Sp H1 Research Articles

Optimization of Bacillus cereus sp H1 amylase immobilization for an eco-friendly approach to textile processing

Optimization of Bacillus cereus sp H1 amylase immobilization for an eco-friendly approach to textile processing

SKRINING DAN IDENTIFIKASI ISOLAT BAKTERI ENDOFIT UNTUK MENGENDALIKAN PENYAKIT HAWAR DAUN BAKTERI PADA BAWANG MERAH

Screening and identification of endophytic bacteria to control bacterial leaf blight disease on Shallot. The experiment was conducted in Laboratory and Green House, from January to June 2012. Laboratory experiment consisted of three steps: (1) isolation of endophytic bacteria from healthy onion roots, (2)In planta /screening of endophytic isolates capable of reducing bacterial leaf blight disease, and (3) molecular identification of potential endophytic isolates. Treatments of in planta test were arranged in Completely Randomized Design. Collected isolates were tested for their capability in controlling bacterial leaf blight disease on shallot. The variables observed were disease incidence, disease severity, and shallot yield. The results showed that out of 82 isolates successfully isolated, 56 isolates (68.29%) were Gram positive, and 26 isolate (31.71%) were Gram negative. All isolates were HR negative and pathogenicity negative. Six endophytic isolates showed better performance in inducing resistance and increasing onion yield. Based on 16S rRNA sequence the six isolates were Bacillus cereus strain P14 , Bacillus cereus strain Se07, Bacillus sp H1, Bacillus sp SJ1 and Serratia marcescens strain PPM4.

Development of albumin microspheres containing Sp H1-DNA complexes: A novel gene delivery system

This study investigated the stability and transfection efficiency of plasmid DNA (pDNA) and sea urchin sperm histone H1 (Sp H1) complexes embedded in albumin microsphere formulations. Sp H1 increased the stability and transfection efficiency of pDNA, while providing a favourable sustained pDNA release profile. Encapsulating Sp H1-complexed pDNA into albumin microspheres further protected the pDNA from physical stress and heparin treatment. When compared with free pDNA encapsulated in albumin microspheres, the Sp H1-pDNA microsphere formulations exhibited decreased hydrophilicity, slower pDNA release profiles, protection against heparin-induced degradation of embedded pDNA and increased stability against physical stress. These results indicate that complex formation of pDNA with Sp H1 facilitates intracellular DNA transfer and that albumin microspheres-Sp H1-pDNA gene delivery formulations are suitable for controlled-release delivery of pDNA while offering protection of the pDNA from degradation and maintaining pDNA biological activity.

Linker DNA destabilizes condensed chromatin

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.Key words: chromatin condensation, sea urchin sperm, chicken red blood cell, nuclei, linker DNA, histone variants, micrococcal nuclease, nucleosome, trypsin, gel electrophoresis.

Linker DNA destabilizes condensed chromatin

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.

Phosphorylation weakens DNA binding by peptides containing multiple “SPKK” sequences

Sea urchin testis-specific H1 and H2B histones (Sp H1 and Sp H2B) are characterized by reversibly phosphorylated N-terminal regions consisting largely of multiple clustered "SPKK" tetrapeptides (serine-proline adjacent to two basic amino acids). This report presents data showing differences in DNA affinities between peptides containing dephosphorylated and phosphorylated N-terminal regions. Sp H1 and its phosphorylated derivative (pSp H1) were purified by hydroxylapatite chromatography. Peptides containing the N-terminal regions of Sp H1 and pSp H1 (NP and pNP, respectively) were produced by digestion with Staphylococcus aureus protease. NP and two forms of pNP differing in phosphate content were purified by DNA-cellulose chromatography. The DNA affinities of the peptides were compared using several criteria. NP was bound more tightly by DNA-cellulose than pNPs. NP precipitated DNA under a broad range of NaCl concentrations; pNPs did not. Both NP and pNPs protected DNA against thermal denaturation, but NP created a more stable DNA-peptide complex. Thirty to sixty times more pNP than NP was required to obtain equivalent inhibition of Hoechst 33258 binding to DNA. NP did not behave as a competitive inhibitor of DNA binding by Hoechst 33258 binding to DNA. We conclude that during spermatogenesis, dephosphorylation of the Sp H1 N-terminal region increases its basicity and thus its affinity for DNA.

Packaging and unpackaging the sea urchin sperm genome.

Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues (‘SPKK’ motifs) and are predicted to have β-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.

Sperm histones and chromatin structure of the “primitive” sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.

6DMAP inhibits chromatin decondensation but not sperm histone kinase in sea urchin male pronuclei

Treatment of sea urchin eggs for 10 min prior to fertilization with the kinase inhibitor 6DMAP (6-dimethylaminopurine) reversibly inhibits swelling and loss of conical morphology of the male pronucleus. Male pronuclei inhibited with 1 m M 6DMAP for 25 min undergo phosphorylation of Sp H1 and Sp H2B histones as fully as do control nuclei. Therefore, Sp histone kinase, whose target sequences resemble those of the M-phase histone kinase, is not inhibited by 6DMAP, and Sp histone phosphorylation, although it may be necessary, is not sufficient for chromatin decondensation.

Interaction of sperm histone variants and linker DNA during spermiogenesis in the sea urchin.

Several physical properties of sea urchin spermatid chromatin, which contains phosphorylated Sp H1 and Sp H2B histone variants, and mature sperm chromatin, in which these histones are dephosphorylated, were compared. Density, thermal stability, average nucleosomal repeat length, and resistance to micrococcal nuclease digestion are all increased in mature sperm relative to spermatid chromatin. Since the chromatins are identical in histone variant subtypes, the altered physical properties are not a consequence of changes in histone primary structure during spermiogenesis. The data are interpreted to mean that dephosphorylation of the N-terminal regions of Sp H1 and Sp H2B in late spermatid nuclei permits strong ionic binding of these highly basic regions to the extended linker, stabilizing the highly condensed structure of sperm chromatin.

Transitions in histone variants during sea urchin spermatogenesis

Transitions in the histone complement of nuclei during sea urchin spermatogenesis were investigated by two-dimensional gel electrophoresis. Nuclei were isolated from male gonads of individuals differing in degree of maturity. Unlike protamines, the sea urchin sperm-specific histone variants Sp H1 and Sp H2B appear early in spermatogenesis, well before spermatid differentiation, as the predominant representatives of their classes. Both proteins are phosphorylated from their first appearance until the last steps of spermiogenesis, when the highly condensed late spermatid nuclei become spermatozoan nuclei. Phosphorylation of serine occurs mostly (Sp H1) or entirely (Sp H2B) on the N-terminal portions of these molecules. We conclude that phosphorylated sperm-specific histone variants in the sea urchin function in spermatocytes during meiosis and are the major histones present during replication and transcription in some spermatogonia as well. We propose that the dephosphorylation of Sp H1 and Sp H2B in late spermatids is not primarily responsible for spermatid chromatin condensation but instead may act to stabilize the chromatin of the spermatozoon or aid in the final shaping of the sperm nucleus.

Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation

Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin.

Transitions in histone variants of the male pronucleus following fertilization and evidence for a maternal store of cleavage-stage histones in the sea urchin egg

A system has been developed for the biochemical study of male pronuclear histones following fertilization. Pronuclei are isolated in high yield and purity from polyspermically fertilized sea urchin eggs. Acid extracts of the pronuclei or chromatin are analyzed by two-dimensional gel electrophoresis. During male pronuclear decondensation, sperm-specific (Sp) H1 is completely replaced by the cleavage-stage (CS) H1 variant and two sperm H2B species are more gradually replaced by two closely migrating species (O, P) perhaps modifications of the Sp2Bs. Histones CS2A and CS2B begin to accumulate on the male chromatin predominantly during DNA synthesis, as does a subspecies of H3 (3″). By the time of chromosome condensation, the histone complement is chiefly CS1; CS2A and Sp2A; CS2B and O, P; three H3s and two H4s. Controls indicate that these patterns are not artifacts of cytoplasmic or maternal pronuclear contamination or modification of sperm histones during isolation. The histone transitions occur even if protein synthesis is inhibited, suggesting a maternal pool of CS histones. Such a store can be demonstrated directly in acid extracts of whole eggs. At least 30 haploid equivalents of CS histones are stored per egg. No α, β, γ, or δ variants appear on the chromatin in the first cycle. This system should allow, for the first time, detailed biochemical study of male pronuclear chromatin.