Dissociation of protamine-DNA complexes by Xenopus nucleoplasmin and minichromosome assembly in vitro

Nucleoplasmin, an acidic thermostable protein abundant in the nucleus of Xenopus laevis oocytes, has been found to dissociate complexes of pUC19 DNA and protein phi 1, an intermediate protamine present in ripe sperm from the mollusc Mytilus edulis. Cruder preparations of nucleoplasmin, such as the amphibian oocyte S150 extract and its thermostable fraction, also dissociate the heterologous DNA-phi 1 complexes and, in addition, promote the assembly of plasmid DNA into a minichromosome displaying regular nucleosomal periodicity, as revealed by micrococcal nuclease digestion. In contrast, purified nucleoplasmin complemented with rat hepatocyte core histone octamers in the presence of DNA topoisomerase I, although capable of inducing nucleoprotein formation onto the complexed DNA, fails to position nucleosomes at the native spacings seen in chromatin in vivo. These data favour the existence of a general mechanism to bring about, in a concerted manner, removal of sperm-specific nuclear proteins and reconstitution of somatic chromatin following fertilization.     

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.     

Molecular phylogeny of phi29-like phages and their evolutionary relatedness to other protein-primed replicating phages and other phages hosted by gram-positive bacteria

The phi29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, phi15, phi29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5'-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the phi29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the phi29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of phi29-like phages, one branch consisting of phages BS32, phi15, phi29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.     

Main-chain conformational features at different conformations of the side-chains in proteins

An analysis of the known protein structures has shown that the main-chain torsion angles, phi and psi of a residue can be affected by the side-chain torsion angle, chi1. The (chi1, psi) plot of all residues (except Gly, Ala and Pro) show six distinct regions where points are concentrated-although some of these regions are nearly absent in specific cases. The mean of these clusters can show a shift along the psi axis by as much as 30 degrees as chi1 is changed from around 180 to -60 to 60 degrees. Because of the lesser steric constraint points are more diffused along the psi axis when chi1 is approximately -60 degrees. Although points are more spread out along the phi axis in the (chi1, phi) plot, the dependence of phi on chi1 shows up in a shortened phi range (by about 30 degrees) when chi1 is around -60 degrees, and a distinct tendency of clustering of points into two regions when chi1 is approximately equal to 60 degrees, especially for the aromatic residues. Based on the dependence of the backbone conformation on its side-chain the 17 amino acids can be grouped into five classes: (i) aliphatic residues branched at the Cbeta position (although Thr is atypical), (ii) Leu (branched at the Cgamma position), (iii) aromatic residues (Trp can show some deviations), (iv) short polar residues (Asp and Asn), and (v) the remaining linear-chain residues, mainly polar. Ser and Thr have the highest inclination to occur with two different orientations of the side-chain that can be located through crystallography. Such residues exhibiting two chi1 angles have their phi and psi angles in a region that is common to the Ramachandran plots at the two different chi1 angles. The dependence of phi and psi angles on chi1 can be used to understand the helical propensities of some residues. Moreover, the average phi, psi values in the alpha-helices vary with the side-chain conformation.     

Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens

Unlike mammals, birds, and most other fishes, winter flounder completes spermatogenesis without replacing its germ cell histones with protamines. Instead, during spermiogenesis, these fish produce a family of high molecular weight (80,000-200,000) basic nuclear proteins (HMrBNPs) that bind to sperm chromatin containing the normal complement of histones. These large, basic proteins are built up of tandem iterations of oligopeptide repeats that contain phosphorylatable DNA-binding motifs. Although the HMrBNPs have no obvious homology to histones, protamines, or other sperm-specific chromatin proteins, we report here the isolation of a clone (2B) from a winter flounder genomic DNA library that establishes a link between the HMrBNPs and histone H1. The 2B sequence contains an open reading frame, which, when conceptually translated, encodes a 265-residue protein. At its N terminus the translation product contains numerous simple repeats that match the oligopeptides contained within the HMrBNPs. Unexpectedly, the C terminus of the putative protein shows 66% identity and 76% conservation to the histone H1 globular domain. This connection suggests that the HMrBNPs may have originated from the extended N-terminal tail region of a testis-specific, H1-like linker histone.     

Recombinant phytochrome of the moss Ceratodon purpureus: heterologous expression and kinetic analysis of Pr-->Pfr conversion

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr-->Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.     

Gene A product of phi X174 is required for site-specific endonucleolytic cleavage during single-stranded DNA synthesis in vivo

A DNA protein complex that is a precursor of mature phi X174 phage was isolated. The complex sedimented with an S value of 50 in a sucrose gradient and contained phage DNA consisting of a replicative form molecule with an extended tail of single-stranded viral DNA. The viral-strand DNA ranged from one to two genomes in length. Proteins coded on the phi X174 genome as well as the host genome were associated with the viral DNA in the 50S precursor complex. Our results indicated that both viral DNA synthesis and cleavage of the growing viral-strand DNA occurred in the 50S complex.     

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.     

Cytokine priming reduces dependence on TNF-R2 for TNF-alpha-mediated induction of macrophage nitric oxide generation

In the presence of interferon-gamma (IFN-gamma), human tumor necrosis factor-alpha (Hu-TNF-alpha), which binds to murine TNF-alpha receptor type 1 (TNF-R1) but not to murine TNF-R2, was effective in inducing nitric oxide (NO) production in spleen-derived macrophages (M phi), albeit at concentrations 12.5-fold greater than those required by murine TNF-alpha (Mu-TNF-alpha), to achieve the same result. Addition of anti-TNF-R1 completely inhibited the Mu-TNF-alpha-mediated induction of NO, demonstrating that TNF-R1 is critical to the IFN-gamma-dependent TNF-alpha-mediated induction of M phi effector function. However, treatment with anti-TNF-R2 resulted in a partial inhibition of M phi activation. Spleen-derived M phi were more dependent on TNF-R2 than RAW 264.7 or peritoneal M phi based on their responsiveness to Hu-TNF-alpha. Priming of spleen-derived M phi with either IFN-gamma or granulocyte-macrophage colony-stimulating factor (GM-CSF) heightened the maximal responses to both TNF-alpha species and increased the overall effectiveness of Hu-TNF-alpha without increasing expression of either TNF-alpha receptor. The dependence of spleen-derived M phi on both TNF-alpha receptors for signaling the induction of effector function supports an active signaling role for TNF-R2 in its synergy with TNF-R1 rather than a passive ligand passing role.     

Structure of the cDNA encoding the precursor for the neuropeptide FMRFamide in the bivalve mollusc Mytilus edulis

FMRFamide immunoreactivity is widespread in the tissues of bivalve molluscs, but some of this immunoreactivity may represent distinct related peptides (FaRPs) rather than the exact tetrapeptide FMRFamide. We have cloned the first full-length cDNA encoding the precursor protein for FMRFamide from this class of molluscs to investigate the possibility that additional peptides may be produced. The precursor contains one copy each of NFLRFamide, FLRFamide, ALAGDHFFRFamide and 16 copies of FMRFamide. This precursor is expressed in all three ganglia of the central nervous system. Since the gene encoding the FMRFamide precursor in pulmonate molluscs is alternatively spliced to give two distinct messages, we searched for evidence that the FMRFamide gene of Mytilus is also alternatively spliced. No evidence of alternative splicing was found.     

Experiences with the application of QSAR in the routine of the notification procedure

IL-8 is a recently described chemokine that increases polymorphonuclear neutrophil infiltration and has been implicated in inflammatory pathology. This study assesses monocyte (M phi) interleukin-8 (IL-8) levels in severe trauma patients (injury severity score > 16) who have elevated levels of M phi cell-associated tumor necrosis factor alpha (TNF alpha), a major marker for systemic inflammatory response syndrome after injury. We demonstrate elevated (p = .0007) levels of M phi IL-8 only in those trauma patients who also have increased (p = .0001) M phi-secreted TNF alpha whereas the patients having normal M phi-secreted TNF alpha levels have normal or even decreased M phi IL-8 production. There is no association between M phi IL-8 production and cell-associated TNF alpha levels. M phi induction by Fc gamma RI cross-linking, a common induction pathway in trauma patients' M phi that increases the production of both cell-associated and secreted TNF alpha, can also increase (p = .0022) M phi IL-8 levels. Therefore, post-trauma elevation of M phi IL-8 levels may be associated with increased secreted TNF alpha resulting from, at least in part, Fc gamma RI cross-linking stimulation in vivo.     

Phage phi29 protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo

Protein p6 from Bacillus subtilis phage phi29 (Mr = 11 800) binds in vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole phi29 DNA in vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 microM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K2) of approximately 2 x 10(5) M-1. The intracellular concentration of protein p6 (approximately 1 mM) was estimated measuring the number of copies per cell (7 x 10(5)) and the cell volume (1 x 10(-15) L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K2,6 = 3.2 x 10(8) M-2) or with an isodesmic association of the dimer (K = 950 M-1), because the apparent weight-average molecular mass (Mw,a) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/fo) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in vivo.     

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.     

Oligomerization-dependent folding of the membrane fusion protein of Semliki Forest virus

The spikes of alphaviruses are composed of three copies of an E2-E1 heterodimer. The E1 protein possesses membrane fusion activity, and the E2 protein, or its precursor form, p62 (sometimes called PE2), controls this function. Both proteins are, together with the viral capsid protein, translated from a common C-p62-E1 coding unit. In an earlier study, we showed that the p62 protein of Semliki Forest virus (SFV) dimerizes rapidly and efficiently in the endoplasmic reticulum (ER) with the E1 protein originating from the same translation product (so-called heterodimerization in cis) (B.-U. Barth, J. M. Wahlberg, and H. Garoff, J. Cell Biol. 128:283-291, 1995). In the present work, we analyzed the ER translocation and folding efficiencies of the p62 and E1 proteins of SFV expressed from separate coding units versus a common one. We found that the separately expressed p62 protein translocated and folded almost as efficiently as when it was expressed from a common coding unit, whereas the independently expressed E1 protein was inefficient in both processes. In particular, we found that the majority of the translocated E1 chains were engaged in disulfide-linked aggregates. This result suggests that the E1 protein needs to form a complex with p62 to avoid aggregation. Further analyses of the E1 aggregation showed that it occurred very rapidly after E1 synthesis and could not be avoided significantly by the coexpression of an excess of p62 from a separate coding unit. These latter results suggest that the p62-E1 heterodimerization has to occur very soon after E1 synthesis and that this is possible only in a cis-directed reaction which follows the synthesis of p62 and E1 from a common coding unit. We propose that the p62 protein, whose synthesis precedes that of the E1 protein, remains in the translocon of the ER and awaits the completion of E1. This strategy enables the p62 protein to complex with the E1 protein immediately after the latter has been made and thereby to control (suppress) its fusion activity.     

Immunochemical investigations of cytochrome P450 forms/epitopes (CYP1A, 2B, 2E, 3A and 4A) in digestive gland of Mytilus sp

Western blot analysis of microsomes and partially purified cytochrome P450 (CYP) from digestive gland of Mytilus edulis was carried out using polyclonal antibodies to hepatic Perca fluviatilis CYP1A, Oncorhynchus mykiss CYP3A and rat CYP2B, CYP2E and CYP4A isoforms. Multiple CYP bands were detected in partially purified CYP compared to single bands for microsomes for anti-CYP1A, anti-CYP2B, anti-CYP2E and anti-CYP3A. In contrast, anti-CYP4A showed two distinct bands for both. The apparent molecular weights in kD (mean +/- range or S.D.; n = 2-4) for partially purified CYP were 42.5 +/- 0.5 and 48.1 +/- 0.3 (2 bands, anti-CYP1A); 67.4 +/- 0.7, 52.8 +/- 0.6, 44.5 +/- 2.5 (3 bands, anti-CYP3A); 52.8 +/- 0.7, 48.1 +/- 1.1 and 43.9 +/- 1.1 (3 bands, anti-CYP2B); 52.7 +/- 0.8 and 47.2 +/- 0.2 (2 bands, anti-CYP2E); 50.9 +/- 0.3 and 44.1 +/- 0.2 kD (2 bands, anti-CYP4A). Digestive gland microsomes of Mytilus galloprovincialis from a polluted compared to a clean field site showed higher levels of bands recognised by anti-CYP1A, anti-CYP2E and anti-CYP4A, but not anti-CYP2B and anti-CYP3A (P < 0.05), indicative of independent regulation of different CYP forms. Overall, the apparent molecular weight and field studies indicate at least five different digestive gland CYP forms.     

The role of the interaction between macrophages and mesangial cells in the progression of IgA nephropathy

IgA nephropathy is the most common form of glomerulonephritis in the world. Approximately, 20 to 30% of IgA nephropathy patients progress to end stage renal failure in 20 years. However, the mechanism (s) underlying the progression of IgA nephropathy has not been fully understood. The purpose of this study was to elucidate the role of the interaction between macrophages and mesangial cells in the progression of IgA nephropathy. Renal biopsy specimens from 40 patients with IgA nephropathy were examined to investigate the relationship between glomerular infiltration of monocytes/macrophages (Mo/M phi), glomerular expression of monocyte-specific chemoattractant, and their clinicopathological findings. The results led to the following conclusions. 1. The localization of Mo/M phi within the glomerulus was identified as intracapillary or intramesangial by immuno-cytochemistry with monoclonal antibody against CD68. 2. Close relationships between mesangial expressions of M-CSF and MCP-1, and mesangial localization of Mo/M phi strongly suggested that mesangial production of these factors, in concert with glomerular ICAM-1 expression, enhances the recruitment and survival of Mo/M phi in the mesangium. 3. It was suggested that Mo/M phi localized in the mesangium play an important role in the progression of IgAN through increasing the production of matrix by mesangial cells.