Evangelos N. Moudrianakis

The compaction of DNA helices into either continuous supercoils or folded-fiber rods and toroids

We have investigated by electron microscopy the mechanism of DNA compaction and have found that the double helix has the intrinsic potential to direct its own packaging into two distinctly different and mutally exclusive modes. The mode of DNA packaging is determined by the electrostatic charge density and water activity of the immediate microenvironment of the helix. The two basic structures formed by both linear and covalently closed-circular DNA are: a left-handed supercoil characteristic of minimally charge-shielded DNA, and a smooth rod characteristic of fully charge-shielded DNA. We propose that in the supercoil, the double helix is overwound (increased turn-angle), while in the rod, the helix is folded back and forth on itself. Variation of these two basic structures are the beaded fiber of DNA obtained with partially charge-shielded DNA and the toroid formed by the bending of the DNA rod and fusion of its ends in the presence of certain cations. We compare the DNA packaging inside these in vitro generated structures to DNA packaging in chromatin and viral capsids, and conclude that the packaging of DNA brought about by the use of salts and alcohol closely mimics the packaging behavior of the DNA in vivo, where it is usually complexed with histones or polyamines.

An Asymmetric Model for the Nucleosome: A Binding Site for Linker Histones Inside the DNA Gyres

Histone-DNA contacts within a nucleosome influence the function of trans-acting factors and the molecular machines required to activate the transcription process. The internal architecture of a positioned nucleosome has now been probed with the use of photoactivatable cross-linking reagents to determine the placement of histones along the DNA molecule. A model for the nucleosome is proposed in which the winged-helix domain of the linker histone is asymmetrically located inside the gyres of DNA that also wrap around the core histones. This domain extends the path of the protein superhelix to one side of the core particle.

Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymus

Abstract We have investigated by means of both spectrophotometric and fluorometric techniques the binding of the fluorescent dye, ethidium bromide, to DNA, to deoxyribonucleoproteins (DNP) and to DNPs from which specific kinds of proteins have been removed. The data from equilibrium binding experiments are analyzed according to Scatchard (1949) theory, and the number of available binding sites and the magnitude of the association constants are compared for whole and partial deoxyribonucleoproteins. As protein is removed from DNP, an increase in the association constant as well as an increase in the number of avaiable binding sites is observed. Furthermore, the magnitude of the changes of both of these binding parameters is largest when the proteins removed from DNP are those extracted by 0.6 m -NaCl which primarily include histone f1. The association constant calculated from the binding of ethidium bromide to native DNP differs significantly from that reported for the binding of the dye to artificially reconstituted DNA-f1 complexes and therefore may reflect important structural differences between native and artificial nucleoproteins. The nature of the environment of the binding sites is further studied by examining the fluorescence emission spectrum and the quantum yield of dye bound to the various nucleoprotein samples. No differences in either of these parameters have been observed. In addition, the effect of ionic strength on ethidium bromide binding is examined fluorometrically and discussed in terms of the probable binding mechanism. The manner by which chromosomal proteins limit the dye-binding capacity of DNA in DNP is discussed with respect to various models of their organization along the DNA. Furthermore, it is suggested that either a specific kind or critical amount of protein is responsible for the reduced affinity for ethidium bromide of whole DNP compared to that of partial DNPs and DNA. This altered dye binding capacity of DNP may be a manifestation of an incomplete restriction of available binding sites or of the appearance of new, structurally distinct sites which do not change the absorption or fluorescence properties of bound dye molecules.

Histone octamer function in vivo : mutations in the dimer–tetramer interfaces disrupt both gene activation and repression

Within the core histone octamer each histone H4 interacts with each H2A–H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A–H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site‐directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature‐sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt‐ and Sin‐phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer–tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression.

A chromatin-bound proteolytic activity with unique specificity for histone H2A

A protease associated with purified calf thymus chromatin has been found to act exclusively upon histone H2A, yielding a single new protein species, cH2A. This fragment migrates faster than H2A in acrylamide gel electrophoresis under denaturing conditions. The cH2A was purified and subjected to amino acid analysis and partial sequencing by the use of carboxypeptidase A. These studies demonstrated that cH2A had been derived from the removal of fifteen amino acids from the carboxy-terminal end of the intact H2A molecule, and that valine114 was its new carboxy-terminal residue. This cleavage does not occur under low ionic strength conditions, where H2A is believed to approximate a random coil; rather, it requires high ionic strength conditions similar to those under which the H2A molecule undergoes radical secondary and tertiary structural changes. This dependence upon ionic strength implies that the proteolytic cleavage is conformation- as well as sequence-specific. The H2A-specific protease is of nuclear origin, since isolation of nuclei by methods designed to maximize or minimize cytoplasmic contamination does not affect the level of proteolytic activity associated with purified chromatin. This nuclear protease appears to be tightly associated with the chromatin in vivo, for 0.6 M NaCl will not free it from isolated chromatin. A concentration of 1.2 M NaCl is required to dissociate the protease as well as its substrate from chromatin. The relationship of this enzyme to previously reported chromatin-bound proteases is discussed.

Fractionation and comparative studies of enzymes in aqueous extracts of spinach chloroplasts.

Abstract Water and dilute salt solutions were employed gor the hypotonic rupture and repeated extraction of proteins from spinach chloroplasts. The extracts were partially fractionated with ammonium sulfate, and the fractions so obtained were resolved by sucrose-gradient centrifugation and by electrophoresis on acrylamide gels. These techniques physically separated several enzymes of phosphate metabolism which are interdependent upon common substrates, co-factors, and reaction products, and which may function critically in photophosphorylation or its reversal. These enzymes are a Ca ++ -dependent ATPase ( S 20 w 12.7; DTT stimulated; active as coupling factor in photophosphorylation; morphologically identical to quantasomes), a discretely different Mg ++ -dependent ATPase ( S 20 w 6.0; entirely different from mitochondrial ATPases; does not require activation but is stabilized by reductants), and ATP-ADP exchange enzyme ( S 20 w 7.0), adenylate kinase ( S 20 w 4.2), and an alkaline inorganic pyrophosphatase (associated with fructose diphosphatase activity). In addition, a similar interacting system of CO 2 -fixing enzymes, comprised of ribulose diphosphate carboxylase, ribose phosphate isomerase, and phosphoribulokinase was separated. The fractionation and isolation conditions described were chosen to preserve all of these activities, and to provide an environment suited to a uniform comparison of their physical and enzymatic properties. These properties are compared with those of enzymes having similar functions, purified from chloroplasts by other means.

Binding of adenine nucleotides to the purified 13S coupling factor of bacterial oxidative phosphorylation

Abstract The 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis forms an unusually stable complex with ADP which can be isolated by simple gel filtration. Most preparations of enzyme exhibit an apparent binding ratio of 1 mol of ADP per mol of enzyme with a dissociation constant of approximately 15 μ m . One mol of adenylyl imidodiphosphate (AMP-PNP) also binds, with a dissociation constant of about 3 μ m . A constant could not be obtained from ATP binding studies because this nucleotide is hydrolyzed by the enzyme. Competition studies suggest that both ADP and AMP-PNP bind to the same site. Bound nucleotides are in a very slow equilibrium with free nucleotides, with a turnover time of 1–2 h. The rate of radionucleotide dissociation from the isolated enzyme-nucleotide complex increases when unlabeled nucleotide is added, suggesting that binding of nucleotide to one site on the enzyme allosterically promotes dissociation of nucleotide from another site. A nucleotide-induced “flip-flop” type of oscillation of the properties of the nucleotide binding sites on the coupling factor is proposed. From a comparison of the kinetic parameters of the intrinsic adenosinetriphosphatase activity and the nucleotide binding parameters of the enzyme population in toto, it is suggested that the enzyme exhibits functional polymorphism.

Molecular cloning of the ets proto-oncogene of the sea urchin and analysis of its developmental expression

The locus SU(Lv)-ets-2 of the sea urchin Lytechinas variegatus related to the oncogene v-ets of avian erythroblastosis virus E26 has been molecularly cloned. The cloned DNA was found to contain a region with a high degree of homology to E26 v-ets. The sea urchin homology with v-ets starts at a consensus splice acceptor sequence and stops at the point where homology between v-ets and human c-ets ends. This region corresponds to the Hu-ets-2 homologous sequences defined by Watson et al. (1985, Proc. Natl. Acad. Sci, USA 82, 7294-7298). Ninety-one out of 97 (or 94%) predicted amino acids are identical between sea urchin c-ets and E26 v-ets over the region of homology. This degree of homology exceeds the maximum homology previously found between any oncogene and an invertebrate homolog. A somewhat weaker homology with the Hu-ets-2 sequences continues beyond, for 13 codons, ending at a common termination codon. Northern blot analysis of mature unfertilized eggs and early embryos from sea urchins of the species Strongylocentrotus purpuratus revealed a single 6.8-kb ets-related RNA that is expressed at a maximum level during the early stages of embryonic development. This RNA species is polyadenylated indicating that it is the message for the sea urchin ets-2 gene.

Hill Reaction site in chloroplast membranes: Non-participation of the quantasome particle in photoreduction☆

Abstract The site of the Hill reaction in spinach chloroplast membranes has been studied, primarily by electron microscopy. Evidence is presented that the membrane-bound particles, known as quantasomes, in salt-extracted preparations are not Hill reaction sites. The membrane-bound particles failed to deposit locally an electron-dense tetrazolium salt, INT, used as a Hill oxidant. Rather, entire membranes appeared to participate uniformly in INT deposition. It was found that specific INT deposition on chloroplast membranes results from photo-reduction. Competition experiments with Hill reaction substrates suggest that INT is reduced at the same site as DCPIP. EDTA extraction yielded particle-free membranes fully active in photoreduction. A non-chlorophyllous protein species is recoverable from the EDTA extracts.

The localization of histone H3.3 in germ line chromatin of Drosophila males as established with a histone H3.3-specific antiserum

Abstract. A rabbit antiserum, specific for the histone H3.3 replacement variant, was raised with the aid of a histone H3.3-specific peptide. Immuno blot experiments demonstrated the specificity of this polyclonal antiserum. In addition, we showed on immuno blots that two monoclonal antibodies isolated from mice with systemic lupus erythematosus (SLE) display strong reactivity with the H3.3 histone, but not with its replication-dependent counterparts. Our observations indicate that histone H3.3 might play a role as autoantigen in SLE. We used the histone H3.3-specific antiserum to characterize the germ line chromatin in cytological preparations of Drosophila testes, because our previous studies had shown that a histone H3.3-encoding gene is strongly expressed in the germ line of Drosophila males. The antiserum reacted with some of the lampbrush loops in spermatocytes and with chromatin of the postmeiotic germ cells of males. Our data indicate that histone H3.3 is not evenly distributed throughout the chromatin of germ cells, but is concentrated in distinct regions. Histone H3.3 disappears from the spermatid nuclei, along with the other core histones, during the late stages of spermatogenesis. In Drosophila polytene chromosomes, however, a rather uniform distribution of the histone H3.3 was observed. The possible role of histone H3.3 is discussed.