A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus.

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.

Ricin B-chain promotes the internalisation of liposomal contents into rat hepatoma cells.

Ricin B-chain covalently attached to liposomes has been shown to promote the binding of the liposomes to rat hepatoma cells through its galactosyl binding site. Internalisation of the bound liposomes is demonstrated by the cytotoxicity of methotrexate-containing liposomes, the transfection of cells with targeted liposomes containing pSV2-neo DNA and the intracellular activity of an enzyme encapsulated in liposomes targeted with the ricin B-chain.

Identification of nuclear envelope proteins and glycoproteins which co-isolate with the nuclear protein matrix.

Nuclear envelopes and nuclear matrices were isolated from rat liver nuclei. Although differences in polypeptide composition of the structures are evident on SDS gel electrophoresis, they have an almost identical distribution of concanavalin A-binding glycoproteins. These matrix-associated concanavalin A-binding glycoproteins derive entirely from the nuclear envelope and are recovered almost quantitatively in the matrix. They constitute easily identifiable markers for nuclear envelope association with matrix or other nuclear subfractions. Surface labelling of nuclei with 125I using solid-phase lactoperoxidase further confirmed that a large number of envelope-associated nuclear surface proteins co-isolate with the matrix. Protein kinase activity, as well as endogenous substrates for the kinase(s) are shown to be the same in both envelopes and matrix. Envelope-derived proteins and glycoproteins may comprise a substantial proportion of total matrix protein.

Plant histone 2 from wheat germ, a family of histone H2a variants. Partial amino acid sequences.

1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.

A 46 kDa NTPase common to rat liver nuclear envelope, mitochondria, plasma membrane, and endoplasmic reticulum.

A 46 kDa ATP binding polypeptide of the nuclear envelope, virtually identical to the nuclear envelope NTPase putatively involved in mRNA efflux [6], is present in all rat liver cell membranes. Its presence in nuclear envelope is not the result of cross contamination during isolation.

The partial amino acid sequences of the two H2B histones from sperm of the sea urchin Psammechinus miliaris.

Two new histone H2B variants have been isolated from sperm cells of the sea urchin Psammechinus miliaris. They have been designated sperm histone H2B(1) Psammechinus and sperm histone H2B(2) Psammechinus. Both histones are highly homologous to the previously described sperm histones from Parechinus angulosus (Strickland et al. (1977) Eur. J. Biochem. 77, 263--275 and 277--286). The amino acid sequences of the Ps. miliaris sperm histones, though highly homologous, are not identical to the amino acid sequence derived from the codon sequence of a histone H2B gene, characterized from the same organism by Birnstiel et al. ((1977) Nature 266, 603--607).

Histone H2B variants from the erythrocytes of an amphibian, a reptile and a bird.

Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.

The identification by sequence homology of stage-specific sea urchin embryo histones H1.

The histone H1 fraction from gastrula of the sea urchin Parechinus angulosus consists of a multitude of polypeptides with different electrophoretic mobilities. The synthesis of these proteins is programmed. Amino acid composition, electrophoretic properties and sequence homologies identify these as isohistones H1. One of these isohistones atypically binds the non-ionic detergent Triton X-100.

Negative supercoiling and nucleosome cores. I. The effect of negative supercoiling on the efficiency of nucleosome core formation in vitro.

The efficiency of nucleosome core formation in vitro as a function of DNA topology was investigated. We show that the reconstitution of nucleosome cores by urea/salt dialysis on both negatively supercoiled and linearized plasmid proceed co-operatively, and that negatively supercoiled molecules are reconstituted significantly more efficiently compared with linearized molecules. The free energy of supercoiling, related to the square of the linking deficit, is further shown to be sufficient to account for this difference, which is particularly pronounced at low molar reconstitution ratios of octamer: DNA. At these low molar ratios the average number of cores formed per negatively supercoiled molecule is equal to the input ratio of octamer: DNA, in contrast to linearized molecules, where few if any cores are reconstituted under identical experimental conditions. The possible contribution of supercoil-stabilized non-B-DNA structural transitions to differences in core-DNA interactions on supercoiled and linearized DNA was also investigated. We show that the change in the nuclease susceptibility of a d(A-G).d(C-T) run in the free and reconstituted supercoiled plasmid is consistent with the reversion of the poly(purine).poly(pyrimidine) stretch from an H-DNA form to a B-DNA form following reconstitution of the negatively supercoiled plasmid into nucleosome cores. The biological significance of the supercoil-dependent efficiency of core formation is discussed, and the results related to other work.

Negative supercoiling and nucleosome cores. II. The effect of negative supercoiling on the positioning of nucleosome cores in vitro.

The influence of unrestrained negative superhelical stress on nucleosome core positioning was investigated in vitro for a core located on a section of the early H1-H4 histone gene spacer of Psammechinus miliaris. We show that the position of this core on a reconstituted molecule occupied by 11 nucleosome cores is identical on a linear DNA molecule and a circular DNA molecule in the absence of unrestrained negative superhelical stress. This position is also identical to that previously found on a 337 base-pair fragment of corresponding sequence. We conclude that the core position is determined primarily by the DNA sequence, and is not influenced by core-core interactions or spatial constraints imposed by an altered geometry of the DNA molecule. This finding is supported by the identical positions assumed by the nucleosome core after altering the angular orientation of the DNA molecule, and presumably that of adjacent cores, on either one or both sides of the test core. It is further demonstrated that the core on the histone spacer region assumes identical positions on circular DNA molecules in both the presence and absence of excess negative superhelical stress equivalent to sigma = -0.03. This result indicates that conservative levels of negative supercoiling do not induce a shift in the positions of nucleosome cores. The biological implications of the experimental results are discussed and related to the findings of other workers.

Stage- and adult tissue-specific expression of a homeobox gene in embryo and adult Parechinus angulosus sea urchins.

We report the isolation of a gene (PaHbox6), encoding a homeobox-containing protein of the South African sea urchin, Parechinus angulosus. Sequencing identified an Antennapedia-class gene encoding a homeobox that is the homologue of the Hawaiian sea urchin Tripneustes gratilla homeobox gene. Extensive restriction-fragment length polymorphism surrounds the gene. RNase-protection analyses revealed expression of PaHbox6 in mesenchyme blastula embryos at maximal levels of 44 +/- 8 transcripts/embryo. Four adult tissues examined (testes, ovary, intestines, Aristotle's lantern) showed expression of PaHbox6, though at greatly differing levels, with testes highest at eleven transcripts/10 pg RNA. Two transcripts of 5.2 and 5.7 kb were identified in adult tissue.

Carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate.

We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.

The quantitation of biotinylated compounds by a solid-phase assay using a 125I-labelled biotin derivative.

The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with 125I. This analogue has been used in conjunction with immobilized streptavidin in an assay which detects as little as 1 fmol biotin or biotinylated molecules in solution. The determination of biotinylated insulin in a tissue extract and the quantitation of a transcription assay are given as examples.

Mr determination of histones from their electrophoretic mobility in acidic urea gels in the absence of detergents.

The Mr of histones can be determined from their electrophoretic mobility at pH 2.3, 8 M urea in a polyacrylamide gel by correcting for differences in their charge density and properties of the gel matrix. The applicability of this method to other proteins is considered.

The reconstitution of histone H3-H4 tetramer from acid extracted histones in the absence of urea.

Simple mixing of acid purified histones H3 and H4 in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high Mr aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H3-H4 tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H3-H4 tetramer of closely similar structure.

Fluorescent labelling of histone H3: effect on histone-histone interaction and core particle assembly.

Substitution of Cys 110 of chicken histone H3 with N-iodoacetyl-N1-(5-sulpho-1-naphthyl)ethylenediamine or iodoacetamide prevents octamer formation in 2 M NaCl but does not prevent polyglutamic acid-mediated core particle assembly.

The effect of Ca2+ on the stability of chicken erythrocyte histone octamers.

Histones can be extracted from chicken erythrocyte chromatin with 2 M CaCl2 10 mM Tris (pH 7.4). The core histones so extracted exist as H3-H4 tetramers and H2A-H2B dimers since calcium at greater than 0.5 M result in dissociation of the histone octamer. Reconstitution of octamers occurs on removal of the Ca2+ by dialysis. Although less than 0.5 M calcium do not result in octamer dissociation, perturbations in the structure can be detected by CD and tyrosine fluorescence spectroscopy.

Octamer reconstitution from acid-extracted chicken erythrocyte histones.

Histone octamers have been reconstituted from acid-extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido-ester cross-linking, the reconstituted octamers have a structure identical to that of salt-extracted octamers.