Biomedical subjects

J Ausio

Publications and source records attributed to J Ausio.

At least 19 recordsLinked to original sources

PubMed2001

Magnesium-dependent association and folding of oligonucleosomes reconstituted with ubiquitinated H2A.

The MgCl2-induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mM MgCl2. These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association.

Animals↗

PubMed2001

Effects of histone acetylation on the solubility and folding of the chromatin fiber.

The folding ability of chromatin fractions containing approximately identical nucleosome numbers and the same linker histone composition, but with different extents of core histone acetylation, were analyzed by analytical ultracentrifugation. It was found that the acetylated fractions consistently exhibited a relatively small but significantly lower extent of compaction than that of their native nonacetylated counterparts. This was regardless of the extent of the size distribution heterogeneity of the fractions analyzed. Furthermore the acetylated chromatin fibers exhibited an enhanced solubility in both NaCl and MgCl(2), which is neither the result of a differential binding affinity of the linker histones to chromatin nor of an alteration in the relative amounts of the histone H1 variants.

Acetylation↗

PubMed2001

Intrinsically disordered protein.

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Models, Molecular↗

PubMed2000

Polyploidy in a natural population of mussel, Mytilus trossulus.

We have analyzed natural polyploidy in a population of Mytilus trossulus from Vancouver Island (British Columbia, Canada) by means of cytogenetic techniques. Results obtained are the first reporting on this type of numerical chromosome aberrations in mussels.

Animals↗

PubMed1999

Protamine-like sperm nuclear basic proteins in the primitive frog Ascaphus truei and histone reversions among more advanced frogs.

Sperm nuclear basic proteins (SNBPs) that condense chromatin are very diverse. In animals, evolution of SNBPs has proceeded from lysine-rich histone H type in sponges to more arginine-rich protamine-like PL and protamine P types. Yet sporadic PL/P to H reversions are known to occur in both protostomes and deuterostomes. To determine why this is the case, we have examined SNBPs in eleven anuran species. We find that sperm of the primitive, internally fertilizing archeobatrachian frog A. truei (family Ascaphidae) has PL/P type (42 mol % arginine), with an electrophoretic profile similar to SNBPs in another archeobatrachian, externally fertilizing Leiopelma hochstetteri (family Leiopelmatidae). Cytochemistry of sperm nuclei in the advanced, externally fertilizing neobatrachian frogs Crinia signifera and C. deserticola (family Myobatrachidae) indicates that they have reverted to H type SNBPs. This is also known to be the case in externally fertilizing Rana (family Ranidae) and Silurana, a subgenus of Xenopus (family Pipidae). Such a trend, from PL/P type SNBPs in two archeobatrachians to sporadic reversions to H type in more advanced frogs, parallels the ultrastructural simplification from complex A. truei introsperm to neobatrachian aquasperm that Jamieson et al. (1993. Herpetologica 49:52-65) attribute as a secondary reversion to external fertilization.

Animals↗

PubMed1998

Periplasmic and fimbrial SefA from Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.

Bacterial Proteins↗

PubMed1998

The histone binding protein nucleoplasmin does not facilitate binding of transcription factor IIIA to nucleosomal Xenopus laevis 5S rRNA genes.

In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. In this study, it was shown that, despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore, it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin, and TFIIIA from Xenopus laevis.

Animals↗

PubMed1998

Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin.

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Aeromonas↗

PubMed1998

Persistent interactions of core histone tails with nucleosomal DNA following acetylation and transcription factor binding.

In this study, we examined the effect of acetylation of the NH2 tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH2 tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH2 tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

Acetylation↗

PubMed1997

Isolation of histones and related chromatin structures from spermatozoa nuclei of a dasyurid marsupial, Sminthopsis crassicaudata.

The spermatozoa of a dasyurid marsupial, Sminthopsis crassicaudata, have two distinct nuclear regions: uniformly electron-dense chromatin (C1) in the interior and fissured chromatin (C2) at the periphery. To investigate whether the differences in morphology are due to incorporation of different packaging proteins, spermatozoa nuclear proteins were characterised by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) and fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The main protein component was protamine I, but a complete histone complement (H1, H2A, H2B, H3, and H4) was also detected. Immunocytochemistry showed localisation of H4, H2B, and H2A histones to the periphery of the nuclei, a region that corresponded to the C2 chromatin. The fissures in the chromatin of this region disappeared following incubation with fish protamines, indicating that the nucleohistone C2 region may be incompletely condensed relative to nucleoprotamines. This observation is consistent with the view that 60% of phosphodiester charges remain negative in nucleohistone DNA, whereas all DNA charges are neutralised in highly compact nucleoprotamines. Treatment of spermatozoa with micrococcal nuclease showed that the C1 chromatin was resistant to digestion, whereas the C2 region was cleaved into 30- to 38-nm agglomerates and 11-nm nucleosomal-size structures. Thus, this study demonstrates that spermatozoa nuclei of this marsupial species contain peripherally localised histones, and the nucleohistone chromatin accounts for the different morphology of the C2 region compared with the rest of the nucleus.

Amino Acid Sequence↗

PubMed1997

Histone H1 variants as sperm-specific nuclear proteins of Rana catesbeiana, and their role in maintaining a unique condensed state of sperm chromatin.

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization.

Amino Acid Sequence↗

PubMed1995

Two highly specialized histone H1 proteins are the major chromosomal proteins of the sperm of the sea anemone Urticina (Tealia) crassicornis.

The sperm nuclear basic proteins (SNBPs) from a sea anemone (Urticina crassicornis) have been isolated and characterized for the first time. They consist of two sperm-specific members of the histone H1 family with Mr 22,700 and 24,600. They amount to about 60--70% of the total chromosomal sperm proteins. Their amino acid composition and the primary structure of their trypsin-resistant core indicate a strong relation to histone H5 from the nucleated erythrocytes of birds and amphibians as well as to other high sperm-specific H1-like (PL-I) proteins from phylogenetically distant groups. The major presence of histone H1-like protein in the sperm of an organism belonging to such a low phylogenetic group provides experimental support to the hypothesis that SNBPs may all have evolved from a primitive histone precursor.

Amino Acid Sequence↗

PubMed1995

Modulation of chromatin folding by histone acetylation.

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J. (1992) TIBS 197, 187-191) as well as its biological relevance are discussed.

Acetylation↗

PubMed1995

Nucleoplasmin-mediated decondensation of Mytilus sperm chromatin. Identification and partial characterization of a nucleoplasmin-like protein with sperm-nuclei decondensing activity in Mytilus californianus.

We have been able to induce sperm nuclear decondensation in the mussel Mytilus californianus (mollusc) using either egg extracts or pure nucleoplasmin from Xenopus (amphibian). The nuclear decondensation involves removal of the sperm nuclear basic proteins (SNBPs) which bind to nucleoplasmin. An attempt has been made to isolate an ooplasmic factor from Mytilus with a similar sperm-chromatin decondensing activity. An acidic, thermostable protein with a molecular mass of 58,000 has been purified and partially characterized.

Amino Acids↗

PubMed1995

Complete sequence and characterization of the major sperm nuclear basic protein from Mytilus trossulus.

We have characterized the major protamine-like protein (PL-III) from the sperm of the mussel Mytilus trossulus. The molecular mass of the main protein component of PL-III, determined by mass spectrometry using fast atom bombardment and matrix-assisted laser desorption ionization was 11304 +/- 6 Da. The complete protein sequence was established by Edman degradation and the molecular mass derived from this sequence coincides with that experimentally determined. The protein has a sedimentation coefficient s20,w = 1.15 +/- 0.5 S which is consistent with a random coil of radius of gyration 34.3 +/- 1.4 A.

Amino Acid Sequence↗

PubMed1994

On the evolution of protamines in bony fish: alternatives to the "retroviral horizontal transmission" hypothesis.

Fish protamines are highly specialized molecules which are responsible for chromatin condensation during the last stages of spermatogenesis (spermiogenesis). However, not all fish contain protamines in their sperm nuclei; rather, there seems to be a random distribution of protamines within this group. The origin of this sporadic presence of protamines in the sperm and its significance have not yet been precisely determined. In this paper we have conducted an exhaustive survey of the literature available on the different types of nuclear protein composition of the sperm of teleost fish in order to try to correlate these data with what is presently known about the taxonomy of this group. The results of this analysis have allowed us to make the following observations. The divergence between protamines and histones has occurred several times during the evolution of the bony fish. However, the relative frequency of this divergence is almost negligible during the differentiation of genera and species (intrafamily variation) and is very small during the differentiation of families (interfamily variation). Nevertheless, the divergence is very noticeable among the different orders. It is therefore possible to conclude from all this that the sporadic distribution of protamines in bony fish is not a random event as initially believed. Furthermore, such a heterogeneous distribution of protamines cannot be easily accounted for by a mechanism of horizontal retroviral transmission through repeated and independent acquisition of a protamine gene as has been recently proposed (Jankowski, Stater, Dixon (1986) J Mol Evol 23:1-10). Rather, it could possibly be explained by a repeated and independent loss of the expression of the protamine gene (or loss of the gene itself) which mainly occurred during the diversification of the orders of this group.

Amino Acid Sequence↗

PubMed1993

Physical and chemical characterization of the oligomerization state of the Aeromonas hydrophila lipase/acyltransferase.

Aeromonas glycerophospholipid:cholesterol acyl transferase undergoes a conformational transition upon activation by treatment with trypsin. Chemical cross-linking and sedimentation velocity analysis showed that the lipase dimerizes due to removal of a region near its C-terminus. The lipase monomer has a sedimentation coefficient s20.w = 2.83 S, whereas the dimer has s20.w = 3.65 +/- 0.22 S. Hydrodynamic analysis using these sedimentation values and the masses determined by mass spectrometry indicated that the monomers are aligned side-by-side in the dimer. An important change occurs in the apparent partial specific volume of the molecule upon activation.

Acyltransferases↗

PubMed1993

Dimerization stabilizes the pore-forming toxin aerolysin in solution.

Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.

Amino Acid Sequence↗