0022-538X/88/041125-07$02.00/0
Copyright
C 1988, American Society forMicrobiology
Regulation of Interferon Gene Expression:
Mechanism of Action of the If-i
Locus
LUC DAIGNEAULT, BENOIT COULOMBE, ANDDANIEL SKUP*
Institutdu Cancer de Montreal, CentreHospitalierNotre-Dame, 1560rueSherbrooke Est,Montreal,
Quebec, Canada H2L 4M1
Received 18 June 1987/Accepted 23 December1987
Wehave examined themechanism of action of theIf-]interferon(IFN) regulatory locus. This locus controls the level of circulatingIFN produced in inbred mice inresponsetointravenous
injection
of Newcastledisease virus. Mice carryingtheIf-lh
(high) allele show circulating IFNlevels 10-to15-foldhigher than thosecarrying theIf-i' (low) allele. In thisreportweshow that inducedsplenocytesfromIf-ih
andIf-11
mice produce IFNatlevels whichareinthesameproportionsasthosefound inthecirculation.Higher levels ofIFN-specificmRNA
were observed insplenocyte populations fromIf
Ih
animals. Thiswasduetoincreasedtranscription of IFNgenes. At the same time, the high- and
low-producing
populations showed no significant difference in the number of IFN mRNA-containingcells.We conclude that the effect ofIf-]in the spleen istocontrol thelevels oftranscription of theIFNgenesin individualinducedsplenocytes.The interferons (IFNs) are the products ofa family of
inducible genes whichare ubiquitousto all vertebrates and which possess varied and potent biological effects on cul-tured cells oranimals (40). In general, the IFNsare
subdi-vided into the type Iorviral IFNs and thetype II, immune
(or
y)
IFNs, accordingtothenatureof induction. ThetypeI IFNs are further subdivided into aand ,B. In humans this subdivision into antigenically distinct subtypes is reinforced by the fact that the production of IFN-a and IFN-1 is segregated, with different cell types producing predomi-nantlyone orthe other (40). Incontrast,cultures of murine cells as a rule produce both IFN-a and IFN-,B (9, 15). The use of recombinant DNA technology has shown that theIFN-agenes constitute afamily madeup ofalarge number
of highly homologousgenesboth in humans (3) and in mice
(25, 28, 38). One human
IFN-P
gene has been well defined(17, 41), but evidence for other human IFN-1 genes exists
(26, 36, 37, 42). In themouse, cDNAscorrespondingto the
IFN-P13
have also been cloned and have been shown torepresenta single copy gene(24).
Generally speaking, uninduced cells ororganisms donot secrete detectable levels of IFN (40). Afterexposure toan
inducer, typically a virus or a double-stranded RNA, IFN
production can occurbothin tissue culture and in the animal (40). TheonsetofIFN production is due to transcriptional activation of the IFN genes (34). Several groups have characterizedinduction-regulatoryelements in the 5' flank-ing regions ofthe human IFNgenes(18-20, 33, 43, 44).Both indirect, and recently direct, evidence fora role for
trans-acting transcriptionfactors in the induction mechanism has been obtained (19, 44).
Regulation of IFN production occurs at another level besides the on-off regulation of induction/repression. In-deed, the levels of production of IFN in the mouse are
regulated by a series of factors. These include age (7), hormones (30, 35), and the genotype (7-14). This last is of particular interest. It has been found that different inbred
mousestrains produce highorlowlevelsofcirculatingIFN inresponse to various inducers (7-14).The lociinvolved in regulatingthisresponsehave been calledtheIfloci andhave
*Correspondingauthor.
been studied extensively from the viewpoint of classical genetics (7-14). The responsetodifferent inducers is
regu-lated by independently segregating loci (12). The level of IFN in the circulation of mice in response to Newcastle disease virus (NDV) is regulated by the locus
If-]
(7-14). Two alleles have been described: If-I and If-i',correspond-ingto high and low levels of circulating interferon,
respec-tively. In mice carrying the
If-ih
allele (e.g., theC57BL/6
strain),
circulating IFN levels after intravenous injection ofNDVare 10-to15-foldhigher than in strains whichcarrythe
If-i' allele (e.g., the BALB/c strain) (7-14). Recombinant inbred andcongenic strains for If-i have been obtained (11), and these mice have been used to assign If-i to murine chromosome 3(31).
As is often the case for regulatory loci, the lack of
information on the site of action ofIf-] has hampered the development of further studies. The cells of the lymphatic system are largely responsible for IFN production after
intravenous injection of viruses (13) and can be carriers of
the If-i phenotype (13). For this reason we decided to examine the action ofIf-] in the spleen.
Themechanism of action ofaregulatory locus suchasIf-i
could ofcoursebeduetoanyofmanydifferentpossibilities. Higher IFN levels in
If-ih
mice could be due to greater specific activityof IFNs translated fromequivalentamounts ofIFN-specific mRNA. On theotherhand, higher levelsof such mRNAcould be present in thehigh-producinganimals. These levels could inturnbecontrolled transcriptionally orposttranscriptionally. Finally, the effect could be due to differencesin the number ofIFN-producingcells (whichwe
call acellular mechanism)ortohigherlevels ofproduction
percell(whichwe callamolecular mechanism).
In the present report we have addressed the problemof the mechanismofIf-i action inonetargetorgan,thespleen. We conclude thatsplenocytes from
If
Jh mice showhigher levels oftranscription of IFN mRNAand that the numbers ofIFN-producingcells inIf.)" andIf-i' spleensareequiva-lent.
MATERIALSAND METHODS
Animals, cell lines, and virus stocks. Male mice of the
C57BL/6,
BALB/c, and HW81 (B6.C H-28c; If-i) (7, 11)1125
on November 10, 2019 by guest
http://jvi.asm.org/
1126 DAIGNEAULT ET AL.
lines, aged from 5 to 6 weeks, were obtained from the Jackson Laboratory, Bar Harbor, Maine.
L cells were grown in Eagle minimal essential medium supplemented with 10% fetal bovine serum. Vesicular sto-matitis virus, Indiana strain,waspropagatedon Lcells, and titers were determined by plaque assay on the same cells. The Kumarov strain of NDVwas propagatedonprimary or
secondary cultures of chicken embryo fibroblasts after the virus was seeded at approximately 1 PFU/cell. Titers of NDV were determined by plaque assay on secondary cul-tures ofchicken embryo fibroblasts.
IFN inductions and titrations. For in vivo inductions,
200-RI
volumes ofEagleminimal essentialmedium, contain-ing approximately 108 PFU ofNDV, were injected into the extraorbital sinus of mice. Sera and spleens were taken 6 h after infection.For the preparation of splenocyte suspensions, spleens
were removed and dissected onto aMillipore filter support
(AP 32; 75-mm diameter).The filter support wasdiscarded, and the suspension was centrifuged and washed twice in Eagle minimal essential medium without fetal bovineserum.
Isolated single-cell suspensions of splenocytes were in-fected at a multiplicity of infection of 100 PFU/cell. Virus
was left in contact with the cells for 2 h, removed, and replaced with Eagle minimal essential medium.
IFN titrations were carried out by the method of cyto-pathic effect reduction in L-cells infected with vesicular stomatitis virus as previouslydescribed (4).
Isolation and analysis of RNA. RNA from splenocyteswas
isolated either by the guanidine hydrochloride method de-scribed previously (39) or by the method ofMurphy et al. (32). For thepreparationofpolyadenylated RNA,total RNA preparations were passaged twice through
oligo(dT)-cel-lulose (type 7, Pharmacia).
For Northern (RNA) blot analysis, RNA samples were
fractionatedon 1.5% agarose gels containing4 mM
methyl-mercury hydroxide (1). RNA was transferredby capillarity
to Pall Biodyne membranes. Hybridizations and washes
were done by the methods of Coulombe and Skup (4). Probes used were pM33, which contains a cDNA for the mouse
IFN-P1
(MuIFN-1l)
gene (24), kindly provided byY. Higashi and Y. Kawade, and pMIF1204, which contains acDNA of the MuIFN-_x2 gene (25). These plasmids were
labeled with
[32P]dCTP
bynick translation (4).RNase protection mapping of mRNA was essentially as
described by Zinn et al. (43). The probe was a 32P-labeled
cRNA complementary to the MuIFN-3 mRNA. This probe
was synthesized using the
MuIFN-P,
(24) cDNA reclonedinto plasmid pGEM-1 (Promega Biotec) linearized with HindlIl and transcribed with the T7 RNA polymerase (27, 29). Hybridizations (to 2 ,ug of polyadenylated RNA) were
carried out at45°Cfor 17 h, and RNase digestion wasfor 1 hat 30°C.
Transcriptioninisolated nuclei. Isolatedsingle-cell
suspen-sions ofsplenocyteswereinduced for2and 4h, nucleiwere
isolated, and nascent transcripts wereelongated essentially asdescribed by Greenbergand Ziff(21),exceptthat 0.5 mM
MnCT2,
2.5 mM dithiothreitol, and 2 U of RNasin per ml were present in the reactions. The labeled nascenttran-scripts were isolated by the method of Groudine et al. (22) exceptthatthe unincorporated nucleotideswereremoved by
centrifugation through a Sephadex G-50 column.
Labeled transcripts were suspended in the hybridization
bufferat42°Cand hybridizedtoPall Biodyne filterscarrying 5jigofplasmids for72 hat42°C. Hybridization and washes
were done by the method of Hannigan and Williams (23).
Filters were
exposed
toKodak
XAR-5 film with CronexLightning-Plus
intensifying
screens(Du
Pont)
forautoradi-ography.
Multiple
exposures of thefilters
were taken and werequantified by densitometry
(GS
300densitometer,
Hoefer
Scientific
Instruments).
Allsignals
observedcorre-sponded
totranscription
which waslargely
sensitive tocx-amanitin.
Insitu
hybridization.
Probes for in situhybridizations
were3H-labeled
cRNAssynthesized
using
theMuIFN-P,
(24)
andMuIFN-a,2
(25)
cDNAsreclonedinto
plasmid pGEM-1
(Pro-mega
Biotec),
linearized
withBgIII,
andtranscribed
with the RNApolymerases
of
bacteriophages
SP6 orT7(27,
29).
Samples
of 50Il of
suspension
at aconcentration of108
cells per mlwereputonto
slides,
rapidly
airdried,
fixed withCarnoy
fixative(methanol-acetic acid, 3:1),
and air dried for at least afurther 2 hafter
dehydration.
Hybridizations
wereby
the method of Cox et al.(5).
Hybridization
buffer
was50%
formamide-0.3
MNaCI-10
mMTris
hydrochloride
(pH 8)-i
mMEDTA-1x
Denhardtsolution (16)-500
,ugof
yeast tRNA perml-10%
dextransulfate. Probe
concentration
was0.2 to0.3pg/ml
perkilo-base of
probe
complexity.
Hybridizations
were carried outin
moistchambers
for 36 to48 h at42°C.
Slides were washed twice in 2x
SSC
(lx
SSC is
0.15 MNaCI
plus
0.015 M sodiumcitrate)
at roomtemperature(30
min
perwash),
four
times with 2x SSC at65°C
(15
min perwash),
and twice with0.1x
SSC at650C
(15
min perwash).
After
washes,
slides
weredehydrated
with
twotreatmentsof
10 min eachwith
70% ethanol
and two treatmentswith
95%
ethanol,
all at room temperature.Dehydrated
slides weredipped
inKodak
Nuclear Track NTBemulsion
type 2and
stored
at4°C in
the dark.After
development,
slides
were stained with Giemsaand
mounted.
RESULTS
If-]
action insplenocytes.
Thefirst
question
that wead-dressed
waswhetherornotIFN
production
in thespleens
of
inbred
mice
wasregulated by
theIf-I
locus.
Forthis
pur-pose,mice of
theC57BL/6, BALB/c,
and HW81lines
(the
lattera
congenic
linewhich carries
theIf-I1
alleleof
BALB/c
mice
on aC57BL/6
genomic
background; 11)
wereinjected
intravenously
with NDV. IFN levelsweredetermined
inthe
seraand in the culturefluids of
isolated
splenocytes
from the infected mice. Theexpected
differencein
IFN levels be-tween theIf-I`
and theIf-f
mice
wasobtained
(Table 1).
Splenocytes
removed from theinfected
micecontinued
toproduce
IFNforsometime,
andthoseof
high-producer
mice made more, inroughly
the sameproportions
asfound
forcirculating
IFN. The HW81congenic
mice behaved in
exactly
the same way as their BALB/c counterparts,indi-cating
that the difference in levels of IFNproduced
by
spleens
is indeed due toIfl-.
Similar results were
obtained
when thesplenocytes
of uninfected micewere induced in vitro with NDV(Table
1).
Although higher
titers were obtained for bothstrains,
the cells fromC57BL/6
miceproduced roughly
10-fold more IFN than did thosefrom BALB/c mice.The differences in the levels of IFN
produced by
the animalswhich carry thedifferent
If-f
allelesare notdueto a shiftinthekinetics of the induction. IFN accumulated in the seraof thehigh-
andlow-producing
animals withanidentical timecourse(Fig.
1A).
Thiscorresponds
tothe resultsof the groupof DeMaeyer
(10).
A timecourseof
IFNproduction
J. VIROL.
on November 10, 2019 by guest
http://jvi.asm.org/
TABLE 1. IFNtiters in sera and in culture media of spleen cell suspensions from mice inoculated intravenously with NDV (in vivo) orfromspleen cell suspensions induced with NDV (in vitro)
Source of IFN Titersa
BALB/cserum... 240, 768,480
C57BL/6serum... 4,800, 7,680, 9,600
HW81serum... 240, 720, 720
BALB/c
spleen
(invivo)... 24, 36, 18bC57BL/6
spleen
(in vivo)... 192,384, 576bHW81
spleen
(in vivo)... 12, 18,24bBALB/c
spleen
(in vitro)... 4,800, 9,800, 2,400cC57BL/6
spleen
(in vitro) ...48,000 36,000, 24,000'aAlltiters are in laboratory units. In this system 1 U corresponds to 1 IU
asmeasured by reference MuIFN preparation no. G-002-904-511. Each figure is the average from three mice of each strain.
bTiters were determined after 16 h in culture. In all cases, the titer at the time incubation was begun was <6.
Titers were determined after 6 h of induction. NDV stock used was a new onewhichyieldshigher responses.
by splenocytes induced in vitro (Fig.
1B) alsoshows that theinduction occurred
with similar kinetics, although theC57BL/6
IFN seemed to be degraded more rapidly, leadingto a
less
significant difference
atlate
times.
Levels of IFN-specific mRNA in the spleen. Figure 2 shows
* A j
22
HOURS
-HOURSCIIONIONAFTER
--0_ ~0- o
2 4 10 12
HOUTRSAFTERvuINSAJECTION(h)
10. K t 7n
.08~~~~~~~~~~~~~~~~~~~~~~o
V
7~~~~~~~~~
sionsweereardfrm plen f 5 ic.ThEAFTERsuspenion(h)
W3
2-1 0~~~~~-
0=~=--35 7, 9 11 24 TIMEAFTERIOICTUTKN th)
FIG. 1. Kinetics ofaccumulation'of MuIFN. (A)Six micewere
injectedwith 200 p.1 eachof NDVfor each point. (B)Cell
suspen-sionswere
prepared
fromspleens
of 15 mice. Thesuspensions
wereinfected with NDV, and
supernatants
werecollected at the times indicated. Symbols:0,
C57BL/6 mice;0,
BALB/c mice. Insets show the kinetics ofaccumulation ofIFN for BALB/c mice onadifferentscale.
A B
-FIG. 2. Northern blot analysis of poly(A)+ RNA from the spleens of NDV-induced mice. Lanes: 1, 5
jig
of RNA from C57BL/6; 2, 5 ,Lg of RNA from BALB/c; 3,5,ugof RNA from HW 81;4, 0.5 ,ugofRNAfrom NDV-induced c243 cells. (A) Probe was nick-translatedpMf33;
(B) probe wasnick-translatedpMIF1204. The markers were PstI-digested pBR322 (4.3 kilobases) and Taql-di-gested pUC8 (1.4, 0.7, and 0.5 kilobases).Northern blots of poly(A)+ RNA
from
spleens of the same animals used toobtain
the datain the first part of Table 1. In nocase was any signal obtained when RNA from the spleensof uninduced animals
wasused (data
notshown). Thesignal
obtained with both
MuIFN-P,B
(Fig. 2A)
andMuIFN-a2(Fig.
2B)
specific
probes was much stronger for thehigh-pro-ducing C57BL/6
mice thanfor
either theBALB/c
or theHW81
strain. This indicates
that theeffect
ofIf-]
in thespleen is
to causehigher levels of IFN-specific
RNAin
If-lh
than in
If-l' mice.
The identityof
the results obtained from HW81 orBALB/c mice confirms
that we areindeed dealing
with If-i and
not some other IFNregulatory locus.In
the
splenocytes
which
wereinduced
invitro,
the levelsof
IFN-specific
mRNA werealsohigher in
thecellsfrom
thehigh-producing If-Ih mice throughout the length of
thein-duction
(Fig. 3). Kinetics of accumulation
wereagain
simi-lar,
and themaximal accumulation occurred
at 3 h afterinduction. With longer
exposures,signals became detectable
at6and 9 h
of induction (data
notshown), and thedifference
between
thehigh-producing C57BL/6 mice
and thelow-producing BALB/c mice
wasmaintained.
Analysis
ofIFN-specific
gene transcription. Todetermine
whether the
difference in
theaccumulation
of IFN-specific
mRNA
between
C57BL/6 mice and
BALB/c mice
is due to ahigher
transcriptional
levelof
the IFN genesin
theformer,
we
performed nuclear
run-ontranscription experiments
withnuclei isolated from induced splenocytes.
The
relative
transcription
rateof
the MuIFN genes(Fig.
4)
was
three
tofive times
higher in
thenuclei of
inducedsplenocytes
of C57BL/6 mice
than in thoseof
BALB/c
mice.Determination of numbers ofIFN-producing cellsin popu-lationsofinduced
splenocytes. Higher
levelsof
transcription
of
IFN genesin
thesplenocyte
populations
obtained
fromC57BL/6
mice could be due to ahigher
numberof
IFN-producing
cells inthis
population
or tohigher
levelsof
transcription
perIFN-producing
cell.
Wecarried
outin situ
hybridization experiments
with cRNA
probes
corresponding
to
the MuIFN-,
genetocomparethenumber ofsplenocytes
producing
IFN mRNA. The firstexperiments
were carried outontissueslices.Positive
cellswerefound
only
inspleens
of
induced animals with theappropriate
probes
(data
not shown), butquantitation
of the number ofpositive
cellswasdifficult.
Toget a better
quantitation
of the results obtainedby
in situhybridization,
hybridizations
were carried out onsple-nocytes isolatedas acell
suspension
fromspleens
ofnormal
on November 10, 2019 by guest
http://jvi.asm.org/
[image:3.612.361.516.81.195.2] [image:3.612.85.278.375.675.2]1128 DAIGNEAULT ET AL.
. L.
?; SjAL L I. ..; 4
OI, IL 4,
am
FIG. 3. Kinetics ofaccumulation of
MuIFN-P,B
mRNA in sple-nocytesitduced in vitro. RNasemappingwith 2,ugof polyadenyl-ated RNA fromsplenocytesatthe timesindicatedpostinfectionwascarried out as described by Zinn et al. (44). The probe was a
32P-labeled
cRNAcomplementarytoMuIFN-13
mRNA.Numbersatthetopindicatetime in hours.Thesignal obtained correspondsto
the predicted 680-nucleotide band.
The
negative controls weretRNA (25
jig)
and0.5,ugofpoly(A)+ RNAfrom uninducedLcells (NI) mixed with the probe. The positive control was 0.5jig
of poly(A)+RNA from L cells induced with NDV(I) (8hofinduction). Markers are the 1-kilobase DNA ladder from Bethesda Research Laboratories.and NDV-infected
mice and
onsplenocytes isolated from
uninfected mice and induced in vitro with
NDV(Fig.
5).
Inno case were
positive
cells obtained
whenthe
splenocytes
wereisolated
from uninduced animals.
However,significant
numbers
of
positive
cells
werefound when splenocytes
wereinduced, either in vivo
orin
vitro.
Hybridization
with labeledRNA
of
the samepolarity
as the mRNA or with RNAcontaining
nonspecific
(X-phage)
sequences neveryielded
signals
(data
notshown).
The
resultsof
analysis of
alarge
number
of cells of both the
BALB/c
(If-i')
andC57BL/6
(Iflh)
strains
areshown
inFig.
6.The insetsconfirm
that nocells that
could
be
classified
aspositive
wereobtained
in theuninduced
populations.
Ininduced
populations
the totalnumber of
TEN-positive
cellsfrom
C57BL/6
andBALB/c
mice wasnot
significantly different,
and the number of highlypositive
cells
wasobviously
greater
in
thepopulation from
If
Ih mice. Comparison of Fig.
6A and 6B shows thatIf-i
action
was equivalentboth
in vivo and in vitro.levels
obtained
corresponded
to thoseexpected
for thegenotype
of
thedonor
(13). If-i
is alsoexpressed
inperito-neal
macrophages (10).
Here we haveshown that
IFN
production
in splenocytes is also controlledby
this locus(Table
1)after both
invivo and
in vitro induction. Thedifferences
observed
arenotduetoshifts in kinetics(Fig.
1)
in
either
case,and
the results with the HW81congenic
line(11)
confirm
that theeffect is
indeed
due toIf-l.
ThusIf-]
action in cells
of
thelymphatic
systemseemstobe
ageneral
phenomenon.
The groupof
DeMaeyer has
reported
that
mouse
embryo fibroblasts from C57BL/6
and BALB/c mice produceidentical
amountsof
IFN in response to NDV(10),
and
wehaveobtained
thesameresult(data
notshown).
Thisindicates that
theIf-i
phenotype
is notexpressed
in allcell
types.
Examination of
the levels of IFN mRNA in thespleen
cells of
theinducedanimals showed
thatthey
correlated wellwith the levels
of
IFNactivity
(Fig.
2). The sameresult
wasfound
for
RNAfrom
splenocytes induced in vitro
(Fig. 3).
Thus the
differences
in levelsof
IFNactivity
arein all
probability
due to a realphysical difference
in the amountof
IFN
produced from
anincreased
amountof translatable
message. The
difference
in thelevels
of
IFN-specific
mRNAin
splenocytes of induced
C57BL/6 and
BALB/c mice is due
to
increased
transcription
in theformer,
asshown
by
run-ontranscription (Fig. 4).
The
question of
whether or not alarger
numberof
IFN-producing cells
inIf-f1
mice could
accountfor
thehigher
levels of
IFNgenetranscription observed
wasanswered
by
in
situ
hybridization on isolated splenocytes.
In all casesin
the
induced
population,
nosignificant
differencein
thenumber
of
cellsproducing
IFN-specific
mRNA was foundwhen
BALB/c
andC57BL/6 mice
werecompared. Cells
obtained
from
C57BL/6 mice
showedhigher signals
than didthose obtained from
theBALB/c mice.
In theseexperi-ments, we have
concentrated
onobtaining,
asfar
aspossi-ble,
themaximum number of
positive
cells
tocomparetheir
numbers.
Thus exposure times were such that theoverall
tendency
wasfor
theC57BL/6 cells
toshow
adistribution
40 .
z
0
CL
cc
I-:i 30,
20
10
DISCUSSION
In
this
report, we have investigated the site and mode ofaction of
theIf-] IFN regulatory locus. We have identifiedthe
spleen
as asite of actionand
shown that in thisorgan the effectof the
If-] locus is to cause greater or lesser levels ofIFN
mRNA in splenocytes
forthe high- and low-producingalleles, respectively.
These differences in mRNA levels areat
least
in
part due totranscriptional
regulation.It
had been shown previously
(13) thattransplantation ofbone
marrowcells from
If-jh
orIf-i'
miceinto irradiated ratsrestored the
ability of
the rats to produceIFN.
TheIFN
BALB/c C57BL/6 BALB/c C57BL/6
2HOURS 4 HOURS
FIG. 4. Relative rateof transcriptionof the IFN genes inisolated nuclei frominducedsplenocytes. Nuclei from approximately 2x 107 cells were preparedfrom isolated splenocytes from C57BL/6 and BALB/cmiceat2 and 4 hafterinduction with NDV. Results were quantitated by densitometry,and all results werestandardized to the level oftranscription of RNA corresponding to ribosomal protein L27' (2), which can be considered as a housekeeping gene. The relativetranscriptionratesofIFNgenes in theinducednuclei were evaluated by the subtraction ofthe basal level observed in the uninducednuclei(30 to
40%
oftheribosomal protein L27').J. VIROL.
:,
-14on November 10, 2019 by guest
http://jvi.asm.org/
[image:4.612.99.264.84.305.2] [image:4.612.323.549.489.646.2]ACTION OF
If-]
INTERFERON REGULATORY LOCUS 1129A
p.
C
_.
-...
4
A *11.
P.S.
S. .
_p
S.
G
a
-t
1I
:Iv
i I ;.
.9
'a.,
.D
qp-
|._
'_.
4.
*-E_ r,s ,,2 4
z ¢N w * ^w. eS.**
^:*
...x
u R} 6- ^
4'J
- wJe
,:s:,
-s |
I .~~~~~~~~~~~~~~~..
'.A
-a _,-.
a
._ _k S_
Aim-
.i.
w
.:S. ....
Xp...
_,~~~~~~4
_:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~::..H
.--
0
_'
*
'w
4
|
*
W.-.
FIG. 5. In situhybridizations on isolated splenocytes. (A) NDV-infected and (B) uninfected C57BL/6mice;(C) NDV-infected and (D) uninfected BALB/c mice; (E) in vitroNDV-infected and (F) uninfected C57BL/6 mice; (G) in vitro NDV-infected and (H) uninfectedBALB/c mice. Hybridizations were for 48 h at 42°C with 10 ng of3H-labeled
MuIFN-P1
cRNA(specific activity, 6.5 x 107Cpmlp.g).
Autoradiographic exposurewasfor 25 days. Bars, 25p.m.
which
wasshifted
towards cells with more silver grains, butthis
wasnotnecessarily
inproportion
tothe ratio in mRNAlevels. In sum, we
interpret
ourresults asmeaning that in thespleens
ofIf-
1 andIf-Pl'
miceroughly equal numbers of cellsrespond
to NDVby producing IFN; thus the cells from thehigh-producing
animals transcribe higher levels of IFNmRNA.
If-1
actsin trans. This is evidentasthestructuralgenes for both IFN-a andIFN-p
are on chromosome 4 (6,25,
28), whereas theIf-f
locus has been mappedgenetically
toVOL.62, 1988
19
f.3
Alk
,,L 7
1.,.
E
O'
vq-7 ...14, -.4.,.
.JIUL
ID.
Adn'.
.IWr
'W,4w-Abr...wwW
vvlw
I
k
on November 10, 2019 by guest
http://jvi.asm.org/
1130 DAIGNEAULT ET AL.
NQ OFGRAINS PERCELL
-J
20 (0-3) (4-7)(8-11)(12-5) (16-9)(20-23)
201
s.I
O) NO.OF GRAINSPER CELL
U.
0
2
(0-3) (4-7) (8-1)) 012-15) M)-19) (20-23)0*2427)0(8-21) 032-35)MO-29 No43)N-4&fl) 51)(52-55)
NQ OF GRAINS PERCELL
FIG. 6. Analysis of silvergrain distribution in isolated spleno-cytes. (A) Cells removedfrom NDV-infected mice. (B) Cells
re-moved from normal mice and subsequently infected in vitro with NDV. Insetsrepresent uninfectedcell populationsforbothcases.
Symbols: Solid bars, C57BL/6; open bars, BALB/c. At least 400
cellswerecounted foreachset.
chromosome 3 (31). We have also found that an X-linked
gene which is coinduced with IFN is regulated by
If-]
(B.Coulombe,
L. Daigneault, D. Skup, and B. R.G. Williams,manuscript in preparation). Furthermore, De Maeyer-Guignard andco-workers have shown, using micecongenic forchromosome 4, that theorigin of the structuralgenes is notimportantfor
If-]
regulation (14). The fact thatIf-]
actsintranstoexertadirect effectontranscription indicates that
the If-i product may be a specific transcription factor or someotherfactor involvedin IFN induction.
In conclusion, wehave shown that the mode of action of the If-i regulatory locus is through a molecular event.
Individual IFN-producing cells in spleen transcribe higher
levels of IFN-specific mRNA in If-qh mice. This fact, in conjunction with the findings that splenocytes induced in
vitroproducehigh levels ofIFN and that this production is
regulated by If-i, will now permit study of the molecular
mechanisms of If-i action.
ACKNOWLEDGMENTS
We thankG. PaternoandP. Belhumeur for useful discussions,R.
Duclos for photography, D. Houle for animal handling, and D.
Forgetfor technicalassistance.
This work has been supported by the National CancerInstituteof Canada, and D.S. holds a Development Grant from the Medical Research Council, Canada. L.D. holds a studentship from the Cancer Research Society of Canada, and B.C. holds one from the Fonds deRecherche en
Sante
duQuebec.
LITERATURE CITED
1. Bailey, J. M., and N. Davidson. 1976. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal. Biochem. 70:75-85.
2. Belhumeur, P., G. D. Paterno, G.
Boileau,
J. M. Claverie, and D. Skup. 1987. Isolation and characterization ofa murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene. Nucleic Acids Res. 15:1019-1029.3. Brack, C., S. Nagata, N. Mantei, and C. Weissman. 1981. Molecular analysis of the human interferon-a gene family. Gene 15:379-394.
4. Coulombe, B., and D. Skup. 1986. Expression of a synthetic human interferon-a gene with modified nucleotide sequence in mammalian cells. Gene 46:89-95.
5. Cox, K. H., D. V. DeLeon, L. M. Angerer, and R. C. Angerer. 1984. Detection of mRNA in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101: 485-502.
6. Dandoy, F., K. A. Kelley, J. De Maeyer-Guignard, E. De Maeyer, and P. M. Pitha. 1984. Linkage analysis of the murine interferon-a locus on chromosome 4. J. Exp. Med. 160: 294-302.
7. De Maeyer, E., and J. De. Maeyer-Guignard. 1968. Influence of the animal genotype and age on the amount of circulating interferon induced by Newcastle disease virus. J. Gen. Virol. 2: 445-449.
8. De Maeyer, E., and J. De Maeyer-Guignard. 1969. Gene with quantitative effect on circulating interferon induced by New-castle disease virus. J. Virol. 3:506-512.
9. De Maeyer, E., and J. De Maeyer-Guignard. 1979. Interferon, p. 205-284. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Com-prehensive virology, vol. 1. Plenum Publishing Corp., New York.
10. De Maeyer, E., and J.DeMaeyer-Guignard. 1979. Consideration on mouse genes influencing interferon production and action, p. 75-100. In
I.
Gresser (ed.), Interferon 1. Academic Press, Inc. (London), Ltd., London.11. DeMaeyer, E., J. De. Maeyer-Guignard, and D. W. Bailey. 1975. Effect of mouse genotype on interferon production.
I.
Lines congenic at the If-1 locus. Immunogenetics 1:438 443. 12. De Maeyer, E., J. De Maeyer-Guignard, W. T. Hall, and D. W.Bailey. 1974. A locus affecting circulating interferon levels induced by mouse mammary tumor virus. J. Gen. Virol. 23:209-211.
13. De Maeyer, E., P. Jullien, J. De Maeyer-Guignard, and P. Demant. 1975. Effect of mouse genotype on interferon produc-tion. Distribution of If-1 alleles among inbred strains and transfer of phenotype by grafting bone marrow cells. Immuno-genetics 2:151-160.
14. De Maeyer-Guignard, J., F. Dandoy, D. W.
Bailey,
and E. De Maeyer. 1986. Interferon structural genes do not participate in quantitative regulation of interferon production by If loci as shown in C57BL/6 mice that are congenic with BALB/c mice at the alpha interferon gene cluster. Journal of Virol. 58:743-747. 15. De Maeyer-Guignard, J., M. G.Tovey,
I.
Gresser, and E. DeMaeyer. 1978. Purification of mouse interferon by sequential affinity chromatography on poly(U)- and antibody-agarose col-umns. Nature (London) 271:622-625.16. Denhardt, D. T. 1966. A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23:641-646.
17. Derynck, R., S. Content, E. De Clerq, G.
Volckaert,
J. Taver-nier, R. Devos, and W. Fiers. 1980. Isolation and structure of a human fibroblast interferon gene. Nature (London) 285:542-547.18. Fujita, T., S. Ohno, H. Yasumitsu, and T. Taniguchi. 1985. J. VIROL.
on November 10, 2019 by guest
http://jvi.asm.org/
[image:6.612.61.303.79.437.2]Delimitation and properties of DNAsequencesrequired for the
regulated expression of human
interferon-p
gene. Cell41:489-496.
19. Goodbourn, S., H. Burstein, and T.Maniatis. 1986. The human p-interferongene enhancer is undernegative control. Cell 45:
601-610.
20. Goodbourn, S., K. Zinn, and T. Maniatis. 1985. Human
p-interferongeneexpression is regulatedbyaninducibleenhancer
element. Cell 41:509-520.
21. Greenberg, M. E., and E. B. Ziff. 1984.Stimulationon3T3cells induces transcription of the c-fos proto-oncogene. Nature (London) 311:433-438.
22. Groudine, M., M. Peretz, and H. Weintraub. 1981. Transcrip-tionalregulation of hemoglobin switching in chicken embryos. Mol.Cell. Biol. 1:281-288.
23. Hannigan, G., and B. R. G. Williams. 19b6. Transcriptional regulation ofinterferon-responsive genes is closely linked to
interferonreceptoroccupancy. EMBO J. 5:1607-1613. 24. Higashi, Y., Y.Sokawa, Y.Watanabe, Y. Kawade,S. Ohno,C.
Takaoka,and T.Taniguchi. 1983. Structure and expression ofa
cloned cDNAformouseinterferon-P.J. Biol. Chem. 258:9522-9529.
25. Keiley, K. A., C. A. Kozak, F. Dandoy, F. Sor, D.Skup,J. D. Windass, J. De Maeyer-Guignard, P. M. Pitha, and E. De Maeyer. 1983. Mapping of murine interferon-a genes to
chromosome4.Gene 26:181-188.
26. Kohase,M., D.Henrikson-Destefano,L. T.May, J. Vilcek, and P. B.Sehgal. 1986. Induction ofP2-interferonbytumornecrosis factor: ahomeostatic mechanism in the control of cell prolifer-ation.Cell 45:659-666.
27. Krieg, P. A., and D. A. Melton. 1984. Functional messenger
RNAs are produced by SP6 in vitro transcription ofcloned
cDNAs.Nucleic AcidsRes.12:7057-7069.
28. Lovett,M., D. R.Cox, D.Yee, W.Boll,C. Weissmann, C. J. Epstein,and L. B.Epstein. 1984. Thechromosomal location of
mouseinterferon-agenes. EMBO J. 3:1643-1646.
29. Melton, D. A., P. A. Krieg, M. R.Rebagliati, T. Maniatis, K. Zinn, and M. R. Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probesfrom plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035-7056.
30. Mendelson,J.,and L. A.Glasgow.1966.The in vitro and in vivo effectsofcortisoloninterferonproductionandaction. J.
Immu-nol. 96:345-352.
31. Mobraaten, L. E., H. P. Bunker, J. De Maeyer-Guignard, E.
De Maeyer, and D. W. Bailey.1984. Locationof histocompati-bility and interferon loci on chromosome 3 of the mouse. J. Hered. 75:233-234.
32. Murphy, D., P. M. Brickell, D. S. Latchman, K.Willison, and P. W. J. Rigby. 1983. Transcripts regulated during normal embryonicdevelopment and oncogenictransformation share a repetitive element. Cell35:865-871.
33. Ohno, S., and T. Taniguchi. 1983. The5'flanking sequence of human interferon-p gene is responsible for viral induction of transcription. Nucleic Acids Res. 11:5403-5412.
34. Raj, N. B. K., and P. M.Pitha. 1983. Two levels of regulation of
p-interferon
geneexpression in human cells. Proc. Natl. Acad. Sci. USA80:3923-3927.35. Rytel, M. W., and E. D. Kilbourne. 1966. The influence of cortisone onexperimentalviralinfection. VIII. Suppression by cortisone of interferon formation in mice injected with New-castledisease virus. J. Exp. Med. 123:767-776.
36. Sagar, A. D.,P. B.Sehgal, L. T. May, M. Inouye, D. L. Slate, L. Shulman, andF. H.Ruddle. 1984. Interferon-p-relatedDNAis dispersed in the human genome. Science 223:1312-1315. 37. Sehgal, P. B., and A. D. Sagar. 1980.Heterogeneity ofpoly(I).
Poly(C)-induced human fibroblast interferon mRNA species. Nature(London) 288:95-97.
38. Shaw, G. D., W. Boll, M. Taira, N.Mantei, P. Lengyel, and C. Weissmann. 1983. Structure and expression of cloned murine IFN-agenes. Nucleic Acids Res. 11:555-573.
39. Skup, D., H. Zarbl, and S. Millward. 1981. Regulation of translation in L-cells infected with reovirus. J. Mol. Biol. 151: 35-55.
40. Stewart, W. E., II. 1979. The interferon system. Springer-Verlag, Vienna.
41. Taniguchi, T.,S.Ohno,Y.Fujii-Kuriyama,and M.Muramatsu. 1980. The nucleotide sequence of human fibroblast interferon cDNA. Gene 10:11-15.
42. Weissenbach, J., J. Chernajovsky, M. Zeeni, L. Shulnan, H. Soreq,U. Nir, D.Wallach,M. Perricaudet,P. Tiollais, andM. Revel. 1981. Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies. Proc. Natl.Acad. Sci. USA 77:7152-7156.
43. Zinn, K., D. DiMaio, and T.Maniatis. 1983. Identification of two distinctregulatory regions adjacenttohuman P-interferon gene. Cell 34:865-879.
44. Zinn, K., and T. Maniatis. 1986. Detection of factors that interact with human P-interferon regulatory regionin vivo by DNAase Ifootprinting. Cell 45:611-618.