In oduc ion
Regula ion o ansc ip ion by euka yo ic RNA polyme ases
equi es many accesso y ac o s ha di ec speci ic p o ein-
DNA ecogni ion and p o ein-p o ein in e ac ions (G umm ,
1989). In con as o RNA polyme ases II and III, RNA
polyme ase I ecognizes and ansc ibes om only one ype
o p omo e , which di ec s he ansc ip ion o he andemly
a ayed genes encoding he la ge ibosomal RNA ( RNA)
p ecu so s ha in in e phase cells ha e been localized o
he nucleolus (Jan zen e al. 1990). Each andem epea con-
ains a ansc ibed egion, which codes o he ibosomal
RNA p ecu so , and a non- ansc ibed egion, which codes
o he non- ansc ibed space (NTS). In mos e eb a es
he egion wi hin 200 bp o he ansc ip ion ini ia ion si e
con ains he p omo e ha di ec s he syn hesis o p e- ibo-
somal RNA (Smi h e al. 1990). In mammals, his p omo e
consis s o a leas wo in e ac ing elemen s e e ed o as
he co e p omo e elemen (CPE), which con ains sequences
essen ial o speci ic ini ia ion, and he ups eam p omo e
elemen (UPE), which enhances ini ia ion by up o a ac o
o 10 o 100 in an o ien a ion- and dis ance-dependen
manne (Bell e al. 1988; O’Mahony and Ro hblum, 1991).
Despi e ex ensi e conse a ion o RNA sequences om
di e se o ganisms, RNA gene p omo e s om di e en
species show only e y limi ed sequence simila i y and a e
no ecognized by he ansc ip ion machine y o he e olo-
gous species unless hey a e closely ela ed (G umm , 1989;
Jan zen e al. 1990; Pikaa d e al. 1989). This species-
speci ic p omo e ecogni ion appea s o be media ed by
one o mo e auxilia y ansc ip ion ac o s a he han by
RNA polyme ase I, which is unc ionally conse ed (Bell
e al. 1988; Pikaa d e al. 1990). Accu a e and e icien an-
sc ip ion ini ia ion by e eb a e RNA polyme ase I equi es
a leas wo DNA-binding p o eins. One o hese ac o s,
e e ed o as SL1, is an essen ial componen o econs i-
u ing ansc ip ion, and can con e p omo e speci ici y on
a he e ologous ex ac (Lea ned e al. 1985). Howe e , SL1
does no show any de ec able sequence-speci ic DNA-bind-
ing ac i i y on i s own, bu i can o m an ini ia ion com-
plex on he DNA empla e in he p esence o a second
ac o , he ups eam binding ac o (UBF), ha binds speci -
ically o he UPE and CPE o he RNA gene p omo e o
ac i a e ansc ip ion in a binding si e-dependen manne
(Jan zen e al. 1990). Some UBF homologues ha e been
isola ed om di e en e eb a e species, hUBF om
1053
Jou nal o Cell Science 103, 1053-1063 (1992)
P in ed in G ea B i ain © The Company o Biologis s Limi ed 1992
We ha e used an i-NOR se um om a pa ien wi h
heuma oid a h i is, o s udy i s eac i i y on di e en
phylogene ically sepa a ed species such as p o ozoa,
highe plan s, bi ds and mammals. The biochemical
cha ac e is ics o he an igens de ec ed a e applying
mono- and wo-dimensional elec opho esis and elec-
opho e ic ans e s con i m ha hey co espond o
he RNA polyme ase I ansc ip ion ac o UBF. We
ha e demons a ed he di e en molecula sizes,
depending on he cell complexi y, bu he same neu al
isoelec ic poin s in whole cell ex ac s o he di e en
species. We ha e also demons a ed an immunolocal-
iza ion o his ansc ip ion ac o o he ib illa com-
ponen in all he species s udied. These esul s sugges
a high conse a ion o UBF h oughou e olu ion and
he possibili y o using his an i-NOR se um as a ool
o he s udy o he s uc u e, nucleola o ganiza ion
and unc ional oles o he di e en nucleola compo-
nen s.
Key wo ds: an i-NOR se um, elec opho esis, immunoblo ing,
immunolabelling, nucleola componen s, nucleolus, DNA
ansc ip ion, RNA polyme ase I ansc ip ion ac o .
Summa y
Cha ac e iza ion and immunolocaliza ion o RNA polyme ase I
ansc ip ion ac o UBF wi h an i-NOR se um in p o ozoa, highe plan
and e eb a e cells
ROSA M. RODRIGO1, M. CARMEN RENDÓN2, JOSÉ TORREBLANCA1, GREGORIO GARCÍA-HERDUGO1and
FRANCISCO J. MORENO1,*
1Depa men o Cell Biology, Facul y o Biology, Uni e si y o A enida Reina Me cedes no. 6, 41012-Se ille, Spain
2Depa men o Cell Biology and Animal Biology, Facul y o Sciences, Uni e si y o Cadiz, Cadiz, Spain
*Au ho o co espondence
1054
human cells (Bell e al. 1988; Jan zen e al. 1990; O’Mahony
and Ro hblum, 1991), xUBF om Xenopus lae is ( P i k a a d
e al. 1989), UBF om a cells (Pikaa d e al. 1990; Smi h
e al. 1990), and i has also been pu i ied om mouse cells
(O’Mahony and Ro hblum, 1991; Schnapp and G umm ,
1991). Compa ison o hese homologues has led o he
obse a ion ha he UBF DNA-binding speci ici y has been
s ongly conse ed om ogs o humans, s eng hening he
conclusion ha UBF homologues wi h iden ical DNA-bind-
ing speci ici y should be ound in all e eb a e cells, and
sugges ing ha he sequence mo i s in he p omo e s mus
be mo e conse ed han is eadily appa en and ha he mol-
ecula cause o species-speci ici y mus eside in some o he
pa o he ansc ip ion machine y, no in he UBF p omo e
ecogni ion (Pikaa d e al. 1990).
Impo an ools ha could acili a e u he analysis o
UBF would be an ibodies di ec ed agains his ansc ip ion
ac o . In a p elimina y s ep in his di ec ion we ha e used
an au oimmune se um, he an i-NOR se um, om a pa ien
wi h heuma oid a h i is, and ound i o eac speci ically
wi h a 92-88 kDa p o ein double wi h an isoelec ic poin
o a ound 7.5 in ex ac s o whole HeLa cells; his clea ly
co esponds o hUBF (Rendón e al. 1992). Fu he mo e,
a e immunoelec on mic oscopic s udies we ha e local-
ized he an igen o he nucleolus, which is a highly o de ed
nuclea componen whe e ibosome biogenesis occu s,
in ol ing he ansc ip ion o RNA genes, he associa ion
o nascen anscip s wi h speci ic p o eins and he mul i-
ple s eps o ma u a ion o p ima y ansc ip s leading o he
o ma ion o he p e- ibosomal subuni s (Fakan, 1986;
Goessens, 1984; He nandez-Ve dun, 1986; Schee and
Bena en e, 1990). These di e en s eps o ibosome bio-
genesis ake place wi hin mo phologically well-de ined
componen s, namely he ib illa cen es and dense ib illa
componen , which cons i u e he ib illa componen , and
he g anula componen (Jo dan, 1984). We ha e ound
immunolabelling wi h an i-NOR se um localized o he ib-
illa componen o HeLa cell nucleoli (Rendón e al. 1992),
he si e whe e ibosomal RNA ansc ip ion is belie ed o
occu (De enzini e al. 1990; Fakan, 1986; Goessens, 1984;
He nandez-Ve dun, 1986; Jo dan, 1991; Pu ion-Du illeul e
al. 1991; Raska e al. 1990; Schee and Bena en e, 1990;
Wa chle e al. 1989).
The aim o his pape has been o inc ease ou unde -
s anding o he ac i i y o he abo e-men ioned human an i-
NOR se um h ough he s udy o i s eac i i y wi h di e -
en phylogene ically sepa a e species using p o ozoan,
highe plan , a ian and mammalian cells as sou ces o
speci ic an igens. I s biochemical cha ac e is ics s ongly
con i m ha i co esponds o he UBF RNA ansc ip ion
ac o , hus sugges ing ha he e has been high conse a-
ion o UBF h oughou e olu ion and he possibili y o
using an i-NOR se um as a nucleola ma ke o s udy he
s uc u e and nucleola o ganiza ion in hese e olu iona ily
e y dis an cells.
Ma e ials and me hods
Human se um
Au oimmune se um was ob ained om a pa ien wi h heuma oid
a h i is.
Ma e ial p epa a ion
The cilia e His iculus similis was mass cul u ed a 20 (± 1)°C in
a 1l E lenmeye lask con aining mine al wa e . The g een alga
Chlo ogonium sp. was used o eed he p o ozoa.
Roo me is ems o Allium cepa L. we e ob ained om onion
bulbs g own unde s anda d condi ions a 15°C (Co és e al.
1982).
P ima y cul u es o chicken chond ocy es we e ob ained om
he s e na o 14-day-old emb yos, diges ed wi h 0.25% (w/ )
ypsin, 0.2% (w/ ) ype IA collagenase, and 1000 uni s/ml ype
II collagenase in PBS. Cells we e cul u ed in Ham’s F-12 medium
supplemen ed wi h 10% oe al cal se um and 0.002 M L-glu a-
mine on 35- and 60-mm dishes coa ed wi h gela in. Chond ocy es
we e ed e e y o he day and 60 min be o e s a he expe imen s.
P K1cells we e cul u ed in Dulbecco’s medium wi h 0.85 g/l
sodium bica bona e supplemen ed wi h 10% oe al bo ine se um
(FBS), 2% glu amine, 1 mM sodium py u a e, 100 i.u./ml peni-
cillin-s ep omycin, 1% Fungizone and 1% ylocine (an iPPLO).
TG cells we e g own and main ained in cell cul u e lasks con-
aining Dulbecco’s medium supplemen ed wi h 10% FBS, 2 mM
glu amine, 1 mM sodium py u a e, 100 i.u./ml penicillin-s ep o-
mycin, 1% Fungizone and 1% an iPPLO.
Ma e ials we e p ocessed o mo phological and immunocy o-
chemical elec on mic oscopic s udies. Fo he mo phological
s udy, cells we e ixed in 1.6% glu a aldehyde in 0.1 M cacody-
la e bu e , pH 7.2, o 60 min a 4°C, excep o Allium cepa
cells ha we e ixed in 4% glu a aldehyde. They we e all pos -
ixed in 1% osmium e oxide o 60 min a 4°C. Samples we e
dehyd a ed in ace one a p og essi ely highe concen a ions and
embedded in esin ollowing he me hod o Spu (1969).
Fo elec on mic oscopic immunolocaliza ion, cells we e ixed
in a mix u e o 4% pa a o maldehyde and 0.1% glu a aldehyde in
0.1 M cacodyla e bu e , pH 7.3, o 60 min a 4°C.
Cul u ed cells we e ixed di ec ly in he cul u e lasks, subse-
quen ly sc aped wi h a ubbe policeman, and pelle ed by cen-
i uga ion.
Samples we e washed in 0.1 M cacodyla e bu e , pH 7.3, and
incuba ed in 0.5 M NH4Cl o 4 h a oom empe a u e o block
ee aldehyde g oups. They we e hen washed in he same bu e
again.
Lowic yl K4M-embedded sec ions
Low- empe a u e embedding wi h he hyd ophilic esin Lowic yl
K4M (Chemische We ke Lowi., Waldk aibu g, FRG) was pe -
o med, wi h sligh modi ica ions, acco ding o he me hod
desc ibed by Ro h (1983). Samples we e dehyd a ed in a se ies
o g aded me hanols a p og essi ely lowe empe a u es. Du ing
in il a ion wi h Lowic yl K4M a −20°C, 90% me hanol was used
as he dehyd a ion agen . Lowic yl was polyme ized by indi ec
long-wa e (360 nm) UV i adia ion om a 15 W Philips luo es-
cen lamp o 24 h.
Ul a hin sec ions we e cu on a Reiche -Jung Ul acu E ul a-
mic o ome, moun ed on 300-mesh nickel g ids and pho og aphed
in a Philips CM-10 ansmission elec on mic oscope (Se icio de
Mic oscopía Elec ónica, Uni e si y o Se ille).
Immunocy ochemical labelling
G ids we e incuba ed by loa ing hem, cell sec ions down, on a
d op o 0.1 M PBS, pH 7.4, con aining 1% BSA, 0.05% T i on
X-100 and 0.05% Tween 20 (PBTT), o 5 min a oom empe -
a u e, and washed in PBS. The nex s ep o he ea men was an
incuba ion in an i-NOR se um dilu ed 1:100 o 1:1000 in PBS o
60 min a oom empe a u e. A e washing in PBS, he second
incuba ion was in P o ein A-gold complex p epa ed ollowing
Bendayan e al. (1980), dilu ed in PBTT un il an abso bance o
R. M. Rod igo and o he s
1055Immunode ec ion o UBF in di e en cell ypes
0.06 (525 nm) was eached, o 60 min a oom empe a u e (Ro h
e al., 1989). G ids we e washed wi h dis illed wa e and con-
as ed wi h 2% u anyl ace a e in dis illed wa e . To demons a e
he speci ici y o he labelling, con ols we e pe o med by omi -
ing he incuba ion wi h he an i-NOR se um o by using a no mal
human se um du ing he i s incuba ion s ep.
Mono-dimensional gel elec opho esis (SDS-PAGE)
Chond ocy es, P K1and TG cells we e ypsinized om cul u e
lasks a e a aining 75% con luence, washed in PBS and pelle ed
a e cen i uga ion a 55 g. Cul u ed His iculus similis cells we e
also pelle ed a e gen le cen i uga ion. Ex ac ion o p o eins was
pe o med by ea ing he pelle s wi h SDS-PAGE sample bu e
o 10 min a 97°C (Laemmli, 1970).
Ex ac ion o p o eins om Allium cepa oo me is em cells was
pe o med a e ea men wi h 32% pec inase in ci ic-ci a e
bu e , pH 4.2, con aining 1 mM PMSF. A e washing in PBS,
oo s we e pul e ized in a liquid ni ogen-cooled mo a . We con-
inued by solubiliza ion o he p o eins in SDS-sample bu e o
10 min a 97°C (Laemmli, 1970).
Elec opho esis was pe o med, as desc ibed by Laemmli
(1970), on 7.5% o 12% SDS-polyac ylamide gels using he Mini-
P o ean II elec opho esis cell (Bio-Rad., Richmond, CA). A se
o molecula mass s anda ds we e ob ained om Bio-RAD Lab-
o a o ies: myosin (200 kDa), β-galac osidase (116.25 kDa), phos-
pho ylase B (97.4 kDa), bo ine se um albumin (66.2 kDa), o al-
bumin (42.69 kDa), ca bonic anhyd ase (31 kDa), soybean ypsin
inhibi o (21.5 kDa), and lysozyme (14.4 kDa). When elec-
opho esis was ca ied ou subsequen ly o pe o m ans e s o
p o eins o ni ocellulose pape , a se o p e-s ained molecula
mass s anda ds we e used (Sigma Chemical Co.): α2-mac oglob-
ulin (180 kDa), β-galac osidase (116 kDa), uc ose-6-phospha e
kinase (84 kDa), py u a e kinase (58 kDa), uma ase (48.5 kDa),
lac ic dehyd ogenase (36.5 kDa), and iosephospha e isome ase
(26.6 kDa).
To al p o eins in gels we e e ealed wi h Coomassie blue s ain-
ing.
Two-dimensional gel elec opho esis
His iculus similis,Allium cepa, P K1and TG cells and chond o-
cy es we e ea ed as o mono-dimensional SDS-PAGE. We used
he O’Fa ell (1975) lysis bu e as he isoelec ic ocusing (IEF)
sample bu e . The concen a ions o p o eins in he IEF sample
bu e we e de e mined wi h a modi ied B ad o d assay, which
allows quan i a ion o p o eins in he p esence o u ea, ca ie
ampholy es, non-ionic de e gen s, and hiol compounds (Ramagli
and Rod iguez, 1985). The olume o sample loaded pe capilla y
gel ube con ained a concen a ion o a ound 30 µg o al p o eins.
Two-dimensional polyac ylamide gel elec opho esis (2D
PAGE) was pe o med acco ding o O’Fa ell (1975) using he
Mini-P o ean 2-D elec opho esis cell (Bio-Rad) as desc ibed p e-
iously (Mo eno e al. 1990). The second-dimensional gel elec-
opho esis in he p esence o sodium dodecyl sul a e (SDS) was
ca ied ou as o mono-dimensional SDS-PAGE elec opho esis.
To al p o eins in gels we e e ealed by using he Bio-Rad sil e
s ain Ki .
Elec opho e ic ans e and immunoblo ing
P o eins we e ans e ed elec opho e ically om polyac ylamide
gels o ni ocellulose pape (Towbin e al. 1979), and incuba ed
in 3% gela in in TBS (15 mM T is, 200 mM NaCl) o 24 h a
oom empe a u e. A e wo washes o 10 min each in TBS con-
aining 0.05% Tween-20 (TTBS), an incuba ion in an i-NOR
se um dilu ed 1:100 in TTBS con aining 1% gela in was pe -
o med o 24 h a oom empe a u e. Blo s we e insed in TTBS
and incuba ed in pe oxidase-labelled goa an i-human IgG
(Boeh inge Mannheim Biochemicals) dilu ed 1:2000 in TTBS,
o 2 h a oom empe a u e. A e washing in TTBS and TBS,
he eac ion was isualized wi h chlo onaph hol subs a e (50 ml
TBS con aining 0.03 g 4-chlo o-1-naph hol in 10 ml me hanol and
20 µl 30% hyd ogen pe oxide). Colou de elopmen was s opped
in dis illed wa e and blo s we e allowed o ai d y. Con ol ials
we e pe o med by omi ing he incuba ion wi h he an i-NOR
se um.
Pho og aphs o gels and ans e s we e aken wi h an AGFA
Copex Rapid A.H.U. ilm p ocessed o 9 ASA.
Resul s
A he ul as uc u al le el, His iculus similis ege a i e
cells showed a oluminous mac onucleus consis ing o he -
e och oma in wi h a e icula e dis ibu ion and in e spe sed
euch oma in. Be ween he condensed ch oma in h eads
nume ous nucleoli wi h a ounded mo phology could be
obse ed in hin sec ions. These s uc u es we e composed
o se e al mode a ely elec on-dense small ib illa a eas,
simila o he ib illa cen es o animal cell nucleoli, o ally
o pa ially su ounded by s ands o dense ib illa com-
ponen , embedded in a ine g anula componen . They e-
quen ly showed la ge acuoles o in e s ices, consis ing o
a ma e ial simila o nucleoplasm. The p esence o eplica-
ion bands was equen ly obse ed in he mac onuclei o
hese ege a i e cells (Fig. 1).
When we p oceeded o immunolocaliza ion wi h a 1:500
an i-NOR se um dilu ion we obse ed ha labelling wi h
P o ein A-gold was es ic ed o nucleoli. Gold pa icles
we e ound mos ly dis ibu ed o e he ib illa componen
s ands. Fine g anula ma e ial was de oid o labelling (Fig.
2).Nucleoli o Allium cepa we e o med o a h ead-like
o ganized ib illa componen imme sed in he g anula
componen . Inside he ib illa componen , some clea a eas
could be obse ed, con aining homogeneous ma e ial and/o
dense inclusions. Some in e s ices co esponding o he
nucleola acuoles could be ound in he g anula compo-
nen (Fig. 3).
Immunolocaliza ion wi h an i-NOR se um in Allium cepa
showed gold pa icles dis ibu ed only o e he nucleola
h ead-like ib illa componen . Clea a eas, wi h and wi h-
ou inclusions, and g anula componen we e ee o
labelling (Figs 4 and 5).
Chicken chond ocy es equen ly disclosed e icula e
nucleoli cha ac e ized by a ne wo k o h eads, o nucle-
olonema. Some ib illa cen es could be de ec ed o ally o
pa ially su ounded by dense ib illa componen . In e -
s ices usually appea ed be ween he nucleolonema h eads
(Fig. 6). When we used he an i-NOR se um we ound he
an igen immunolocalized o he ib illa cen es and dense
ib illa componen o chond ocy e nucleoli (Fig. 7).
P K1nucleoli in cycling cells showed nume ous small
ib illa cen es pa ially su ounded by connec ed s ands
o dense ib illa componen , si es whe e we ound immuno-
labelling wi h an i-NOR se um, o ming a ne wo k embed-
ded in he g anula componen (Figs 8 and 9).
A he ul as uc u al le el TG cells disclosed nucleoli
wi h a compac mo phology, o med by ib illa cen es ha
we e ound o be a leas pa ly su ounded by dense
1056
ib illa componen , and he g anula componen (Fig.
10).
The p ecise localiza ion o an igens ob ained wi h an i-
NOR se um was de ec ed o e he ib illa cen es and
dense ib illa componen o TG cell nucleoli (Figs 11 and
12). The g anula componen , ch oma in, nucleoplasm and
R. M. Rod igo and o he s
Figs 1-5. Fo legends see p. 1058
1057Immunode ec ion o UBF in di e en cell ypes
cy oplasm we e de oid o labelling. We did no obse e
gold pa icles in any o he con ols pe o med.
To cha ac e ize he nucleola an igens ecognized by he
an i-NOR se um we ca ied ou mono-dimensional gel elec-
opho esis o whole cell ex ac s ob ained om he di e -
en species s udied, ollowed by elec opho e ic ans e .
Figs 6-9. Fo legends see p. 1058

1058
We ound wo polypep ides which, espec i ely, mig a ed
a 92 and 88 kDa immunoblo ed wi h an i-NOR se um in
TG and P K1cells and chicken chond ocy es. A simila p o-
ein double eac ed in His iculus similis cells, bu wi h a
molecula mass o 58-56 kDa, which comig a ed wi h a
single polypep ide in whole Allium cepa oo me is em cell
ex ac s. Co esponding Coomassie-s ained gels o cell
ex ac s showing he ull complemen o ex ac ed p o eins
and a nega i e con ol assay in which an i-NOR was
excluded oge he con i med he blo ing immunospeci ici y
(Fig. 13).
In o de o cha ac e ize u he he polypep ides e ealed
by he an i-NOR se um we ca ied ou wo-dimensional
elec opho esis ollowed by elec opho e ic ans e s o
whole cell ex ac s o he di e en species s udied. We
ound, a e immunoblo ing wi h an i-NOR se um, speci ic
an igens wi h app oxima e molecula masses o 92 kDa and
isoelec ic poin s o a ound 7 in TG cells, P K1cells and
chond ocy es. In Allium cepa oo me is em cell ex ac s,
h ee polypep ides wi h molecula masses o a ound 58 kDa
immunoblo ed wi h an i-NOR se um, wi h isoelec ic
poin s o app oxima ely 7 (Figs 14-17).
Discussion
The nucleolus is a domain o he in e phase cell nucleus
whe e ibosomal RNA ( RNA) syn hesis akes place and
whe e se e al non-his one p o eins a e ound (Goessens,
1984; He nandez-Ve dun, 1986; Jo dan, 1991). I is gene -
ally ag eed ha ansc ip ion o DNA genes and he i s
s eps o RNA p ocessing occu a he ib illa componen
o he nucleolus. In e es ingly, by using au oan ibodies om
di e en pa ien s wi h au oimmune diseases, di e en p o-
eins ha e been localized o he ib illa componen , such
as RNA polyme ase I (Reime e al. 1987), DNA opoiso-
me ase I (She o e al. 1986), nucleolin (Mino a e al. 1991)
and ib illa in (Ochs e al. 1985; Reime e al. 1987). O he
au oimmune se a ha e also been shown o eac agains
speci ic an igens localized o he ib illa cen es o dense
ib illa componen o nucleoli om di e en cell ypes
(He nandez-Ve dun e al. 1988, 1991; Masson e al. 1990;
Rod iguez-Sanchez e al. 1987). The inding ha some o
hese p o eins ha e been conse ed du ing e olu ion indi-
ca es ha hey migh pa icipa e in some undamen al unc-
ion (He nandez-Ve dun, 1991).
In a p e ious s udy, we used an au oimmune se um om
a pa ien wi h heuma oid a h i is and de ec ed a p o ein
double a 92-88 kDa in whole HeLa cell ex ac s, which
co espond o hUBF, and we immunolocalized i o he ib-
illa componen o he nucleolus o HeLa cells (Rendón e
al. 1992). Some au ho s ha e shown ha , despi e he ex en-
si e sequence di e gence o polyme ase I p omo e s, he e
exis s an e olu iona ily s ong conse a ion o he p ima y
UBF DNA-binding speci ici y in e eb a es (Pikaa d e al.
1990). This UBF ansc ip ion ac o has been shown o
consis o wo polypep ides wi h appa en molecula masses
o 97-94 kDa in mammals (Bell e al. 1988; Jan zen e al.
1990; O’Mahony and Ro hblum, 1991; Pikaa d e al. 1990;
Smi h e al. 1990; Schnapp and G umm , 1991) and 85-82
kDa in ogs (Pikaa d e al. 1989).
We ha e ex ended hese s udies o di e en cell ypes in
mammals (TG and P K1), bi ds (chicken chond ocy es),
highe plan s (Allium cepa oo me is em cells) and p o o-
zoa (His iculus similis). A e mono-dimensional elec-
opho esis o whole cell p o eins, elec o ans e o ni o-
cellulose and immunoblo ing wi h an i-NOR se um, we
ha e de ec ed speci ic an igens wi h molecula masses o
92 and 88 kDa in TG cells, P K1cells and chicken chon-
d ocy es, while wo polypep ides a 58 and 56 kDa ha e
been localized in His iculus similis and a single polypep-
R. M. Rod igo and o he s
Fig. 1. Po ion o a His iculus similis nucleus wi h nume ous
ounded nucleoli o med by ib illa componen (a ows) and
g anula componen . Replica ion band (R). Ma e ial ixed in 1.6%
glu a aldehyde, pos ixed in 1% osmium e oxide and embedded
in Spu esin. ×15500.
Fig. 2. Immunolabelling wi h an i-NOR se um localized o he
ib illa componen s ands o a His iculus similis nucleolus ixed
in 4% pa a o maldehyde - 0.1% glu a aldehyde and embedded in
Lowic yl K4M. G anula componen (G). ×95700.
Fig. 3. Allium cepa oo me is em cell nucleolus o med by a
h ead-like ib illa componen (F) embedded in he g anula
componen (G). Clea a eas wi h inclusions (a ows) and wi hou
inclusions (a owheads) can be obse ed. Roo s ixed in 4%
glu a aldehyde, pos ixed in 1% osmium e oxide and embedded
in Spu esin. ×18850.
Figs 4 and 5. The localiza ion o gold pa icles a e an i-NOR
se um was ound o e he ib illa componen h eads o Allium
cepa nucleoli ixed in 4% pa a o maldehyde - 0.1%
glu a aldehyde and embedded in Lowic yl K4M. Clea a eas
wi hou inclusions (a owheads), wi h inclusions (a ow), g anula
componen and he e och oma in a e de oid o labelling. Fig. 4.
×23900. Fig. 5. ×46300.
Fig. 6. Re icula e nucleolus o a chicken chond ocy e disclosing
ib illa cen es (a owhead) su ounded by dense ib illa
componen (a ows). G anula componen (G). Fixa ion in 1.6%
glu a aldehyde, pos ixa ion in 1% osmium e oxide and
embedding in Spu esin. ×47500.
Fig. 7. Immunolabelling wi h an i-NOR se um o e he ib illa
cen es (a owheads) and dense ib illa componen (a ows) in a
chicken chond ocy e nucleolus ixed in 4% pa a o maldehyde -
0.1% glu a aldehyde and embedded in Lowic yl K4M. ×47000.
Fig. 8. Nucleolus o a P k1cell cons i u ed o small ib illa
cen es (a owheads) su ounded by s ands o dense ib illa
componen (a ows). G anula componen (G). Ma e ial ixed in
1.6% glu a aldehyde, pos ixed in 1% osmium e oxide and
embedded in Spu esin. ×33000.
Fig. 9. Immunolocaliza ion o an igens wi h an i-NOR se um o e
ib illa cen es (a owhead) and dense ib illa componen
(a ows) in a P k1nucleolus ixed in 4% pa a o maldehyde - 0.1%
glu a aldehyde and embedded in Lowic yl K4M. ×41600.
Fig. 10. Compac nucleolus o a TG cell showing wo ib illa
cen es su ounded by dense ib illa componen , and imme sed in
g anula componen . Cell ixed in 1.6% glu a aldehyde, pos ixed
in 1% osmium e oxide and embedded in Spu esin. ×45500.
Figs 11 and 12. Immunolabelling wi h an i-NOR se um in TG
cells ixed in 4% pa a o maldehyde - 0.1% glu a aldehyde and
embedded in Lowic yl K4M.
Fig. 11. Gold pa icles a e localized o he ib illa cen es and
dense ib illa componen . G anula componen and nucleoplasm
a e de oid o labelling. ×46750.
Fig. 12. An igens immunolocalized inside a ib illa cen e and
o e he po ions o dense ib illa componen su ounding i .
×121300.
1059Immunode ec ion o UBF in di e en cell ypes
ide a 58 kDa in Allium cepa oo me is em cells. A e
wo-dimensional elec opho esis hese an igens showed iso-
elec ic poin s o a ound 7, hus con i ming he esul s o
ou p e ious s udy wi h HeLa cells (Rendón e al. 1992).
No wi hs anding he di e gence in he appa en molecu-
la masses o he an igens speci ically de ec ed in whole
cell ex ac s, we ha e obse ed ha immunolocaliza ion
wi h an i-NOR se um always emains es ic ed o he
1060 R. M. Rod igo and o he s
Fig. 13. Coomassie blue s aining o 12% PAGE-sepa a ed
p o eins p esen in whole cell ex ac s o (A) His iculus similis,
(B) Allium cepa, (C) chicken chond ocy es, (D) P K1and (E) TG
and he espec i e cha ac e iza ion by immunoblo ing o he
an igen ecognized upon p obing wi h 1:100 dilu ed an i-NOR
se um. Two polypep ides, 58 and 56 kDa, a e e ealed in
His iculus similis (A′), a single p o ein a 58 kDa in Allium cepa
(B′), and a p o ein double o 92 and 88 kDa in chicken
chond ocy es (C′), P K1(D′) and TG cells (E′). (F) The
co esponding con ol assay in which an i-NOR se um was
omi ed.
Figs 14 and 15. Cha ac e iza ion o he an igens by means o wo-
dimensional elec opho esis using 12% SDS-PAGE, ollowed by
elec opho e ic ans e o whole cell ex ac s in Allium cepa oo
me is em cells (Fig. 14) and chicken chond ocy es (Fig. 15). (A)
Pa e n o o al p o eins s ained wi h sil e . (B) Immunoblo ing
wi h a 1:100 dilu ion o an i-NOR se um. The an igens wi h
isoelec ic poin s o a ound 7 (a ows) can be obse ed.
A B C D E F
1061Immunode ec ion o UBF in di e en cell ypes
nucleola ib illa componen o he di e en species s ud-
ied, sugges ing a uni o mi y in he unc ional ole o his
p o ein. The pe sis ence o he an igen a he ib illa com-
ponen a e ea men wi h he RNA syn hesis inhibi o
AMD, and he exis ence o labelling a he NORs du ing
mi osis (Rendón e al. 1992) demons a e ha his p o ein
emains joined o he DNA unde si ua ions o low an-
sc ip ional ac i i y. These da a ha e been e y ecen ly co -
obo a ed by O’Mahony e al. (1992), who ha e shown ha
he le el o exp ession o UBF does no change in esponse
o se um dep i a ion in CHO cells. Howe e , hey ha e
demons a ed ha he deg ee o phospho yla ion o se ine
esidues o his phosphop o ein is educed ollowing se um
dep i a ion and ha , a e e eeding, his le el inc eases
d ama ically. These au ho s ha e also obse ed ha unde
he condi ions o se um s a a ion, he subcellula dis ib-
u ion o UBF shi s om being p edominan ly nucleola o
a dispe sed nucleola /nuclea /cy oplasmic dis ibu ion and,
a e e eeding, i e u ns again o being immunolocalized
o he nucleolus. This ansloca ion om he nucleolus o
he nucleoplasm, which o igina ed a e se um s a a ion,
also occu s wi h he nucleola phosphop o ein B23 o NO38
(Busch e al. 1987). Mo eo e , O’Mahony e al. (1992)
sugges ha he phospho yla ion s a e o UBF may a ec
i s ansac i a ion p ope ies and ha he e also is a possi-
bili y ha he phospho yla ion o UBF is necessa y o i s
nucleola localiza ion. Besides ha , his DNA-binding p o-
ein con ains DNA-binding domains homologous o HMG
Figs 16 and 17. Cha ac e iza ion o he an igens by means o wo-dimensional elec opho esis using 12% SDS-PAGE, ollowed by
elec opho e ic ans e o whole ex ac s o P K1cells (Fig. 16) and TG cells (Fig. 17). (A) Pa e n o o al p o eins s ained wi h sil e .
(B) Immunoblo ing wi h a 1:100 dilu ion o an i-NOR se um. The an igens can be obse ed disclosing isoelec ic poin s o a ound 7
(a ows).