Evolutionary Probes

Glycg!ytic Genes from Escher-lchla Coli as .

Evolutionary Probes

Newfoundland By

Depart ment

o. r

Biochemistry

September UI86 S~~diesinpartialrUlCillm~nt.of-t~e

requireme~tsCorthedegree of M~ter^OCScieDce

<:

MemorialUniversity,crNewrcuedlend

. . . /

-'':''~,_ ^{. . A.}

thesis submitted

to

^the

SC~Graduate

.> " .

ISBN 0_ 3 15 - 450 9 6-7

'c "

~.,.

. '

' ,( "

Abstract

, .." , - ' " "--." .

~omparativestudiesbast!0D the,struct ureor ribosopl e5a~dthein:omponents ..

.suggestedthat ther earethr ee primar ykingdoms:eubecterle,archaebll.c,terla-end

urkar yot'es.,:However^Ithevalidityof thishypoi hesisisbeing"questioned..ln.crder toresolv,he

eV~~i1tio~~I)',

^{.releticaehipeamong}

.bac,te;i~

^itis

' ~ee,es!a;y '

^to

~O~l\~e

the primar y struct ure8? r.mo) eculesothe r then those'Of'the'tro.uslati ooo.l .apparati;s."

.Th~

main objectiv;-DC

th~ pr~j~ct was

todeteemlnewhether'oc"n-ot hybrid

re~6mbiDa~t pla'smid~

^thatcontain

~~queDces to~, glycoly~iri~yni~~ 'rro~

..

'E8cheri~hia

^co,ti

coul~

^beused

to _~et~ct.' corr~sp~ntling s~quetic~5

ⁱⁿ

p'ripa~;tfOni

or genomi<

DN~

^.

r~6m

^-

dist~~t1y r~la~ed C?r~aD~ms: ',"·~h:'~~( ~I~mids: fro~ t~e::

CIa.rk,~CarbonE.c~li'genomiclibrar ywere chosen fof this study:"The plesmld

. ~LC 16.4· ~contains

^thegenesfor

hiose-·pbospba.~e ' iso~erase

^and

PhosphOfr~~t()o

kiaese , whereasit had'

b~~D

^{reporte.dthat'pLC}

i~4T

^and

P~C>l1·8:

^bothcontain

the genefor enolase':'Restr iction_.nn:"~~'.'...."",!,,~~~""'''''_J'LJn.'''_----,~-j

J

pl:ismid~,.and^{theJocationofitie}-~loning^vectorwas determ inedin each'eese by Sou t hern

bl~ttilig

and probing withCorEL"Comp arisonof the r,eStriction,mnps

I - ' , , " .

ofpte10-47 andpLe 11·8indicated , that therewer enoseque ncesin commonin, • the',genomic insertsof these plesmlds. Subsequent studies'at'~prot~i,o^level..

she wedthat oolypLe10-47ecat elne a gene,coding forenolase..GenomicDNA'·

was'prepared from

represe·ntll.ti~

^{species ofthe}

Enteroba.cteri~'ue,

^,othetgram-

negative:

b~cte~ia.'gra~-posi tive

^bacteria,andarcbae "n cteria.

Th~' g~n~mic, DN~··/

was,subject ed to Sout hern'blotting and.probed:withradioaf tively-lil.belle4I

'.'pLCIo--t7-or

pi.~

^'16-4.lThe

r~~~~s

^fromthese:hybridization studies

indic,o.~e'd :

th,!,t,genese,~C~iDg^{glycclytlcenzymes in}

E :

cof~^wet,·n,~t,a~le'~croSNeac~/~i~h .D~AIrom species that had di:erged CromE.coli moretlia.n4Q,milliotlre~r~ago.

Therefore ,'the~eDes,^ior'glycoly~icen:rm~will^{not be}o~^use:~ 1~,II'g^,:Q.uge

~lutioQaryprobes." - J / '

-

." .~

\

iv

Table' of Contents

,I 1.Introliu ct lon

l.l."fbeOriginofLde~

L2.lmJ;llications

of

theFossilRecord .'i, I'

13.MorphologicalEvolution \

14.MolecularEvolution

15 TheEvidenceforThreePrimaryKingdoms ~ 16 How Good is theEvidence Cor Three PruvaryKigdoms!' 17.Can GlycolyticEnzymesand TheirGenesSer~eee Evolutionary

. Probes! . ;-,, '

2. ResearchP.ropo8~1 ..

.a.r.Purjose .; . 2.2.Approach of Investigationv 3. Ma te rials"and Met hods .

'a.l.M~terials· - 3.2. St ra ins and Plasmids •. 3.3.GrowthMe.dia'and'Suffers

"- -""3,>.-t .j,j,~e I3t_ion__oE_Pfasrri_id-DN'k

.3:5.RestrictionEndonucleaseDigestions 3.6.Agaros,eGel Electrophoresis 3.?GrowthctPrcka eyctes 3.8.Isolationof Bacterial DNA 3.g.'Southern iJlottint 3.10.NickTranslation

3.11.Hybridization and Washing Conditions 3. 1~.Prepara.tion'or Extracts - 3.13.EnolaseAssaJ'

3.14..-SDS Polyacrylamide GelEleetropbcresls 3.15.,OEAE-Seph"adexChromatogra.phy ______

4.Chuacte rlz a tl on'o~pLC16·4 '~

• 4.1.Background 4.2.Reeulta

::;:~: ::~~~:~o:f~?~I~~~~ ~~c~rlon ~LC

^16-4.

4,3.Discussion . "

.-4.3.1.R~trictioDmap

of

pl.O 16-4

1

\ ~

\

₁₀

:

11 13

16 15 15 17 17' 17 10 10 120

'0

, '0

"

: ',

'3 '3

"

,~

-' 5.

27 '5

2;

.2

55 '5 55

.58

61 51 .68

:82 85 85 85

8' -

88 91

., ^.

.g~

94 94 100.

!,~~

;- '(:

v

. \

4'3.2.Tb'E.

,,IN:J..h

⁰⁰pLC16-4 38

.6. Charad erbat tonof pLC1()"47"an d pLCU·8 t1

5.1.Background'. 41

5.1.1.Propeties

or

enolase '-.... 41

5.1.2.Enolase inE.coli ."'• 42·

S.2..Results " , . ' ' . 43

5.2.1.Restriction Endonuclease Analysis

or

pLC 10-47and pLO.11-8 '43 .5.2.2.Locationsofth~CloningVector

cersi

on pLC10-,47 and "47

pLC11-8 . . •

5.2.~,Furt herSearches forHomologousSequences . 47 5.2.4.PostlbleExplanations.rorResults ObtainedusingRestriction 50

Endonuclease!'

5.2.5~Searchtor MultipleFo'~orEnolaseinE.coli ' ,' 50 5.2.6.Enolase Activityin ExtractsorE.colicootaining"PLC10-47, '52

pLC11-8

or

pLC 16-4 , .' . .,"',..,

I

5.2.7.50S Polyacrylamide Gel Electrcphcreeis(PAGE)ofExtracts .S2

oCE.colicontaining pLC1'0-47,pLO 11-8 or pLC16-4'),

.5,3rDiscussi.on ' ..' • " ' .

f .

5.3.1.EnolaseGenes I

5.3.2-..Hew-Ma ny EnolaseGenes inE.com

6.Evolu ti onary.Studl~l!Iushig pLC,16·4 and pLC ",10-47 as

. H;yb r ld lzatibnPro bes ' ,f

"6'. 1:Background

6.2. Sequence Ocmperjsccs of Glycolyti cEnzymesand' Genes

~ ::;:~: g~:~~:~:,~: ~~S;~:::::s

^;

~2.3~ComparisonorEnolaseSequences

6.3.Potentialor pLC16-4and pLClo.~j

as

Evolutionary.Probes 6,4.Res~lts

6.4.1. Characterization

or

GenomicDNAs 6.4.2.Hybridizo.tion Studies:EnterobacteriaceaeDNAs

6.4.2.1. Hybridization usingK30 cloning vectorasprobe 6.4.2.2.Hybridization withpLe1~4as Probe • 6.4.2.3.Hybridizationwith pl.C.10-4-7asProbe 6,4.3.Hybridization Studies:Prcker yotlcand EukaryoticDNAs.

6.4.3.1.'Hybridizationwith pLC 16-4asProbe ._ 6:4.3,2,Hybridization withpLClO:~7asProbe 6.5:Discussion' .

"6.6.Conclusion

1,References 8,Append ix1

List of T ables

45

58,

.67

i .

Table1-1: UEPstorSeve'talProteins n

.__~.'.Tabl e 3-11

\-sburces

or Bacteria. . 18

Tab le 3-21 RestrictionEedceucleeses aed theirRecognition Sequence» 21 .T';'ble....1: ~roleclliar'Sizes.andDistances Migrated by'')., DNA 30

·Digested with Hind

m

or Ecc RI

labte4-2:.

A.

\'Summa ry

or

th~'Fragm~ot Sizes Generated bY. 33.

~able

^5-1i

~:~~:t;;Do~:e:St~:::~7g~~\r

^pLe-II-S

~~d·~PLC.1O.47

TTa.,bbll••5-e-2,',-Enol~e.Activity^orE.-coliContainingPlasmlds

" Per~~DtAmino Acid aodNucleotide Sequence Dirterences

d r

TP'Is-Crom.E.coli(EC),,B.'8Ieafolhormophilu8.(BS};":.:.~~

Yea.sti~Coelacanth(Coel), Chick,Rabbitand Human _. Table8-2: ferCe~t'AminoAcld-SequeeeeDltrerecceeb;tweenE.

4 oli,

bacillusstearolhermophilu8nod RabbitPFKs" "

Tabl e6-3:"PerCent'AminoAcidSequenceandNucleotideSequence 81

, rreren,,~

between Y,'" and Rat Enol"es

II \

.

-'c•

) \ . I

List of Figyres

75 Figure

l·i:

Figu re1·2:

Figure1·3:

Figure1-4 :

Figure 4-2:

F,lgure4·3:

Figure4-4:

Figure '4.6:

.Flg' r e'4-6:

,

Geological Era and,the Fossi,1Recor~ f 'lI

Evolutionary Progression

or

Eukaryotes a,ndPrckaeyctes Ratesof Protein~volu~ion

I

Ind ependent EvolutionorAlebaebacteria,Eubeeterle aad 12

,!rkaryo~es ' .

r ' .

Figure4·1 : ASemi-logarit hmicPlot 9r. Molecular Weight Marker '31 Versus'DistanceMigrated00"anAgarceeGel

Restr iction EndonucleaseAnidysisof pLC 16-4

1325

FourSite MapofpLe16-4

A RestrictionEndc nueleese Map of pLCl6-4

A RestrictionMapal Col~1 37

Analysis of pl.C16-4,usingResrrlctlou Enzymes and 39 Southern.Blottingwith the Cloning Vector-K30 as

Radioactively-LabelledProbe. •_ -

Figure5-1: Restriction Eod.oDuclease'-Analysis ct pbO11-8.and pl.C 44"

10-47 .. _ l.. - . •

?

Figure6·2: Restriction Endonuclease Maps torpLC~10-47 anl( pl.C 46

11-8 . ' . ' -,

Figure 6--3: Search torSequence Homology betweenpt e 1O·.j7 and '48

-, .pl.C11-8 . \ \

....Figure6-4:'Restrict ionDigest AnalysisoC~10-·11,pte lkB.and 4Q K30usingFour BaseRecc gnlticnEndonucleases

Figure 6--6: Chromato gra phyotE. coliJA200 in 'l>EAE-Sepbadex 51 Fig u re&-6: 50SPAGE AnalysisoCExtractsoCE.coliCarryingpte 54.

10.41',pLeJI-8orpte 16-4. . .

Fig u r e 6-11 TheCompariso n

of

Amino Add SequencesoCTPIfrom. 62 .E. coli, B. ~te.(Jrothermophi/tJs;·Yeast, Coelacanth,

Chi~ken·,J.bbbifandHuma n__ __..

Fig~re6·2: Nucleotide Sequen cesCcmperlson

or

^TPICrom·E,iofi, .64

Yeast and Chicken ' " . _ ,

Figure6·3: Amino acidsequencecomparisonoCPFK ,CromE.coli, 69.

""'8aciltu8 titearoth ermophil u8 and ro.b.bit \ :~

FJgu re 8.41-AminoAcidSeql.\.eo~e,Compar isonotYeastandRo.t 73

EnoltlSes .

Figure6·6: Nucleo tideSequencesoCEnola.s~CromYeast-and Rat.

\

· . .' .')

.

'../"'Viii

'to. I •

Figure6-11! Comparisonor£heAmounto£ SequenceDifr~rence:!lat the 83 Amino Acid and-NucleicAcidLevel .

Figure8-7: Analysis of 'Prokaryotic Genomic DNAs by Gel 86.

Electrophoresis •

'Figure~8: Electr ophoretic Pattern or Genomic DNAs from 87

Enterobacteriaceae Digest edwithHindIll -

""'Flgure6-g: Sout hernBlotor' Enterobacteriaceae DNAprobedwith 80 K30 atHfgbStringency

Fig ur e'6-10:.,s.9.ut bern Blotof.EnterobacteriaceaeDNA probedwith'00 '-' , .- . '01<.30etLow~trin gency

~,Fjgure6~11:,So~Blo~ofEnterobncterill.ceneDNA-PrObed with 02

pLCJ6-4 ' .

Figur~}I-l~: Southe rnBlotOfEnterobacteriaceaeDNAProbedwith 03

. , .pL,C

lo-·n

~ .

. .

,~Figu r e8-13: SouthernBlot ofGenomicDNAs'frobedwithpLe 16--1 0.')

:,.FigW e6·14:'SouthernBlotofGenomicDNAs·ProbedwithpLe10--17 06

' / '

^{.. .. ' " " '}

; ! ,

/ / '

/ '

List of A bbr evi ations

,','

^{" ',,,"'. ,, ' ,}

.,"' ... ".

""'~"",,

5 ,r.\.

absorbanceatxom counts perminute deoxyribonucleic"acid Escherichiacoli ethylillediaminetetraaeeti~.add enolase-gene ~.. -.

glyceraldehyde-3-phosphate dehydrogenase"

kilobasepairs polyacrylamidegel. electrophoresis phosphofructo-kin as eprotein phoephofruetc-kin aaegene ribosomalribonucleicadd sodiumdodecylsulphate'

N,N,N',N'-tetram~thylenediamine triose-phosphate isomerase protein triose-phosphat eisomerasegene unitevolutionaryperiod volts-.

,

.

..

A, cpm DNA

e.o«

EOTA

",#

kbp F;AGE PFK ..

plk..

·rRNA SOS TE~IEO TPI lpl' UEP V eno GAPOH

,.'~..,

' .

^:::'.'',:

Chapter '1 Int r:oduction

1.1.The'Origin

or

Life,

,The

a,;i~

^Iat .lite has,

l~ng be~c-.a· matt~'

^ot

speculiLti~~

^...It·is unrart1,\nate .:

;~:.

that one.maynever be ab.l,etoascerta in't he truth:EvenSD,continuouSresearch using

.

II,variet'y,.of

appro~ches

.. ,. ,'may provide'

valull.bl~' infQrmati~n

, .^upon'which

lnteBfgen t. .speculation may be based..Fcrinstance,it has'. been wellr "istablished· . .. that·our~Iar^{system,includingthe}Ear~,^{.originatedasear'lyas}4.8·b·i1li~n~ear~

ago-andthat the historyatthe Earth~ay'bedelineated.byan orderlysequenceot' fossilsth a.t'isusedto'asllign.geological

eras

and periods (Figu ret-r;Futuyma, IQBJ).Thefirstevidence',afbiochemicalrea ctionsandcellularactivity'cccurain tire Jrecambrian et lf.·T heearliest

P~ecambria~

^{rocks areri;h .i'D}unoxidized.iroD.

co.mp~undsthat couldnot ha.yetermedin:i.hepresenceor,~xygen.This indicntes th:' 'h••tmoiph;"

.

^'

i,

tho'tim. lackedoxygen and^-. oecne. Precernbdeu ,"'k'^{, "}

dat ed 3.7biU.ion year s old~re ~mewh~tdifte,rentio,thnt_t~containpeculiar _~, layers'on ron,~hichalmostcertainl yevincethepresenceoflife (Futuyma.IQS3).

Iossilsotba.eteria.like.Iorme'thatwete.

. ..

sediments. Redlceetlve da.ting orthese

. ' . . ' .

3.2to3,5'b·il.1io~^'yearsold{Dayhoi t,'197~i

,.

_10··

. .

^{~ ,~.}

^I

- ^{T ... ... I}

^,-~

/

'" ,-

~~=

~

- ^{, .}

~

- '

-- ^{'" ....}

- _m

-

. _'" ... - _{~J-}~ .}

" -

- ^{, .} , . - n"""...

~

- ^..

^{~ L}

- - ^{I '='-d}

~

^'

^{.... ~} - ,1 -

...

_~

^{_WiI ... ,}

-L....i

0.-.., ~

1 '· ' - .

F1gqr~ i~l : Geological

E ra.

andtbeFossilRecord

•. ,Source: Futuyme,tQ83

l ./

Doolitt le, 1980), .These inhabitan ts"of the earth were simp le microscopic organisms. )'I~ny^ofthem were.comperablein sizeandcomplexity tomodern bacte riaalt houghthecon,dition~under wh!ch these organismslived.dirrt'rrl'~1 grcntly from those prevailingtoday. Fossils-forme done billio nyearslaterindic ate

.t he presence of,blue-green alga~ (cyanobacterial wh ich were capab le or

pho tos\:ll hesis andtheproductionofoxygen, Rocksprod uceddur ing thisperio,1 >:;

p and,since then.revealanoxygen-etchatmosp here, There fore,it :lp'p'earsth:'!.t

' .

.,

^, \. " \

prokaryotic cellsw~rethefirs tlife-fo rmson Earthandthnt~theydomin at edI1In~t ofthe Precambrlnne ra .

1.2.Implications of,t he Fossil ,Record

Definingth~ti~e^ortheappeara nceor

the

first euka ryotesrrOl'lUh~~'^fosiiil record Is difficult.'T his is~ecnusethe.^{crite ria.bywhi,ch·the.J!}'nrlies t elika ryo tes differredfro m Iheirprckji ryc riecounte rpa rtsarefewa~inm~nyceses open to .debate, S~h?pr( 11l78)has.listed,aseries?~cr~tetbas edonsize,sh ape~nd.'

•morpho logicalcomplexitythat maybe'usedto~d irCl!rentia.~ebetween eukar yo t ic"

, ,

and prokaryotic Icssils. ~tructuralt~aracteristics'oC^{cukaryotesinclude}bran~ h('d^'",

~l3mentswith intern alcrosswalls,complex[e.g.flask-shaped) microfossils,large-' .\

nlgal crsts••internaldense-bod ies resem bling the residues01euknrycr lcorga nelles, endtetr~ds~fcells, possibl yrepresenti ngthe prcd ucu.of meiosis, The presen ce ofthesewelts havebeen

us~d

^{tJmark tbe ori ginoC}

e~kilrrotes.

Unjcrt umet ely, some ofth~sepr~ll.medeuk eeycuest ruct ur esare.dubiouss,incein experiments

, , '. ' I " . ' . f

performe dwitheult ures.cr hving blue-greenalgaeKnoll.!lndBarghoo~(lg75)

.showed thatanorganelle--li~e^massappear ed in degeneratingblue-green ~lg41

.

•4

cells. Oehle rand co-workers(1976)have foundthatblue-green algaecan Corm ooo-meiotictetra di.Theseresults eastdoubton thedireetentdat esthat have been reportedlotthe tirst appear anceoCtheeukeryctic cells. RecentlyVid al (lQS4)indicat edthat theoriginoCeukaryotes begec 1.6billion years ago inaCorm

,

^{' .}

\

orgreenalgae.Itisreasona blethen , tc.eceepethatadiver sity oreuk eryo tlccella was presentbefore0,9billi~arsago, but animal fossils dooat appear in profusionuntil thebeginiog of the Cambrianperiod - 580to6~Omillionyea rs ago.

. " -

', " ,

Based onec mps ratlv e morphology,thefossilrecord seemsto suggest.tha~

" . I,

the proka ryotic-genealogiesare r,at more:aileieni than their eukaryotic-"

.

counterpart s.,~a,resultit,isgenerall

^.

yacceptedwhho,:!tother:evidence, that.

.'eukeryotee evolvedr~om'prokaryotes:-However,th.e,questions of:how.andwhen

this.evolutionaryeventtookplaceremain'unanswered. Thefossilevidence

eecumulet eds~^Carsuggeststhat lireisflionophyl~t.ic:It~at^is,~nliving organisms aredescendedfromanan~ien~^anaerobichet~rotrophic prokarYote,^{'simiiarto.that} of amodernClostridium(Fox etal.,19S0). T'he firsteukeryc ricc~11was'

.' . . - .

^"

.

probably a unicellularalga,derivedCroina.prokaryotic blue-greena~!a,andit -was from this

sl~~le

euka eyot iccell

thlltbiglf~r

plants arose.Algaethatlost.their photo.syntheticability evolvedtoCO,rmorganisms such~protists,fungi

ind

,animals(Figure1-2;~oolit~le'"'UI8D;1~821;'Th e blue-green,~acteriaare viewed as theint:: medlateb~twee~-the'prokaryctesaDd-Iowe; -phOtOSYDih~tic^eukeryctes and thuSrepr,~sent ~^steady~evotutlonary^p:rogre'ssion:

"

:Figure1.~: Evolutio~aryProgression ofEukaryotes alldProk:lryotes.

Source;Doolittle,IgSO

~ ,

-,2BILLION

~ '

:-J, BI LLION

~ .

\ EUKA RYOIES..

ICo)TI plllCells,Wllh N~ C I.II PLANTS FUNGI ANIMAl S

' ~ _~t .f _'~ '~ _·

~~ ,

FI RSTEUKARYOIICCElLS"

' IPROTISTSI'

INANI MATE MATTER . .,".

, 0

.::

"

~4 BILLION

PRO KARYOTES (Simple Cell s.~NoNuclell

-...

NOW BACTE RI ABLUE-GREE N

1

^ALGAE

I

^'~

;,;:

^0'0=";

-I BlLlION

\

.".;. -

f

.

^'

1.3 .Morphological Evolution

,

Atacitas.sumptioD.ot the fossilrecordisthatmorphological changes renect g,enomic change" Itisgenerallyaccepted

~t th~re

^isa.rela tionship between morphologicaiand'genomic evolution, but morphologicalcomplexity'maynot in .teet.bean accurate indexoCthe extent~tgenomicevolution[Sebopft:lai.,HJ7S). . , . .

\,Thisi~suppor ted-b'j evidenc~Jreviewed byWilsont:lat.;19j j j that some vertebrat esexperlenee(aSte~rates~fphenotypicevolution'than others. For

.'. . ' ..

beento.ecmper ethe sequences or specificgeneproductsfrom diCferentorganisms.

- ^.

example,Crogs'have remained esaentially'unchanged morphologicallysince'the

" (irst. ~ro~·~ppe~red

whereee thep;esent'day

ma~mals" show

^a'

gre~t' ~~riety '~L

,sbapes

compar~ .~ith their: comm~~\ncestoh~' both

^Irogs

'~~d

^{mammals .,}

sharethe,same rateof

'e~olutio~

at.the protein

le~~l.

^{Itis'difficult}'to assess'the

.

.

.

^{" "} "

.

relative .ext,ent oC-genomicevolutlon,.ot'organisms (rom compariscne of morphpiogicalchara cter iStics; One approaoh to investigatethisconundrum has

1,<1. Molecular Evolution

.':"""1

Manyproteineequeneeehavebeen determined andcollected.intoav'e,ry

·useCu.t,g;.owingcompendium:~t:'Ailatl""bf.Prot~in^Sequence.!'a'n~5.'ruclures (Dayho(!"1078) , Aminoacid,sequence compariseneofho~ologousproteinsfrom dirrere~ 8p~durevealed thatth~^primarystr uctures.',0.'proteins(li~rer i~':a phylogenetieatly,consistent, manner,' T hatis,themorecloselyrelatedthes"pecie~,

t~e.'·more - sim~l.ar

^'

iJ, t~

^{·t he·aminoacidsequencesottheir}

homOI~g'ou~

^'protei.ns.

Divergenee

ti mes

'"oforg·a.nis~·^{een'beobtainedtro.m the}geologic~re~ord,^Thus. the

r~tes

of amino atid

s~bs ~'it~'tion

can b'e calculated'(or severalproteins.It was

, . .\'.

'.

round thatthe rateofamino acid5~bstitutioois coos'taot·within a particularset or bcmclcgousproteins,but ther~tes~renot nec'essarily the.5a~etorproteins havingdirterentJunctions (WilsontIai.,19,i7'J, Thisis~~Iustratedin Figu're1.3, Theseo~servat~onsIcrm the'basisferthe concept'or theeMoleculnr Clock"

(Dkk~rsqnandGeis,~IDeO),

•The

rat~

^or'prJein evolutionis

_. me~ured ~n te~ms' _"

^orunit

eVOI~tiOnnry

period(UEP},.i.e.the time in million ofyearsrequired for cueper cent ebengein amino acidsequence to accumulate'betweentwo~diverg~ntli~eages...Theg~eater the UEP:thesl~we~the rat e orc~ange:.,Ithasbeen observedthat there isagood C:or.r~la~ionbetween-:the,rates'ofmOlec'ular evolut iono~proteins and-!!l eir b~ologicalIuneticne.Fo~exa~ple, fibri~opeptidesevolve,rasterthan,cyto~hrome .~,·Fibrinopeptid·es hav~little known runctionarter'they~re .cu~ ~uf^orfibri~?gen .~benit~cdnve~tedto'fib~itiin a'-b1ood clot, Vlrtunlly'any aminolici d change thatpe~m.,thepeptidee to,b,ere01:0vedmay be acceptabie. Ontheother hand,.' cytochromee iote.ncts .with.macromoleculessuch,as cytochromeoxidaseend

I,.cytochrornecreductase. Itsrunctionismore sped'fic,andany.e~angeIn the ,st ruct.ure. that.,alters

t~.e

^{ru.actionis d.}

~trim.n'.1.

^{Thererore,'iY[O'hrom.clsmore}

conserved and itsUE?isgreaterth~~that or tibrin,opeptide,Table 1·1shows

theUEPsfor several~teins, .' • '.I ' .

.

.~'"

. '

^{'. ,}

,

Cytoeh~ome

c

b

roundin mitochondria of nUcleatt d ellst;'nd

&\

protein

\

.

hasbeenused to examine the evolutionaryrelntioDships ongeukeryctesT~e amino acidsequencesorcytochromeehavebeen deter laed rormorethan 35

t

speeles end e _ , ,"treebaeedcn • •

,~.f~· ·-····

,,~:ii(;;.,~~~,,~," ':-I~~',;~',~',-,'J,' ,j..A'"_,~,::i~:l>"':;.:""\.:;~•.f.:"':.~,,_,,:,)-..: I.;z~-<.;~,.~,; ~, '~';~;:>:~;:~;";~-':;";'.,,,';~

. ..

• --1.", •

\F.lg~~e

^{1. 3.1"Rates orPrct elaEvolution}

. "soure!:

Di~k!r:so~:

^Ul!l"

, . I

i .

•,1r

i

"1-

.'

"~"

.CAP DHdenotesClyeeraldebyde-3-pb~p.hatedehydrcgenase'

. .. .

Table 1-1: uEPstor

Se~eral Prot~ins

-I.

U,E.P,(mililion years) Protein

Fibrinopeptide

•Albumin Myoglobin Cyetoebromee Triose-phosphateisomerase GAPDH

Histone4

constructed (Dickerson: 1011): This phylogenetic tree isconsistent,with

':i'<i

conventionaltaxonomic relationships that have been~erived fromclassical'

~ethodssuchas comparat iveanatomy,embryology and.paleontology. Thus,

, I

evolutionary data at the;'molecular'level provide an invaluabletool for determiningrelationshipsamong orgi:l:nism's,10 perttcular,itispossibletopredict • whe~org'anisms lastshar~d^{'a common}~ncestor'even in theabsence ofafossil record.:

Althou"gh'so~~'prok'~rY~tes

contain a proteinwithsimilar Iunctionto the->q cyt~rlJ~~me

: ' c

fro,:':eukaryoticmitoch~ndria,'.it:is'not kn::rwhetherthe~~

proteinsore homologous.!herdore;eytoehrome~c~nnoibe':~sedto "determine the evolutionaryrelationshipsbetweenprekarycees and eukaryctes.

~.5.^{,T h e}EVhi'~~e~ro~Th~eePri'maryKingd~ms

The.:bilitytei translatemessengerRNAinto~rotein'^is',acommonre~ture^of

nlllh:ing,otga~isms.~~nce,,"molecule$~ciat~~.wi~tr,ansl~tio~alapparatu s

b'---'-'----,~--'c---

to investigate the..

~volutio'nary

originof prokaryotesand

·eukaryotes. RibosomalRNA (r.RNA)~~mthe smallribosomalsubunit was'onep.r

.theCirst components,10be investigatedindetail[woeeeand Fox,,HI;;), Comparative.enalyses of thestructures'of the res and 18SrRNAsIrorn prokaryotcsandeukeryctes involving ailoligonucleotide catalo gingapproachwere .'.

us~d

^{io examinethe}

Phy;~ge~etic' relation~h~p: ,~~g 't~e:e

orgsnisrns. Each rll.dioa~tiJeIY~labelledi'RNA.was digested with ribonuclease T1and"theresulting fr~gme~tswereresolr ed:bytwc-dimenslonelp~per electrophoresisand.then 's~que,~ced,Thus

a

cataiog•or oligonucleotidesequencescharacterlsticcl a specific o;ganism'was

obta.i~ed. Associati~n

coefficientswere

caiculate~, r~r

^all.thepair-

,_;"1,,

" ~';'bi:.tiOO~

^',or'thm

s~-talo

..,Althougblb. "I.tion,bip b' lw_S,o"b" y

\ 11 • ' . association coerleieate

id

^{the actual}Dumber or nucleotidesequencedlUerencesi!.

UD,kDOwn,,these valuesw~reusedtoecnetruetdendro~ams. Thep~yloge~ythat

•res~ltedi~om^thisa~aIYs~^{iadieatedthat}livingsystemscan becategorized"into

" \

threeprimarylineages:lhreubacteria,theurka.ryotes and the eecbeebeeteele

"

(Woeseand~OX'.1071;

F l ox

^tl al.,IQBO). Itappear! thtlt these Ilneegeeare equidistantCrom"one another ,and thatth~ydivergedindependentlyfrom a

\

'

common'a~cestor(Figure 1

14

^).•

The

eUbaete~ial

^{kinJJ:mcontainsall the}\ypkal bacteria. Itcan

b :

" . I , '

eubdlvldedinto..the cyanobacteria,the_gum_positive,andthegram -neglltive bacteria.The urkaryotes,'

t t; -predec~~r

^or'the

euka.ryot:~, -~are

^defined

-b~ the simi~ariti~

ⁱⁿ

t~e ~ttucturJ I '~r thei~ ",'

cytoplasmid

. . . l~S r~As. . .

'Eubacteria and

. '

·urkarrotes"correspond to pr.okaryoteand,eukaryotein,the cOD,ventiona.!sense.

The thirdkingdom,the"archlebacter ia,compriSemethanogens,

thermoa9;~~Phile9

^.

and halobact eiia{tor

rev'iew~

^'see Doolittle,lOaO}.'T hlshypothesishas,re'ceived suppct tlromiedependen,etudlesonthe three dimmion'lstructure s of ribosoins!

I . :

small s.~buoits

^{(Lake dat,}

lO~2;

^Lake,

lO~).

^{.. (}

, \ "

LO:'HowGoodlatheBvlden eefor Thr eePr imaryKlng d o ma1

, . \ ' -

.

Sequence compariso nsor complete165'or155-like'rR~As(ISS) Cram

, I

org~nisl1l3 repre5entative,oC';the<eubaeteria,.urk~ryotl!!l and.archaebacteria

·reveal~dthat the primary !ltr~cture,o! the'archaebact~rial165rRNAillmore similar to both its'eubecterle land eukeryctlccounterparts, tban these twoareto

I • . . •

oneao~ther^{(Gupt a d}al." 1,983),Thl!!l_ere!l~lts;ugg~t^that'therat~'oCev~lutloD^. in the'_~ a;chaebacteri&l'.

ii~eage

, ^isslower' ^thanin.

th~

^other

t~o

. '^lineages," ^aDd

th~t

;

" ^{", :',-1 .. . .}

" . , ••• 0f-

;·><.i~;;L~l,;~;,,~·~·t:

I'

; UKARYOIIC

cmy ."

•CHl OROPl A.STS NU9{EUS' .MI TOCHONDRIA PROKARYOIES

.J

".1.

'·1

Flgu,re1-41'.IndependentEv6111tioQorArchaebacteria,Euba~teriaand' Urkaryotes; . ,

"·4

BILLION"

YEARS

Souf5e:Doolittle,

loao

,.:.,0" ,"" .,>,,: '."-',":'' """;"~,.",

13

thestru ctureo!the 165 rRN Ainirchaebacter!1.iJclosertothatorthe"commOD ancestor. OneoCt~e^basicBSS}lmptioDsDCphylogeneticst udies at. themolecular levelis thMthe rate orevolut ioniscoostantaloogeach:'lio'e-age. T~eterore,^tbe

~parisoDortp~155rRNA sequeaeeecasu doubt on thedeDdfl!Tgr~msderived

~11he'rRNA catalogi~gexp eriments.

-r.

"'

Otherstudies,DOribosomalcl,mpODl!Db.namely comparisonsorth.eam~D~

~erinin&lsequences.

o r

^{riboso mal}prot eins (Yagucbietal.,H182;Kimura ad'd.

. . I ' ,

Langner, 19S4), a~^{_.question the,}relat ionshipsamoogthe three'prima.ry. .'

~ing~O~s.

^,These

c~~parisons

^{indicated that}

athDeb~cier.i~

^{are.moreclOJely}

rel~~edto eukaryo te'-t hantothe eubacteria. Since'the'dataaccumulatedfrom

co~~~rative

^{stu'dies OD th,} molecules of,the

tr~nslatioJial

^{appa; atus are}

,

iBSurric!en~ ~d~termine

unequivocallythe phylogenetic

~rigins ~f p~~~r)'o:'tes

',/,\ •ead

eukaryo,~ ~olecul~~other

^{those.pre:sent in}

ri~me5

^should

b'~

^exami'ned,

,ThesemacromoleculesmustsatisfY_t~Win,crheri&:,. ..themoleculesmustbe ubiquitcuainnature,thatisbe present inall

organisIIlllregardless oftheirtaxoa..~micorganization,

.~th~lmllstbe conserved molecules,with aslowbut const:1n t rate of

evclutice;and '

.'',

th~,.

^{shouldcon;ain}

jurti~ie~t iDhe;~D't

information,'derivedfrom a commongeneticlocus,.toanowaIo~g rangt1lhYlog:~et~cst udy.

i.T.Can

Glyc~iytic

^{EJ;uiymesandTheir}

G~nes Scr~e

^as

" "Evolutlon~ryProbe~f ^{.' " • .}

.' -""hqco'!ytie

eDZY~es ~eem

^to's atisty the

a~ve

·criteria.··Th;-UEPjof,the

. ~ . ;

.glycolyticenzymes.examinedtodate, are surpwe:,only byth?s~ofhutanes

;>; I

t' .t~:__ .,.

"';~"";-'~.;:.::.:t-'~:~:~.}, ,,,,,,,::;4:'~l1 -;" '~"~':l-.';...:...;:;: ;~'·i\;o. ~~~~~~":::"· ~:j '~.;$& ~"k~

^..

{>;~~!.u:C\>:'>':~'~'i-~~," " ~,~.:

^...

,:-~:;.~.;:.-~

....

.... 14 , '

, ~

'V

.. (Willlonel et.,1077;Fotbergill-G llmct e,1086;Table1-1). Thisia dieeteeth;Lt glycolyt ic enzymes arehighlycon served. In addition, glycolyti c enzymesare found in"311organisms,and are easilyisolated(Sp ring andWold,197 i ;Chinet»t..

19!3la )".

?\-I~{lY

^01theenzymesinvolved

iD~his

^pathway

h~ve

^been

inv~tigated.in

gre~td,tail(Fothergill-Gilmore,1986).

, /

For:,example,the'amine acid seq~nces'orglyceraldebyde-S pbosphate

dehydrogellMe (GAPDH) havebeen det ermined Ior a variety

o r

eukaryctes .including,human,,'pig,rat,~hi~ken,^lobsterandfeast~ rind three prokaryctes (Therm"u8aqua[icu8,Bacill u8', lea rolhef'1tlophilu,'and Escherichi acoli)(Milner

•_.¥.••. • • ",' '. .. . '.

dal.,~983;Branlaot dal.,1983; Hollandetal.,1083;Hock ing.andHarris,1980;)... ....

Thesesequences'areolsim~~arle~gth',~pproXimatelY333a~inoacid residu es,and"

th eiralignmentclearlydemonstr at esthatthey are.homologousproteins(Dayhorr d,al.,lOiBI.Sequence

compa~~ons

^orth e primary.struct ureso·c'G APDHsfrom th e aboveorganismsshowedtha tthe reisapprox·imately.fiftypercent!tomolo~

between theeinyrn~o~prckeryctes andeukaryo tes. Thisind ica te,that this' glycolyt ic enzyme containssur£icient, informat ion to resolvethe relati onship amoDg prokaryotesand eukaryct ee. Moreover, the similarit yin len gt hoC

.

. ^"

. .

glyc olyticenzym esamongorgo\nismsprovid~^an,dditionaledvantegeover rRNAs in which"the"alignment

~r thei~ ~eq~ences

^requires

iilserti~ns '

^andio;

+ . ' . . : . , , '

deletions togive.maxlm um mat ch ing (Brimaeom~,:; 1M" ).T,hus,glrc~IY~ic en zymes andtheir geaeeshouldprcnd ean,e~~elle~t^{mea os.rce}st~,({)'ing^long

. .. ...

. : . " ,- '

^.

\

rangeevo~~tio"n.

\

I

Chaptee.z Rese arch Proposa l

. .

Kr12{str~in C~20;'H/rCllrpA58Im~tB,g~yV,u581 g:.n~micDNA'into,Irngment!

withan average'size018.4.x'lOG daltcna. Thes~',lragmentswere treatedwith~. ~^.'.

,. '..

. . . .

....

-- , .

5'-exoDuclease andtheninsertedintotheEcoRlsiteorthe ColElcloning-vector

. ' . ^.

.;.{;::-

The main purposeM this project~~to investigatethe,possibilityotUSlllg cloned E,,coU~ene!l^encodingglycoly,ti~~nzymestor,long rangeevofu.tion~·rY·

·'studies-.

B~ed

^{on t.heavailabie}p\otein data,itwasanticipated th:t

.the':9~~dy'ot

^..7' these'geneswouldprovjdesomecluestoJ,he..phylogenetic relat!onship8lw,ithin prokaryctee;:and between

proka;~es~

^{'an d}euknfy otes.,This'project

~~

^a'lso

designedto formthefoundationtor tut:urestudiesc~ncernilig'^the,re,g:uiation and e:C,~teSSioD'^{.of'glycolytic}.genesin prokary'otes,.and to deter.mlnehow these

·'proka.ryotic·gen~ ~iffer.(romtheireukaryotic~o.u~terpll.r}s.

. 2 . i.

^Purpose

2.2.ApproachofInveet lgatlon"

Thesourceat~hecloned E,coli glycolyticge'Des wee the91~rke-'CarboO:

·clone b.• nk,·

Th~

^{cion.bank was},,'n,t,nct.dby CI"k.andC.d"m in19'6; H

~ ­

,, is

a

^{gencmielibrarywhichc},ontainshybrid recombib,nnt.pleemidere~resenting^the

,entire' E,coli genome. Thepleernlde wereconstructedby.,~hearingto,tal,E.coli

:, \

' .I )

" , .

"

I

U~iDgP?!ydT·d.Aexte usioue. The resultan t hybr idpla9~idsweretransformed into anotherE.coliK·12 (strainJA200: F+, alrpES,recA,lhr ,leu, iacY)(Clarke andCa~bon.^{1076): Thehybridplasmid!pLe16--4,pLeIlJ-:i7andpL.C}

n-e

wereeeleetedtorthis project.These plasmid!Werefi~gtident ifiedbyThomsonet

,

ai.(lg7(J)tor.,theirabilitytocom plement E.colimuta tionsat the triose- phosphate isomerase(tpi)locus(pte16-'4) or in the enolasegene(pte10-41and pLe 11-8).

Tbi~'proj eet

was

dividedinto twosectio~s.In thefirst part{Chapters4and S} these three plasmid!werecharacte rised.Thisinvolvedcon~tructiogrestr iction. .endon ucleasemap4,lo,dl ingthe positio n,or the1ector ineachhybridrecombina nt

plesmld;anddetermining the app roximate.positionot. the glycolyticgenes. The secondpart,(Ch~pter⁶⁾i~volved^{using.thethe cloned}E.coliglyeolyt lc genes to• examine.the'extent ofrelate dnessb! ;eenorganisms:

G~nomic

^DtiAfrom

represen tat ives01theeubacteria,archaeb acte ria..and euka ryotes'wasdigested witlir~strietion en~oDu.sleasesan? subjectedto^{Sout hernblotanalyses}usi~g^the plasmidspLe

16-.t'~~d

^pLe10-47 as radioacti;ely-Iabelledhybridizat ionprobes.

.~

··'i;

11·

Chapter 3 Materials, and Mjethods

\

3.1.Mater iall!l

Salmon sperm DNA" t\-licroCOCCU8 IY4od~jkticu4 DNA, Clo4lri~ium per/ringens DNA,ribonuclease,pro~einase, K,an~chemicalsunlessotherwise stated were purchased trom"Sigma Chemical

ce.,

St Louie,MO" USA, Acrylamide:bisacrylamide,TEMED,and"a mmooium'pereulphete were BioRad produete (BioRad~an'ada^Ltd"·Mississa.uga~ ~ot.)'.'DEA.E.Sepbadexwa?bought• tra mPharm~ci~(Canad"a)Ltd".Dorval,·Que.Restrict ion enzymeswere'obtll.in~d tram:'Bethesda Resear~b,Labofatc ries (BRL) lac" Gait bersburg,MD.,liSA.

(l32p~dCTP'and reagentstor nicktransla tion were'bco ght"from~er'sh3m, Oakville,Ont:

3.2.Strain sand.P.las m ids

CulturesotE8cheric~iacoli (JA200) andE.coli'containingeithe rpLC 1()"41.pLe'11·8orpLC16-4,,:,ereobtainedfromDr.Barbara'J.Bachmannatthe

: •I ." . . ,

E.coliGenetic'Stock Center,New Haven,CT,~·USA. E.coli (\yAS02'l harbouringCol Elwee a girt tramDr,·L,Visentin,N.R,C,Ot!awa,On't~ The namesandsourcesatotherorganis.nuusedin'these studiesaregive~io Table i.L

",:"

'l"

ATCC denot.e s Americ anTypeCulture Collection

N/A denotes~otAvailabl'e . ,"

,

-

'.:~

:~

) .

~ ' .

. _t~A

,j -:,'i ,,~~

12011 NIA 8090 .25~31^{" ;}

9240 •

EIJ048 E 495 ,. E!"'3S80

E13883 NIl..

N7A.~."

NIA E238S6

7469\"

NI A H/A • ATCC

Biologyuept• , HemorialU~i ve,rs i ty Biology~pt..,,Memo ~ ia lUn ive rs ~ ty Biolog yDept.,MemorialUn iv ers ity. Biology Depl., Memorial Universi t y

Biolo~yDep t.,He~daluni've rsit y .nr,J.Wright.He~r.ialun ~v~rB i t;

Dr•.J."Wright,HelDOrial~Uni v er sity

Dr,

J:

Wright,HemorialUnivers it . Biology'Dept. ,Hemo r i a ~Un ive rsi ty Dr•.J.Wright,,Hemor ial University D.r .J;Wright ,Memorial University Dr.E.Bams.leY~Hemori;l'Urii y ersit y

Dr.E.'Ba m Sl ey,Hemorial,U-rtiversity

Biology 'Dept.,Memorialt:Jniversity . ,Dr.~W',F.,Doo lit't\e .Dalhousie Universii'y

Dr,W. F ."Doo li tt l e,Dal~usie U~iyersid

Sou r c e T.i.ble3-1:.SOurc esofBac',,",ria

, ~r~;~~

S,ll'm,'lll'fl',lrll,'f/'t'lt' ~lI i~

ISalmonella.tlJphiftll/u.um"

CU'l.obac::teJt6~ew1d.U.

Sh..igeUa6DI1I'l"-<' ,Pl(oteubI!Wl.ab.l.Ub·

{tttVlQb~ct~^{a.VlOgelll!.6'} E1UII&Li.acaJtotOV01la SeM.a..ua. IIlo'tJlCUCl!.~

Ktl!.b,iclta. pneumorUal!.

''''''''''''illA

^tMd4

YeJl.&.in.ia I!.ntVl.Oc.ot.i..Uca.

P6w dottanah'pu.t.ida.

Baeil.ltJ.6-6ubt.i1..i.6 Lae tobttc.i.l..lu.!l CMei Halob~c.t~luttobiwn Ha1.obactVLi.wn vofCitttU

-,:,)~

~:'

"

/.

J.

10 3.3.Growth MediaandBurfers

Yeast extract, beeto-tr ypt cee and beeto-egsr were'purchasedtromBritish DrugHouse(Canada)Ltd., Halifax.TherecipestorLB,Mg and othermediaarc giveninAppendix1.-Pheconstituentsorthe commonlyusedbujters(e,g,LamM TE,

sse,

TBE,Denbeedt's solution) aredescribedinAppendixI.'

3.4.Iaolat~onof PleemldDNA

Acult ureor.E.'coli containin gtheplaSmid""~f^{inter estwas}grow~^{overnigb t} ,at

37°C with~ut

shaklng) n 10 PII·LS.1 mlorthe

c~ltu~e w~

^used

~o

^inoculate

250 ml LB and the

E:

^coliweregrownwithvigourous

shakin~310C.

^W'henthe

Aeoo^reached0.5,cblcrempbenie cl wasadded toa finaleoaceut raticn of 170

I .

us/mland'theculture wasallowed toshakeJoranother 12 to. 15'hours.The~ells wereharvestedby centrifugationandplasmid DN.A."W88 prepar edBc~or~ingtothe procedure.of Maniatis.

e l

ai, •(1082),_usi~g ^the_BlkBIi/~DS ^{lysismethod..}

.~ovalently_.dosed circularplasmidDNA

w as

purifiedfromcrudeextrac~by·

equilibrium centrlfugationIn cesium chloride containing-,ethidium bro·mid'e.

PlasmidDNA .wesextrac ted fromtheultracentritug etube-bypuncturingthe side'

witha 21'gauge needle,anddra~iDgthesolutionintoa syringe. Ethidium...

bromidewas removed

fr~m '

theDNAbyisoomyl alcoholextrac'tionand the eeslum -ehlorlde wasremoved by extensivedialysisagainst10m.~lTE. -T he purified plasmidDNA~asrecovered.by ethanolp~ecipit4tionat.2~~CrfOIl~...ed bycentrirugation .The plasmid

DNA'

was dissolved in 10m.\ (TE.Theinte gri'ty orDNAprepar~tionswaschecked by agarose gel elect rophoresis.

20

.3.5.Rea~rletloDE:"~ODUclc_eⁿicellt lo n s

Table3-2,howlthereeopition Seque nces01tber~tri c tioDcndODudeun usedi~ tbilll_pr~ject. S.~ples^orD~~'^weredilnt~d^ovunightat

370t

in30.1:11 reseuee midura. Core"buUe.,(suppliedby thern.aDulactu~er)...a"usedin all restrict ioneD~oDudease^{ru ct}ioDS excepttorSmatwho 20mM

xci.

10rn.'-t' TrisfHCl.pH.8.0,10m,\t:MgCI2and 1mMdit h iot breitol wasused.

3.0. Agarollc GelEle~tropb~'resis

DNA'fragments (cDu ate d by re neteuce endceuejease digestion were separatedbyelectrop horesis~D'0.8or"1.0%ag.roscgels.TheburCers~stemused

. ^{' . ..}

was.TBE.-.4J'tereleetro,phoresis'~he'_ge~-were .tainedin0.5ug/mlethi~iu~

b;om ide tor'balt anhour

.

and.the DNA wes vi.s~~liz^.ed by ult~a.viotet tr'~i.llumiVatioD(~Itr,~violdPro'duets ,tee.,'Sa.nGabri~~l,^?alilorni a.U.S:A.):

The-results wererecorded~y_photographing thegel"usinga Pclar cldMP-4· ·

.

;. . ... . .· · : .01

3.7 .Growi'hor Pro karyo tes.

Bacterial cult u ret.wer e,atarte d (rom siDlle colonies rro~ agar plates.

Enteric bact eria

..a':r

^{BlJeil h,.'e u6tiliewere}grown in'LBat

3t'~.

P,e udomon lJe pulid o

w~

^{gr9wn in}

~

^at

~C.

^Species

~r H~ob""cteria

^{werecult ure dat}

~C·

^.

.inbr~th ~ntainibg^{a hlgb}~oncentration^{or salt}

(S;.

AppeDd~^{.1). .}

\

21

Restrict ion Endonuclease Recogni\ ioliSite G"GATCC GCCNNNN"NGGC A"GATCT .G"AATTC GO"Ce A"AGCTT G"ANTG GGT AC'C

e-cce

CTCGA"C CAG'CT~

GAGOT"C C"T CGA C CCC"GGG T"CGA T"CTAG A

T"ble3-2: Restri ct ion Endonucleases

8 f

^~heirRecognitio~Sequences

•d4!DOt~thesiteatwhic h the endonucl easeeleaves Bamm

Bgll Bgi D EcoR1 Hae m Hindm HinEr KpnI

","pi PstI Pvu'U Sael .SaI l

"SmsI Taql Xb>l

22 3.8:Isol~tion

' or

^BacterialDNA

Bacterialcultu ree'from theorganisms listedinTable3-1'were.allowed to

, I . ",

grow untilstatio~aryphase. The cellswere harvested,Iyzedandgenomic DN!,- was Isolatedby the procedureof Mar mur(ig61). These crudeprepara tions

o r

DNA and.Microcouu!fysodeikll'cu8DNAandClostridiu mperfrill ge1l 8.D~A purchasedfrom SigmaWerediss';lIvedi~^{10 rnM TE}and RNA wasrem~ved,^by ....,. theaddition_~hent-tr eated"ribonucleasea~

a

^linalcopcent ratlcnof 100ug/ro l.-.

Art~r jn.cubating~ror.~:.~?~r ,at

^{3!DC,'thesclutkmwas}

~~ju~ted'

^{to,0.5%5DSand}

109-ug/m l

p.roteinas~

^{K',was,eddedThe}

~ixture, w~ ~urther'

^{Incuba ted}

a t.

500

q

Cor2'hours endt~en:deproteln b edbyextra cti on'with,anequa lvclumeotphenol

sntu~~ted

^with)O

~ T.rislHCli 1 mM E~TA~ O~'5'%

^{SDS,(pH7.5').}

~h~

^aqueous

andphenolp,h~eswe_resepeeatedby centrifugat ion at 4,OOOx'gr~r'li;mi~ut.es'~t.

~OC.

The aqueous laYier. .

'~as a~j'us

~

ted' t~

'^0.2

M _{s6(li~'~ ac'e~ate}

. (pH

~.l),

^andthe

nuele.l;acids'were~reciPitated,~Y,^{theaddit il:m}or two volu mesorethanol. The'"

purified.DN~'wasdissolvedin10~m~TE anddialyze~exte~sively'againstthe

r _ • _, - .

same bufter'berore.use;,'Tbis,procedur e yie!dsbighmolecular,weightD~Awith anA26~fA280ratioOr·1.8·te.,2.~. .

3.9.,S(J,u~hern'Blot tl n g

" . . ,

. .

, . /

.

.Afteragaresegel elect rophoresis, DNACragmenlsweredenaturedinsituby

" soakiDg 'th~ e~t1~

^gel'in'

~oo ~lo'C ^{0 ;5} M

^{NaOH/ 1.5'MNaCI,}

witb" Co~;tant

ag~t~tion ~~room te~per~'tur~. '~he 'geI9:' ~ere incu~~te~

^twicelor45

mi~u'ies.

,

" . .

^" " , ' ^{' " ,} .

.The'~els^were't1ilmne~tr.~U~,edi~:^thepresencef?,~ O.5'MT~b/HCl,^,3MNaCI,p.H

7.5:"~'it~ agita~iori~

^-

T~iS st'e~

^'wasrepeated

tWj~e

^lor

30"~i~~te9 e~ch

time. Alter:\'

.-i·

23

neutralization,"theDNAfragments fromplasmid,were.trlUl5feredtoGen:esci'een.

JNew·E~~I8!'d .Nuel~':"1 BOs~n.

^MASS.,USA)

uSin~.10x sse;

^according

~ th . ,

manuraet~rer·s.lns~ruction,(MethodItCt!a1ogNo.NEF-072).'For_So~the;~

,

blotting

o f

^genomic"DNA,~iod1De^A'Dylon~embr.nes^{(PallUlt rafineFiltration_} Carpe ratio n, GleoCove,N.Y.)werewed. DNAfragm entswere-ru.edODto the__

~

... . .

^~

.

fdtersbybakingthemembtaneir~r2·4 hours atsoGeuDd~rVAcuum.

3.10 .Nick Transla tion.

Plasmid,DNA'Wasradlceetlvely-lebelled with.Q'3~P~dCTf'"usingII.'nick

'trans~lL~io~ ,kit' purcbased rr~~

^{Amersha m. Thereactio n}mixture.wasincubated

at .100Cior gOminute;..nd the reastionw~stoppedbytbeadditionofEOTA

to

,8

t~ai ' con~~D{ratiori

^{"or 100}

ll~; Plu~id

^or.(A,wu.",·eparated.rtom:' u~i~~rporated^Cl32p·dCT P by~btomatographj,in'S"epliadexG-50in10'm.\{

TE

coD~^..iDing 0.1% SDS. The DNA.ltt-belled using thismethod.routla ely bad..'

•specificactivity'or 103 epmpetug DNA...

s.u,

Hyb'rl~latioq·:~Dd.

^Was,blng

CODdltio~1I

The Geneeereeafilterswereprehybridizedina!Oll1tion cO.lltain ing50%

rorm~~de,'

^.5XSSC,5X

De~hardt's

^!Olution,

~

^mM!Odiurt:!:

P~05Pb.ll.te (~H' ~.8j~··

...

:5oo·ugim~

^salmonsp!rm

~NA(!O~icate~,

depurinated,single--stranded)

"lid'

1%

.glycine.

App'r~ximately ~o 'ui ~.r" tb.e

prebYbrid ization solutio n per'cm2 or tilter

·

'are~ w~"added

^{,to"8;sealable}

P~a.!tic

^pouch

cODt~i~;ng

^the

GeneS.c~een.

^{The pouch}

:.Wa.!

he~t~,

sealed and'

iD~bated

- 31

'00 wi~h COD,~a!1t .git~tion ··r~r, ~+36

. hour.J.,·Hybridiu tioDwu carried

~~t

in 25 ul or

hybridi~ation burre~r-..e: m2,or :·

^.

· .. ...' '.''. - .' . "'~ ' .0-.

·filterpl~therad~attiveprobe (2500cp~per unit em ea~rt~e tilter)~a~42C

... .,.--; :,.:"!"~:"'::~'f

'",, '2:'

~.?'_}..-r(::,' ::':.._~...:.•,:,:",. e::'i:\{:;'; ~~_

,

process.

24

for36 hours:The hy bridlearion~urrerconsistedof50% form amide, 5X

sse,

IX \ ' Denhart's ecjuuoe, 20mMsodiumphospha~e(pll 6.8)aD~100ug/ml salmon sperm'ONA.·After'hybridizat iontheIilterswerewashedtwice in2X

sse ,

0.1%

---.

^{. "}

SOS3-uoonr1e~peratu refor 30minutes andthentwiceinl~SSC,.O.l%50Sat :10DCforthe same periodofliine. The filtersweredriedat room temperat ure

. .

.."

nnd,subjectedt~-a'u tqradiograpbyusingKodak X-OmatRPX.r~yIilm (Kodak··

,eanad~,I~c., Toron~o,Canada)for twoto sevendays, at.70,OC.A cassette_with

t

nnint;.nsityingscreen(DupontCrcnexjwasused to enha.ncetb~autoradiograp hic

3.12.Pteparatl~,n,.orExtracts.:

E.·io li .was

~row~'i'n

^Msmedle,

in"'th~ 'i>resence

^orabsenceof glucose. l-q'

o - the former_case,glucosewaSadded-ata,fin.alc~~centr~t.io~^0'(0.4%. Cr ude extractswere prepared fromE.coli(!A200)alone -and fromE. coli.(JA200) _containingpLC10--·i7,pLC11·8 orpLe

l~4.

^{,Celldensity'}

^WM monito;e ~y

mensuringtheab~rbanceat 600nrci.~The'cells wereharyeste~in themidlog phaseof·growth bycent rifugatiop.,,a~dthecells wereresusp ended in50..roM imi<!.l1,zole;H CI,

pH'

6.8 containing10

~

^MgCI2:

Toluen~ w~

^{addedto tbe}

;us·~en.;:~n^toa.'finalconee~tration^of.1~ ~eidin lysing the cells.Furtber lysis was.completedbytwominut es ofso nicationat4~watts, using a'Scnificercell dlsruptor (H'eat'Sy;tems,

Ullta~sOD~C$

^{Ine.,Plainview,N.)'). The};cells were soolcated.,f9!'30eeeonus, followedbycooling on-tee 'water,fo.' Cine minute.The

sonie&ti~~·'~nd ~~JiOg ste~$

^were

rep~~ted:

^{fourtimes.Alter}

s~~ication',

^the'cell

at280nm or 1.

3.13,Enolase A8say

Enolase activity was measuredbyfollowing tbeiaerease inthe absorban ce

,

?r.udeextractsp~ep'ar.edas descr'ibedabovewere dlalyeed overn'igbtat4oC,

.against~ooov~lum~,9~ 'l~\tITrisjHCI,~H-8.5.. ~hedialyzed ex.t,raciwee

applied to acohlmn·(1.5 gm x 20 .cm) ol DEJ\E..Sepbadexequilibrat ed with10- at240urnusingaPYEUNlCAMSP8-100W{V'tsspectrophotometer.The'way mixtureconsistedof 50~timidizolefflC I,'pH·6.8,10fIlI\1~tgCl2And2mM'or 2-phosphoglyceric seld.,Theprot,einc_oncentration ofanextract wuestimated,.

Ircm its absorbanceat 280 ern. Specific activities aregivenin termsor the cbangein

a~sorbi.nce

at 240 om per ;;;inute

p~r

^ml01extract withanab:orban c&

. '',' .

mM TrisjH Cl.pH8.5.'.Thecolumn was washedwith50 mtot thisbutter and.

,then aHneergradi,nt10to,soornMJ(5mMper ml) o-fNa.CIinio m.\[,Trb / HC,.

pH8.5

(y app~ied. Alf~~ t~p gradi~~~ w as compl~ted,

^{I,MNaC! in}

TrIS~HC1,

pH8.5wu a pplied·.toth~ ~olumn"r:.ractioDswere testedtor enolaseactivity

"3_14,

sop

.pOlY~il~~ide

Gel

~l~e~~~Phor eSls

Crudeextractsor·cultures,'prep\"edas~escribedinSection 3.12,were subjectedto·electroph~r~sisunderden.tu ringc~nditionsin'10%polyacrYlamid~

geb~sing

the procedure'or Laemlli (1970).,

Bo~iDese~u)n

^albumin.

rabbi~ enol~e,

chickenlactate debydrogenaseand chymotrypsinogenwere used,as standards.

..r6/' pels were

s~~

with 0.1%-Coomassie Brituant

~lue G25~

^{in 1.75%}

perchlO~iC

acid(or two'htluu

.

J.Ddthen

d~tai~;d

in 7% acetic acid.

3.~5,DEAE-Sephadex.~hromatography

..1

28 .

.usingt~e,tand3!G.way and protein'wasmonitoredby.Collowingthe absorb,anee

at280om.

Chapter 4

Characterization of pLC 16-4

4.1.Backg round

Trjose-phosphate isometa.s~ (TBl)' [E.C. 5.3·1.1.1 ctLtalyzes the

"L.

iDtere~Dversion^ofd~bydroxyaeetone-phosphate^andg!yceraldehyde-3-pbosphnte.

This-prot eini;sone.oC"the

be~t

characterized

·giycolytic ·e.niYin~~. ~he

^amino

~cid'

se~uence^of-thiswo~ein^{h"asbeendeterminedfrom}eUka~yotes^{such'asbUmali,.} r~bbit,ClitaCen,~oelacan~th,^{andyeast ,(Dayhorr:1918;Lu e!al.,1(84)8ood}r~om¹¹ prckerycte,Bacillus8te;rot~ermophUIl9(Artavanis-TsakonasandHerrls,HISO;

Kolb,1980). Comparisonsoftheamino acid sequences showed thatTPIisa. highlyconservedprotei n;_9~ggestingthat all TPIgenes aredescendent! ofasingle primof? ial gene(Straus andGilbel t, 1985).

F;om

genetic'map~~g

^ofth'E.colichromosome,itisknownth'atth,e

geDe~"

encodingTPIand'phosphofructo-ki; ase(PF K)·are tightly linked to oneanother.

Thec~rom~somal1~cation^{orthetpilocusis 87.8mi.Dandthe}pI /: A genemops ot the.87.7 min region or the~bromosome(Bachmann,·l g80).Thomsonet al,(1079) .beveshown

~h~t

^the'hybrid rec6mbinant

pl~mid p~C

^{16-4rtofllthe Clarke-} Car~DclOD~bsnk'(Clarke'andCarbo D,19i 6}:compleme.ntsbot~^TPr·andPFK"

pbeactyp ee.Therefore,itwas'decided to~se

p Le

16-4 u a'soutce

of

the gene

with radioact ively-labelledC~IEl,

)

. :".',.!,',.'.~.

..

28

4.2;Re15ulta

4,2.l:Rest r let lon MaporpLC10-4'

encodingTPI,and PFK rromE.tolitoexamine the extentoCsequence homology at this"locus'among

Pt~ka~otes

and eukeryotes. It was hoped that the ... information derivedIrcmthese studieswould also indicatewhether ornotit.

would be T,easible in theluturetouse hybridpl~mids^{containingE.coli}glycolytic' genesaspr~bes,toisolate the geneslor glycolytic enzymesIrcm seehaebected e.

Beloretherecombinant~plasmidpLe-l~.acouldbeI1sed~forthisevolutionary, studyitwas necessary tocharacterizethe plasmid. A restrictionendonuclease map,~as^c,onstructed usingsix base f'eeognitioD enzymes. Tbelocation,o~^the cloning vector Col El in ptc 1~4was determ inedby comparingther~tr~ction

\ ~aps^{orpLe,16-4 and,}~ol^si,and by probingthe,~ec,ombiDant^{plasmidpLe16-4}

(

The.restrictioD endonuclease map Icrpl.C16-4was constructed from an .analysj:! ofthefragmentsizesproduced by single,double,'andtriple digests of pLC16-4.'.·TheDumber or rJ:agmentsproduced bya restrict ion enzymedepeD~

00thenumberorrestrictioo sites itrecognizes. For 'a circular DNA,the number

.

.. . ' . ~

~r.lragments g~Deratedbya.single.en.zymeisthe~ameas thenumber or cleavage sites mad:'by.thee~z~rewhereasi~.'~d9ubt.digestion,th8total:numbet~C ,rragme·DtS..obtai~edequal! the number orCragments gen,eratedbythefirs~e~zyme

plusthe numb@roCfragm@~tsprooue~d^{by the}sec~n~^enzyme:

.:. DNA.fragmentstra~el'through:.agarose;gel

2 .

inverselyprcpc rt loaeltothe logloofth~irsizes"[Helling dat.,191"). Thus, a larger fragment migrates at'a slowerrate thana smaller fragment. Aplot'

of

..

_{loglO(siJeinb~epairs]}versqe distance migrated bystandard markers enablesthe approximatesize ofthe unknownfragments to beestim::l.t~(Ta b!e4-~i'Figure 4-1).KoOtro'ing the distance travelledbythe DNAr; agment ofioteres t,thenits size can bedetermin~dCromt,hestand'o.rdplot.

The strategyusedf~rcons~ructing^'arestri't~ion^map

.wJS

asroliows'(Referto' Figure

4-?

~ndTable,4-2):Bsln(lane2)cutsthe'plasmidpteiG-oJ.attwo !ites.

to

g~nerate rr;~ents

that migrated'

~6

^mm

a.~d.

^{:109 mm}frpmthepoint"'\?C application. :These·'d.ist.sncescorrespondto·'size.spC11.5kbp'and'2_kb~

!espect ively. When

~

doub'jedigestusing

.~co

^RIand-Bgln

w<~

^carriedout

(I11.n;~

' . .

3),three Iragmea ta withsizes11.5,1.4and 0.6 kbpwereproduced(the0.6 kbp

rragyhe~t wl t~

^smallto be seen all.this'gel,Figure'4-Z), Table4-Z summarizes the sizes ofthe restr iction Iragments ofthe ditest inFigure4-2.

I

This showedthat~co·RIefeevesthe

2

kbp (Bgl

n·

Bgl0)to gi.,e.thetw~

•sm:a.ner

rrag~ents

(Figure

4-3~)J.

A.triple digest using Bg1.II, Eco

R I

and

Hi~d m

(lane 4)generate~Iourrr,agm_en t~lindicatin~that Hind

m

cuts(he11.5:kbp.

(Bgi Il - Bgl 0)into7:5and4.0'kbp;Thetwo possible permutationsorthisdigest·

a;e ,~sho~n

^{inFigure4-3(cl}and (d). The

relati~e

orien't·ation.or

t~e r;t~ict(on

siteswas achieved by-examining the'HindW/ SeoRI doubledigest. lhi.I produced

t~~

^fragmentsof

'~ize

^8.0and5.4

~bp

^',(Figure

4-3(e)I~ ·

^Therero;e,u'

illustra tedin Figure 4-3, the 5.4kbpEtc)R£

~

^'Hind

m

rraVne'nimust coo.tlh" a I

.0

Ta ble"-II MolecularSizes and Di3tanctsMi~Tatedby), DNA Dige;tedwith HindmorEta RI

dbtance(mm) size(kbp)

.'V _xDNA/Hind,

m

.7 23.7

4" 0.5

57 6.7

60 4.a

101 2.a

100 2."0

), DNA/ Eco RI

.7 '21.8

5. 7.6

60 ·S.Q

64 5.2.

~6

.,4

The distance migrafed by 't he DNAfragmentswas measured"in millimeters.

"

.

(mm)from-thepointoto'rigini.e,wher e~heDN~werefirs tload~din tbe~el~

/

FI~u:r;

^{,(,,1: A}Semi-logarit hmic

PI~t

o'r·Mole cularWelgbtMarkerversus

\ Distarice Migrated onaDAgatou Gel Themarkers', \lldw~tegener at edby.d.ige~ting.).DNA with Hindill or EcoRI,and.t\eirsizes andthecorr espondingdlstanc~tro..velled

•arelist~din Table4-1.

..,.-', :.'.. "31a

30

95

)

85 65

Distanc.e(111'11).. 55

" ,

'20

" 10

9

;, 8,

"" 7,

.

^'

~

^{·· t}

t~;;~i;"~~<.w~~~(~:~;.-;:.:;'-~',i,;:,~~~:,;:~ ~"':'«"':t.;:,,~.~:~<~L.~~~.::,,~::~~~~:,;"';:,~.'\~I~~;~~,;~.,;;'t\~~'i: ^.. ~.~~.~;:;,,'li.'~.~':;'~:'

^.;;.j'P

2 3 4 5 6 7 8 9 10 11

•

• ^• - ^- ..

^~

^- - ^{- - - - -} -

^;

^- I - •

- - ^{. .}

Ftg ure4-~: Restr ict ionEadocucleaseAnalysisorpte16-4

ThegelW:l,Selectrophoresed at100 V,in TBE buller . Lanes~-lO correspondtcpte16-1digestedwith:(~JBgiII. (3)B'1;;!U/EcoRL 1'1BglD/E coRlfHindID. (.;)HindID/E , o RI.(6) B<ID/lliod ID.

(7)B<\ U/Sac1.(8)Bgill/SacI/HindIII. 101 SecI/Hind III.

(10) Hind

m.

Lanes13D..!11correspondtoxO:-':.-\/Hindilland ),0:'\.-\/£ <:0Rlma rkers respectiv ely .

Table '-21A Summary or the FrilgmentSizesGeneratedbyRestriction

. f

^Digestllor pLC 16-4. RutrictioDEn,z,...

SilII Bl lIIlEco.HI 811IIlEco HI/Hind 11.

Hi nd III/ECo RI' 8gl.II/HindIII 811II/S ac-i S'llII/BaeI/HindUI Sac t/Kisd:II I Hill.d.'III

Sin(Diltuc.) kbp'

·11,5(48). 2.0'(109)

11.5 (48). 1.4(129). 0.8 • 1.5'(52). 4.0 (11). 1.4 (128),c.s,

•.0 (13),,5.5 (80) 1.5(52), 4.0 (11).2.0(l09) 9.p,(48),' 3.0 (83). 2.11 (11:19) 8.3(61). 3,0'(83).2.0'(l09) i2.5(44). 1.0

13.5(43)

r :

SeeFigure4-2lorthegelcorrespondingto thesedata

h •

..

4-3(d) bee't hecorrect fragment size and orientation. Ina similar manner,o,ther endon~aseswereml>~pedonpLe16-4 (Figure 4-4).•

4.2.2.LoeaUan

or

theClc nlng VeetoronpLC18.."

. .

The hybr idplesmld pLC16-4containsapieceorE.coli DNA insert,ligated intothe cloningvectorCol"EIby polydA • dT tailing. .TheI~ationof the cloningvectorinpLe 16-4Wa!Idetermined bya.combinationoCrestri cli?n mapping-and Southern blotting usingradioactively-labelledCol El as,the

1't.'

hybridiza,tioD?robe"

Dr:L.V19~n.tiD,·N.R.C.,Ottawavery.kindlyprovidedares~~i<:tionmapCor ColEl [Figure 4-5).'ColEl hasnuniquesite CorSma I.MpLC16-4.wuround to haveonly oneSmaIsite, thiS

~uggested~'bat

^the

u~.iqu.e'

^SmaIrest; ;cti.on:iiteis locat ed in the.region containingthevect or,Colsi.Using.tbisrecognitionsiteas areference position,,together'with three Pst land~vu,nrecognition"sites obtai ned trom theCol E;l restrictionmapconst ructedby"'Dr".Visentin, the map torpl.C16-4wa.s'_e.~t"abHsbed(Fi~re4-4).

To

lo~ate

.the position of thecloningvector,

Coi

^EI,Southerntransfer'in"·. conjuilftion"withDNA~DNAhYbridizatjo~'^{was used. The Col EI}pl~mid^was.

usedas a

3~P~labelled

^probeto

ide~tiCy

the:location or.t be

~1~niDg_

^{vector onthe}

"'tr ietio.m.ap.The plasmid

~LC I6-4.

w..

n ,;, ~i.,,'ed ~i'h

^{..ricue,••}

"ieti~. ?

enzymes, as s,eenin,~igur~ 4-16(~). Thett:lg~entsgeneratedweret~ent~/d. .. .. ,: to"Gen.esc:.eeQa.o.~.hYbridi~~dwjtlt.~adio·activeIY~labelledK~.O(,~i~g vecto~~. '.

, probe.T~eprobe b:iD.!~omologous^{to thecloning}vector.annealed,totragm~nts

35

' .

. ," . FIgure:

".~l Fonrrite

Map

o r

pLC·15-4,

B, E.and,Hde~\)te.re9t~ictionsites

o r

Bgi 0, EeaR1eadHind

m

~e,p~e~~vely