From the Departament d'Enginyeria Química,
Escola Tècnica Superior d'Enginyers Industrials de Barcelona,
Universitat Politècnica de Catalunya, Diagonal 647, 08028 Barcelona, Spain, the ¶ Departament Biología Cellular Animal
Vegetal, Facultat Biología, Universitat de Barcelona, 08028 Barcelona, Spain,
URA, CNRS 1309, Chimie des Biomolécules,
Institut Pasteur, 59019 Lille, France, ** Institut Biologie
Moléculaire Cellullaire, UPR 9021, CNRS, 67084 Strasbourg,
France,
Departament d'Enginyeria Química
Bioquímica, Facultat Ciències, Universitat Rovira i
Virgili, 43005 Tarragona, Spain, and §§ Departament
Molecular Cellular Biología, Institut Biología Molecular,
Centro Investigacion y Desarrollo-Consejo Superior de Investigaciones
Científicas, 08028 Barcelona, Spain
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ABSTRACT |
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Sperm chromatin of Murex brandaris (a
neogastropod mollusc) undergoes a series of structural transitions
during spermiogenesis. The DNA-interacting proteins responsible for
these changes as well as the mature protamines present in the ripe
sperm nucleus have been characterized. The results reveal that
spermiogenic nuclear proteins are protamine precursors that are
subjected to a substantial number of small N-terminal deletions that
gradually modify their overall charge. The composition of mature
protamines is remarkably simple in turn, promoting an efficient and
extremely tight packaging of DNA. The pattern of spermiogenic chromatin condensation in M. brandaris clearly departs from that
corresponding to vertebrate chromatin.
The current model for the nuclear changes occurring in
spermiogenesis has been essentially drawn from studies on bony fishes and other vertebrates (1-5). The model considers that in early spermatids, histones become acetylated preceding their replacement by a
highly basic protein (protamine). During histone
displacement, the protamine is found polyphosphorylated, although it
undergoes progressive dephosphorylation in the subsequent spermiogenic
development. The processes of protamine phosphorylation and
dephosphorylation imply a mechanism regulating the interaction with
DNA. This mechanism allows for the orderly substitution of protamine
for the nucleohistones and for the ensuing binding to DNA. The
protamine is a small molecule displaying a very high positive charge
density (6, 7). Consequently, the nuclear replacement of histones by
protamine induces profound transitions in chromatin structure directly
leading to the compactness of the sperm nucleus.
Although these events are well established for some bony fishes and
related vertebrates, there is not a universal pattern, or even a
"more frequent" model, to account for spermiogenic processes. Evolution has generated an enormous diversity of both DNA-condensing proteins (8-10) and structural conformations for spermiogenic and/or
sperm chromatin (11, 12).
Traditionally, electron microscopy studies have focused on
cenogastropod molluscs due to the complexity of their chromatin condensation patterns (13-16). In this regard, Murex
brandaris is a paradigmatic species (17). Chromatin in the early
haploid spermatid displays a somatic-like appearance, which immediately undergoes a peripheral migration, giving rise gradually to granular and
fibrilar structures and finally to 18-nm lamellae (18). The nascent
lamellar structures are first seen in a disordered arrangement but
later become progressively ordered in a regular concentric pattern
surrounding the nuclear axis.
The progression of structural changes in chromatin should be paralleled
by simultaneous modifications of DNA-protein interactions, concurrently
with thermodynamic restrictions on the size of the DNA-protein
complexes (19, 20). It was found in a previous study (18) that the
histone complement of the immature gonads of M. brandaris
becomes replaced by a large set of intermediate proteins that lead to
the final appearance of three small and simple protamine molecules in
the sperm nucleus. It was also observed that these intermediate
proteins reacted positively to antibodies elicited against a sperm
protamine. In this paper, we report the primary structure of the
M. brandaris mature protamines and show that one of them
(protamine P1) is synthesized as a precursor molecule that undergoes a
complex series of partial deletions in its N terminus in conjunction
with a single step of dephosphorylation. The final forms of these
mature protamines generate an almost extreme packing of sperm DNA .
Living Organisms--
Male specimens of the mollusc M. brandaris were collected periodically on the Mediterranean coast
of Spain and moved live to the laboratory in cold sea water.
Nuclear Preparation--
Either free-flowing sperm cells or
gonadal tissue was homogenized separately in ice-cold buffer (0.25 M sucrose, 10 mM MgCl2, 3 mM CaCl2, 10 mM Tris-HCl, pH 7.0, 0.1% Triton X-100, 50 mM benzamidine chloride as protease
inhibitor) and centrifuged at 3000 × g for 5 min.
Crude nuclear pellets were then rehomogenized and resedimented three
times in the same buffer. The purified nuclear pellets were next
homogenized in 10 mM Tris-HCl, pH 7.0, 20 mM
Na2EDTA, followed by centrifugation and then one more time
in the same buffer, omitting the chelating agent. The nuclear sediments
thus obtained were used to extract proteins as well as for x-ray
diffraction analyses.
Protein Extractions--
Proteins were extracted from purified
sperm or gonadal nuclei with 0.4 N HCl, precipitated with 6 volumes of cold acetone, and finally rinsed with acidified acetone
(21). On one occasion, nuclear sediments were reduced with 50 mM Tris-HCl, pH 8.8, 2 mM Na2EDTA,
10 mM dithiothreitol for 1 h at 37 °C under
N2 atmosphere and then alkylated with 12.5 mM
iodoacetamide in the same buffer prior to HCl extraction (22, 23).
Preparation of Antibodies and Immunodetection of the Protamine P1
Precursor--
Polyclonal antibodies against protamine P1 were
prepared as described previously (18) with some modifications. Briefly, outbred New Zealand White female rabbits were multi-injected
intradermally with 750 µg of protamine/animal. The antigen was
previously emulsified with Freund's complete adjuvant. The
anti-protamine P1 antisera was tested by an enzyme-linked immunosorbent
assay with purified protamines.
Western blotting of nuclear proteins from both sperm and gonads was
performed according to Harlow and Lane (24). Following acetic acid-urea
polyacrylamide gel electrophoresis, proteins were electrotransferred
onto nitrocellulose membranes at 500 mA for 1 h at 4 °C in a
solution of 0.7% acetic acid, 10% methanol. The membranes were washed
in PBS, 0.1% Tween 20 (PBS/T), blocked with PBS/T containing 0.1%
gelatin for 2 h, and incubated with anti-protamine P1 antisera for
2 h at 37 °C. Detection was performed with diluted
peroxidase-conjugated anti-rabbit IgG (1:1000) and 4-chloro-1-naphtol
as substrate.
Chromatography--
Chromatographic resolution of proteins in
ion exchange columns was performed on CM-52 cellulose (Whatman).
Protein samples were dissolved in 0.2 N NaCl, 50 mM acetate, pH 6.0, and loaded onto the column. Elution was
carried out stepwise with 0.2 and 0.6 N NaCl, finally
applying a linear gradient of NaCl concentrations from 0.6 to 2.0 N in the same acetate buffer (25). The collected fractions
were dialyzed against 5 mM HCl and lyophilized.
For reverse phase HPLC,1 a
C4, 300-Å Delta-Pack column (25 × 0.46 cm) was used. Proteins
were eluted applying a linear gradient of acetonitrile (0-25%) in
0.05% trifluoroacetic acid (26).
Electrophoresis--
One-dimensional polyacrylamide slab gel
electrophoresis was performed according to Panyim and Chalkley (27)
with the modifications described by Hurley (28).
Protein Analyses--
Amino acid analysis of proteins was
carried out after hydrolysis of the samples in 6 N HCl
(29). Sequencing of the M. brandaris proteins was performed
on a Procise Sequenator/ABI-492 (Perkin-Elmer) using the Pulsed Liquid
2HL program. The molecular mass of proteins was determined by ion spray
mass spectrometry. Samples were dissolved in 200 µl of an aqueous
solution of 20% acetonitrile, 0.1% HCOOH. Ion spray mass spectra were
recorded on a simple quadrupole mass spectrometer API I (Perkin-Elmer),
equipped with an ion spray (nebulized-assisted electrospray) source
(Sciex, Toronto, Canada). The solutions were continuously infused with
a medical infusion pump (model 11, Harward Apparatus, South Natick, MA)
at a flow rate of 5 µl/min. Polypropyleneglycol was used to calibrate
the quadrupole. Ion spray mass spectra were acquired at unit resolution by scanning from m/z 400 to 1200 with a step size
of 0.1 Da and a dwell time of 2 min. Ten spectra were summed. The
potential of the spray needle was held at +4.5 kV. Spectra were
recorded at an orifice voltage of +90 V. A Mac Bio Spec computer
program was used to measure the molecular masses of the protein
samples. Protein alignments were done using the method of Lipman and
Pearson (30).
X-ray Diffraction Analysis--
The x-ray diffraction patterns
were obtained from samples of nuclei or fibrous complexes
(reconstituted complexes). In both instances, samples were sealed in
capillaries containing a drop of a saturated salt solution used as a
control of the relative humidity. The patterns were recorded with
nickel-filtered copper radiation on Kodak film. Either a modified
Philips microcamera or a Statton camera (W. R. Warhus, Wilmington,
DE) was used.
Complexes of M. brandaris protamines with DNA were prepared
by mixing both components in a proportion to achieve complete charge
neutralization. The mixing buffer used was 2 M guanidine hydrochloride, 1 mM Tris-HCl, pH 8.0, 1 mM
EDTA. Mixtures were sequentially dialyzed against solutions of 2, 1, 0.8, 0.4, and 0.2 M guanidine hydrochloride, 1 mM Tris-HCl, pH 8.0, 1 mM EDTA, followed by
extensive dialysis against 1 mM Tris-HCl, 1 mM
EDTA. Complexes started to precipitate when the guanidine hydrochloride concentration was about 0.8 M. The fibrous precipitates
were then pulled with tweezers and allowed to dry under tension in
order to yield fibers suitable for x-ray diffraction analysis (31, 32).
Electron Microscopy--
Electron microscopy analysis was
performed as described previously (18). Either gonadal tissue or sperm
cell sediments were fixed in 2.5% glutaraldehyde, 0.1 M
cacodylate buffer and postfixed in 1% osmium tetroxide in the same
buffer. The samples were next dehydrated and soaked in Spurr's resin.
Sample sections were stained with uranyl acetate and lead citrate and
examined under a Hitachi H-600 transmission electron microscope.
Purification of M. brandaris Protamines--
Sperm cells of
M. brandaris contain electrodense cylindrical nuclei grooved
by axonemes (Fig. 1, inset).
Acid extraction of purified nuclei with 0.4 N HCl yielded
three basic proteins (Fig. 1, lane w). Previous
chemical reduction of chromatin prior to extraction did not solubilize
any additional proteins. Nucleoprotein components were purified by
reverse-phase HPLC eluting in the order P3 Analysis of Protamines--
The amino acid analysis of M. brandaris protamines revealed a set of simple molecules of very
basic nature (Table I). P1 is made up of
six different aminoacyl types, P2 by only four types, whereas P3
appears to be the simplest protamine known to date, involving just
three amino acid residues (Arg, Lys, and Gly). Arginine, lysine, and
glycine are the major components of all three protamines (94.9% in P1,
96.7% in P2, and 100% in P3). Another relevant feature revealed by
the amino acid analyses was the low proportion of phosphorylatable
residues in all of the molecules (2% in P1, 3.2% in P2, and 0% in
P3). This is a striking fact if it is considered that phosphorylation
and dephosphorylation processes regulate the interactions with DNA in
vertebrate protamines. All protamines were repeatedly analyzed by
automated Edman degradation with consistent results. Due to the yields
and purity of the protein samples together with the powerful
methodology employed, it was possible to obtain complete and
unambiguous sequences in single sequencing runs (Fig.
2C).
Molecular masses of protamines P1, P2, and P3 as established by ion
spray mass spectrometry were 8416 ± 1 Da, 6962 Da, and 6474 Da,
respectively (Fig. 2A). The molecular mass of protamine P1
was also determined in mature gonads containing partially ripe spermatozoa (Fig. 2B). In this case, a molecular mass of
8497 Da was obtained for a fraction of the protamine, consistent with the phosphorylated form (8417 + 80 Da). This result suggests that protamine P1 is monophosphorylated in the stages preceding full ripening of the sperm and is coincident with the presence of only one
serine residue in the P1 molecule.
The analytical results so far reported are wholly congruent. First, the
molecular masses derived from the amino acid analyses (P1, 8415 Da; P2,
6960 Da; P3, 6473 Da) are totally coincident with the masses
determined. Second, the number of amino acid residues obtained from the
compositional analyses is practically identical to that afforded by the
primary structures (see Table I).
Protamine P1 Precursors in M. brandaris--
It is relevant to
note the simplicity of the M. brandaris protamine sequences
whose organization will be dealt with later. Protamine P3 appears
exclusively constituted of Arg, Lys, and Gly amino acids, while both P1
and P2 contain a few additional residues. This structural simplicity
together with the low proportion of phosphorylatable residues (only
three serines out of 184 amino acid residues) contrasts with the
elaborated forms of spermiogenic chromatin condensation illustrated in
Fig. 3. We have previously shown that
unripe spermiogenic nuclei of M. brandaris contain a
substantial number of proteins with an electrophoretic mobility intermediate between histones and protamines in denaturing
polyacrylamide gels (18). This abundant subset of intermediate proteins
disappears in very advanced stages of spermiogenesis and becomes wholly
absent from ripe sperm nuclei. Considering both the electrophoretic
behavior and amino acid composition of these intermediate proteins, we surmised that they might direct the complex transitions in the condensation of the M. brandaris spermiogenic chromatin,
modulating the interaction with DNA through a series of
posttranslational intranuclear modifications and acting as precursors
of the ripe sperm protamines.
In order to verify the preceding assertion these proteins were analyzed
in detail. First, the largest intermediate molecule Pr-P1, displaying
the slowest electrophoretic mobility, was purified, and the sequence of
its N terminus comprising the initial 52 amino acid residues was
determined by automated Edman degradation together with the assessment
of its molecular mass by ion spray mass spectrometry (Fig.
4). Moreover, purified Pr-P1 protein
reacted positively with anti-protamine P1 antisera (Fig.
5, lane e). Table
II shows the actual amino acid
composition of the putative precursor molecule and its comparison with
the composition estimated under the assumption that the molecule is a
true precursor of protamine P1. The results indicate that this
intermediate protein (Pr-P1) indeed corresponds to a monophosphorylated
precursor of protamine P1. Thus, the molecular mass of the
monophosphorylated form of protamine P1 (8497 Da in Fig. 2) plus that
corresponding to the 35 initial residues of the N terminus of Pr-P1
yielded a value (12,657 Da) almost identical to the molecular mass of
Pr-P1 obtained by ion spray mass spectrometry (12,662 Da). In addition,
the determined amino acid composition is very similar to the
composition estimated (Table II). Finally, the last 17 sequenced
residues of Pr-P1 are identical to the 17 initial amino acid residues
of P1. The presence of an alanine in position 48 of Pr-P1 confirms that
this molecule truly encompasses protamine P1, since both P2 and P3 lack
the former residue.
It can be seen in Fig. 5 that the anti-protamine P1 antiserum reacts
positively with the bulk of intermediate proteins present in
spermiogenic nuclei (see lane b). This result
suggests that these proteins do contain also the protamine P1 sequence
and therefore can be considered precursor molecules. To confirm this
assumption, the material under the individual peaks in Fig.
4A was subfractionated by HPLC, and the resulting proteins
were directly sequenced by automated Edman degradation. In some cases,
protein yields allowed for additional determinations of the molecular
mass by ion spray mass spectrometry (Table
III). The sequences obtained (Fig.
6) clearly show that the intermediate
proteins analyzed arise from the gradual trimming of the N-terminal
residues of protein Pr-P1. Moreover, the molecular weights determined
(Table III) indicate that these molecules are monophosphorylated. Nine
major precursor forms have been identified; however, the presence of
additional minor components cannot be discounted. These results show
that the M. brandaris Pr-P1 precursor is processed by means
of serial deletions each involving one or a few more amino acid
residues. Although this processing mechanism is not common to other
molluscs (33), it stands comparison with the processing of the P2
precursor protamine in some mammals (34, 35).
X-ray Diffraction Analysis of M. brandaris DNA-Protamine
Complexes--
To determine the capacity of M. brandaris
protamines to compact DNA, protamine-DNA complexes were subjected to
x-ray diffraction analysis. Two sets of experiments were carried out.
In the first series, x-ray diffraction patterns were obtained from
purified sperm nuclei, whereas in the second the analyses were
performed with reconstituted complexes of DNA with purified M. brandaris protamines (see "Materials and Methods"). Nuclei or
reconstituted complexes were analyzed at different values of relative
humidity. Fig. 7 shows the x-ray
diffraction patterns obtained from reconstituted complexes at 76%
relative humidity (Fig. 7A) and from sperm nuclei at 92%
relative humidity (Fig. 7B). The complex in Fig.
7A may be interpreted as a B form of DNA packed in an
orthorhombic lattice with two molecules per unit cell, with approximate
cell parameters as follows: a = 33.2 Å,
b = 24.1 Å, and c = 33.4 Å. The
values reported in Table IV show that
distances between DNA strands observed in reconstituted complexes and
sperm nuclei are highly coincident at each value of relative
humidity.
These experiments show that DNA becomes packaged by M. brandaris protamines in a very tight and compact manner as deduced from the distance between axes of the neighbor DNA strands, which varies between 19.5 and 22.8 Å (see Table IV). These values are similar to those found in squid nucleoprotamine, which contains 79%
arginine (36). It is likely that the charge distribution and large
percentage of glycine (an amino acid with a minimal volume) in the
protamines of M. brandaris generate a compactness of the DNA
to such extremes that the resulting volume of the mature sperm nucleus
becomes comparable with that of the DNA alone when considered as a
cylinder of 20 Å in diameter.
Three protamines of a very simple composition (P1, P2, and P3) are
present in the ripe sperm nuclei of the mollusc M. brandaris. One of them (P3) is the simplest protamine known to
date, being made up of only three types of amino acids (Gly, Lys, and
Arg) and completely lacking phosphorylatable residues. The most
relevant features of these three molecules are their extreme basicity
and the presence of arginine clusters interspersed with very rich GK
tracts. Arginine clusters have been held responsible for the cooperative interaction of protamines with DNA (37), but GK-rich regions are absent in other known protamines. Among the M. brandaris protamines, the GK-alternating residues are particularly
evident near the N terminus of protamine P1 (see residues 8-22 in Fig. 2). It is worth indicating that some other protamines contain substantial clusters of alternating basic/nonbasic residues close to
their N termini, notably the (RS)n repeat (5, 45). Interestingly, a (RS)n motif adjacent to a tract of basic amino
acid residues has been observed in some splicing factors, having been
implicated in intranuclear location (38). The repeating GK dipeptide
might also represent a novel protamine-DNA element, although further
structural studies are required to unambiguously establish its specific
role in the highly efficient and organized packaging of DNA in the
sperm chromatin.
In addition to the amino acids Arg, Lys, and Gly, protamine P1 also
contains a phosphorylatable residue of serine, one alanine and two
cysteine residues. The high content of cysteine usually found in
protamines from mammals and some other species is thought to stabilize
the nuclear structure of sperm by intermolecular disulfide bridging
(39, 40). This notwithstanding, the fact that protamine P1 from
M. brandaris can be extracted from nuclei with no need of
previous chemical reduction of the sperm chromatin strongly supports
the notion that the cysteine residues present in this protein do not
form intermolecular covalent linkages. The compositional and structural
simplicity of the M. brandaris protamines suggests that
these molecules have acquired a defined specialization to perform a
tight and compact packaging of DNA (Table IV).
It has been established in the present work that protamine P1 from
M. brandaris appears as a precursor molecule in the
spermiogenic nuclei encompassing the mature protamine in a
phosphorylated form preceded by an N-terminal precursor peptide. The
precursor sequence is made up of 35 amino acid residues of which nine
are basic (five Arg, three Lys, one His) and seven are acidic (two Asp
and five Glu, four of them in a row, Glu4). The presence of
basic and acidic residues allows the peptide to interact with both DNA
and basic proteins (histones or protamines). It is noticeable that
nucleoplasmin, a protein specialized in remodeling chromatin structure
during fertilization, also interacts with histones or protamines by
means of its clusters of acidic residues (41-44). The successive
deletions of the precursor peptide (see Fig. 6) might modulate the
interactions of the proteins with DNA and might also be instrumental in
the structural transitions undergone by the spermiogenic chromatin (see
Fig. 3). Likewise, the dephosphorylation observed may have a
significant role, although most probably restricted to the final step
of chromatin condensation (coalescence of lamellae). The protamine P1
precursor contains a single serine residue in the sequence. The latter
residue appears phosphorylated throughout the processing of the
molecule (see Table III), undergoing dephosphorylation only in the
fully ripe spermiogenic nuclei.
Finally, the analysis of the internal repeats in M. brandaris protamines (Fig. 8)
suggests a particular evolution for these proteins, which may have
arisen from ancestral peptides having undergone a Lys4
indel (insertion or deletion) together with some duplications.
Ancestral peptides might be represented by
R6(GK)8 tracts in protamine P1 and
R6(GK)3K4(GK)5
stretches in protamines P2 and P3, respectively.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
P2
P1 (Fig. 1).
Protamine P1 (peak 3 and lane
3 in Fig. 1) was repurified by reverse-phase HPLC in the
same conditions to eliminate a small amount of contaminating P2
protamine.
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Fig. 1.
Purification of sperm nucleoproteins from
M. brandaris. Densitometer tracings of the HPLC
chromatographic fractionation of the sperm protamines. Proteins under
chromatographic peaks 1-3 were subjected to acid-urea-polyacrylamide
slab gel electrophoresis (lanes 1-3, respectively).
Lane W corresponds to the total protein
complement extracted from purified, ripe sperm nuclei (Protamines 1, 2, and 3). Inset, electron microscopy of a ripe sperm head
showing the extreme degree of DNA packing (bar, 1 µm;
min, minutes; AN, acetonitrile). Direction of
electrophoresis is from top to bottom in this and
subsequent figures.
Amino acid composition of M. brandaris protamines P1, P2, and P3
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Fig. 2.
Primary structure of M. brandaris
protamines. A, ion spray mass spectrometry of
protamines P3 (6474 Da), P2 (6962 Da), and P1 (8415 Da). B,
ion spray mass spectrometry of protamine P1 isolated from ripe gonads
(gonadal spermatozoa). Note that a fraction of P1 yields a mass of 8497 Da. C, amino acid sequences of protamines P1, P2, and P3
obtained from automated Edman degradation (MW, relative
molecular weight).
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Fig. 3.
Transitions in the spermiogenic chromatin of
M. brandaris. Electron micrographs showing successive
stages of nuclear condensation during spermiogenesis in M. brandaris, illustrative of the main changes undergone by
chromatin. A and B, fiber-granular structures in
early spermatids (B, detail). C, fibrillate structures in
elongating spermatids (transverse (top) and semilongitudinal
(bottom) sections; D, detail). E,
transverse view of relatively disordered lamellae in elongated
spermatids. F, concentric arrangement of chromatin lamellae
(transverse (left) and longitudinal (right)
sections). G, homogeneously condensed chromatin following
lamellae coalescence in the ripe sperm nucleus seen in a transverse
view. Bars, 250 nm.
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Fig. 4.
Purification and characterization of the
protein Pr-P1. A, CM-cellulose ion exchange
chromatography of proteins extracted from nuclei of ripe M. brandaris gonads (left). The material of the fraction
marked with an arrow was subjected to additional HPLC
purification (right). The inset shows the
electrophoretic patterns of the gonadal nucleoprotein complement
(right) and the purified Pr-P1 protein from HPLC (*).
Migration ranges are indicated for core histones (H),
intermediate proteins (I), and protamines (P).
B, ion spray mass spectrometry of the Pr-P1 protein (12,662 Da). C, sequence of the amino-terminal region of purified
protein Pr-P1 obtained by Edman degradation (initial 52 amino acid
residues of the N terminus). The final 17 residues of Pr-P1 sequenced
coincide entirely with the leading amino acids of the amino-terminal
sequence of protamine P1 (see Fig. 2). AN,
acetonitrile.
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Fig. 5.
Immunoreactivity of protein Pr-P1 to anti-P1
antisera. Western blot analysis of M. brandaris
proteins with polyclonal antibodies after acetic
acid-urea-polyacrylamide gel electrophoresis is shown. Lane
a, blot of the nucleoproteins extracted from whole gonads stained
with Amido Black following electrotransfer onto nitrocellulose filter.
Lane b, same strip as in A immunoreacted with
anti-P1. Lanes c and d, electrophoretically
resolved gonadal nucleoproteins (lane c) and purified
protein Pr-P1 (lane d), blotted to membranes and stained
with Amido Black. Lane e, strip containing purified protein
Pr-P1 reacted with anti-P1 antibodies. Migration ranges are indicated
for histones (h), intermediate proteins (i), and
protamines (p).
Compositional analysis of Pr-P1
Precursor forms of protamine P1
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Fig. 6.
Primary structure of precursor forms of
M. brandaris protamine P1. Amino-terminal sequences
obtained from intermediate nucleoproteins during the spermiogenesis of
M. brandaris, Pr-P1 and Pr8 to Pr1.
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Fig. 7.
DNA packing by M. brandaris
protamines. X-ray diffraction patterns of reconstituted
complexes of DNA with M. brandaris protamines P1, P2, and P3
at 76% relative humidity (A) and whole nuclei at 92%
relative humidity (B).
DNA packing by M. brandaris protamines
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 8.
Alignment analysis of M. brandaris
protamines. Putative repeated motifs and derived consensus
sequences for protamines P1, P2, and P3. Vertical
bars indicate the identities to the respective consensus
sequences.
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FOOTNOTES |
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* This work was supported in part by Spanish Dirección General Interministerial de Ciencia y Tecnologia Grants PB93-1067 (to J. A. S. and M. C.) and PB97-1136 (to L. C.) and by European Economic Community Grant SC1-CT91-0693 (to S. M. and L. C.).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.
§ Recipient of a fellowship from the Generalitat de Catalunya.
¶¶ To whom correspondence should be addressed: Dept. d'Enginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Diagonal 647, E-08028 Barcelona, Spain. Fax: 34-934017150; E-mail: chiva{at}eq.upc.es.
The abbreviation used is: HPLC, high pressure liquid chromatography.
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