0022-538X/79/04-0014/07$02.00/0
qinlOl: Promoter Mutation Which Allows the Constitutive
Expression of the Late Genes
C.DAMBLY,* M.DELSTANCHE,tAND A. M. GATHOYE
LaboratoiredeGinetique, DepartementdeBiologieMoltculaire, UniversiteLibre de Bruxelles, 1640
Rhode-St-Genese, Belgium
Received forpublication 13 September 1978
We describe the isolation and characterization ofa mutant (XqinlOl) which
rendersX growth Q independent. Wehave shown that this mutation creates a
newpromoter,located between genes P and Q, whichresultsin the constitutive expression of the entire Q late region.
Transcription of the late genes of
bacterio-phage A is assumed to occurby two pathways.
Thefirstis amajor pathwaywhich ispositively
controlled bythe Q product (8, 15, 20). The Q
geneproductappears toact asan
antitermina-tor, allowingtheextension, overthelategenes,
of a short RNA (6S) initiated at the late pro-moter(18) (Fig. 1).
The secondisaminorpathway which is
com-pletely Q independent ("leakiness" of the
Q-mutants) (6). We have shown that this minor
transcriptional pathway operates only in the
presenceoftheNactivator and in the absence
ofimmunity. It consistsof theprolongation,over
the lategenes, oftranscription initiated inthe
immunity region (promoter pR
[Fig.
1]). Thisminor transcription occurs to about 5% of the
level ofthe major pathway and is obscured in
the presenceoftheQproduct. Byitself(that is, inQ-mutants), it is tooslowto result inplaque
fornation; however, inliquid medium, Q-
mu-tantsfinally produce nornal phage yields.
Toelucidatethemechanismsofprolongation
andantitermination, wehavelooked, by
differ-entmethods, formutantsabletotranscribe the
lategenes at ahighrate, evenin the absence of
theQ product. Asimilarapproachhas been used
by Herskowitz and Signer (14) and Sato and
Campbell(19).
MATERILS
ANDMETHODS
Bacterial strains. All bacterial strains used are listed inTable 1 and are derivatives of Escherichia coli K-12.The strains were used as suchand, for some ofthem, aslysogenderivatives.
Bacteriophages. Xc+, Ximm21c+ (16) c17 (17) is located in region y; it expresses constitutively genes
clI, 0,and P andreplicates even in thepresence of immunity.
tPresent address: Institut National de laSanteet de la RechercheM6dicale,Unit6 de Recherches de Virologie U 102, Lille,FranceF-59045.
Those phageswere used as such or labeled with various amber mutations:Qam2l, Aamll,and Ram5 (4); Sam7 (13);Qam2O3andRam216(22).Most ofthe phages used to infect sensitive hosts carry the cII2002 mutation to diminishlysogenization frequency.
bypE (3) and byp3 (our laboratory) are located between genes P and Q. AQ-h80 (Q- derivative of
Xhy6 (21) is aAX-480 hybridwith the leftarm (A-.
red) from 80 and therightarm (red-- R) from A.
A(QSR)21Kam24 (constructed by L. Desmet and F.
Salomon) is a X-21 hybrid phage with the A -* P
regionfromXand theQ-*Rregionfrom21.Kam24 wasisolatedby Campbell (4) and introducedin the hybrid.
Methods. (i) Infectionexperiments. The infec-tionexperiments weredescribedpreviously by Dam-blyand Couturier(6).Exponentialcultures(lysogens ornot) wereinfectedbyone ortwophages,eachat a
multiplicity of 3, diluted 100-fold, and incubated at
370C. At 100 min afterinfection, the dilutionswere
treated withCHC13, and adequatedilutions were ti-tratedat370C.
(ii)Endolysinassay. Theendolysinassaywas as described by Dambly and Couturier (6) except the indicatorbacteria were treated withCHC13inbuffer (0.05MTris-hydrochloride,pH 7.8).
(iii) Lysogenization experiment. A Lysogens wereselectedandlysogenization frequencieswere de-terminedasdescribedbyGottesman andYarmolinsky
(12) except that the selection was done on EMBO platesseeded with bothAred3int6cIh+A andAcIh+80.
RESULTS
Isolation and characterization. The
qin-101 mutationwasisolated from
XQam2O3Ram-216asaspontaneous plaque-forming phageon alawn of the host HB15.The infecting phage
carries, in addition to a Q- mutation, an R-mutationto be able to further distinguish be-tweentheqinmutants
(XqinQ-R-)
andtheQ'
revertants (XQ+R-) by transactivation
experi-ments. The HB15 host bacterium was chosen since itcarriesanochresuppressor(supB) able to restore, to a sufficient level, the R function
14
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Imm. 21
NEAD TA L
int red N ci cro y d O P (byp)Q S R A F K J
tL pL pR tRinl'TERclNATOitA2 LATF
6SPROMOTEA
aoactivation
[image:2.501.89.429.55.185.2]FIG. 1. Geneticmap of Aprophage.Arrows indicate the origin and direction oftranscription.Theblock represents the region ofA-imn2l nonhomology. Markers given are those relative to this study. Symbols: V, transcription terminationsites; X,promoter sites.
TABLE 1. Bacterial strains
Bacterialstrain Genotype and relevantproperties Source/reference
H F-argHlac amsup+ Ghysenand Pironio(10)
H15 IsogenictoHbut carries theronmutation(defined Ghysenand Pironio (10) bythe number 15)
HB15 supBderivative ofH15 Ghysenand Pironio(10)
W3110 F-sup+ LederbergviaWeigle
594 F-gal- strAsup' Campbell(4)
N100 recAgal- derivative of W3350 Meselson via Yarmolinsky;
Gottesman and Yarmolinsky (12)
RH1413 RH1422 Thermoresistant derivatives of M5020G Castellazzietal.(5) (ANNcI857)
M5020G sup' (lac,pro)strARsgly ProvidedbyJ. Davies
CA244.1 Cured derivativeof CA244 Thislaboratory
CA244 HfrHBr-lac-trp-sup' Brennerand Beckwith(2)
CA244.1recA recA derivative of CA244.1
C600 supII, pernissivefor ambermutations Appleyard(1)
CA161 supHl, usedinsteadof C600(which is resistantto Brenner and Beckwith(2) 480) in the experiments involving phages h+A
andh+80
CA161/A h+A-resistant derivativeof CA161 Thislaboratory
but not the Q function and the ron mutation
whichstrongly decreases the leakiness of the
Q-mutations[compare AQ-productions after
infec-tionsof H (ron+) and H15 (ron-) (Table 2)]. We could isolate only one mutant (XqinlOl) ableto grow onstrain HB15 which plates with the sameefficiencyonsup'hostsand on hosts
carrying an amber suppressor. However, the
plaques formed on the sup' hosts are small,
whereas thoseformedonthe hosts carryingan ambersuppressor arealso
normal
butclear.Preliminary characterization of the
Xqin-1O1Q-R- phage showedthat it isnot arevertant
orpseudorevertantofthe AQ-R- phage.Indeed,
itfailedtotransactivatetheexpression of the R+
gene of a prophage, showing that it does not synthesize active Q product in detectable amounts. Furthermore, whentheqinlOl muta-tionwascombined with other Qam mutations,
such as t57, Qam2l, Qam501, or Qam73, the
resulting phages behave inthe same way as the qin derivative ofQ203.
Burstsizesof
XqinlOlQ-R+
phageondifferentsup'
strainsare giveninTable2.ItisapparentthatXqinlOl
Q-
growsaswellontheH15(ron-sup')
and the H (ron+sup')
strains as onthe HB15(ron-supB)strain,theyieldsbeing
about105 timesgreater than those of
AQ-
platedonH15. The yield of
XqinlOlQ-
onsup'
strainsseems, however, to be
impaired by
mutationsconferring streptomycin
resistance,
insofar asone canjudgefromthelower
yield
onthe strAstrains594and N100.
Tohave a more
quantitative
measureof theeffect of the qinlOl mutation on late gene
expression,wemeasureddirectlytheexpression
of the late gene Rby endolysin titrations. We found that inW3110(ron+ sup+hostbacterium)
XqinlOlQ- producedtwo tothreetimesasmuch
endolysinasdid
XQ-,
reachingavalue of 10% ofthat synthesized by
XQ4
phage(Table
3). We,therefore, conclude that the qinlOl mutation
permits growth, in Q-
conditions,
by
only
in-creasingthe lategene
expression
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[image:2.501.62.460.229.434.2]DAMBLY, DELSTANCHE, GATHOYE
TABLE 2. Growth of XqinlOlQ on different bacterialstrainsa
Yield(phage/bacterium) at 100 min after infection with: Bacterialstrain
AQ-R+ \qinlOlQ-R+
AqQnlOlQ4R+
HB15(ron- supB) 3 75 52 45
H15 (ron- sup+) 5.4x10-4 70.4 58 55
H (ron+sup') 3.5 56.1 50 52
W3110 (ron+sup+) 2.7 66.7 75 60
CA244.1 (ron+sup+) 3 20.4 69 65
594(ron+sup+strA) 2 6.4 33 40
N100(ron+sup'strA) 1.75 7.1 35 29
aTheinfection
experiments
wereperformed as described in the text. Phage productions were measured on the C600 indicatorstrain.AQ-R+isXcII2iooQam2O3, andAqinlOlQ-R- isAcII2emqinlOlQam2O3Ram216;XQ+ andXqinlOlQ+ alsocarry thecII2oom mutation.2 to 3.
Mapping of the qinlOl mutation. Precise
localization of the qinlOl mutation has been
inferredfrom three types ofcrosses.(i)Ina cross
of Xc+qinlOlQ7R- with Ximm21Q-R- phages,
imm21qinlOlQjR- recombinants [selected on
HB15 (X)] are found with a frequency of 1%,
indicating thattheqinlOlmutation is localized
outside the imm2lregion.
(ii) A moreprecise mappingwasobtainedby
crossing Ac+qinlOlQ-R- phage with various X
prophage deletionmutants.The deletions used
enter the prophage from the left and end at
different points in the P-Q region. For these
crosses,theclear phenotype given bytheqinlOl
mutationwasusedasmarker, and therecovery
ofturbid-plaque Arecombinants indicated that
the qinlOl+ region was present within the
de-leted prophage. The crosses between
Xc+qin-1O1Q-R- and differentdeletedprophages
local-ized qinlOl between the endsof the deletions
RH1417 and RH1415(Table 4).
(iii)We alsomapped the qinlOl mutationin
respect to two mutations located in the P-Q
region:bypE (1) and byp3
(previously
isolatedinthislaboratory). Thesetwomutations,although
theyhave the sameproperties of N
independ-encefor the relief of the
tr2
terminator sitesandofclear-plaque phenotype,aredistinctontheA
map: byp3 is within the RH1415 deletion and
bypE is not. Crosses between
Ac+qinlOlQ-R-phage andXimm2lbypE (Fig. 2) show that the turbid recombinants(v =
5.10-2)
presentalmost all (97%) the imm2l and the Q-R- mutations. On the contrary, the same cross made withXimm2lbyp3 (Fig. 2) gives turbid recombinants
withamuchlower frequency (v=6.10-3)which have indifferently immA or imm2l, the am or
am'character. This shows that the qinlOl and
byp3 mutations are very close. The presence withequal probability of one or the other outside marker isprobably due tohighnegative inter-ference. From theseresults,weinferred that the
TABLE3. Endolysinsynthesis byAqinlOl phage infectingasup'straina
Endolysin activity W3110 infectedwith: (units/100ploflysate
at100min)
XQ- 36
XqinlOlQ- 95
XQ+ 595
XqinlOlQ+ 950
aAll
thephagescarry anadditionalcII2moo
mutation. Q- isQam2O3.Endolysinwasassayedas describedby Damblyand Couturier (6).TABLE 4. DektionmappingoftheqinlOlmutationa Clear Turbid Ratio of
Host amn+ am+ c am
(%)b (%)b (%)/c
can (%)b
RH1422(Q-/R+) 100 lo-l 10-3
RH1415(PI/Q+) 100 1o-l 10-3
RH1417(P7/Q+) 51.8 48.2 0.93
RH1416(PI/Q+) 38 62 1.62
RH1413(PI/Q+) 33.65 66.35 1.97
QR4 3
RH1415
I-RH1417
RH1416
RH14
aThe deleted
strains derive
from M5020G
(sUp+
strA) (see text). The blocks represent the deleted prophageregion.Theam'recombinantsweretitrated on 594 (sup+ strA).Thepercentageof clearam' (or
turbidam+) representsthe fraction ofam' recombi-nantswhichpresentaclear(orturbid)phenotype. Q
isQam2O3;R- isRam216.
bAfter infection with
Xc+qinlOlQ-R-.
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[image:3.501.259.451.228.331.2] [image:3.501.258.453.384.585.2]qinlOl: CONSTITUTIVE EXPRESSION OF LATE GENES 17
qinlOl mutation is located to the left ofbypE,
verynearbyp3.
Dominance-recessivity test. To test the dominantorrecessive, cisortranscharacter of the qinlOl mutation, we coinfected strain H15
withXqinlOlQ-h+A (able togrow onH15) and
XQ-h+80 (unabletogrow onH15)and measured
the production of eachphage. The h+80 char-acter wasusedto testfora transeffect of the qinlOl mutation on late gene expression since
thepresence of theh+80 late proteinresults in
ahostrangedifferent from that ofX.
We observed that themixed infection results in high, comparable yields ofboth XqinlOlQ
and XQ- phages (Table 5). The difference in XqinlOlQ- yields betweenmixed andsingle in-fections(comparelines 3 and 1 of Table5) may
beexplained by phenotypic mixing; aswill be-comeapparentlater,there isnosynthesisof late 4)80 proteins, and the coats made by XqinlOlQ-h+X packagenotonlytheirownDNA
butalsoXQ-h+80DNA. Thiscompetitionlimits the amount ofXqinlOlQ-h+X phage packaged.
AcoiNxIQR
A imm2l bypE
XcQINix R[
R-Aimm2lbyp3
c 0
imm21
Indeed,wecansee (Table 5) that,among
prog-eny,the number ofh+80 phages whichpresent
thehostrangeof 480[h+80 (h+80)]remainsvery
low. Asexpected, thisproduction is again high when the qinlOlderivative is Q+ (Table 5).
All of these results showthat, in mixed infec-tion, AqinlOlQ- phage continuestoexpress its ownlategenesbut isunabletoinduce intrans
the late genes of the coinfecting phage. We,
therefore, conclude that qinlOl isacis-dominant
mutation.
Nature of the qinlOlmutation.Asalready described, the qinlOl mutation gives a clear
phenotype in Q+ conditions (phageXqinlOlQ+
orXqinlOlQ- ona sup am host).In Q-
condi-tions(phage XqinlOlQ- ona
sup'
host), plaques are verysmall, and the phenotype isnotaseasytodetermine. To determine thephenotype, we
measured thepercentageoflysogenization after infection ofa
sup'
hostwith differentderivativesofXqinlOl and found that XqinlOlQ- lysogenizes
normally (33%), in contrast to XqinlOlQ+ (<0.05%; Table 6). The observation that the
IINIOI Q0 R
Aimm21C'QR recombinants
[image:4.501.52.448.314.593.2]byp3
FIG. 2. Mappingof qinlOl inrespecttobyp3andbypE.
TABLE 5. Cis-dominant effectof the qinlOl mutationa
Phage production (phage/bacterium)at90min
Phage inl Aor Q-hk)r
Total q (h+80)A) (h+80) AQ-h+80(h+80)
AqinlOlQ-h+A 35 35
AQ-h+80 2x10-1 2x10' 2x 10'
XqinlOlQ-h+X+AQ-h+80 24 13 11 2x10'
XqinlOlQ+h+X 28
AqinlOlQ+h+X+AQ-h+80 42 25 17 12
aStrainH15(asensitive hosttobothh+Xandh+80 derivatives)wasinfectedbyeachphageatamultiplicity
of3.Thismixedinfectiongave(4)classes ofphagesfollowingtheirgenotype(h+Xorh+80)and theirphenotype [(h+X) or(h+80)]: the twoparental types,h+A (h+X)andh+80 (h+80), and twotypes duetothephenotypic
mixing, h+X (h+80) andh+80 (h+A). Among the fourprogenytypes,onlythephages which areh+80 (h+80)
should beabletoformaplaqueonastrainresistanttoAandthusmaybe titrated.XQ-h+80isaQ- derivative
ofAhy6 (see text).Totalphage productionwastitratedonCA161, AqinlOlQ-weretitratedonH15,XqinlOlQ+ weretitratedon594,andXQ-weretitratedbythe difference betweenthe total andXqinlOl yields.XQ-h+80
(h+80)productionwastitratedonCA161/X.All thephagescarrythecII2002mutation.
imm21 bypE
C GIN101 R
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DELSTANCHE,
frequency of lysogenization by XqinQ+ is im-proved(5%) in thepresenceofmutationsS and
R- (Table 6) and almost reaches the normal level(26%) withanSR-A- triplemutant(Table
6) shows that these functions areexpressed by
XqinlOlQ+ under immunity andthus indicates that theqinlOlmutationconstitutesapromoter
abletoinitiate,evenunderimmunity,the
con-stitutive expression ofgeneQ (in Q+ conditions)
and ofallthelate genes. InQ- conditions, this
additional transcription does not provoke the killing of thelysogens but sufficestoallowphage
growth.
To obtain more direct evidence supporting
thisconclusion,we measured the expression of
twolate genes (R and K) by aAqinlOl phage
infectinganimmune host. In the R experiment,
theprophage only provides the immunity, and
we tested whether, despite this immunity, the
superinfecting XqinlOlQ+ phage is ableto
syn-thesizeendolysin. Resultsareshown inTable7,
part1.We observeda smallbutsignificant
pro-duction of endolysin when the homoimmune phage carries the qinlOl mutation, production whichdisappears entirely iftheAqinlOl phage isQ- (Table 7). This production ofendolysin is low(11 units), aswould beexpected in view of
the low dosage ofgene R+ present in the cell. Indeed, thepresenceof thec17 promoter
muta-tion,which enables thephagetoreplicateeven
in the presence ofimmunity, greatly improves
the endolysin production (Table 7). Moreover, the Xcl7qinlOl phage gives high phage bursts under immunity and is Nindependent for its development (data notshown). We also
exam-ined the trans effectofthe Q product synthe-sizedbythe homoimmuneXqinlOlQ+ phageon an R+ gene located on the prophage. In this
experiment, the strain islysogenic forAR+
pro-phage, and the AqinlOl infecting phagecarries
an R- mutation (Table 7,part 2).Itwasfound
that AqinlOl Q+R- phage completely fails to elicit endolysin expression in trans. However, thepositiveresult obtained with the c17 deriv-ative shows that this trans expression is
[image:5.501.266.461.285.663.2]com-pletelydependentonthe numberofXqinlOlQ+
TABLE 6. Lysogenization by XqinlOlderivativesa CA244.1infected with: Lysogeny(%)
Ac+orXc+Q- 35
Xc+qinlOlQ- 33
Ac+qinlOlQ+ <0.05
Ac+qinlOlS-R- 5
Ac+qinlOlS-R-A- 26
aTheprocedureused isdescribed in thetext.The
percentage oflysogenyrepresentsthe numberof
ly-sogensdividedbythenumber ofinfectedcells.Q- is Qam2O3,S- isSam7,R- isRam5,and A-isAamll.
copies present. The fact that this transactivation isnotseenwith the productof a single Q+ gene shows thatQactspreferentially in cis, in agree-ment withprevious observations(7, 9).
In theKexperiment, the prophage constitu-tively provides the K gene product, and we tested the growth characteristics of a heteroim-mune superinfecting phage which was neither able to synthesize the K product itself (being
K)norabletotransactivate theK+ gene of the Aprophage (since its Q product has a different specificity: Q21). Thus, we infected the
Xinm21c+qin1O1Q+SR7A-K`
lysogen with ahybridphage A(QSR)21K7 and looked for
com-plementation of the Kdeficiency by the K+ gene ofthe prophage, inAK progeny. This comple-mentationbyauniqueK+gene iseasily detect-able in view of thecatalytic character of the K function. The yield of the superinfecting
X(Q)+21K-
phagewasgreatlyimproved when theprophage
carriedtheqinlOl
mutation(Table
8)
TABLE 7. R experimenta
Endolysin activity Strain (units/100 ug of ly-sate) atloomin Part 1: CA244.1 recA (Ac'R)
in-fected with:
Ac+ <2
Ac+qinlOlQ+R+ 11
Xc+qinlOlQ- <2
Acl7qin1O1Q+R+ 570
Ac17 <2
Part 2: CA244.1 recA (Ac+R+) in-fectedwith:
XqinlOlQ+R- <2
Acl7qinlOlQ+R- 8
aInfection
experiments
were performed as de-scribed in the text. Q- isQam203,and R- isRam216.TABLE 8. Kexperimenta
Yield(phage/bacterium)after infectionwith
A(QSR)2,K-phage
Bacterial strain
re-Super- combinants infecting or
type revertants
CA244.1 0.1 0.02
CA244.1(Ximm2l) 0.5 0.3
CA244.1 (21c+) 7 0.3
CA244.1 9 0.28
(Ximm21qin1O1S-R-A-)
A K J immA Q S R
aSuperinfectingphage productionwastitrated on C600(21c+),andXK+ productionwastitratedon594
(21c+).Heterologoussubstitutions of the superinfect-ing phage DNA withADNA are given by the dark area.
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[image:5.501.67.253.560.625.2]and even reached theoptimal values obtained
afterK' transactivation (Table 8).
The results of these experiments show that the late functions tested are expressed under immunity in the presence of the qinlOl muta-tion, confirming the promoter character of this mutation.
DISCUSSION
qinlOl isapromoter mutation located in the
P-Q regionwhich allows expression of Q and of
the late genes. The levels of endolysin
synthe-sized by XqinlOlQ-phage suggest that late gene
expression, in the presence of this promoter,
results in a two- to threefold increase in the level of expression over that observed with Q- phage (residual levels of endolysin are synthesized by phage as a result of the leakiness of the
Q-mutation). Although the increase of late
tran-scription broughtabout by the qinlOl mutation
isrelativelysmall, it is nevertheless sufficient to
make qinlOlQ- phage plaque formers, in con-trast to Q- phage. Moreover, the fact that the
XqinlOlQ- phagecontinues tomake plaques in
a ron host, which completely eliminates the
leakiness of the Q- mutation, shows that the
level of late transcription, resulting from the
presenceofthe qinlOl promoter, is sufficient to allow the growth ofaQ- derivative. This
con-stitutive late expression does notalterthe
via-bility of qinlOlQ- lysogens. Late expression,
which isnormallyamplifiedbyactive replication inthe absence ofimmunity, is notamplified in
theXqinlOlQ- lysogenas aresultofthe presence
of immunity.
Endolysin titrations with XqinlOlQ+ phage
showed that, after superinfection ofnormal
ly-sogens (and transactivation experiments), this
phage produced significantamountsofQ
prod-uct.It is thereforenotsurprisingthatqinlOlQ+
lysogens are nonviable. However, qinlOlQ+
ly-sogenswithmutationally inactivated S,R, and A genesareviable.We have also shown that the
qinlOlQ+phage under immunity constitutively
expressesthelategene K.
Howcan weexplainthe molecular mechanism
bywhichtheqinlOlmutation
gives
risetolategene expression? We propose that the
qinlOl
mutation, by promoting a reinitiation of
tran-scription before gene Q, increases
Q
mRNAsynthesisto alevelwhichresultsin the
produc-tionofasmallamountofQ productdespitethe presence of the Qam mutation. These few Q
moleculeswould then allow thepartialrelief of
the lateterminator,resultinginlate gene
tran-scription.
This explanation is consistent with the
ab-sence of growth of qinlOlQ- phage on strA
hosts. Indeed, sinoe the strA mutation greatly decreases the degree of misreading of codons in general and of amber codons in particular (11),
onewould expectXqinlOlQamphageto synthe-sizeless Q product in the strA host and conse-quently fewer late proteins. Furthermore, our explanation is consistent with the absence of growth ofXqinlOl phage containingtwoamber mutations in gene Q (data not shown). The presence of such adouble mutation in gene Q should reduce the amount ofQ proteins synthe-sized.
ACKNOWLEDGMENTS
We aregratefultoR.Thomas forhelpfulsuggestionsand constructive criticism of themanuscript.We also thank M. Couturier and F. Salomon forstimulatingdiscussions,L. Des-metforconstructingdifferentphagederivatives,and 0. Dou-bleday for correcting the paper.
ThisworkwascarriedoutundercontractEuratom-U.L.B. 156-761BIOB and an agreement between theBelgian Govern-mentand theUniversiteLibre de Bruxelles "Actions de Re-cherchesConcertees."
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