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J Ausio

Publications and source records attributed to J Ausio.

At least 37 records · Page 2Linked to original sources
PubMed1993

Sequence and characterization of a sperm-specific histone H1-like protein of Mytilus californianus.

The major protein fraction of the protamine-like PL-II* (phi 2B) from the sperm of Mytilus californianus has been sequenced and characterized. Immunological and sequence analyses unequivocally show that this protein is indeed a member of the histone H1 family. Along with proteins of the histone H1 class, the protein also shows cross-reactivity and sequence identity, in its NH2-terminal region, with the major protamine-like protein component of Mytilus sperm: PL-III (phi 1), of smaller molecular mass. Indeed it is the unusual repetitive sequence motif of the NH2-terminal domain of PL-II* that bestows to this protein its protamine-like nature. Fourier-transform infrared spectroscopy spectroscopy indicates that the protein contains considerable secondary structure: 18% alpha-helix, 21% beta-sheet, 39% turns and bends, 22% random coil. At the higher levels of structure, PL-II* exhibits ionic strength-dependent folding which is indistinguishable from that of histone H5, as monitored by fluorescence anisotropy.

Amino Acid Sequence↗
PubMed1993

Post-translational cleavage of a histone H1-like protein in the sperm of Mytilus.

Starting with total RNA from spermatogenic cells of Mytilus trossulus and using random priming, we have cloned and sequenced the c-DNAs corresponding to two variants of the sperm-specific protein PLII* (phi 2B). DNA sequencing in conjunction with mass spectrometry and protein sequence data have allowed us to establish that of the three sperm-specific proteins present in the sperm of Mytilus (PL-II*(phi 2B), PL-III (phi 1), PL-IV (phi 3)), the first and the last one are the result of post-translational cleavage of a common precursor. This common precursor is a member of the histone H1 family, and it exhibits inter- and intraspecific microheterogeneity.

Amino Acid Sequence↗
PubMed1992

Presence of a highly specific histone H1-like protein in the chromatin of the sperm of the bivalve mollusks.

Chromatin organization in the sperm of the bivalve mollusks results from the interaction between a discrete number of protamine-like proteins (PL) and DNA. A small variable amount of histones is also present. An extensive study carried out on a relatively large number of species, within the class Bivalvia, has shown that it is possible to arrange these mollusks into five major categories on the basis of their PL composition (Ausio, J. Comp. Biochem. Physiol. 85, 439-449, (1986) [1]). In the present work, we have extended this analysis to a larger number of species and found that in spite of the inter- and intra-specific similarity of all PL proteins in their chemical composition, they exhibit different degrees of structural variability. Moreover one of these PL proteins is present in all the species analyzed, and bears an enormous resemblance to histones of the H1 family. The evolutionary significance of this finding is discussed.

Amino Acid Sequence↗
PubMed1992

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

An oligonucleosome 12-mer was reconstituted in the absence of linker histones, onto a DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Ly-techinus variegatus (Simpson, R. T., Thoma, F. S., and Burbaker, J. M. (1985) Cell 42, 799-808). The ionic strength-dependent folding of this nucleohistone complex was compared with that of a native oligonucleosome fraction obtained from chicken erythrocyte chromatin, which had been carefully stripped of linker histones and fractionated in sucrose gradients. The DNA of this native fraction exhibited a narrow size distribution centered around the length of the 208-12 DNA template used in the reconstituted complex. These two complexes displayed very similar hydrodynamic behavior as judged by sedimentation velocity analysis. By combining these data with electron microscopy analysis, it was shown that the salt-dependent folding of oligonucleosomes in the absence of linker histones involves the bending of the linker DNA region connecting adjacent nucleosomes. It was also found that selective removal by trypsin of the N-terminal regions ("tails") of the core histones prevents the oligonucleosome chains from folding. Thus, in the absence of these histone domains, the bending of the linker DNA necessary to bring the nucleosomes in contact is completely abolished. In addition to the complete lack of folding, removal of the histone tails results in an unwinding at low salt of a 20-base pair region at each flanking side of the nucleosome core particle. The possible functional relevance of these results is discussed.

Animals↗
PubMed1992

Chromatin dynamics and the modulation of genetic activity.

Chromatin, the genetic material of eukaryotes, is a dynamic macromolecular assembly that continuously changes its composition and conformation to accommodate different stages of genetic activity, e.g. transcription and replication. Evidence is accumulating that the dynamic behavior of chromatin has important functional roles in the modulation of genetic activity, largely due to the intrinsic properties of arrays of nucleosome cores.

Animals↗
PubMed1992

Analytical sedimentation studies of turkey gizzard myosin light chain kinase and telokin.

Sedimentation equilibrium and velocity studies were performed with turkey gizzard myosin light chain kinase (MLCK) and telokin, a small protein apparently corresponding to the sequence of the COOH-terminal end of MLCK. The measurements carried out with MLCK give values for the monomer molecular weight (M(r)), sedimentation coefficient (S20 degrees,w), and virial coefficient (A2) of 108,000, 3.74 S, and -1.95 x 10(-4) mol.ml.g-2, respectively. In the case of telokin, M(r) = 18,500; S20 degrees, w = 1.63 S; and A2 = 5.81 x 10(-4)mol.ml.g-2. Combination of the results of the two kinds of experiment shows that MLCK is a rod-shaped molecule (a/b = 18.9) with a Stoke's radius of 69 A. Telokin is also elongated (a/b = 8.3) with a Stoke's radius of 29 A. MLCK apparently exhibits self-association, with 15% of the protein sedimenting as a dimer in the experiments.

Amino Acids↗
PubMed1991

Microheterogeneity and interspecific variability of the nuclear sperm proteins from Mytilus.

We have used acetic acid-urea-triton (AUT) gel electrophoresis and ionic exchange chromatography in order to analyze the interspecific variability and microheterogeneity pattern of the protamine-like (PL) proteins of the sperm of 4 different species of the bivalve mollusc, Mytilus. We have found that based upon these 2 criteria, it is possible to unambiguously distinguish each species from the rest. We have thus been able to corroborate the identity of M. trossulus. We have also analyzed the amino acid composition of some of the PL components for each different species. In the case of the PL-II* fraction, we have analyzed the composition of its major protein subcomponents.

Amino Acids↗
PubMed1990

Analysis of the changes in the structure and hydration of the nucleosome core particle at moderate ionic strengths.

In order to better understand the conformational changes induced in the nucleosome core particle by changes in the ionic strength of the media in the range from 0.1 to 0.6 M NaCl, we have conducted a very detailed structural analysis, combining circular dichroism, DNase I digestion, and sedimentation equilibrium. The results of such analysis indicate that the secondary structure of both DNA and histones exhibits small (approximately 5%) but noticeable changes as the salt increases within this range. In the case of DNA, the data are consistent with a trend toward a more relaxed secondary structure. The DNase I pattern of digestion is also altered by the salt and suggests a DNA relaxation around the flanking ends. From the hydrodynamic measurements, we also observe a significant change in the virial coefficients of the particle as the salt increases, which in turn are in very good agreement with the theoretically expected values. Furthermore, the preferential hydration parameter is also found to increase with the salt. We believe that the self-dependent conformational change of the nucleosome core particle is the result of the conjunction of all these subtle changes. Yet, from the present data, their exact relationship to the tertiary structure of the whole particle at the different ionic strengths cannot be exactly defined.

Circular Dichroism↗
PubMed1989

Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1.

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.

Animals↗
PubMed1989

Turkey gizzard caldesmon: molecular weight determination and calmodulin binding studies.

Sedimentation equilibrium and sedimentation velocity measurements demonstrate that turkey gizzard caldesmon is an elongated molecule of molecular mass 75 +/- 2 kDa. The frictional ratio (2.14) is consistent with a prolate ellipsoid of axial ratio 24, corresponding to an apparent length and width of 516 and 21.5 A, respectively. As was previously determined for chicken gizzard caldesmon [Graceffa, P., Wang, C.-L.A., & Stafford, W.F. (1988) J. Biol. Chem. 263, 14196-14202], this molecular weight is appreciably smaller than the value (approximately 135,000) estimated from the results of NaDodSO4 gel electrophoresis experiments. However, a significant difference between the true molecular weights of turkey and chicken gizzard caldesmons--75,000 versus 93,000--also points to probable molecular weight variations within the subclass. Binding measurements, based on perturbation of the intrinsic tryptophan fluorescence of caldesmon in the presence of calmodulin, show that the interaction between the two proteins is strongly ionic strength and temperature dependent. Dissociation constants of 0.075 and 0.38 microM were determined in solutions containing 0.1 and 0.2 M KCl, respectively, at 24.3 degrees C. Fluorescence emission spectra and fluorescence anisotropy excitation spectra indicate that the tryptophanyl residues of caldesmon are located in solvent-accessible regions of the molecule, where they exhibit a high degree of mobility even when calmodulin is bound.

Animals↗
PubMed1989

Sequence and characterization of the sperm-specific protein phi 3 from Mytilus californianus.

We have shown that the sperm-specific protein phi 3 from Mytilus californianus (Conrad) exhibits compositional microheterogeneity. For the first time, we have isolated and characterized the three major components of this protein. These fractions display different electrophoretic mobilities on Triton/urea/acetic acid polycrylamide gels. However, they have a very similar molecular mass of 5 +/- 0.1 kDa as measured by sedimentation equilibrium in the analytical ultracentrifuge. All of them show a marked trend toward aggregation. We have also established the sequence for each of these three fractions. The sequencing data suggest an even greater extent of microheterogeneity for this protein. The predicted secondary structure from the sequences, as well as infrared analyzes carried out on the native protein, suggest a structure organization into an alpha helix.

Amino Acid Sequence↗
PubMed1989

Use of selectively trypsinized nucleosome core particles to analyze the role of the histone "tails" in the stabilization of the nucleosome.

Using immobilized trypsin and an appropriate fractionation procedure, we have been able to prepare, for the first time, nucleosome core particles containing selectively trypsinized histone domains. The particles thus obtained: [(H3T-H4T)2-2(H2AT-H2BT)].DNA; [(H3-H4)2-2(H2AT-H2BT)].DNA; [H3T-H4T)2-2(H2A-H2B)].DNA (where T means trypsinized), together with the non-trypsinized controls have been characterized using the following techniques: analytical ultracentrifugation, circular dichroism, thermal denaturation and DNAse I digestion. The major aim of this study was to analyze the role of the amino-terminal regions (the histone "tails") on the stability of the nucleosome in solution. The data obtained from this analysis clearly show that stability of the nucleosome core particle to dissociation (below a salt concentration of 0.7 M-NaCl) is not affected by the presence or the absence of any of the N-terminal regions of the histones. Furthermore, these histone regions make very little contribution, if any, to the conformational transition that nucleosomes undergo in this range of salt concentrations. They play, however, a very important role in determining the thermal stability of the particle, as reflected in the dramatic alterations exhibited by the melting profiles upon selective removal of these tails by trypsinization. The melting data can be explained by a simple hypothesis that ascribes interaction of H2A/H2B and H3/H4 tails to particular regions of the nucleosomal DNA.

Animals↗
PubMed1988

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone.

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.

Amino Acids↗
PubMed1987

A reexamination of the reported B----Z DNA transition in nucleosomes reconstituted with poly(dG-m5dC).poly(dG-m5dC).

Polynucleosomes with poly(dG-m5dC).poly(dG-m5dC) have been reconstituted, and well-defined nucleosome core particles from these have been prepared. Upon addition of MgCl2 to the levels used to induce the B to Z transition in this highly methylated DNA, significant changes in the circular dichroism spectrum are observed in solutions of these particles. However, such core particles also exhibit a noticeable instability when compared to chicken erythrocyte core particles under the same conditions. The change in circular dichroism can be entirely accounted for on the assumption that only free nucleotide, released by core particle dissociation, undergoes the B----Z transition. Therefore, no evidence has been found for "Z nucleosomes" in these solutions. In fact, the histone-DNA interaction in the nucelosome seems to partially inhibit the B to Z transition of the DNA. The analysis of our results is consistent with a model in which all of the DNA that remains bound to the histone octamer retains the B form.

Animals↗
PubMed1987

A dual chromatin organization in the sperm of the bivalve mollusc Spisula solidissima.

Most of the DNA in the sperm of the bivalve mollusc. Spisula solidissima, is found to be associated with a specific high-molecular-mass, protamine-like component, sharing features common both to protamines and to histones. We have found that this component coexists, in the mature sperm nucleus, with a complete set of histones, including an H1-like histone. Such histones account for approximately 20% of the whole protein content in the sperm chromatin, the overall protein/DNA ratio (w/w) being 0.87. These data, together with micrococcal nuclease digestions in combination with salt fractionation, have allowed us to propose a structural model for this chromatin in which short nucleosomal domains are interspersed in a highly saturated protamine-DNA complex.

Animals↗
PubMed1987

Structural characterization of the trypsin-resistant core in the nuclear sperm-specific protein from Spisula solidissima.

Trypsin digestion of the protamine-like protein from Spisula solidissima has revealed the existence of an internal resistant core. The peptide contains 75 amino acid residues, and its primary structure shows some conserved sequences that are common to those found in the core of the somatic histone H5 from chicken erythrocytes. The secondary structure of this core exhibits 33% antiparallel beta-sheet, 18% beta-turns, 37% random coil, and only 10% alpha-helix, in contrast to histone H5. Hydrodynamic measurements indicate a compact globular assembly for the tertiary structure of this peptide, when compared to the more extended shape observed for the whole protein. The possible relatedness of this protein to the histone H1 family is discussed.

Amino Acid Sequence↗