Note: Descriptions are shown in the official language in which they were submitted.
J~N.29 '9~ 17:35 613-Z32-8440 P.~5
do ot ~1.
2088379
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
VSV 217
C~n~t-
Tol:(604~-8~6-8034
Don~ld Jonor
S-6630 SUtlo~ Al~enuo
~by, ~.C.
VSH 3C6
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.
2088379
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.
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.
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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
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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)
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