DNA, recombination, interactions, and repair : proceedings of the FEBS Symposium on DNA, Liblice, 1979

556 Pages·1980·18.492 MB·English

by  S Zadrazil, J Sponar, Federation of European Biochemical Societies

FederaotfEi uorno peBaino chemSioccaile ties
(FEBvSo)l umpeusb libsyhP eedr gamPorne ss
Proceedionftg hse 1 1thF EBSM eeting
RegulatMoercyh anisomfCs a rbohydrate Metabolism
Volume4 2
GeneE xpression
Volume4 3
BiochemiAcsaple cotfsN ewP roteFiono d
Volume4 4
MembranPer oteins
Volume4 5
RegulatoifFo ant tAyc iadn dG lycerolMieptiadb olism
Volume4 6
Regulatory PrEontzeyomleyastn idtc h eiIrn hibitors
Volume4 7
GrowtFha ctors
Volume4 8
FunctioofnA sl ternaTteirvmei nal Oxidases
Volume4 9
Albumi-Snt ructuBrieo,s ynthFeusnicst,i on
Volume5 0
Proceedionftg hse 1 2thF EBSM eeting
GeneF unction
Volume5 1
ProteSitnr:u ctuFruen,c tiaonndI ndustArpipalli cations
Volume5 2
ProcessainndTg u rnovoefPr r oteainndsO rganelilnte hse C ell
Volume5 3
CyclNiucc leotainddeP sr otePihno sphorylianCt eiolRnle gulation
Volume5 4
RegulatoifSo enc ondaPrryo duacntd P lanHto rmonMee tabolism
Volume5 5
MolecuDliasre ases
Volume5 6
AntimetaboilnBi itoecsh emisBtiroyl,oa gnyd M edicine
Volume5 7
TrendisnE nzymoloPgryo:c eedionftg hse F EBSS peciMaele tinogn
Enzymes
60 EnzymRee gulatainodMn e chaniosfmA ction
Volume
IndustarnidaC ll iniEcnazly mology
Volume6 1
Forf urthdeert aipllse aswer itteoy ounre arePsetr gamoonffi ce.
DNA - RECOMBINATION
INTERACTIONASN D REPAIR
Proceedionfgt sh eF EBSS ymposiuomn D NA
Libli1c9e7,9
Editors
S.Z ADRAZIL
J.S PONAR
PERGAMONP RESS
OXFORD NEW YORK TORONTO SYDNEY · PARIS FRANKFURT
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FEBSS ymposiuomn D NA,
Lib/i1c9e.7 9
DNA,r ecombinaitnitoenr,a catnido ns
repai(rF.E BSp ublicatviooln.s6;3 ).
1. De·o xyribonuaccli-edCi ocn gresses
I. Title IL ZadrazSi l,
IllS.p onaJr , IVF.e deratoifoE nu ropean
BiochemiScoacli eties
V. Ceskosloveanksakdae mvieed VI. Series
574.8'732 QP624 79-42641
ISBN0 -08-025494-2
Ino rdetro m aket hivso lumaev ailaabsle ec onomically
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PREFACE
The thirds ymposiumo n DNA, organizedb y the instituteo f the
CzechoslovaAkc ademyo f Sciencesa nd sponsoredb y FEBS,b rought
togethers peicalistfsr om the Europeanc ountriest o exchangei nformation
in a rapidlyd evelopinga rea of research. These symposiah ave
traditionallbye en focussedo n the relationshibpe tweent he structure
and functiono f the geneticm aterial. In this meetingp resentations
were relatedt o four topics:D NA recombinatioinn vivoa nd in vitro,
DNA interactionasn d DNA repair.
For the past five yearst hese problemsh ave been studiedi ntensively
usingn ew methodologicaalp proachesa nd newlyd evelopedt echniques
for cloning,p hysicalm appinga nd sequencingo f DNA. This,t ogether
with the potentialf or more exacta nalysiso f the complexp rocesses
in which DNA and other biologicalm acromoleculeasr e involved,i n
the coursoef storagea nd expressiono f genetici nformationh,a s
provideda basis fofru rtherg enetics tudiesa t the molecularl evel.
Thus,p roblemsr elatedt o the geneticc omplexityo f highero rganisms,
relationshipbse tweent he structureo f geneticm ateriala nd the
regulationo f expressiono f genetici nformationa nd genetic
engineerinogf microorganismsh,a veb ecomea menablet o experimental
investigation.T hesep roblemsa re closelya ssociatedw ith endeavours
to understandt he molecularf oundationosf livingp rocessest o create
a rationalb asisf or treatingg enetica nd virald iseases.
The resultso f more than 25 yearso f researchw ork devotedt o the study
of DNA, sinceW atsona nd Crick's discoveorfy i ts structure,p rovide
greath opesf or a usefule xploitatioonf all thesef indingsf or the
benefito f mankindi n termso f improvingh is geneticb ackgrounda nd
his environment.T he resultso f the symposium,p resentedi n this
volumeo f FEBS publicationsa,r e a modestc ontributiotno thesei ssues.
S. Zadrazil
J. Sponar
xiii
STRUCTURAL AND FUNCTIONAL
ANALYSISO F CLONED BACTERIAL
rRNA GENES
VenetiIa.Bn oerro,s C,s ordas-TK6itshs,,
P. E. A.
I.K isasn d Sain
8.
InstiotfuB tieo chemisBtiroyl,o gRiecsaela rCcehn teSrz,e ged,
Hungary
ABTSRACT
Thes eevnr RNAo peron(seg ne)s ofE .c oliw erep hysiaclyl
mappebdy r estirctioenn dnouclseea,s usign theS outehrn
blotitngt echnqiue. Twcoo pmleter RNAo peron(srr nBa nd
rrnh'De rei solatebdy t her eocmbiannt DmNeAt ohd, usnig
thep BR322p lamsidc loning ivceelh.T her RNAo preon
carribeydt het ransducipnhga gt.ed ar1o5E2w as fountdo
bea hyrbido f trhren Da ndr rnEo peorn.s The DNA
sequencoef t hep roomterre igonf orr rnB wdaeset rmined.
TheR NAp-olyemrasbein dign siteosn t hec lonedr RNA
opeornsw erel ocailzedb yf itler-bindainndeg le crton­
micrsocpoict echniqsu.er RNAt rasncirptiowna si nvseti­
gatebdy e lcetronm icorscopya ndh ybriidzatioann aylsis
oft hei nv itrot rasncripst.A ll thesfein dingwse re
correlatweidt ht heD NAs equecne, anda tentatiev model
wasp rpoosde to pelxaitnh eu nusuparlpo ertioefsr RNA
proomtres.
INRTODUICOTN
Oneo ft hem aina dvnatageosf t her ecmobinaDnNtA
technooglyi st hati t laolwst hed etailemdol ecular
analysiosf g enewsh icahr en ota menabtloec laiscsal
genteicm ethod.s Ther ibomsaolR NAg eneosf Escherichia
colbie lontgo t higsr ou,p nom utanhta se verb een
isolateidn t heMT.h et hreseta blerR NA( 61 S,2 3 S)
s, S
compnoenst oft heb acteriraiolbs omea res ynthiesetda s
a 30 precruso,r which siubsse qunetlpyr ocsesedt hrough
s
a copmlexs erieosfr eactnis.o Theg ense (poeorns) coding
fort his3 0S transcriptr edaurned ,a snetveraclop ies
arel ocatsecda tteroendt heb atceriaclh roomsmoe.
3
P. Venetiaaln.e r
et
These are the most active genes of bacteria. l.lthough
they wake up only about 0.8 % of the genome, more than
50 % of all transcription takes place on them in expo­
nentially growing cells. This high rate of transcription
is subject to several intricate control mechanisms. The
problems raised by this interesting system can ultimately
be solved only by a detailed molecular analysis of the
rRNA genes, especially their regulatory regions. In this
report we summarize our work, done in the past few years
on this system.
RESULTS
By using the so-called Southern-blotting technique
(Southern,1 975) we were able to deterl'line the copy
number of E. coli rRNA genes. BrieflyE:. coli DNA was
digested with BamHI restriction endonuclease,w hich does
not cleave into the rRNA operon, and the DNA was hybrid­
ized to rRNA after electrophoresis.A s seven distinct
hybridizing bands of approxi�ately equal intensity were
detected, it was concluded that the number of rmJA
operons is seven (Kiss, Sain, Venetianer, 1977). This
approach was then extended, and E. coli DNA was similarly
analyzed with 9 different restriction endonucleasesa nd
36 different double and triple combinations of these
enzymes (Boros, Kiss, Venetianer, 1979). These results
unambiguously established that the copy number is indeed
seven, and allowed the determinationo f the physical map
of the vicinity of all seven genes. This map, shown on
Fig 1.w as very helpful in the further analysis of rRNA
genes. First: it helped to identify cloned rRNA genes
(see below); second: it allowed the detection of hitherto
unknown rearrangements.F or instance, it was supposed
that the transducing phage AdaroE152 carries the bacte­
rial rrnD operon (Jorgensen, 1976). A comparison of the
physical map of this phagew ith the E. coli map suggests
that the RNA operon of this phage is a "hybrid", re­
sulting from recombinationb etween the rrnD anrdr nE
operons.
For detailed analysis of individual rRNA genes, and
esrecially their promoter regions we decided to clone
them. First the rrnB gene was cloned, starting from the
transducing phage Adrifdl8 (Kiss and coworkers, 1978).
ElectronmicroscopicR -loop mapping and in vitro transcrip­
tion experiments verifietdh at the 7.1 kB Barr.HI fragment
cloned in plasmid pBR 313 indeed carries the intact rRNA
gene of the phage.
Then we attempted to clone all seven genes starting from
the bacterial chromosome, but this attempt had been only
4
AnalyosfiC sl oneBda cterriRaNlAG enes
partilayls ucecssufl. Outo f2 000s creeedn rebcioanmnst
onlys eevnc lnoes containbeadce triarlR NAg eens. By
usnigt hep hysicmaalp s howonn F ig1 .i tw as eastyo
estbalisht hats ix of thecsleoe nsc arritehde r rnDg ene
and ocnoen tnaeidr rn.B Thisl attecrl oen howevperro ved
to breat herun stabel,t hem aintaenncea ndl areg-scale
preparatoifot nh er ecobmianntp lasmid temredp BK1 7)
(
wasd ifficult. Iti si nteerstingt on otet hatt hel oss
oft hep lamsida ppearteod bger auda,l througshp onta­
neosu deleitonss tartnigf romt hec lnoedb acterifarla­ g
men,t butg oing itnhteov ectorp lasidmi tesl.f Another
intertesingo bservationi:n severacla setsh ed isappear­
anceo fa nr RNAg enec arrygi pnlamsidw asa ccomapniebdy
thea ppearaonfca e n ew, eightrhR NA geinnte h ec hromo­
som.e Thseee xtrac opiewse rea los unstable.
rrAn El
:i'� ';E ;
-"'0 f.� "'=c. {� n.�+:r 8. -!..::t:..-.��- •.. ..-l..;n- -cr:Jt..� .. !;;_ia= -�:i x�0 --a:'"8
rr9n i s
8."' 5 a: ."0-!:o
:x: + ill IV>
rrnC � i = ;;
-iro � "'� "'0E c."' "'0 -i! Ii
.... x .... ..
rrn 0
-..;g.......ttla:..-�. o£°tl -..� ;r
-
E a
rrn -= ;:: ;;;
;ji;g l ;r i"Oc ilJlli
-
rrnFlorG) iE = -+
"'c.
rmG lorFl
-=
8.
ii
- :r
-15 -10 -5 0 5 10 15K B
Fig.l. Physcialm apo ft hes evenr RNAo peroonfsE . coli
5
P.V enetianer
eta l.
Forf urhtera naylsiwse choset her rnBg en.e Fig2 .s hows
thep hyiscla mapo f thtweo r eocmbniantp lasmidsc aryrign
rrn.B Fporr acticraeals ontsh es equenncgiw orkw asd one
onp lamsid2 1I2. An 1.6 kB longH indI II -BamHfIr agment
wasp reparferdo mt hisp lamsid. Itc ontaienda llt heD NA
ofb acterioarli gifnr omt hea ttr egiotnil lt he8 0ht
nucelotideo ft hem atur1e6 S rRNsAeq uenc,e thusi tm ust
haev contianedt hep romtoerr egi.o Anftetrh ed etermina­
tiono ft he deatiled physicamla po ft hisr eigo,n se­
quenicngw asc arreido utb yt het ehcniquoef M axaamn d
Gilbte r(1797),a ccodringto t hes trategoyut lniedo n
Fig2 .
;:11E" 1 :r0." E:rn E :f Q... :r11E" 1
i • H • t
235 165
pBK 17
23s 2/12
p BR3 13
:3 ...... :::: ::1 -- ---
:X:� 11�1.z::C '.:I:� �'.:I:'.:[� �::C�ID :J<C ��111..8'.:l: ::C
+H t H t t Ht t
100 0 -100 -200 -300 -400 -500 -&00 -700B P
Fi.g 2.P hysailcm apo ft her ecobminantp lamsidsp BK1 7
and2 /1a2n do ultineo ft hes equencisntrga tegy
Incideanltl,y itm ustb en otetdh ata tt heo there ndo f
(
thisf ragmetnhte s equencweas alsod eetrmineadn dt his
sequecned efnieda seocndarya ttachmesnitte f orp hage
lambda, Csordas-Th6t,B oors, Veneatnier,s ubmittefdo r
publiactio.n The7 00b pl ongs equenciem mdeiateplrye ­
)
cedingt hem aturreR NAs equnecet husp rseumabliyn cluding
thee nitrep romotreerg ioins s hwono nF ig
3.
6
A•GC TGAGACGGTAGTTTCTGACTTGGCACTAAAACGTATCCTACGCCCAGATGGAGGTCTCA£GGACACATACGGCAAGTTTGTATTGGG
-700 -680 -660 -640 -620
TTGCACCATTTCACATGCAGAAACAACCGTiT ACAAG!GCTGClAG AGGGAATATCCCTCGGGGCCGTACGTAGTGTGCGCGGTCGCCGAAC
-600 -580 -560 -540
G�G CCTGGTTGTT,AGAACAlT GAATAGAGCACACTACCTCGGCGCGATG!CAGATATGC�TTTTGTATGGCGCC�AA GACTTGGAACACAATT
-520 -500 -480 -460 -440
ATGTCCCGTTTTACAGCGTTCTGAAAACCGGGCCTTCGAAAACATGGCAGTTATGTGCTGATTTGGTTGAATGTTGCGCGGTCAGAAAAT
-420 -400 -380 -360
TATTTTAAATTTCC��CCTbGTAGATTACACATGCGctrCATAAdcTGCt:_CCACTGACACGGAACAACGGCJGAGAGATCGAC CAGCCGC
-340 -320 -300 -280 -260
I
CGGGGTTCTCCTGGAGGAACGCAATGCAAAAAGAACATAAGACTTTCGTACTGTAGCGGGAATATGAGTCACACCCCGCGCCGCT
C qr (J::
�- - - - L_
-240 -220 -200 -180
'
GAGAAAAA GCAGAGCGGCACTTGTCACTAA.ACTTTTATCAGACAATCTGTGTGAGTGACCAGCGTACTGTACATGTAACGTCGCAAGACG
-160 -140 -120 -100 -80
AAATAGAATAAACGCTCTCAAGAGTGAACACGTAATTCATTACGAAGCTAATATCATATTTTTCATATATTGTAGGACAAGATGTTTGAT
-60 -40 -20 -1 +17
Fig Ther rnBp romtoer sequnec.e Nubmerig nstratsa tt heb eginninogf t hem ature
3. 16 s
rRNAs euqenc.e Onlyo ne stranids s hwon.A rrowisnd iactet her estritcione ndo­
nulceaes cleavagsei teuss edi ns euqenicng.T heP ribwn-oseuqecnesc orrsepodning
tot het wop romotersa reb oexd,b rokena rrowisnd ictaet hes ietso fi nitiaiton.
Thep olymerraesoceg nitisoeqnu encaers eu ndreliend.
rrnB TTTGTATGGCAATGACGCACGCTGAACAATTATTGCCCGTTTTACAGCGTTACGGCT� CGAAACGCT C�AAAA 1CTGGCAG
rrnD GGCCCCGTGCAAGTCTTTTAG TATGCAAAAAA GCA CC TTTTGTGTGCG
rrnX GATTGGGTGT��..AATAGCCT GGCAGACCTGCCGCAAGC
-4�0 -460 -440 -420
rrnA CCTCTTGTCAGGCCGGAATAACTCC
rrnB TTTT CCTCTTGTCAGGCCGGAATAACTCC
rrnD ATT GCA.U\..lt<R-"1'1� fth-""-""� TACTTGTGCAAAAAATTGGGATCC ATAA
rrnE CTATTGCGGCCTGCGGAGAACTCC ATAA
CGCTTGTCTTCCTGAGCCGACTCCA TAA
-340 -320
rrnA --,T GCCACCACTGACACGGAA CAACGGCAAACACGCCGCCGGGTCAGCGGGGTTCTCCTGAGA ACTCCGGCAGAGAAAGCAA
rrnB GCCACCACTGACACGGAACAACGGCAAACACGCCGCCGGGTCAGCGGGGTTCTCCTGAGAACTCCGGCAGAGAAAGCAA
rrnD T GCCTCCGTTGAGACG AGAACGTGAAACACTTCACAGGATG CTCGGAACA AC GAA GAGAAAAAAA TCC
rrnE GCCTCCATCGACACGGCGGAT GTGAATCACTTCACACAAACAGCC GGTTC GGT TGAA GAGAAA AATCC
rrnx�;;i=GC=:::::CTCCATCGACACGGC�G T gTGAATCACTT�A�ACAAACAGCC GGTT� GGT TGAA GAGAAA AATCC
-300 -280 -260 -240
L
rrnA AAATAAATGCTTGACTCTGTAGCGGGAAGGC AC ACCCCGCGCCGCTGAG AAAAAGCGAAGCG GC ACTGC
rrnB AAATAAATGCTTGACTCTGTAGCGGGAAGGC AC ACCCCGCGCCGCTGAGA AAAA GCGAAGCG GC ACTGC
rrnD TGAA ATTCAGGGTTGACTCTAAGA GAGGAA AGC GCCACCTCGCGACAGTGAGCTGAAAGCC GCGTCGCAACTGC
rrnE TGAAA TTCAGGGTTGACTCTGAAAGAGGAAAGC GCCACCTCGCGACAGTGCG�TA AAGC GCGTCGCAACTGC
rrnX TGA1\ATTC �GgGTTGACTCTG� GAGGAAAGC _G£CACCTCGCGA�AGTGAgcTGAAAGCC GCGTCGCAACTGC
---==----==�
-220 -200 -180L_ -160
Fig 4.T hef iev rrnp roomterre gionasl ignetdom axizmeih omolioeg.s