. ~.P.~ T~U~enjC A~o~6 IccZ Jlrctns of ~C rDil ~o~tota C~norllabdi~ a~ blolo~lo~l m~ni~or~ ~t ~nvironn cnl~l ~rrou"~
~U~mlllCa.
I~. SallloD, ~F, ~n~ l~rc~t, S, Tht DN~ o~ Ca~n~orh~d~lw ~ a~ erlclic~
10~ (197~)-13. Hclel~-y-mc, ~., Shir~l~hl, H. cn~ Su~ , Y. "~onltorl~ ol Ihc ov-r-ll pc~iold~ IOaiOi-y ar rivor ~lcl lo ~9L~lic or~obrn~ n~ ~toJhw~tol ~hrtn~, Pu4tye co~ o imp~Ylu", Cbo~oa~erc 2~. 229-23~ (~991~.
14. ~lo~y, ~.P., ~o~lu, C.J., O~ , Rl. o~ ~leluy, M.CI, "~enthic InvortcO
bi~eu~y~ ~ilh toxi~ sedin~ nd pre ~IOr~. ~nviron. T~xicol. Chon- ~ 233~24 (~ 990~.
. . . :
. . : .
J~N.29 '93 17:43 613-Z32-~440 P.14 ~ C) C~do et ~1, 2 0 8 ~ 3 ~ 9 15 B~borowsSi, M Rac~ho~v61~i, U ~ ~wch-~, H "Molbot ~Ind app~r~ for ~o~rminlnB ~JIar ~oxlolly ~y lhe tc~ ion Jn tho Ien~lh or ~ h ll~y 1~ tho ~ut Ocr~un p~tonI DD 2~85 ycz~nov. ~ Y Kr~l~yui~ov~, ~ N a~t V~ze~O, A C; "D~vloo tor b~1~ic-1 e~tiln~tion o~ c to~icity" So~b~ p~tom 5U ~46027, 17. "S~ble~l blw~sa,Y for ~uYi~oamcnul g~ ", World Pa~o~t No wO ~013023 1~, R~onthd, N L~ "Method~ ~D E~ymology", S~. ~or~a ~nd AR KirNne1. et~, Ac~t~mic Pro -. L~ 704 7~0 (19~7~
19. FIJO, ~, ~'lnte~ti~ mn~orm~tlon or C~lorh~dl~lJ ~l~g~ns" ~l~O ~ ~, 2673-26~0 (l9~6) . . :. :
;
~' . ' :'' : .
- : . ... , . -- - . - . . .. .. . -
Claims (21)
1. A method of detecting toxins or pollutants in a sample comprising:
- providing a transgenic organism having a stress-inducible control region linked to a gene encoding a detectable protein, wherein said control region regulates the expression of said detectable protein;
- exposing said organism to said sample; and - determining the amount or detectable protein produced.
- providing a transgenic organism having a stress-inducible control region linked to a gene encoding a detectable protein, wherein said control region regulates the expression of said detectable protein;
- exposing said organism to said sample; and - determining the amount or detectable protein produced.
2. The method according to claim 1 wherein said organism is a nematode.
3. The method according to claim 1 wherein said organism is a member of the genus Caenorhabditis.
4. A method according to claim 1 wherein said organism is Caenorhabditis elegans.
5. A method according to claim 4 wherein said Caenorhabditis elegans is selected from the group consisting of PC71, PC72 and PC73.
6. The method according to claim 1 wherein said control region includes a stress-inducible promoter derived from a heat shock gene.
7. A method according to claim 6 wherein said heat shock gene is hsp 16.
8. The method according to claim 1 wherein said second gene consists of E. coli lacZ gene.
9. The method according to claim 1 wherein said sample is an environmental sample selected from the group consisting of water, soil and sludge.
10. A kit for detecting toxins or pollutants in a sample comprising (a) a test chamber containing at least one transgenic organism having a stress-inducible control region linked to a gene encoding a detectable protein, wherein said control region regulates the expression of said detectable protein; and (b) means for detecting the detectable protein.
11. The kit according to claim 10 wherein said organism is a nematode.
12. The kit according to claim 10 wherein said organism is a member of the genus Caenorhabditis.
13. The kit according to claim 10 wherein said organism is Caenorhabditis elegans.
14. The kit according to claim 13 wherein said Caenorhabditis elegans is selected from the group consisting of PC71, PC72 and PC73.
15. The kit according to claim 10 wherein said control region includes a stress-inducible promoter derived from a heat shock gene.
16. The kit according to claim 15 wherein said heat shock gene is hsp 16.
17. The kit according to claim 10 wherein said second gene consists of E. coli lacZ gene.
18. The kit according to claim 10 wherein said sample is an environmental sample selected from the group consisting of water, soil and sludge.
19. A transgenic organism having a stress-inducible a control region linked to a gene encoding a detectable protein wherein said control region regulates the expression of said detectable protein.
20. The transgenic organism according to claim 19 wherein said organism is a member of the genus Caenorhabditis.
21. The transgenic organism according to claim 19 wherein said organism is selected from the group consisting of PC71, PC72 and PC73.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2088379 CA2088379A1 (en) | 1993-01-29 | 1993-01-29 | Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2088379 CA2088379A1 (en) | 1993-01-29 | 1993-01-29 | Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2088379A1 true CA2088379A1 (en) | 1994-07-30 |
Family
ID=4151062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2088379 Abandoned CA2088379A1 (en) | 1993-01-29 | 1993-01-29 | Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2088379A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998028971A2 (en) * | 1997-01-03 | 1998-07-09 | University Technology Corporation | Transgenic animals for in vivo visualization of beta-amyloid toxicity |
| WO1999011772A1 (en) * | 1997-08-28 | 1999-03-11 | Consiglio Nazionale Delle Ricerche | Transgenic animals for the study of biological, physical and chemical toxic agents |
| EP0969100A1 (en) * | 1997-09-29 | 2000-01-05 | HSP Research Institute, Inc. | Method for screening heat shock protein expression induction regulators |
| WO2000001846A2 (en) * | 1998-07-03 | 2000-01-13 | Devgen N.V. | Characterisation of gene function using double stranded rna inhibition |
| US7005423B1 (en) | 1999-07-02 | 2006-02-28 | Devgen Nv | Characterization of gene function using double stranded RNA inhibition |
-
1993
- 1993-01-29 CA CA 2088379 patent/CA2088379A1/en not_active Abandoned
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998028971A2 (en) * | 1997-01-03 | 1998-07-09 | University Technology Corporation | Transgenic animals for in vivo visualization of beta-amyloid toxicity |
| WO1998028971A3 (en) * | 1997-01-03 | 1998-10-29 | Univ Technology Corp | Transgenic animals for in vivo visualization of beta-amyloid toxicity |
| WO1999011772A1 (en) * | 1997-08-28 | 1999-03-11 | Consiglio Nazionale Delle Ricerche | Transgenic animals for the study of biological, physical and chemical toxic agents |
| EP0969100A1 (en) * | 1997-09-29 | 2000-01-05 | HSP Research Institute, Inc. | Method for screening heat shock protein expression induction regulators |
| EP0969100A4 (en) * | 1997-09-29 | 2005-03-16 | Hsp Res Inst Inc | Method for screening heat shock protein expression induction regulators |
| WO2000001846A2 (en) * | 1998-07-03 | 2000-01-13 | Devgen N.V. | Characterisation of gene function using double stranded rna inhibition |
| WO2000001846A3 (en) * | 1998-07-03 | 2000-06-15 | Devgen Nv | Characterisation of gene function using double stranded rna inhibition |
| GB2349885A (en) * | 1998-07-03 | 2000-11-15 | Devgen Nv | Characterisation of gene function using double stranded RNA inhibition |
| GB2349885B (en) * | 1998-07-03 | 2003-01-29 | Devgen Nv | Characterisation of gene function using double stranded RNA inhibition |
| US8114980B2 (en) | 1998-07-03 | 2012-02-14 | Devgen Nv | Characterisation of gene function using double stranded RNA inhibition |
| US7005423B1 (en) | 1999-07-02 | 2006-02-28 | Devgen Nv | Characterization of gene function using double stranded RNA inhibition |
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