From the Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Via Celoria 26, 20133 Milano, Italy
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ABSTRACT |
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The CCAAT-binding activator NF-Y is formed by
three evolutionary conserved subunits, two of which contain putative
histone-like domains. We investigated NF-Y binding to all CCAAT boxes
of globin promoters in direct binding, competition, and supershift
electrophoretic mobility shift assay; we found that the ,
, and
proximal
CCAAT boxes of human and the prosimian Galago bind avidly,
and distal
CCAAT boxes have intermediate affinity, whereas the
and
sequences bind NF-Y very poorly. We developed an efficient
in vitro transcription system from erythroid K562 cells and
established that both the distal and the proximal CCAAT boxes are
important for optimal
-globin promoter activity. Surprisingly, NF-Y
binding to a mutated distal CCAAT box (a C to T at position
114) is
remarkably increased upon occupancy of the high affinity proximal
element, located 27 base pairs away. Shortening the distance between
the two CCAAT boxes progressively prevents simultaneous CCAAT binding, indicating that NF-Y interacts in a mutually exclusive way with CCAAT
boxes closer than 24 base pairs apart. A combination of circular
permutation and phasing analysis proved that (i) NF-Y-induced angles of
the two
-globin CCAAT boxes have similar amplitudes; (ii) occupancy
of the two CCAAT boxes leads to compensatory distortions; (iii) the two
NF-Y bends are spatially oriented with combined twisting angles of
about 100°. Interestingly, such distortions are reminiscent of core
histone-DNA interactions. We conclude that NF-Y binding imposes a high
level of functionally important coordinate organization to the
-globin promoter.
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INTRODUCTION |
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The CCAAT box is a widespread regulatory sequence found in promoters and enhancers of several genes (1), whose functional importance has been well established in different systems (2-12). NF-Y (also termed CBF) has an almost absolute requirement for these five nucleotides and a strong preference for additional flanking sequences (13, 14). Based on supershift experiments with anti-NF-Y antibodies, on competition analysis with the original Ea Y box oligo,1 or on the heteromeric nature of the DNA-binding complex, NF-Y has been identified as the CCAAT box activator in over 100 promoters (7-11, 14, 15). The CCAAT consensus derived statistically by Bucher (1) (RRCCAAT(C/G)(A/G)) fits well with the optimal NF-Y-binding site.
NF-Y is a ubiquitous heteromeric protein composed of three subunits,
NF-YA, NF-YB, and NF-YC, all necessary for DNA binding (16, 17). The
cloning of NF-Y genes from several species including yeast, maize,
lamprey, and sea urchin, evidenced highly conserved domains (16-22).
The NF-YA homology domain can be divided into subunit association and
DNA-contacting subdomains (20). The N-terminal contains a hydrophobic
and Gln-rich activation surface (18). The NF-YC gene has been recently
cloned and is specular with respect to NF-YA, since the homology domain
is at the N terminus, whereas the C-terminal 180 amino acids are rich
in glutamines and hydrophobic residues. NF-YB and NF-YC tightly
interact with each other, and their association is a prerequisite for
NF-YA binding and sequence-specific DNA interactions (16, 22). Both the
NF-YB- and NF-YC-conserved domains contain putative histone fold
motifs. This motif, common to all core histones, is responsible for the
formation of the histone octamer (24) and is composed of three
-helices, separated by short loops/strand regions, enabling histones
to dimerize with companion subunits (24, 25). Recent experiments on the
yeast HAP3 (26), NF-YB/CBF-A (27), and NF-YC/CBF-C (28) indicate that
this 65 amino acid long motif is necessary for subunit interactions and
DNA binding. NF-Y has additional interesting features as follows: (i)
CCAAT boxes are not able to activate alone even if multimerized,
but they increase the activity of neighboring enhancer motifs. (ii)
NF-Y appears to increase the affinity of transcription factors for
their target sequence (29). However, the exact mechanisms of
transcriptional activation by NF-Y are still elusive.
Globin genes are transcribed in a tissue-specific and developmentally
regulated manner by means of various regulatory elements in their
clusters (30). In addition to the locus control regions which have been
characterized in transgenic mice, the promoters of each globin gene
contain sequences that usually impart tissue-specific control in
transfection experiments. Several lines of reasoning point to CCAAT
sequences as important elements in globin gene expression as follows:
(i) they are present in all globin promoters; (ii) they have been
remarkably conserved in different species at a fixed distance from the
cap site; (iii) genomic footprinting of all globin promoters in
erythroid cells showed invariable protection of CCAAT sequences
in vivo, indicating binding of activators (31); (iv) the
functional importance has been documented in the -,
-,
-, and
-globin promoters (2-5, 15); (v) the
-globin duplicated CCAAT
boxes are the target of mutations affecting developmental silencing of
-globin expression in adult life. In particular, strong genetic
evidence suggests that point mutations in the CCAAT box region are
causative of the HPFH syndromes, characterized by increased fetal
globin levels in adults (32-37).
Several studies investigated factors binding to CCAAT; CDP, NFE3, and
c/EBP appear to recognize different globin promoters (3, 32-36). One
CCAAT-binding protein, -CP1, was purified to homogeneity using an
-globin CCAAT box affinity column and found to be a trimeric factor
most likely identical to NF-Y (15). Moreover, competition and
supershift EMSA experiments performed with nuclear extracts determined
that a CCAAT binding activity similar to NF-Y (termed CP1 in the globin
field) binds to the
-globin promoters (32, 33). However,
identification of CCAAT binding activities in other promoters is less
certain; therefore, we wished to definitely determine which of the
globin CCAAT boxes represents a bona fide NF-Y-binding site and
establish a hierarchy of affinities. We then focused our attention on
the highly regulated
-globin duplicated CCAAT box region. Because of
the histone-like nature of NF-Y and its ability to distort DNA, we
examined the possibility that NF-Y organizes this region.
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MATERIALS AND METHODS |
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Plasmid Constructions-- The starting plasmid for the circular permutation assays was pBend2 (14). The proximal and distal CCAAT box oligos described in Table I were inserted by blunt end ligation into the XbaI site. The double CCAAT and the phasing analysis oligos were also cloned into the XbaI site.
Plasmids for in vitro transcription (pAG1 and mutants thereof) were obtained by inserting theElectrophoretic Mobility Shift Assay (EMSA)-- Labeled oligonucleotides (10,000 cpm) containing the different globin CCAAT boxes were incubated with K562 nuclear extracts (5 µg) for 30 min at 25 °C. Binding reactions for NF-Y were performed incubating labeled oligonucleotides for 20 min at 20 °C, in a buffer containing 5% glycerol, 50 mM NaCl, 20 mM Tris, pH 7.5, 0.5 mM EDTA, 5 mM MgCl2, and 1 mM dithiothreitol, and run in 4% polyacrylamide gels (acrylamide/bis-acrylamide ratio of 29:1) at 4 °C. Supershift experiments were performed as described in Ref. 12.
Competition experiments were performed following two different incubation procedures as described in Fig. 6: increasing concentrations (1, 3, 10, and 25 ng) of cold competitors were incubated either before addition of the labeled oligos (6000 cpm) or after 10 min of incubation of NF-Y with the probe. Recombinant NF-YA9 and purified NF-YB/C were prepared as in Refs. 22 and 39. For circular permutation assays, NF-Y was incubated with end-labeled fragments generated by cuts with different enzymes (2000 cpm in each reaction). Fragments generated by the central XhoI digestions were used for the phasing analysis.Calculations of Bending Angles-- Location of the points of flexure and amplitudes of the bending angles were described previously (14, 40). Briefly, the mobilities of the NF-Y-DNA complexes were normalized to the mobilities of the corresponding free DNA fragments; bending angles were calculated considering the ratio between the fastest and the slowest migrating complexes in EMSA, according to the formula mM/mE = cos a/2, where mM is the relative mobility of the complex exactly in the middle, mE is the relative mobility of the complex at the end of the fragment, and a is the angle of deviation. To determine bending centers, the normalized mobility of each NF-Y-DNA complex was plotted as a function of the distance between the center of the CCAAT sequence and the end of the DNA fragment; the bend was determined as the position at which the NF-Y-DNA complex was at a minimum.
In the phasing analysis, the mobility of the upper bands of the +3,In Vitro Transcription-- Preparation of transcriptionally competent K562 nuclear extracts and in vitro transcription reactions were detailed in Ref. 38. Two independent CsCl plasmid preparations of the pAG vectors were used in different sets of experiments.
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RESULTS |
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NF-Y Binds to CCAAT Boxes of All Globin Promoters--
To
determine which of the globin CCAAT boxes is recognized by NF-Y, we
labeled oligonucleotides (see Table I)
for EMSA experiments with nuclear extracts, challenging the resulting
complexes with anti-NF-YB- and anti-NF-YA-purified antibodies (12).
Bands of different mobilities and intensities are generated with all
oligos (Fig. 1), with the (lanes 4-6),
(lanes 8-10), h
P (human
proximal, lanes 16-18), g
P (prosimian Galago
crassicaudatus
proximal, see Ref. 36, lanes
28-30), h
D (human
distal, lanes 20-22) and
g
D (G. crassicaudatus
distal, lanes
32-34), one predominant shifted band is visible; these complexes
are supershifted by specific anti-YB and anti-YAc antibodies (12). In
each case we ran parallel migrations of the Y box oligo incubated with
recombinant NF-YA and purified NF-YB/NF-YC; as shown in Fig. 1, the
bands have electrophoretic behaviors identical to the endogenous K562 bands shifted by the anti-NF-Y antibodies. On the other hand, with the
and
CCAAT multiple bands are visualized (lanes
12-14 and 24-26); for the
a weak band comigrating
with NF-Y and supershifted by the antibodies is observed, and for the
the four major bands detected have different mobilities compared
with the Y/NF-Y band, and no supershift is evident. These data show
that NF-Y binding is readily visualized on all globin CCAAT boxes and
is the most prominent binding protein, with the exception of
and
, which show a much higher affinity for proteins unrelated to
NF-Y.
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Both -Globin CCAAT Boxes Contribute to Promoter Activity in
Vitro--
We next focused our attention on the developmentally
regulated
-globin promoter. We fused the minimal tissue-specific
-globin promoter (
299 to +35) to a rabbit
-globin reporter gene
(plasmid pAG1) and generated mutants in the proximal or in the distal
CCAAT boxes (see scheme in Fig.
3A). The wt and mutated
constructs were tested in a functional in vitro assay with
transcriptionally competent erythroid K562 extracts. RNA was purified
and hybridized to a single-stranded end-labeled DNA probe; subsequent
S1 mapping allowed the determination of qualitative and quantitative
changes in the transcription rate. As an internal control we added a
plasmid containing the adenovirus major late promoter TATA box devoid of any activating sequences and fused to the same reporter gene. Fig.
3B shows that our system efficiently transcribes the
-globin promoter and faithfully reproduces the correct start site
used in vivo. We tested the different mutants: alteration of
the proximal CCAAT box or destruction of the distal by a 13-base pair
deletion (pAG3 and pAG4) decreases transcription 3-4-fold (Fig.
3C, lanes 3 and 4). Swapping the weak
-globin
CCAAT box into the distal CCAAT box position partially restores
transcription when the proximal CCAAT is mutated, while having minor
effects when the proximal CCAAT is intact (pAG2 and pAG5, respectively;
Fig. 3C, lanes 1, 2 and 5). A mutant promoter
containing mutations in both CCAAT boxes (pAG6) was also compared with
wt pAG1 and resulted in the lowest transcriptional rate (6-fold down,
compare Fig. 3C, lanes 6 and 7). Note that the
signals in Fig. 3C result from a 4-h exposure. These data
indicate that both CCAAT elements contribute to the optimal promoter
activity. However, other important activators are probably operating,
since mutations in the CCAAT boxes do not abolish transcription
completely.
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Binding of NF-Y to the -Globin Double CCAAT Boxes
Region--
By having shown that both
-globin CCAAT boxes bind NF-Y
and are important for in vitro promoter activity, we
investigated their interplay; we labeled a long oligo encompassing the
two CCAAT boxes, incubating increasing concentrations of recombinant NF-Y, containing wt NF-YA and NF-YB and the homology domain of NF-YC.
Fig. 4 shows that two bands of different
electrophoretic mobility are generated (lanes 1-4); to
ascertain whether the slow migrating complex corresponds to DNA
fragments bound by two NF-Y molecules, we used fragments mutated in the
distal CCAAT (C
114), in the proximal CCAAT (the
corresponding C to T mutation at
87), or in both. We have
deliberately chosen this mutation because genetic evidence strongly
associates it with HPFH syndromes in humans (34). Mutations in such
position are known to essentially abolish NF-Y binding to all CCAAT
boxes tested so far, including the
-globin (13-15,
40).2 The faster complex is
only modestly affected by mutations in the distal CCAAT; the slower
complex is greatly diminished in the
87 CCAAT mutant and in the
double mutant (compare lanes 4, 12 and 16).
Surprisingly, the
114 mutant exhibited a considerable level of the
upper complex (compare lanes 4 and 8 and see the calculated ratios in Fig. 4B). With the double mutant, both
the slower and the faster complexes were also crippled (lanes
13-16). These data suggest that the faster band corresponds to
NF-Y binding to either the proximal or the distal site, whereas the
upper one results from double occupancy of the two CCAAT boxes.
Interestingly, consistent with the cross-competition experiments,
binding of NF-Y to the proximal CCAAT is predominant and compensates
for a crippling mutation in the distal CCAAT, whereas the reverse is
not true.
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NF-Y Induces Bending of -Globin CCAAT Boxes--
We have
recently shown that NF-Y is able to induce distortions in the double
helix, with angles that vary depending on the surrounding sequences
(14). By using the circular permutation assay we checked the degree of
distortion of the proximal and distal CCAAT boxes separately. We cloned
the two oligos of identical length in the pBend2 vector (14); we then
cut with different enzymes so that the CCAAT boxes were at different
distances from the extremities of the fragments, and we performed EMSA
with NF-Y. To calculate precisely the angles, we maximized the
differences in mobilities of the protein-DNA complexes, using the small
NF-YA9 mutant and purified endogenous NF-YB/NF-YC. We have shown that such combination does not alter significantly the distortion angles of
four NF-Y sites (14). Clear indication that NF-Y induces distortions on
both
-globin CCAAT boxes was evident from the different
electrophoretic mobilities of the fragments (Fig.
7). Calculations of the angles gave
similar results for both sites, and the values, 66° and 72° for
distal and proximal, respectively, are similar to those observed for
the murine sarcoma virus and
-globin and slightly lower than for the
Ea Y box and HSP70 NF-Y-binding sites. As expected, the flexures are
indeed centered on the CCAAT sequences (Ref. 14 and data not
shown).
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Phasing Analysis of CCAAT Boxes--
To verify whether NF-Y
induces directed bends, as suggested by the previous experiment, and to
seek further information about the relative distortions of the CCAAT
region, we performed phasing analysis. This assay is usually tackled
with appropriate vectors containing a fixed angle of known curvature,
obtained exploiting the distortions caused by short AT-rich sequences
spaced by one turn of the helix (Ref. 40 and references therein).
Having fixed one angle, the binding site is rotationally moved on
different sides of the double helix, by increasing the distance from
the fixed angle, taking into account that one turn corresponds to 10.5 base pairs. The relative mobilities of the protein-bound complexes give
clues about the orientation of the angles. Rather than adopting this
strategy, which would give information about one isolated CCAAT, we
took advantage of the two -globin-binding sites with their
previously calculated distortions. We cloned in pBend2 the
3,
5,
8 oligos, and a +3 oligo, in which we added 3 bp between the two
CCAAT. Cutting the resulting plasmids with any of the enzymes generates
nearly identical fragments with NF-Y-binding sites rotationally
displaced with respect to the wt situation. EMSA analysis of the
central XhoI fragments is shown in Fig.
8, and comparable amounts of protein
generated the single occupancy band with identical electrophoretic
mobility for all fragments. The double occupancy band is visible with
the +3, wt, and
3 fragments, with the
5 only after prolonged
exposures, and is absent in the
8. The mobilities of the latter
bands are different, a clear indication that indeed the DNA is oriented
upon NF-Y binding (40). The slowest fragment should be the one in which
the two bends are in phase with respect to the helical turn of DNA,
whereas the fastest is the one in which the two bends are facing
opposite sides and counteract each other; the +3, in which the two
CCAAT boxes are only 1.5 bp away from perfect alignment on the same side of the helix, is the slowest, and the
3 and
5 are the
fastest. This is consistent with overall distortion angles of about
100° among the two CCAAT in the wt configuration (see Fig.
9). These results are indeed confirmed by
experiments with a "mini" NF-Y protein (22) that is able to
generate double bands in the
8 as well, which give a phasing
"period" of about 8 bp, corresponding to a rotation of about
100°.3
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DISCUSSION |
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In this study we undertook a systematic analysis of NF-Y binding
to the CCAAT boxes of globin gene promoters and established that most,
but not all, contain bona fide high affinity sites. We focused then on
the human -globin, and we developed an efficient in vitro
functional assay with K562 extracts and provided for the first time
evidence that both CCAAT boxes add to the overall level of expression.
NF-Y binds better to the proximal than to the distal site, and DNA
binding is neither cooperative nor mutually exclusive. The bending
angles of the isolated CCAAT boxes are similar, and double binding
induces compensatory alterations; the combined twisting angles are
about 100°.
NF-Y and the Globin CCAAT Boxes--
Previous experiments with
nuclear extracts identified several proteins binding to the CCAAT boxes
of various globin promoters. CDP binds to the duplicated -globin
CCAAT box region (32, 33), NF-E3 to the
distal CCAAT box and to the
box (32, 36), and c/EBP to the
CCAAT (3). The
CCAAT box
region is bound by a number of factors (36). Binding of NF-Y has been
more than suspected; the
-globin CCAAT box has been shown to
recognize a multimeric factor (
-CP1) which by several criteria is
NF-Y (15). The
CCAAT boxes were also shown to be cross-competed by
the Ea Y box (32, 33). By performing a systematic EMSA analysis on all
globin CCAAT boxes with NF-Y, we found that the
,
, and proximal
CCAAT bind NF-Y efficiently, the distal
CCAAT well, whereas the
affinity for
and
is so low that we consider it unlikely that
they are in vivo targets for NF-Y. A good binding site for
NF-Y requires two additional conserved nucleotides at the 5' and three
at the 3' (13, 41). As in all high affinity sites, two purines are
present in all globin sites at the 5' end, indicating that sequences at
the 3' end are responsible for the dramatic variations in affinities;
the T at second position of the 3' end of
deviates from the
consensus and is severely underrepresented in over 178 bona fide
NF-Y-binding sites (41). Within the
-globin promoters, both in human
and in the prosimian G. crassicaudatus, we note that the
third position at the 3' end is the only different nucleotide among the
distal and proximal CCAAT of human and Galago (see Table I),
surprisingly pointing to this position, relatively distant to the core
pentanucleotide, as important in affinity determination.
Role of CCAAT Boxes in -Globin Transcription--
The efficient
in vitro transcription system for the
-globin promoter
allowed us to determine the role of the CCAAT boxes. Our data are
consistent with the idea that both NF-Y-binding sites contribute to the
optimal activity, and mutations in both CCAAT resulted in transcription
levels that were lower (6-fold) than alterations in either CCAAT alone.
A point mutation in the proximal and removal of the distal CCAAT box
led to a comparable 4-fold decrease in transcription; and swapping an
-like CCAAT box, which is a poor NF-Y-binding site, in the place of
the distal
CCAAT weakens the promoter both when the proximal CCAAT
is intact (2-fold) and even more if it is mutated, as in pAG5. Thus,
from the functional point of view, the twin CCAAT boxes seem to have an
additive rather than a multiplicative effect, a result that is in line
with the lack of cooperativity between the two proteins observed in the binding assays. Either CCAAT is nevertheless sufficient to impart rather efficient levels of transcription. This might be entirely due to
strong TATA box and basal elements or to the activity of additional
upstream factors, as suggested by challenging transcription with
anti-GATA antibodies.5
NF-Y Binding to Duplicated CCAAT Boxes--
CCAAT boxes show a
strong position bias within promoters being usually positioned between
60 and
100 (1). Functional experiments indicated that a single
CCAAT box is not able to increase transcription over basal levels, and
multimerized CCAAT boxes also fail to do so. NF-Y can greatly improve
the activity of diverse upstream transcription factors, and in some
cases, it has been proved that it does so by dramatically improving the
affinity of neighboring factors for their target DNA sequence (29, 42). Since no data were available on the binding of NF-Y in promoters harboring more than one CCAAT box, the
-globin represents an excellent model to study their relationship. Several findings in our
study bear implications for other systems as follows: (i) bringing two
CCAAT sequences closer than 24 base pairs essentially abrogates double
binding, and (ii) NF-Y complexes are more stable on the DNA only with
the
5 mutant, a deletion bringing the two CCAAT boxes on the same
side of the helix. This suggests that cooperativity is possible,
provided that the correct rotational position is respected. From the
long list of CCAAT-containing promoters activated by NF-Y, a growing
number contains two or more sites (see Table
II); we note that in all, bar the
gp91phox, the distance between the two NF-Y sites is greater
than 27 nucleotides; in three cases, cdc2, H2b, and TK, it is of 30-32
bp, so that the two CCAAT face the same side of the helix. Most
promoters have more distant sites (40/80 bp apart), and it is not known at present whether NF-Y complexes could influence each other at such
distances. The only apparent exception is the gp91phox
promoter, in which two CCAAT boxes are separated by only 14 nucleotides; however, while the proximal CCAAT box has been formally
proven to be a bona fide NF-Y site, the distal CCAAT box appears to
bind NF-Y poorly and is possibly targeted by other proteins. In
conclusion, wherever CCAAT boxes closer than 24 base pairs are found,
binding of NF-Y should be considered as mutually exclusive, or one of them is presumably activated by proteins other than NF-Y. Simultaneous binding of NF-Y to sites with multiples of 10.5 bp might be considered as more stable. Experiments aimed at verifying such hypotheses with our
EMSA systems are currently underway. The assays described here,
binding, bending, and phasing, will also be used in testing the effect
of HPFH mutations in the distal CCAAT region on NF-Y binding.
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ACKNOWLEDGEMENT |
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We thank L. Cairns for reviewing the manuscript.
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FOOTNOTES |
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* This work was supported in part by grants from Murst (to R. M. and S. O.), Telethon Grant E596 (to A. R.), EEC Biotec, and from Fondazione Italiana L. Giambrone per la Guarigione della Talassemia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Fondazione L. Giambrone.
§ To whom correspondence should be addressed. Tel.: 39-2-26605239/26605225; Fax: 39-2-2664551; E-mail:mantor{at}imiucca.csi.unimi.it.
1 The abbreviations used are: oligos, oligonucleotides; HPFH, hereditary persistence of fetal hemoglobin; bp, base pair(s); EMSA, electrophoretic mobility shift assay; MHC, major histocompatibility complex; wt, wild type.
2 A. Ronchi, unpublished observations.
3 C. Liberati, S. Ottolenghi, and R. Mantovani, manuscript in preparation.
4 M. C. Motta and R. Mantovani, manuscript in preparation.
5 R. Mantovani, unpublished observations.
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REFERENCES |
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