Micrococci

Ann Rech Vet, 1983, 14(4), 493 - 501
Lysozyme, an abomasal enzyme in the ruminants; Pahud JJ et al.; A strong lytic activity against Micrococcus luteus was demonstrated in abomasal secretions from calf, adult cattle, goat and sheep . This bacteriolytic activity was undetectable in other secretions . Bacteriolysis was caused by a glycosidase displaying endo-N-acetylmuramoylhydrolase specificity (EC 3.2.1.17) and was further characterized in the calf . This lysozyme also displayed significant chitinase activity . Immunofluorescence microscopy confirmed the secretion of lysozyme by abomasal gastric glands exclusively . Electrofocusing revealed multiple molecular forms, the predominant one (more than 80%) being characterized by Mr approx . 15,000, pH optimum 5.0, pl 7.5 and remarkable conformational stability . The lytic activity of lysozyme was ionic strength dependent and competitive inhibition was observed with both N-acetyl glucosamine and N-acetyl-muramic acid . Amino-acid analysis demonstrated common characteristics with known lysozymes, i.e . four disulphide bridges, two proline and N-terminal lysine . Structural homology between the three ruminant lysozymes was established by immunological cross-reactivity.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1983, (3), 86 - 90
{Taxonomic position of the genus Mycococcus}; Dobrovol'skaia TG; An attempt to elucidate the taxonomic position of the genera Mycococcus has been made . For this purpose the full range of the taxonomically important properties were investigated among microbial cultures preliminary identified as Mycococci . As a result of comparison Mycococcus with Micrococcus the taxonomic status of the genus Mycococcus was no longer confirmed . On the grounds of the published data and the author's experimental results it was concluded that genus Micrococcus was closely related to genus Arthrobacter . The possibility to place the genus Micrococcus within the group of coryneform bacteria is being discussed.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1983, (10), 4 - 17
{Radioresistance mechanisms of Micrococcus radiodurans}; Samoilenko II; The modern conceptions on the molecular mechanisms of Micrococcus radiodurans viability under the action of ionized radiation have been considered . Factors providing a high level of the bacterium radioresistance-the peculiarities of the cell wall structure, membranes, DNA, the redundancy of genetic information, the multiplicity of sites of DNA attachment to the membrane, a high level of antioxidant and antiradical systems-have been analysed . It has been shown that the efficiency of accurate, error-free, well balanced DNA repair system in connection with M . radiodurans properties mentioned provides a high radiation resistance of this microorganism.

Mol Cell Biochem, 1983, 55(2), 159 - 76
Association of thyroid hormone receptors with chromatin; Jump DB et al.; A large body of circumstantial evidence indicates that receptors located in nuclei of T3 responsive tissues represent a site of initiation of thyroid hormone action at the cellular level . Partial characterization of T3 receptors indicates that these proteins are monomeric structures in nuclei and are chromatin-associated non-histone proteins . Treatment of rat liver nuclei with either pancreatic DNase I or micrococcal nuclease releases T3 receptors from nuclei in two forms: a predominant (95 400 Mr; 5.5-6.0S) and a minor (265 000-365 000 Mr; 12.5S) nucleoprotein complex . Similar structures are excised from rat kidney, brain, and heart nuclei and from GH1 pituitary cell nuclei by micrococcal nuclease digestion . These endonuclease-excised receptor-containing complexes are significantly larger than the salt-extracted receptor (50 000 Mr; 3.5S) . The presence of DNA and other non-receptor proteins in these structures indicates that T3 receptors probably function within multimeric complexes in vivo . Although T3 receptors appear to be associated with DNA between nucleosomes, i.e . linker DNA, it is not entirely clear whether all or only a fraction of T3 receptors interact with nucleosomal components . The 12.5S receptor-containing nucleoprotein complex may represent T3 receptors in association with linker DNA and nucleosomal components . T3 receptors do not appear to be uniformly distributed to all chromatin fractions, but are associated with structures having characteristics of transcriptionally active chromatin . They are found in a region of chromatin which is enriched in RNA polymerase activity, rapidly labeled RNA and non-histone proteins, and depleted of histone Hl . This region is also highly sensitive to both micrococcal nuclease and pancreatic DNase I digestion . The association of receptors with transcriptionally active chromatin, however, must be considered provisional until additional details of the precise receptor-chromatin interaction have been established . The recent demonstration of a 20-fold increase in a specific hepatic mRNA four hours following administration of T3 to hypothyroid rats indicates that thyroid hormone potentially has very rapid effects on hepatic gene expression . However, significant changes in nuclear protein phosphorylation, nuclear protein composition, and chromatin structure have not been detected within this four-hour period . Thus, effects of T3 on hepatic gene expression are brought about by local and presumably subtle changes in nuclear function.

Proc Natl Acad Sci U S A, 1983 Jan, 80(1), 75 - 9
Discriminatory inhibition of protein synthesis in cell-free systems by vaccinia virus transcripts; Coppola G et al.; The effect of vaccinia virus early transcripts on cellular (globin, HeLa, Chinese hamster ovary) and viral (vaccinia, encephalomyocarditis) mRNA function was studied in reticulocyte and wheat germ cell-free protein-synthesizing systems . Vaccinia virus transcripts of two size classes (8-10 S and 4-7 S), generated in vitro by viral cores, inhibited function of cellular and encephalomyocarditis virus mRNA but not that of vaccinia virus in reticulocyte lysate systems . Mild alkaline hydrolysis or micrococcal nuclease treatment of vaccinia virus in vitro transcripts resulted in a loss of their ability to inhibit protein synthesis directed by HeLa cell RNA . Vaccinia virus in vitro transcripts also selectively inhibited HeLa cell protein synthesis in wheat germ systems, suggesting that double-stranded RNA is not involved in this inhibition of protein synthesis . The addition, to the reticulocyte translating system, of cytoplasmic RNA obtained from infected cells in conjunction with cellular mRNA (globin, HeLa) resulted in the inhibition of synthesis of the globin or HeLa polypeptides with little or no effect on the translation of the vaccinia virus proteins . RNA extracted from vaccinia virions inhibited cellular but not vaccinia virus mRNA function when added to the reticulocyte lysate systems with uninfected or infected HeLa cell cytoplasmic RNA.

Mol Cell Biochem, 1983, 56(1), 73 - 80
Topography of the subunits of Micrococcus lysodeikticus F1-ATPase; Mimbrera A et al.; The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with {125I} applied to membrane-bound or soluble pure F1-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its alpha, beta, gamma and delta subunits within the protein molecule and with respect to the plane of the membrane . The beta subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role . The alpha and gamma subunits lie in an intermediate layer between the beta subunits and the membrane, in which the gamma subunit occupies a central position within the F1-ATPase molecule in contact with the alpha subunit . The delta subunit appears to be tightly bound to the F0 component of the ATPase complex, probably buried in the membrane bilayer . A molecular arrangement of M . lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry alpha 3 beta 3 gamma 2 delta 2 (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.

Biokhimiia, 1983 Jan, 48(1), 104 - 10
{Submergence of Micrococcus lysodeikticus F1-ATPase into the hydrophobic phase of the membrane, using 2,4,6-trinitrobenzosulfonate and 12-0-(azidoformyl) stearic acid methyl ester}; Mileikovskaia EI et al.; The accessibility of F1-ATPase from Micrococcus lysodeikticus in solution and in the membrane for the specific water-soluble NH2-group reagent, 2,4,6-trinitrobenzosulfonate (TNBS), was studied . Incubation of the soluble factor F1 with 50 mM TNBS pH 8.3 results in incorporation of 58.6 +/- 4.4 trinitrophenyl residues per mole of enzyme . At the same time F1-ATPase isolated from TNBS-pretreated membranes contains 27.2 +/- 2.0 TNP-residues per mole of enzyme . It is assumed that the different accessibility of F1-ATPase for TNBS in solution and in the membrane is due to incorporation of F1-ATPase into the membrane . Study of membrane F1-ATPase interaction with the radioactive lipid-soluble photoreactive label, 12-0-(azidoformyl) stearic acid methyl ester demonstrated that F1-ATPase does not immediately interact with the lipid phase of the membrane . It is suggested that membrane F1-ATPase may be enveloped by hydrophobic proteins.

Acta Microbiol Pol, 1983, 32(2), 139 - 45
Lysine production from hydrocarbon by Micrococcus varians 2Fa; Sen SK et al.; A bacterium isolated from Assam (India) soil was found to accumulale l-lysine from hydrocarbon and was identified as a strain of Micrococcus varians . The strain is able to grow and accumulate lysine in a purely synthetic medium though supplementation of the synthetic medium with casamino acids significantly improves the yield . The yield of l-lysine under optimal conditions was found to be 2.6 g X 1(-1) of the compound isolated in crystalline form.

DNA, 1983, 2(1), 9 - 13
Chromatin structure and expression of amylase genes in rat pituitary tumor cells; Levy-Wilson B; Amylase-coding sequences were detected in the population of cytoplasmic RNA molecules from cultured rat pituitary GH3 cells . The amylase genes in these cells are in a micrococcal nuclease-sensitive, transcriptionally active chromatin conformation . The amount of cytoplasmic amylase mRNA appears to be controlled by small molecules provided by the fetal calf serum component of the culture medium.

Biochim Biophys Acta, 1982 Dec 31, 699(3), 241 - 6
Organization of the newly replicated chromatin in the vicinity of the replication fork; Pospelov V et al.; Ehrlich ascites tumour cells were pulse-labelled with {3H}thymidine for 1 min or were treated with cycloheximide and labelled with {3H}thymidine for 45 min . The kinetics of digestion with micrococcal nuclease of both pulse-labelled and cycloheximide chromatins showed that they exhibited increased susceptibility towards the enzyme . At the same time their release from the nucleus was retarded and this was interpreted to mean that, unlike the bulk of chromatin, they were tightly bound to a fixed nuclear structure . When subjected to an equilibrium metrizamide-triethanolamine density gradient centrifugation both pulse-labelled and cycloheximide chromatins banded at higher density than control chromatin, which was an indication of their higher protein to DNA ratio . After a mild trypsinization, eliminating H1 and the nonhistone proteins, the pulse-labelled chromatin sedimented to the same density as control chromatin, and the cycloheximide chromatin sedimented to a density which was intermediate between those of control chromatin and free DNA . This result showed that the newly replicated chromatin had the same, and the cycloheximide chromatin half the amount of core histones present in control chromatin.

J Biol Chem, 1982 Dec 25, 257(24), 14610 - 2
Hexaprenyl pyrophosphate synthetase from Micrococcus luteus B-P 26 . Separation of two essential components; Fujii H et al.; Hexaprenyl pyrophosphate synthetase was detected in extracts of Micrococcus luteus B-P 26 . During the course of purification the enzyme was resolved into two components, each of which had no catalytic activity but restored the hexaprenyl pyrophosphate synthetase activity when combined with each other . Both fractions, designated components A and B in the order of their elution from hydroxyapatite, were purified free of farnesyl pyrophosphate synthetase co-occurring in the same bacterium . They appeared to be proteins of molecular weights of approximately 20,000 (component A) and 60,000 (component B) . Component A was more stable as compared with component B which was easily destroyed by relatively mild heat treatment . The hexaprenyl pyrophosphate synthetase reconstituted of these two components catalyzed the synthesis of all-trans-hexaprenyl pyrophosphate from isopentenyl pyrophosphate and all-trans-farnesyl or all-trans-geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate.

Biochemistry, 1982 Dec 21, 21(26), 6746 - 51
Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes; Boiteux S et al.; Heat treatment of poly(deoxycytidylic acid)-{poly(dC)} induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III . The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC) . The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase . The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions . However, the alteration of E . coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites . There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites . The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B . The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro . Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.

Nucleic Acids Res, 1982 Dec 20, 10(24), 7977 - 91
Chromatin structure of histone genes in sea urchin sperms and embryos; Spinelli G et al.; The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P . lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes . In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin . In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed . Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region . As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.

Lancet, 1982 Dec 18, 2(8312), 1356 - 61
Detection of RNA complementary to herpes-simplex virus in mononuclear cells from patients with Behçet's syndrome and recurrent oral ulcers; Eglin RP et al.; Viral DNA probes were used in in-situ hybridisation to detect the complementary RNA in mononuclear cells from the blood of normal subjects and patients with Behcet's syndrome and recurrent oral ulcers . Hybridisation of 125I-labelled DNA probes was detected by means of autoradiography and quantitated with a video image-analysis technique . Hybridisation between herpes-simplex virus type 1 (HSV-1) DNA and the complementary RNA in mononuclear cells was significantly greater in 10 of 20 patients with Behcet's syndrome than in controls . Consistently, mononuclear cells from fewer patients showed significant hybridisation with the HSV-2 probe, and the grain counts were also lower . Control DNA probes from adenovirus 2, bacteriophage lambda, and Micrococcus lysodeikticus did not show significant hybridisation . Further separation of the patients into the four types of Behcet's syndrome revealed that mononuclear cells in 8 of 10 patients with the ocular or arthritic types showed significant hybridisation of RNA to the HSV-1 DNA probe, compared with 2 of 10 patients with the mucocutaneous or neurological types . Mononuclear cells from 4 of 8 patients with minor but not major aphthous ulcers also showed significant RNA hybridisation to HSV-1 DNA . The results suggest that at least part of the HSV genome is present and transcribed in peripheral-blood mononuclear cells--probably lymphocytes--of patients with the ocular and arthritic types of Behcet's syndrome and minor aphthous ulcers . The immunopathogenesis of these diseases might therefore be associated with HSV-1.

Nucleic Acids Res, 1982 Dec 11, 10(23), 7593 - 608
DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena; Bonven B et al.; A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena . The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites . The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA . The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions . Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages . Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.

J Bacteriol, 1982 Dec, 152(3), 976 - 82
Rescue of mitomycin C- or psoralen-inactivated Micrococcus radiodurans by additional exposure to radiation or alkylating agents; Hansen MT; The processing of damaged DNA was altered in a mitomycin C-sensitive mutant (mtcA) of Micrococcus radiodurans . Even though the mutant retained resistance to 254-nm UV radiation, it did not, in contrast to the wild-type strain, show any excessive DNA degradation or cell death when incubated with chloramphenicol after sublethal doses of either UV light or mitomycin C . The results suggest the constitutive synthesis of an enzyme system responsible for wild-type proficiency in the repair of mitomycin C-induced damage . An alternative system able to repair damage caused by mitomycin C was demonstrated in the mtcA background . In this strain, additional damage inflicted upon the cellular DNA effected a massive rescue of cells previously inactivated by mitomycin C . Rescue was provoked by ionizing radiation, by UV light, or by simple alkylating agents . Cells treated with psoralen plus near-UV radiation could be rescued only when inactivation was due primarily to psoralen-DNA interstrand cross-links rather than to monoadducts . The rescue of inactivated cells was prevented in the presence of chloramphenicol . These results can be interpreted most readily in terms of an alternative repair system able to overcome DNA interstrand cross-links produced by mitomycin C or psoralen plus near-UV light, but induced only by the more abundant number of damages produced by radiation or simple alkylating agents.

Can J Biochem, 1982 Dec, 60(12), 1085 - 94
Hyper(ADP-ribosyl)ation of histone H1; Aubin RJ et al.; Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei . Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes . Electrophoresis of {32P}ADP-ribosylated histones on first-dimension acid-urea or acid-urea-Triton gels and on second-dimension acid--urea--cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H1(0) . Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid . The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations . Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.

Proc Natl Acad Sci U S A, 1982 Dec, 79(23), 7278 - 82
Electron paramagnetic resonance crystallography of bacterial catalase: g-Contour mapping method of analysis; Dickinson LC et al.; Single crystals of bacterial catalase from Micrococcus luteus have been examined by EPR at 77 K . X-ray perfect crystals gave a set of four prominent resonances in all three orthogonal planes which yielded eight heme direction cosine matrices to an accuracy of +/- 2 degrees as expected for the P4(2)2(1)2 space group and unit cell parameters previously determined . These matrices are related by D4 symmetry operation of the space group . There were additional weaker resonances only resolved in two or even one plane . A method of g-contour mapping was devised to solve for the orientations of hemes that give rise to these weaker resonances . Three additional sets of heme orientations, also following D4 symmetry, were determined . All of the above sites have the same principal g values, 2.0, 5.4, and 6.6 . The EPR crystallographic results imply that several conformational substates may be trapped at 77 K.

J Gen Microbiol, 1982 Dec, 128 (pt 12), 3037 - 50
Isolation, properties and nucleolytic degradation of chromatin from Escherichia coli; Sjastad K et al.; A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli . The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes . The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA . The protein content of the chromatin was almost three times greater than the nucleic acid content . Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters . The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect . The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S . Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle . The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp . The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes . Several low molecular weight proteins (12000-27000) were found in the DNA-protein complex . The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

Mol Biol Rep, 1982 Nov 30, 8(4), 203 - 11
Distribution of chromatin proteins between fractions of hamster liver chromatin differing in their susceptibility to micrococcal nuclease; Kilianska Z et al.; Hamster liver nuclei were fractionated by digestion with micrococcal nuclease into nuclease released (SP) and nuclease resistant (PP) fractions varying in chemical composition and transcriptional activity . Electrophoretic analysis of histones from SP and PP showed no qualitative and quantitative differences . Apparently chromatin-bound protease activity can be found in both fractions . Nonhistone chromatin proteins isolated from SP and PP under mild conditions were fractionated by hydroxyapatite chromatography into NHCP1, NHCP2, NHCP3 and molecular heterogeneity and specificity were tested by SDS-polyacrylamide gel electrophoresis . The differences observed in nonhistone proteins are mainly of quantitative nature, however some specific polypeptides for SP and PP are observed.

J Biol Chem, 1982 Nov 25, 257(22), 13465 - 74
Enzymatic repair of pyrimidine dimer-containing DNA . A 5' dimer DNA glycosylase: 3'-apyrimidinic endonuclease mechanism from Micrococcus luteus; Grafstrom RH et al.; A pyrimidine dimer-DNA glycosylase has been purified 20,000-fold from Micrococcus luteus . The enzyme is a single polypeptide chain with Mr = 18,000 that acts specifically on pyrimidine dimers, preferring those in double-stranded DNA to those in single-stranded DNA . The glycosylase cleaves the 5' residue of a pyrimidine dimer generating an apyrimidinic site and a mixed pyrimidine/pyrimidine nucleotide dimer . Under conditions of substrate excess, dimers containing a 5'-thymine are preferred to those with a 5'-cytosine residue . The glycosylase has an associated apyrimidinic/apurinic (AP) endonuclease that prefers apyrimidinic sites at the site of glycosylase action to either apurinic or apyrimidinic residues . This endonuclease is a Class I AP endonuclease in that it cleaves 3' to the AP site generating a 3'-deoxyribose moiety and a 5'-phosphate.

Biochim Biophys Acta, 1982 Nov 24, 719(2), 292 - 8
NMR field-cycling relaxation spectroscopy of bovine serum albumin, muscle tissue, Micrococcus luteus and yeast . 14N1H-quadrupole dips; Winter F et al.; The frequency dependence of the proton spin lattice relaxation time of bovine serum albumin, muscle tissue, Micrococcus luteus and yeast has been measured by the aid of the field-cycling technique . In all systems 14N1H-quadrupole dips have been observed . The conclusion is that amide groups are the dominating relaxation centers up to approx . 10(7) Hz . This finding can be understood by the fact that protein backbone fluctuations and, if possible, tumbling of the whole molecule rather than side group motions are the relevant mechanisms in this frequency range . A proton relaxation scheme for cells and tissue is presented.

Eur J Biochem, 1982 Nov 15, 128(2-3), 435 - 43
NMN adenylyltransferase: its association with chromatin and with poly(ADP-ribose) polymerase; Uhr ML et al.; The nuclear location of NMN adenylytransferase, which catalyses the formation of NAD and pyrophosphate from ATP and NMN, has been examined to ascertain if the enzyme is bound to the domains of chromatin which undergo poly(ADP-ribos)ylation . This latter reaction utilizes much of the cellular NAD . A radioisotope assay using {alpha-32P}ATP was developed to enable precise measurement of picomole amounts of NAD . With this assay, it appeared that the reaction catalysed by NMN adenylyltransferase proceeded with a rapid, early 'burst' of NAD before steady-state velocities were established . From this it was calculated that there could be 10- active sites of NMN adenylyltransferase per HeLa nucleus in asynchronously growing cells: that is, approximately one per 10-20 nucleosomes . Very little enzyme activity was liberated by digesting HeLa nuclei with micrococcal nuclease in 80 mM NaCl, and the enzyme which was solubilized was not bound to oligonucleosomes separated by electrophoresis on polyacrylamide gels . In contrast, poly(ADP-ribose) polymerase activity was clearly demonstrated on these particles . The enzyme was readily liberated by DNase I digestion, especially when the digestion was carried out in low-ionic-strength buffer . The results demonstrated that the enzyme was neither bound to oligonucleosomes nor part of the nuclear envelope or matrix . Preliminary results suggested that there could be some direct channelling of NAD between the two enzymes in intact nuclei . It appears that NMN adenylyltransferase is bound within rather than to chromatin.

J Biol Chem, 1982 Nov 10, 257(21), 13101 - 7
Reassociation of histone H1 to H1-depleted polynucleosomes; Klingholz R et al.; A new procedure is described for the reassociation of histone H1 to rat liver polynucleosomes selectively depleted of H1 and nonhistone proteins . The fidelity of reconstitution was scrutinized by nuclease digestion and sedimentation studies monitoring the regeneration of structural features which disappear following removal of H1 . The results demonstrate that the 166-base pair barrier of digestion with micrococcal nuclease was restored in polynucleosomes reconstituted at the same ratio of H1 per octamer as that found in native nuclei, while association of twice that amount of H1 produced a barrier of digestion at about 178-180 base pairs . Polynucleosomes associated with polylysine exhibited a similar barrier as H1-depleted polynucleosomes . Digestion of H1-reconstituted polynucleosomes with DNase I produced preferentially dinucleosomal DNA fragments in the same manner as that of untreated polynucleosomes . The rate of digestion of H1-reconstituted polynucleosomes by micrococcal nuclease and DNase I was also comparable to that of untreated polynucleosomes . Sedimentation of H1-reconstituted polynucleosomes in a quasi-physiological ionic milieu revealed the regeneration of disassembly-refractory particles, a structural feature of untreated polynucleosomes . We conclude that the nucleosomal and supra-nucleosomal structure of polynucleosomes was reconstituted with fidelity.

J Biol Chem, 1982 Nov 10, 257(21), 13018 - 27
Hormonal regulation of the conformation of the ovalbumin gene in chick oviduct chromatin; Bloom KS et al.; We have examined the effects of steroid hormones in the chromatin sensitivity of the ovalbumin gene to micrococcal nuclease and have attempted to define the importance of the nucleosome core, higher order chromatin folding, and transcription in the maintenance of the nuclease-sensitive conformation of the ovalbumin chromatin . Solution hybridization studies demonstrated that the sensitivity of the ovalbumin gene in oviduct nuclei to micrococcal nuclease paralleled the hormone-dependent transcription of the ovalbumin gene in the immature chick . Blot hybridization analysis also revealed a hormone-dependent change in this chromatin region since ovalbumin DNA fragments from nuclease-treated hen and estrogen-stimulated chick oviduct nuclei exhibited nucleosomal repeat patterns that were less discrete than those observed for the ovalbumin specific fragments from liver and hormone-withdrawn oviducts . This transcription-related conformation was not the result of enhanced sensitivity of the ovalbumin-containing nucleosomal cores since the bulk of the nucleosomes associated with the ovalbumin chromatin were not preferentially cleaved internally by micrococcal nuclease . Rather, an analysis of the fragmentation of the ovalbumin chromatin as a function of digestion extent suggested a mechanism in which the heightened sensitivity resulted from the collective expansion of the nuclease cutting sites in the linker regions of the ovalbumin chromatin because the gene was in an unfolded conformation . The transcription-specific conformation was not merely a consequence of RNA synthesis per se since the selective sensitivity of the gene was unaffected by treatment of oviduct nuclei with alpha-amanitin, actinomycin D, or RNase . In addition, the presence of the transcriptional complex on the ovalbumin chromatin was presumably not required for selective nuclease recognition since preferential cleavage was observed under conditions expected to deplete oviduct nuclei of template-bound RNA polymerase and nascent RNA chains . These results are consistent with a model in which the expressed ovalbumin gene is in an unfolded polynucleosomal structure whose formation is related to transcriptional activity but not dependent on the transcriptional process.

J Biol Chem, 1982 Nov 10, 257(21), 13001 - 8
Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system; Tyagi JS et al.; Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts . The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template . At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts . The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos . The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease . A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C . These data suggest that it is a small, hydrolphilic compound probably organic in nature . The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates . The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II . In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.

J Biol Chem, 1982 Nov 10, 257(21), 13088 - 94
Isolation of high mobility group-containing mononucleosomes from avian erythrocyte nuclei and their sensitivity to DNase I; Kootstra A; Conditions have been established which have led to the isolation of mononucleosomes which contain the high mobility group (HMG) proteins, in particular HMG 14, from mature chicken erythrocyte nuclei after extended micrococcal nuclease digestion . This selective enhancement of HMG-containing mononucleosomes appears to be due to their preferential solubilization at a time when other mononucleosomes, i.e . those containing H5 and H1 which represent the bulk of the mononucleosomes, were no longer soluble . Isolation of "early" mononucleosomes and subsequent analysis of these mononucleosomes after DNase I digestion showed that the DNA of these "early" mononucleosomes was more accessible to DNase I and that those mononucleosomes which contained HMG 14 were more soluble when the DNA became extensively degraded by DNase I . The resulting pattern of single-stranded DNA fragments suggests that the NH2 termini of the core histones no longer bind strongly to the nucleosomal DNA of the "early" mononucleosomes, and thus enhance the rate of DNase I digestion, while the presence of HMG 14 increased the solubility of these mononucleosomes . These two properties are probably the basis for the increased DNase I sensitivity of the transcriptionally active chromatin.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1982 Nov, 253(2), 253 - 64
{Optimization of lysozyme determination: comparative study of preparations of test cultures of Micrococcus luteus (M . lysodeikticus Fleming)}; Stelzner A et al.; About 6000 single measurements of the susceptibility of different preparations of Micrococcus luteus (M . lysodeikticus Fleming) cells were performed in order to assess their suitability for the determination of various muramidase concentrations . In particular, a turbidimetric method was compared with an agar-diffusion technique under varying experimental conditions . From our results it can be concluded that M . luteus cells washed with ether and acetone or living lyophilized cells are most suitable for turbidimetric determinations of lysozyme activity . The optimum conditions for turbidimetric assays comprised an initial transmission of 20% and a test time of 5 min . For the agar-diffusion technique fresh living or living lyophilized cells were most suitable . For a given incubation time of 3 h an incubation temperature of 50 degrees C yielded the best results.

Mutat Res, 1982 Nov, 106(1), 179 - 89
Excision repair in Cockayne syndrome; Mayne LV et al.; Cockayne syndrome (CS) is a genetic disorder showing cellular sensitivity to the lethal effects of UV-irradiation . No defects in unscheduled DNA synthesis or in daughter-strand repair have been detected after UV-irradiation of CS cells . We have studied several aspects of excision repair, particularly at early times after UV-irradiation, and with one exception, we have not been able to detect any difference in the response of normal and CS cells to UV-irradiation, by measuring: (1) the rate of formation of incision breaks in the presence of 1-beta-D-arabinofuranosylcytosine (araC); (2) the amount of repair replication as measured by equilibrium centrifugation; (3) the ligation of repaired DNA to pre-existing DNA; (4) the digestibility of repaired DNA after treatment of nuclei with micrococcal nuclease . The single exception was a pair of CS strains from sibling donors in which the rate of uncoupled incision due to the presence of either araC or the specific inhibitor of DNA polymerase alpha, aphidicolin, was slightly faster than in other cells studied . This effect was absent in the heterozygous parents . However, since this was not seen in two other CS strains in the same genetic complementation group, we can not attribute this increased rate of incision to the defective CS gene . We conclude that, within the limits of resolution of these techniques, CS cells do not have a detectable defect in excision repair.

Biochem Pharmacol, 1982 Nov 1, 31(21), 3379 - 86
Alteration of chromatin structure induced by the binding of adriamycin, daunorubicin and ethidium bromide; Grimmond HE et al.; The results reported in this paper show the changes in chromatin structure caused by the binding of adriamycin (ADR), daunorubicin (DR) and ethidium bromide (EtdBr) to DNA in chromatin, either isolated or in nuclei or whole cells . Micrococcal nuclease was used as the structural probe of chromatin . The binding of the drugs to chromatin DNA induced two structural changes . First, it produced an unfolding of the overall chromatin structure as evidenced by the increased production of acid-soluble oligonucleotides for the drug-treated samples above the level of the control sample . Second, it caused a disruption of the core particle structure with increased production of DNA of subnucleosomal size and smearing of the nucleosome pattern . The effects were greatest for duanorubicin, followed by adriamycin and ethidium bromide.

J Gerontol, 1982 Nov, 37(6), 673 - 9
Influence of disulfide-reducing agents on fractionation of the chromatin complex by endogenous nucleases and deoxyribonuclease I in aging mice; Tas S et al.; Age-related alterations in chromatin were evaluated by a fractionation procedure involving limited digestion of liver cell nuclei from young adult and old mice with endogenous nucleases and deoxyribonuclease I . The results suggest that in the chromatin the bulk of the nuclear deoxyribonucleic acid (DNA) may be folded or compacted into domains or loops and associated at a few points with a nuclear protein skeleton structure represented by the final pellet fraction which is resistant to deoxyribonuclease I . Disulfide bonds appear to play a role in the linkage of regions of DNA with this nuclear protein skeleton structure . The amount of DNA that could be released from the pellet fraction by disulfide-reducing agents was significantly greater with old than with young adult mice . In addition, the amount of chromatin material released into the first soluble fraction decreased and that in the second soluble fraction increased with age . Treatment of the nuclei with disulfide-reducing agents did not correct this particular age-related change . Previous results had shown a similar alteration with age of chromatin subjected to digestion by micrococcal nuclease . The combined results suggest the existence of a supranucleosomal alteration in chromatin structure during aging.

Can J Microbiol, 1982 Nov, 28(11), 1252 - 60
Ovine ill-thrift in Nova Scotia . 9 . Production of experimental quantities of isocyanide metabolites of Trichoderma hamatum; Brewer D et al.; Laboratory cultures of Trichoderma hamatum produce metabolites that are characterized by an isocyanide functionality . Three such metabolites predominate . One is the known compound trichoviridin (I) . The other two, described here for the first time, are 3-(3-isocyano-6-oxabicyclo{3,1,0}hex-2-en-5-yl)acrylic acid (II) and a very unstable compound 3-(3-isocyanocyclopent-2-enylidene-)propionic acid (III) . Production of these three metabolites by a random sample of wild isolates of the fungus has been examined . At least one of these isocyanides was isolated from all cultures in which the culture broth inhibited the growth of Micrococcus luteus . The relative amounts of the three isocyanides produced by individual isolates were not the same and cultures were found in which I, II, or III was the main product . The isocyanide III was produced by all wild isolates which had antibiotic activity in their culture broth, and it was present in the concentration range 2-40 mg X L-1.

J Virol, 1982 Nov, 44(2), 603 - 9
Intracellular forms of simian virus 40 nucleoprotein complexes . IV . Micrococcal nuclease digestion; Coca-Prados M et al.; The structures of DNAs present in various intracellular forms of simian virus 40 (SV40) nucleoprotein complexes were analyzed by micrococcal nuclease digestion . The results showed that the 70S SV40 chromatin was completely sensitive to nuclease digestion, whereas CsCl gradient-purified mature virion was completely resistant . Virion assembly intermediates with different degrees of virion maturation showed intermediate resistance, and three products were found: nucleosomal DNA fragments, representing the fraction of intermediates that were sensitive to nuclease; linear SV40 genome-sized DNA, representing the more mature intermediates that contained one or limited defects in the capsid shell; and supercoiled SV40, which was derived from mature virions . These digestion products, however, remained associated with capsid shells after nuclease digestion . These results were consistent with the model in which maturation of the SV40 virion is achieved through the organization of capsid proteins that accumulate around SV40 chromatin . Mild digestion of SV40 nucleoprotein complexes with micrococcal nuclease revealed the difference in nucleosome repeat length between SV40 chromatin and virion assembly intermediates . A novel DNA fragment of about 75 nucleotides was observed early in nuclease digestion.

Proc Natl Acad Sci U S A, 1982 Nov, 79(21), 6484 - 8
Spermidine-condensed phi X174 DNA cleavage by micrococcal nuclease: torus cleavage model and evidence for unidirectional circumferential DNA wrapping; Marx KA et al.; Spermidine-condensed phi X174 replicative form (RF) II DNA was digested with micrococcal nuclease to yield seven identifiable DNA bands forming an arithmetic fragment-length series . The DNA monomer unit length was found to be 780 +/- 80 base pairs . This result is most consistent with a proposed model for micrococcal nuclease cleavage of a DNA torus organized by the unidirectional, circumferential wrapping of B-geometry DNA . By a topological consideration, the blunt-end-rod-fusion model for torus formation {Eickbush, T . H . & Moudrianakis, E . N . (1978) Cell 13, 295-306} is shown to be inconsistent with our empirical solution results . We propose a continuous, circumferential DNA wrapping model in which a significant fraction of the collapsed circular phi X174 RFII DNA molecules form regular toruses comprised of seven complete, unidirectional double-helical wraps.

J Gen Virol, 1982 Nov, 63 (Pt 1), 131 - 40
Poliovirus-induced inhibition of host RNA synthesis studied in isolated HEp-2 cell nuclei; Bossart W et al.; Nuclei isolated from uninfected HEp-2 cells synthesized RNA for 60 to 90 min . The individual RNA polymerase activities were determined by alpha-amanitin differential inhibition and the RNA products characterized by electron microscope (EM) autoradiography and sucrose gradient centrifugation . In nuclei prepared from poliovirus-infected cells, the capacity to synthesize RNA in vitro decreased with time after infection . RNA polymerase II activity (hnRNA synthesis) was preferentially inhibited more than was the polymerase I activity (rRNA synthesis) . Poliovirus-infected cytoplasm (S-30) inhibited in vitro RNA synthesis in uninfected nuclei by selectively affecting the polymerase II activity . Selective inhibition of hnRNA synthesis by the crude extracts could be monitored by EM autoradiography directly . Determinations of individual RNA polymerase activities by differential alpha-amanitin inhibition were done only after treatment of the infected cytoplasm with micrococcal nuclease to abolish virus RNA replication . Selective inhibition of hnRNA synthesis depended on preincubation of the nuclei together with the infected cytoplasm, indicating that inhibitory substances from the infected cytoplasm entered the nuclei . Isolated nuclei therefore provide a useful system for studying the nature of the inhibitor(s) and of host RNA synthesis inhibition by picornaviruses.

Proc Natl Acad Sci U S A, 1982 Nov, 79(21), 6458 - 60
Micrococcal nuclease cleavage of nucleotide linked to glutamine synthetase yields phosphotyrosine at the ligation site; Martensen TM et al.; The activity of micrococcal nuclease was studied on a novel substrate, denatured adenylylated glutamine synthetase {L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2}, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP . The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase . The Km of the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used for assay of the enzyme . Native adenylylated glutamine synthetase was not deadenosylated by micrococcal nuclease under the conditions that permit rapid deadenosylation of denatured glutamine synthetase . Failure to attack native glutamine synthetase is probably not due to steric factors because the native enzyme is deadenylylated by snake venom phosphodiesterase under identical conditions . The inability of micrococcal nuclease to deadenosylate native glutamine synthetase may be due to the formation of an inactive complex because the native protein inhibited the nuclease activity on the denatured protein.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5835 - 52
Analysis of chromatin structure and DNA sequence organization: use of the 1,10-phenanthroline-cuprous complex; Cartwright IL et al.; Limited treatment of Drosophila nuclei with the 1,10-phenanthroline-cuprous complex leads to rapid production of nucleosomal ladders indistinguishable from those obtained by micrococcal nuclease digestion . An investigation of the preferential sites of cleavage of protein-free DNA at locus 67B1 surprisingly indicated that both reagents recognized very similar features . Thus, a virtually identical pattern of preferential cleavages was generated over a 12 kb fragment encoding four transcripts at this locus . The distribution of cleavage sites was highly non-random, with major sites falling in the spacers between the genes . Both reagents cleaved certain chromatin-specific sites near the 5' ends of the genes . However, an analysis of preferential cleavages at the sequence level did not reveal the same close correspondence . We suggest that both reagents can recognize some localized secondary structural features of the DNA and that the particular distribution of sequences present at this locus results in a distinctive pattern of cleavage sites that delineates gene and spacer segments.

Nucleic Acids Res, 1982 Oct 11, 10(19), 5823 - 34
Analogous cleavage of DNA by micrococcal nuclease and a 1-10-phenanthroline-cuprous complex; Jessee B et al.; We have examined the DNA cleavage specificity of a 1,10-phenanthroline-cuprous complex and find this agent to recognize the same sites and cleave with the same relative preferences as micrococcal nuclease . In contrast, DNase I and bleomycin-ferrous complex cleave the same 5000 bp D . melanogaster histone gene DNA with different specificities, although some of the sites appear to be recognized and cleaved by all four reagents . Our results suggest that the reagents used probably detect discrete conformational perturbations along the DNA double helix.

J Biol Chem, 1982 Oct 10, 257(19), 11784 - 90
Chromatin fragments containing bovine 1.715 g ml-1 satellite DNA . Nucleosome structure and protein composition; Weber JL et al.; Some of the properties of chromatographically purified satellite chromatin are compared with those of unfractionated, control chromatin . Nucleosomes were present in the purified satellite chromatin as verified by digestion with micrococcal nuclease and DNase I and by electron microscopy . Average nucleosome DNA repeat lengths of 186 +/- 7 and 193 +/- 5 base pairs were obtained through micrococcal nuclease digestion of the purified satellite chromatin and control chromatin, respectively; nucleosome spacer lengths were equally heterogeneous for the two chromatin samples . The distribution of Eco RI-produced chromatin fragments of different size in the satellite chromatin was the same as that calculated assuming random cleavage at each Eco RI site, consistent with the notion that nucleosomes do not have specific locations on the 1.715 g ml-1 satellite DNA . The purified satellite chromatin contained little non-histone protein, but did contain all five histones and all detectable histone sequence variants . Amounts of the core histones were identical in the satellite chromatin and the control chromatin, but the amount of histone H1 was 30% less in the satellite chromatin than in the control . Although the molar ratios for the major sequence variants of both histones H3 and H2A differed between kidney and thymus, the ratios were the same for satellite and control chromatin isolated from a single tissue.

J Cell Sci, 1982 Oct, 57, 151 - 60
Chromatin from the unicellular red alga Porphyridium has a nucleosome structure; Barnes KL et al.; We have isolated a crude nuclear preparation from the unicellular red alga Porphyridium aerugineum and investigated the structure of Porphyridium chromatin . Electrophoresis of deproteinized DNA fragments produced by micrococcal nuclease digestion of Porphyridium nuclei gives a typical ladder pattern, indicative of a repeating structure . The DNA repeat-length, calculated from plots of multimer length against multimer number, varies somewhat between different digestions, ranging from 160 to 180 base-pairs (average 173) . We interpret this as evidence of heterogeneity in repeat-length; the calculated repeat-length depends on the extent of digestion because chromatin sub-populations with longer repeat-lengths are on average digested earlier . Polyacrylamide/sodium dodecyl sulphate gel electrophoresis of basic proteins purified from Porphyridium nuclear preparations gives a pattern characteristic of core histones . Although our interpretation is complicated by some degradation, the result strongly suggests that Porphyridium chromatin contains each of the four core histones and that they are similar to those of higher eukaryotes . This, together with the micrococcal nuclease digestion results, demonstrates that Porphyridium chromatin is not fundamentally different from that of higher eukaryotes.

J Cell Biol, 1982 Oct, 95(1), 262 - 6
Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster; Hill RJ et al.; The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster . A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem . Biophys . Res . Commun., 52:504-510) yielded native Drosophila salivary chromosomes . These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope . Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter . Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.

Proc Natl Acad Sci U S A, 1982 Oct, 79(19), 5803 - 7
Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units; Chrysogelos S et al.; Electron microscopy shows that complexes of the single-strand DNA binding protein (SSB) of Escherichia coli and phage fd DNA appear as beaded fiber loops containing an average of 38 beads, 1 per 170 bases of DNA . Extensive digestion of native unfixed SSB-fd DNA complexes with micrococcal nuclease reveals a protected DNA fragment of 145 bases, while shorter digestion periods result in a sequence of fragments in multiples of 160 +/- 25 bases . Digestion of these complexes with DNase I produces a repeating pattern of bands, multiples of approximately 15 bases with strong bands at 60, 105, 118, 130, 145, 150, and 210 bases . Isopycnic banding in CsCl solution yields densities of 1.272 and 1.700 g/ml, respectively, for SSB alone and for fd DNA and, after fixation, of 1.388 g/ml for fd DNA-SSB beaded fibers and 1.373 g/ml for the individual protein-DNA beads . Based on these data and the molecular weights of SSB and fd DNA, we suggest that the nucleoprotein chain consists of eight molecules of SSB bound to 145 bases of DNA, with these units linked by roughly 30 bases of protein-free DNA . The excellent concord between results obtained by enzyme digestion of unfixed native samples and, after fixation, by electron microscopy and density banding supports the conclusion that SSB organizes single-stranded DNA in a manner similar to the organization of duplex DNA by histones.

J Biochem (Tokyo), 1982 Oct, 92(4), 1205 - 12
Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione; Nakano I et al.; ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography . Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion . The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction . The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine . These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.

J Virol, 1982 Oct, 44(1), 189 - 98
Evidence that vesicular stomatitis virus produces double-stranded RNA that inhibits protein synthesis in a reticulocyte lysate; Thomas JR et al.; Cell-free protein synthesis by reticulocyte lysates was inhibited by a polyadenylated RNA fraction extracted from HeLa cells infected with vesicular stomatitis virus (VSV) but not by polyadenylated RNA from mock-infected HeLa cells . A similar inhibitor of cell-free protein synthesis was found in a polyadenylated fraction of RNA transcribed in vitro by VSV but not in untranscribed nucleocapsids . Fractionation of the VSV transcription product showed that the translation inhibitor segregated with nucleocapsids containing newly transcribed polyadenylated or non-polyadenylated RNA, as determined by oligodeoxythymidylate-cellulose chromatography . The inhibitors present in VSV-infected HeLa cells and in VSV in vitro transcripts both appeared to be double-stranded RNA, as judged by the characteristics for inhibition of reticulocyte cell-free protein synthesis described by Hunter et al . (J . Biol . Chem . 250:409-417, 1975) . The double-stranded nature of the VSV RNA inhibitor was supported by the finding that the translational inhibitory effect was inactivated by melting the inhibitor in the absence of salt and by micrococcal nuclease.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 307 - 9
Prenatal appearance of a short nucleosomal DNA repeat length in neurons of the guinea pig cerebral cortex; Brown IR; The organization of chromatin in neurons of the cerebral cortex of the guinea pig brain was analyzed by digesting isolated nuclei with micrococcal nuclease . During development, cortical neurons were observed to undergo an alteration in chromatin structure which results in an atypically short nucleosomal DNA repeat length of 164 bp . This change in chromatin organization occurs postnatally in certain mammals but in the guinea pig it takes place prior to birth between days 32 and 44 of fetal development . This suggests that the appearance of the short nucleosomal DNA repeat length in cortical neurons correlates to a particular stage of differentiation of cortical neurons rather than to the event of birth.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 295 - 302
The roles of different repair mechanisms in the ultraviolet resistance of Micrococcus luteus; Zherebtsov SV et al.; In ultraviolet-irradiated Micrococcus luteus wild type the replication of DNA was not interrupted at every pyrimidine dimer, in contrast to that in ultraviolet-sensitive G7 and some other mutants . The contribution of uninterrupted replication to the ultraviolet resistance of M . luteus proved to be equal to the contributions of excision repair and inducible postreplication repair . It was found that some postreplication gaps could be filled by constitutive pathways of postreplication repair when inducible pathways were suppressed by chloramphenicol . Prolonged treatment with chloramphenicol was shown to block not only inducible repair but also other processes essential for ultraviolet irradiation survival.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 237 - 42
Normal response of fanconi's anemia cells to high concentrations of O2 as determined by alkaline elution; Seres DS et al.; In this investigation, normal and Fanconi's anemia fibroblasts were exposed to high concentrations of oxygen and the effects of this treatment on DNA were analyzed by alkaline elution . No DNA single-strand breaks were detected in either cell type with up to 20 h incubation in high(50-95%) concentrations of O2 . No evidence of DNA damage by O2 could be detected with an endonuclease preparation from Micrococcus luteus . Cells which have been treated with various DNA-damaging agents in the presence of the polymerase inhibitor cytosine arabinoside have been shown to accumulate DNA single-strand breaks during DNA excision repair . When cells were treated with the polymerase inhibitor in 50 or 95% O2, a low level of DNA single-strand breaks accumulated in both cells types . However, no significant differences in the frequency of DNA single-strand breaks were detected between normal and Fanconi's anemia cells after exposure to high O2.

Biochim Biophys Acta, 1982 Sep 27, 698(3), 287 - 94
An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease; Tomilin NV et al.; The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers . In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M . luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair . A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme . The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination which ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one.

J Biol Chem, 1982 Sep 25, 257(18), 11160 - 5
Chromatin reorganization during spermatogenesis in the winter flounder; Kennedy BP et al.; During spermatogenesis in the winter flounder, the average repeat length of nucleosomal DNA in the testis increases from 195 +/- 2 base pairs in prespermatid nuclei to 222 +/- 3 base pairs in sperm . This increase in repeat length apparently occurs in the linker region since there is no change in the pattern of DNA fragments produced during micrococcal nuclease digestion of the nucleosome core . The timing of the increase coincides with the loss of phosphate from the high molecular weight basic nuclear proteins and histones H2A and H4 . When prespermatid nuclei are digested with micrococcal nuclease to the point where 10% of the DNA is acid-soluble, mononucleosomes and higher oligomers are readily released . However, when sperm chromatin is digested to the same extent, these products are no longer soluble and only traces of H1 and small DNA fragments are released . This situation is not changed in sperm chromatin that has been depleted of H1 by extraction with 0.4 M NaCl . However, if nuclease-treated sperm chromatin is lightly digested with trypsin, mono- and oligonucleosomes are released . At this level of proteolysis, the high molecular weight basic nuclear proteins are completely broken down, but the core histones are largely intact . These data are consistent with a model in which the unphosphorylated high molecular weight basic nuclear proteins function in cross-linking nucleosomes together within the sperm nucleus.

Nucleic Acids Res, 1982 Sep 25, 10(18), 5533 - 52
Conversion of simian virus 40 DNA to ordered nucleoprotein structures by extracts that direct accurate initiation by eukaryotic RNA polymerase II; Sinha SN et al.; Interaction of SV40 DNA with three different HeLa cell extracts capable of directing correct initiation of transcription leads to the formation of ordered nucleoprotein complexes that are structurally similar to SV40 minichromosomes and eukaryotic chromatin . These nucleoprotein complexes can be conveniently purified by band sedimentation or gel filtration . Their sedimentation and elution properties resemble those of SV40 minichromosomes . Electron microscopy of purified complexes shows beaded structures that are sensitive to proteases, resulting in recovery of naked, largely undegraded DNA . Contour lengths and compaction ratios of these nucleoprotein complexes are similar to those of authentic SV40 minichromosomes . Their digestion patterns with micrococcal nuclease and pancreatic DNase I resemble those of SV40 minichromosomes . Such nucleosome-like structures can also be obtained with linear SV40 DNA . Unlike nucleosomes, no histones could be detected in the purified nucleoprotein complexes . Non-histone chromosomal protein fractions (high mol . wt . and free of high mobility group proteins) prepared from the HeLa cell extracts can also generate similar ordered structures . We conclude that ordered nucleoprotein structures with certain common characteristics can be formed by interaction of DNA with non-histone chromosomal proteins as well as with histones . Only the former structures are generated in currently used cell-free transcription systems . It appears that only those purified nucleoprotein complexes containing the promoter can be actively transcribed in the presence of additional cell-free extract, suggesting that such structures and their protein components may be important in transcription.

Biochim Biophys Acta, 1982 Sep 17, 718(1), 42 - 8
Lactoferrin binding to lysozyme-treated Micrococcus luteus; Perraudin JP et al.; When the cell lysis of Micrococcus luteus by hen egg white or human lysozyme is performed in the presence of bovine or human lactoferrin, a temporary increase of the turbidity of the solution as followed at 450 nm is observed . Examination of the suspension under light microscopy has proven that the protoplasts produced upon lysozyme action are agglutinated by lactoferrin . The rate of agglutination depends on pH, lactoferrin, lysozyme and cells concentrations . Agglutination is maximal at pH 5.5 . Around 1.4 X 10(6) binding sites for lactoferrin per cell have been determined through a Scatchard plot analysis . The binding to the cells is not mediated by the glycosidic moiety of lactoferrin but rather by a charge-to charge interaction as succinylation of about four out of the 39 lysines of lactoferrin completely abolishes its ability to agglutinate the cells . Binding does not depend on ionic iron nor on the iron content of lactoferrin itself.

J Biochem (Tokyo), 1982 Sep, 92(3), 749 - 55
Characteristic distribution of two forms of chromatin-bound RNA polymerase II in rat liver nuclei; Hatayama T et al.; We have shown that, in rat liver nuclei, the chromatin-bound RNA polymerase II is released as two different forms on digestion with micrococcal nuclease or DNase I (peak 1 and peak 2) . To elucidate the origin of the two forms of the enzyme, we examined their distribution in fractionated chromatins obtained by mild micrococcal nuclease digestion of the nuclei . About half of the total peak 2 activity was recovered in a nuclease-sensitive chromatin fraction which contained DNA enriched in the sequences appearing in the polysomal polyadenylated mRNA . On the other hand, four-fifths of the total peak 1 activity was recovered in a nuclease-resistant chromatin fraction which contained DNA comprising only two thirds of the transcribed sequences . Furthermore, during the nuclease digestion, peak 2 activity was rapidly released from the chromatin, whereas peak 1 activity was gradually released . These results indicate that the two forms of RNA polymerase II are distributed differently in the cell nuclei.

Biokhimiia, 1982 Sep, 47(9), 1540 - 6
{Comparison of endogenous nuclear RNA-polymerase II at different stages of mycoplasmodial growth}; Kostiuchenko DA et al.; The nuclei of Physarum polycephalum isolated from the 48- and 96-hour-old growing microplasmodium differ 7-8 fold by the rates of {3H}UTP incorporation in vitro accompanied by a slight (approximately 1.6 fold) change in concentration of endogenous RNA-polymerase II . Using mild fragmentation of chromatin nuclei by micrococcal nuclease and DEAE-Sephadex chromatography the bound enzyme was shown to consist of two forms differing in the degree of their binding to the template and in functional significance . Transcription in the Physarum polycephalum nuclei can be regulated by changes in the correlation of these forms.

Biofizika, 1982 Sep-Oct, 27(5), 768 - 71
{Structure of polylysine-DNA complexes}; Khachatrian AT et al.; By the methods of nuclease digestion and electron microscopy the complexes of poly-I-lysine with phage T2 DNA were studied . At micrococcal nuclease digestion of complexes polylysine--phage T2 DNA structures resistant to nuclease and containing fragments of DNA with lengths of about 300, 600, 1200 base pairs were revealed . On the electron micrographs structures superficially resembling nucleosomes were detected.

Cell, 1982 Sep, 30(2), 567 - 78
The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition; Nasmyth KA; The two yeast mating-type alleles MATa and MAT alpha each produce two mRNAs that are transcribed in opposite and diverging directions from central promoters . Silent copies of MATa (HMRa) and MAT alpha (HML alpha) contain identical DNA sequences throughout the transcribed region, yet are not transcribed, except in sir- strains . Since SIR represses not only transcription from a silent copy but also its ability to act as a recipient in a mating-type interconversion, we have investigated whether it might act by regulating the entire chromatin structure of a silent locus . We have therefore compared the profile of DNAase I and micrococcal nuclease cleavage at HML alpha with MAT alpha and HMRa with MATa in sir- and SIR+ strains . We find that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT . One particularly striking change that SIR induces provides a simple explanation for one of its biological properties: control of directionality of switching . SIR causes the disappearance of a DNAase I-hypersensitive site at Y-Z boundary (found at MAT or HM sir-) that is coincident with a double-strand cleavage possibly created by HO in the initiation of a mating-type switch.

J Exp Med, 1982 Sep 1, 156(3), 860 - 72
Naturally induced auto-anit-idiotypic antibodies . Induction by identical idiotopes in some members of an outbred rabbit family; Binion SB et al.; Naturally induced auto-anti-idiotypic (AAI) antibody responses specific for antimicrococcal antibody idiotypes were detected in 42% of the rabbits in a family immunized with Micrococcus lysodeikticus . The natural AAI response of each rabbit recognized only a portion (11-41%) of that individual's total antimicrococcal antibody population . Cross-reactions of idiotypes were observed within the group of rabbits exhibiting natural AAI responses . Examination of the basis for the cross-reactions showed that the natural AAI antisera recognized identical idiotopes on the antimicrococcal F(ab')2 fragments from each rabbit that made an AAI response . The cross-reactive idiotopes were shown to be of paternal origin and were found in the antimicrococcal antibodies of each offspring . The data strongly support the idiotypic network concept that naturally induced AAI responses may occur routinely in outbred normal individuals as a result of antigenic stimulation . Further, the data suggest that the induction of regulatory AAI antibody responses in outbred rabbits may depend on the expression of particular germ line idiotopes.

Biochemistry, 1982 Aug 31, 21(18), 4338 - 43
Poly(7-deazaguanylic acid), the homopolynucleotide of the parent nucleoside of queuosine; Seela F et al.; Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield . The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum . No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution . From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide . In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex . However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand . This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G) . RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested . Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.

Biochim Biophys Acta, 1982 Aug 30, 698(2), 218 - 21
Initiation reactions in the mRNA-dependent reticulocyte lysate; Kay JE et al.; Reticulocyte lysates depleted of mRNA by digestion with micrococcal nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes . Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested . Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete . As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.

Biochim Biophys Acta, 1982 Aug 30, 698(2), 167 - 72
Effect of thiostrepton and 3'-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis; Bhuta P et al.; The effect of the antibiotics thiostrepton and micrococcin on EF-Tu-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3'-terminus of aminoacyl-tRNA)(System I) or by methanol ('uncoupled GTPase', System II) was investigated . In System I, thiostrepton increases the binding affinities of the effectors to the EF-Tu.GTP.70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the KmGTP is virtually unchanged . Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases VmaxGTP, whereas KmGTP remains unaffected . Micrococcin is without any effect in both systems . The 'uncoupled GTPase' (in System II) is also strongly inhibited by C-A-Phe . The results indicate the crucial role of the EF-Tu site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis . It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-Tu for GTP.

Nucleic Acids Res, 1982 Aug 25, 10(16), 5059 - 72
Influence of nonhistone chromatin protein HMG-1 on the enzymatic digestion of purified DNA; Shastri K et al.; The effect of chicken erythrocyte High Mobility Group protein 1 (HMG-1) on the enzymatic hydrolysis of purified double-stranded and single-stranded bacteriophage lambda DNA was studied . HMG-1 was found to inhibit the digestion of single- and double-stranded DNA by S1 nuclease and DNase I, respectively . HMG-I increased the rate of hydrolysis of double-stranded DNA by micrococcal nuclease, particularly at low HMG-1/DNA ratios, and had little effect on the hydrolysis of single-stranded DNA by micrococcal nucleases, even at high HMG-1 DNA ratios . We also present a semi-quantitative estimate that HMG-1 and HMG-2 occur in chromatin from rapidly dividing, cultured rat hepatoma cells at about 8 times the level that they occur in adult rat liver chromatin.

Nucleic Acids Res, 1982 Aug 11, 10(15), 4655 - 69
Eukaryotic ternary transcription complexes . II . An approach to the determination of chromatin conformation at the site of transcription; Sargan DR et al.; Digestion of rat liver nuclei by endogenous nucleases or micrococcal nuclease releases a chromatin fraction containing RNA polymerases I and II bound to DNA fragments in ternary transcription complexes . To label the DNA in these transcription complexes, the polymerases were allowed to add radioactively labelled ribonucleotides in vitro to in vivo-initiated RNA chains . During this transcription step, nucleic acids were photochemically cross-linked using 8-methoxypsoralen . Nucleic acids in transcription complexes were then sized by gel electrophoresis . Under conditions where RNA polymerases I and II were active in vitro, most of the labelled DNA was found in a series of fragments of sizes which were multiples of approximately 200 base-pairs . When polymerase I alone was active, the smallest member of this series carried the bulk of the label; when polymerase II also was active, a significant proportion of the label was carried on the dimer and higher oligomers . Proteins other than polymerase alone are shown to be responsible for the pattern of DNA fragments protected from nucleases . Therefore active RNA polymerases I and II in vivo are in close proximity to structures protecting DNA fragments, the sizes of which are similar to those found in nucleosomes . We have yet to establish that these structures are composed of histones.

J Biol Chem, 1982 Aug 10, 257(15), 9086 - 92
Role of non-histones in chromosome structure . Cell cycle variations in protein synthesis; Adolph KW et al.; As part of a study of the role of non-histone proteins in chromosome structure, the synthesis of non-histones associated with interphase chromatin was investigated . Synchronized suspension cultures of HeLa cells were pulse-labeled with {35S}methionine, and chromatin was prepared by mild micrococcal nuclease digestion . Two-dimensional polyacrylamide gel electrophoresis, in addition to one-dimensional electrophoresis, was used to resolve the patterns of incorporation of radioactive label . Significant variations in non-histone synthesis were seen during the cell cycle . A strong correlation was not found between DNA synthesis in mid-S phase and variations in non-histone synthesis . The non-histone proteins of purified metaphase chromosomes were also characterized by two-dimensional gel electrophoresis and compared to the proteins of interphase chromatin . The pattern of non-histones is not identical with that of interphase chromatin, although a number of major species may be shared by interphase chromatin and metaphase chromosomes . The HeLa nuclear scaffold, the framework that maintains the overall morphology of the interphase nucleus, shows relatively few proteins on two-dimensional gels . The synthesis of nuclear scaffold proteins was quantitated by excising each of 19 proteins from two-dimensional gels and determining the incorporated radioactivity by scintillation counting . Substantial variations in protein synthesis were found, with several species showing changes of about 2-fold in the percentage of incorporation.

Arch Microbiol, 1982 Aug, 132(2), 185 - 8
Utilization of phthalate esters by micrococci; Eaton RW et al.; Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source . They have been separated into four groups by their esterase content and nutritional characteristics . The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups . Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.

J Microsc, 1982 Aug, 127 (Pt 2), 127 - 38
The correlation averaging of a regularly arranged bacterial cell envelope protein; Saxton WO et al.; An adaptation of the 'correlation averaging' method is described which allows reliable and almost fully automatic image averaging in the case of near-periodic structures notwithstanding the presence of substantial crystal imperfections; methods for assessing resolution and symmetry without reliance on crystallinity are also discussed . Electron micrographs of negatively stained and rotary shadowed preparations of the HPI-layer protein from the cell envelope of Micrococcus radiodurans have been averaged using the method, and the projected structure is described to a resolution of about 1.9 nm.

Biochimie, 1982 Aug-Sep, 64(8-9), 735 - 8
Comparison at the molecular level of uracil-DNA glycosylases from different origins; Leblanc JP et al.; A nuclear and a cytoplasmic uracil-DNA glycosylase have been purified from epithelial cells derived from a rat hepatoma (H4 cells) cultured in vitro . They have different optimum pH, molecular weight, isoelectric points, activation energy, Km . Uracil acts as a non competitive inhibitor towards the nuclear enzyme while it is a competitive one for the cytoplasmic enzyme . Comparison of the properties of the two mammalian enzymes with those of the enzymes isolated from Escherichia coli and Micrococcus luteus shows that they all behave differently . The following criteria were studied: molecular weight, optimum pH, isoelectric point, inhibition by uracil analogs, modulation of their activity by polyamines or by intercalating drugs . The only common properties shared by these four enzymes are: an activity twice as high on single stranded DNA than on double stranded DNA and no requirement for divalent cation for maximal activity.

J Biol Chem, 1982 Jul 25, 257(14), 8507 - 15
Association of newly synthesized histones with replicating and nonreplicating regions of chromatin; Annunziato AT et al.; Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoresis mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA . The separation efficiency of these two methods is approximately 70% . Following micrococcal nuclease digestion, chromatin was fractionated by salt elution . 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further down to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions . The remaining chromatin fractions, solubilized with 0.4-0.6 M NaCl, were several-fold enriched in nascent DNA (Annunziato, A . T., Schindler, R . K., Thomas, C . A., Jr., and Seale, R . L . (1981) J . Biol . Chem . 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones . This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA . In contrast, histone H1 showed little tendency toward deposition onto new DNA . Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin . We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.

Nucleic Acids Res, 1982 Jul 24, 10(14), 4321 - 37
Alignment of nucleosomes along DNA and organization of spacer DNA in Drosophila chromatin; Karpov VL et al.; A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat . 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position . Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp) . The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments . The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/ . We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix . Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA . Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.

Biochim Biophys Acta, 1982 Jul 14, 689(1), 173 - 6
Evidence for the incorporation of a fluorescent anthracene fatty acid into the membrane lipids of Micrococcus luteus; Welby M et al.; 9-(2-Anthryl)-nonanoic acid, a newly synthesized photoactivable molecule, is shown to be incorporated into the membrane lipids of the bacterium Micrococcus luteus, through the regular metabolic pathway . This incorporation, which occurs at the sn-1 position exclusively and without any degradation or elongation of the anthracene fatty acid, is accompanied by an upward shift of the chain length of the other fatty acids.

Nucleic Acids Res, 1982 Jul 10, 10(13), 4107 - 17
Chromosome-bound mitotic factors: release by endonucleases; Adlakha RC et al.; Additional evidence is presented to support our recently reported conclusion that the mitotic factors of mammalian cells, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus laevis oocytes, are localized on metaphase chromosomes . Chromosomes isolated from mitotic HeLa cells were further purified on sucrose gradients and digested for varying periods with either the micrococcal nuclease or DNase II . At each time point of digestion the amount of mitotic factors released was determined by injecting a supernatant of these fractions, obtained by high-speed centrifugation, into oocytes . The amount of DNA rendered acid soluble under the conditions of digestion used was 3% ot 5% of the total chromosomal DNA . The extent of release of mitotic factors with both nucleases was estimated to be about 30% to 40% as evidenced by the reextraction of the undigested chromosomal pellet with 0.2 M NaC1 . Similar results were obtained when nuclei from G2 cells were digested under identical conditions . The release of these chromosome-bound mitotic factors by mild digestion with these nucleases though only partial, clearly demonstrates that a significant proportion of these factors are localized on metaphase chromosomes.

Nucleic Acids Res, 1982 Jul 10, 10(13), 3951 - 65
Deoxyribonuclease II as a probe to sequence-specific chromatin organization: preferential cleavage in the 72 bp modulator sequence of SV40 minichromosome; Shakhov AN et al.; T prove sequence-specific chromatin structure of SV40 minichromosome, we further modified previously described hybridization mapping . Actually (i) the digestion patterns by two nucleases (micrococcal and DNAase II) were compared and (ii) the kinetics of nuclease digestion was analyzed from early time points when only a fraction of minichromosomes was cleaved once to longer digestions when oligo- and mononucleosomal bands appeared . DNAase II is shown to possess certain sequence specificity different from that of micrococcal nuclease . The major finding is that DNAase II preferentially cleaves the SV40 minichromosome at a distinct region of the genome known as 72 bp modulator element . Other hypersensitive sites are located near the replication origin and T-ag binding site II and also near BamHI site where termination of replication and "late" transcription occurs . Micrococcal nuclease splits the BglI-Hpaii region in a different manner.

J Biol Chem, 1982 Jul 10, 257(13), 7730 - 6
Chromatin structure of the chicken beta-globin gene region . Sensitivity to DNase I, micrococcal nuclease, and DNase II; Wood WI et al.; We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene . The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion . We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA . The sensitivity factors were determined by a least squares curve fitting method based on target analysis . In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II . In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences . There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity . This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain . Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain . These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.

Biochem J, 1982 Jul 1, 205(1), 15 - 21
Localization of testis-variant histones in rat testis chromatin; Rao MR et al.; Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column . The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S . Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4 . The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B) . It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA.

J Bacteriol, 1982 Jul, 151(1), 48 - 57
Metabolism of dibutylphthalate and phthalate by Micrococcus sp . strain 12B; Eaton RW et al.; Micrococcus sp . strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source . A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp . strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate) . Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate . Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate . Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway . Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes . The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product . During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters . One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway . Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.

J Bacteriol, 1982 Jul, 151(1), 465 - 7
Metabolism of dimethylphthalate by Micrococcus sp . strain 12B; Eaton RW et al.; During growth of Micrococcus sp . strain 12B with dimethylphthalate, 4-carboxy-2-hydroxymuconate lactone (CHML, X) and 3,4-dihydroxyphthalate-2-methyl ester (XI) were isolated from culture filtrates . CHML is the lactone of intermediate 4-carboxy-2-hydroxymuconate (IX) . Accumulation of XI which is not a substrate for 3,4-dihydroxyphthalate-2-decarboxylase in strain 12B afforded an easy access to the preparation of 3,4-dihydroxyphthalate.

Proc Natl Acad Sci U S A, 1982 Jul, 79(13), 4050 - 4
Mass mapping of a protein complex with the scanning transmission electron microscope; Engel A et al.; A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy . Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging . The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining . The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries . Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons . The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data.

Biochim Biophys Acta, 1982 Jun 30, 697(3), 381 - 4
Adaptive repair of cross-links in DNA of Micrococcus radiodurans; Kitayama S; Cross-links in the DNA of Micrococcus radiodurans induced by mitomycin C were repaired during post-incubation . This repair process was inhibited in cells post-incubated in the presence of chloramphenicol . However, the removal of cross-links in DNA was almost normal, even in the presence of chloramphenicol, if the cells were pretreated with lower concentrations of mitomycin C.

Nucleic Acids Res, 1982 Jun 25, 10(12), 3627 - 45
Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei; Weischet WO et al.; In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites . In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele . Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences . Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence . The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.

Biochemistry, 1982 Jun 22, 21(13), 3167 - 74
Replicative conformation of parental nucleosomes: salt sensitivity of deoxyribonucleic acid-histone interaction and alteration of histone H1 binding; Schlaeger EJ; The transiently altered DNA-histone interaction of parental chromatin during replication was studied by micrococcal nuclease digestion . A large amount of nuclease-resistant pulse-labeled DNA and a small fraction of nonreplicating DNA are released from chromatin fragments by treatment with 0.5 M NaCl and appear as protein-free DNA . As shown by reconstitution experiments, the salt sensitivity of digested nascent chromatin is most probably a consequence of the shorter DNA fragment size (55 +/- 15 base pairs) in these complexes . This new DNA is associated with parental chromatin fragments which are structurally changed in such a way that parts of nucleosomal DNA were more susceptible to nuclease attack . The core histones of these particles are probably not distinct from those of salt-stable nucleosomes . However, histone H1 and probably high-mobility group proteins appear to be more weakly bound during replication as shown by electrophoresis under nondenaturing conditions . The results agree with the assumption that the transient alteration of nucleosomal conformation describes a state in which DNA could be replicated without leaving the associated core histone complexes . A possible attachment of pulse-labeled chromatin with nuclear matrix is discussed.

Biochem J, 1982 Jun 15, 204(3), 653 - 62
Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen; Lebeau MC et al.; Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen . Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease . After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with {3H}-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S . After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak . After injection of anti-oestrogen alone or together with oestradiol benzoate, no {3H}oestradiol-binding or 4-hydroxy{3H}tamoxifen-binding peaks were detected in the chromatin . Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor . Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction . Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone . The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.

Cell, 1982 Jun, 29(2), 395 - 402
A transcriptionally active, covalently closed minichromosome of cauliflower mosaic virus DNA isolated from infected turnip leaves; Olszewski N et al.; Purified nuclei from turnip leaves infected by cauliflower mosaic virus (CaMV) have been shown to contain a fraction of CaMV DNA that consists of covalently closed circular molecules; possesses a nucleosome structure, based on sensitivity to micrococcal nuclease; and contains nuclear RNA polymerase II that selectively transcribes the coding strand of CaMV DNA in vitro . Our results suggest that the transcriptionally active CaMV DNA is in the form of a minichromosome and that this DNA does not contain the site-specific discontinuities characteristic of the virion.

Biokhimiia, 1982 Jun, 47(6), 999 - 1006
{Protein composition of chromatin fractions differing in their attachment to nuclear structures at low ionic strength}; Lobanenkov VV et al.; Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +70 microM beta-mercaptoethanol) and the nuclei were purified by sedimentation through 2.2 M sucrose with the same components as in TMS . Then the nuclei were resuspended and washed 3 times in TMS . After that the nuclei were resuspended to 1 mg DNA per ml in TM buffer (10 mM Tris-HCl, pH 7.6, 0.2 mM MgCl2) followed by centrifugation at low speed . About 60% of total nuclear DNP was recovered by this extraction . The protein/DNA ratio in the extracted chromatin fraction (DNPs) was about 1.1 . The bulk of the non-extracted in TM residual chromatin fraction was released from the nuclear pellet after treatment with micrococcal nuclease . This matrix-associated chromatin fraction (DNPm) is significantly enriched in non-histone proteins as compared with the DNPs; hence the protein/DNA ratio of DNPm is at least two times higher than that of DNPs . The protein components of DNPs are represented by five histones containing negligible non-histone admixture . One of them was identified as protein A24, another--as non-dissociated from DNA in 0.6 M NaCl acid-soluble protein with m . w . of about 42,500 . The possible structural features of these two distinguishable chromatin fractions are discussed.

Mutat Res, 1982 Jun, 94(2), 263 - 76
Measurement of M . luteus endonuclease-sensitive lesions by alkaline elution; Fornace AJ Jr; The UV-endonuclease approach to detect DNA damage has been combined with the alkaline elution technique with a resultant marked increase in sensitivity compared to the conventional method using alkaline sedimentation . DNA from UV-irradiated cells was digested on an inert filter with an extract from Micrococcus luteus and then analyzed by alkaline elution . Endonuclease-sensitive sites (endo-sites) were measured after doses of 0.08-0.7 Jm-2 of UV-radiation . An estimate of endo-site production with UV radiation, 0.27 endo-sites/10(8) daltons of DNA/0.1 Jm-2, was similar to that usually seen at higher doses by others . With repair incubation, approx . 50% of the endo-sites were removed in 4 h by normal human fibroblasts after 0.2 or 0.4 Jm-2; no appreciable repair was seen in xeroderma pigmentosum fibroblasts from complementation group A after 24 h of repair incubation . No photoreaction of UV damage due to 0.4 Jm-2 was detected in normal human fibroblasts . The endonuclease preparation also recognized DNA damage produced by ionizing radiation or an alkylating agent . Approx . 0.4 endo-sites/10(8) daltons of DNA were detected after a dose of 1 krad and 1 endo-site/10(8) daltons was observed after exposure of human cells to 2.5 microM MNNG for 1.3 h . The lesions detected after MNNG treatment by the endonuclease preparation decreased with post-treatment incubation--T1/2 8 h . The kinetics of removal of the endo-sites induced by MNNG were similar in normal cells and human cells of the mer- phenotype which has been shown to be more sensitive by cell killing to alkylating-agent damage . This should prove to be a useful approach to study DNA damage and repair since the entire assay can be done in several hours and a very low level of damage (1 endo-site/2 x 10(9) daltons of DNA) can be detected.

J Steroid Biochem, 1982 Jun, 16(6), 811 - 6
Concentration and preservation of nuclear androgen receptor by lyophilisation; de Larminat MA et al.; Nuclei from rat ventral prostate were disrupted by sonication and treated with micrococcal nuclease to precipitate nuclear proteins including the androgen receptor . The precipitate was dissolved in 0.6--1.2 M ammonium bicarbonate buffer, pH 7.6, with no loss of receptor when compared to the conventional Tes buffer, pH 7.0 containing 0.6--1.2 M NaCl . Lyophilisation of the solubilised protein did not produce any qualitative or quantitative differences in the recovery of receptor relative to a non-lyophilised control preparation, both of which were analysed for binding properties by Sephadex G-25/G-100 dual-column chromatography . Over longer periods of storage at -80 degrees C, the rate of inactivation of receptor was found to be 6% per week . The stability of the lyophilised receptor was improved by the inclusion of MgCl2 and SH-reducing agents in the ammonium bicarbonate buffer . Recovery improved also with increasing ionic strength of the buffer used to dissolve the lyophilised receptor.

J Virol, 1982 Jun, 42(3), 1099 - 107
Minor coat protein composition and location of the A protein in bacteriophage f1 spheroids and I-forms; Lopez J et al.; The filamentous bacteriophage f1 can be transformed into a spherical particle (spheroid) or an intermediate shortened filament with a flared end (I-forms) by exposure to a chloroform-water interface at 22 or 4 degrees C, respectively . The protein composition of bacteriophage f1 spheroids and I-forms was examined by separating the proteins from the purified . {35S}cysteine-labeled particles by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis . Quantitation of the radioactivity on the gels showed that I-forms and spheroids contain the same complement of minor coat proteins as do untreated f1 phage . This composition is unchanged after removal of the DNA, either by digestion with micrococcal nuclease or by centrifugation of the particles through CsCl density gradients, indicating that none of the minor coat proteins is held in the particles solely through an interaction with the DNA . We also examined the location of the A protein in I-forms by decoration with ferritin-conjugated antibodies and examination under the electron microscope and found that the A protein is located specifically at the flared end of the I-form particle, through which the DNA is extruded and at which contraction into spheroids begins . The implications of these results with regard to the orientation of the DNA within the capsid and the process of infection are discussed.

J Nutr, 1982 Jun, 112(6), 1203 - 11
Analysis of rat liver chromatin and nuclear proteins after nutritional variation 1,2; Castro CE et al.; Chemical composition of liver chromatin was determined for rats fed a complete stock diet, or a diet lacking protein or fat . High carbohydrate, fat-free (diet 1) and low carbohydrate, protein-free (diet 2) diets were selected because they elicit structural alteration in chromatin as measured by incubation with micrococcal nuclease (E.C . 3.1.4.7) . In the present study, either dietary treatment caused an increase in mass ratios of RNA:DNA and nonhistone:DNA, relative to control ratios . The nonhistone-DNA ratios in liver of rats fed diet 1 or diet 2 were 2.4-fold and 3.5-fold, respectively, larger than control ratios . The histone:DNA ratio remained relatively constant among all three dietary treatments . Liver nuclei were purified from rats fed each dietary treatment and were solubilized in 9 M urea . The nuclear proteins were analyzed by two-dimensional electrophoresis and visualized with a silver treatment that stains proteins in color . The electrophoretograms presented show preferentially proteins with low molecular weights and acidic pIs, two characteristics of nonhistones . The two-dimensional protein patterns are nearly identical for nuclear proteins from all three treatments . Analysis of the electrophoretograms indicates that the diet-induced increased nonhistone:DNA ratios are apparently not attributable to new species of protein, but rather to increased relative abundance of many proteins in the existing populations.

Endocrinology, 1982 Jun, 110(6), 2118 - 23
Effect of thyrotropin on the sensitivity of thyroid nuclear deoxyribonucleic acid to digestion by micrococcal nuclease; Abe Y et al.; Little is known about the mechanisms of action of polypeptide hormones on chromatin structure and nuclear function . We have employed micrococcal nuclease to examine the effect of TSH on the accessibility of DNA in thyroid nuclei . Brief digestion of nuclear suspensions with 0.05-0.2 U/ml micrococcal nuclease at 26-28 C decreased their opacity at 600 nm . The decrease in opacity was linear with increasing nuclear concentration up to 0.2 mg/ml DNA . This response to nuclease was enhanced in nuclear suspensions prepared from thyroid slices that had been incubated with TSH (50 mU/ml) for 5 h (P less than 0.001) . To determine whether TSH also increased the digestion of DNA, we measured the amount of DNA released into 1200 X g supernatants by nuclease treatment of nuclei prepared from control and TSH-treated slices . When TSH-treated nuclei (110 micrograms/100 microliters) were digested with 0.2 U micrococcal nuclease/ml at 37 C for 30 sec, a mean of 12.6 micrograms +/- 3.6 (SD) DNA appeared in the supernatant, as compared to 8.4 micrograms +/- 1.98 DNA from control nuclei (P less than 0.05) . This increase in the insensitivity of nuclear DNA to micrococcal nuclease may reflect some conformational change in chromatin in response to TSH . Since micrococcal nuclease sensitivity may reflect transcriptional competence of DNA, we speculate that polypeptide hormones may enhance the accessibility of DNA to RNA polymerase or to endogenous stimulators of transcription.

Eur J Biochem, 1982 Jun, 124(3), 427 - 33
Species specificity of promoter recognition by RNA polymerase and its transfer by the sigma factor; Ernst H et al.; RNA polymerase holoenzyme from Micrococcus luteus synthesizes in vitro a run-off transcript of 85 nucleotides from a DNA fragment containing part of gene E of bacteriophage phi X174 . This RNA starts with GTP as the 5' terminus 18 nucleotides downstream from the start of gene E on the viral (+)strand . Transcription does not occur when the fragment is cleaved 36 nucleotides upstream of the initiation site . No transcript is obtained with RNA polymerase core or holoenzyme from Escherichia coli . Other DNA fragments containing the three major E . coli promoters of phi X174 are transcribed by both enzymes although much less efficiently by M . luteus RNA polymerase . When subunit sigma in E . coli RNA polymerase is replaced by sigma from M . luteus the resulting hybrid enzyme actively transcribes the DNA fragment containing the inner region of gene D with formation of the same run-off transcript which is obtained with M . luteus holoenzyme . In the presence of sigma from E . coli this RNA is not synthesized . The hybrid enzyme also transcribes a DNA fragment containing the gene A promoter of phi X174 with even higher efficiency than RNA polymerase holoenzyme from E . coli.

Cell, 1982 Jun, 29(2), 305 - 17
Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes; Bloom KS et al.; We have examined the chromatin structure of the centromere regions of chromosomes III and XI in yeast by using cloned functional centromere DNAs (CEN3 and CEN11) as labeled probes . When chromatin from isolated nuclei is digested with micrococcal nuclease and the resulting DNA fragments separated electrophoretically and blotted to nitrocellulose filters, the centromeric nucleosomal subunits are resolved into significantly more distinct ladders than are those from the bulk of the chromatin . A discrete protected region of 220-250 bp of CEN sequence flanked by highly nuclease-sensitive sites was revealed by mapping the exact nuclease cleavage sites within the centromeric chromatin . On both sides of this protected region, highly phased and specific nuclease cutting sites exist at nucleosomal intervals (160 bp) for a total length of 12-15 nucleosomal subunits . The central protected region in the chromatin of both centromeres spans the 130 bp segment that exhibits the highest degree of sequence homology (71%) between functional CEN3 and CEN11 DNAs . This unique chromatin structure is maintained on CEN sequences introduced into yeast on autonomously replicating plasmids, but is not propagated through foreign DNA sequences flanking the inserted yeast DNA.

Biochim Biophys Acta, 1982 May 31, 697(2), 134 - 47
Fractionation of chromatin, released by nuclease digestion, on ECTHAM-cellulose . Separation of active and inactive chromatin; Smith AJ et al.; Chromatin released by two nucleases under various ionic conditions has been fractionated by chromatography on ECTHAM-cellulose . Mg2+ -soluble chromatin, which according to Gottesfeld and Partington is enriched in transcribed DNA sequences (Gottesfeld, J.M . and Partington, G.A., (1977) Cell 12, 953-962) and produced by DNAase II digestion at intermediate ionic strength, comprises material eluting from ECTHAM-cellulose at 80-100 mM Cl-, pH 6.8-7.0, whereas bulk, Mg2+ -insoluble chromatin comprises more tightly binding material . Free hnRNP particles elute at 30 mM Cl-, pH 6.8 . Oligonucleosomes, which according to Dimitriadis and Tata are enriched in transcribed sequences (Dimitriadis, G.J . and Tata, J.R . (1980) Biochem . J . 187, 467-477) and produced by micrococcal nuclease digestion at physiological ionic strength, also elute predominantly at 80-100 mM Cl-, pH 6.8-7.0 . When liver nuclei are digested with micrococcal nuclease at low ionic strength, the most rapidly released chromatin is enriched in nascent RNA and hnRNP particles, and binds weakly to ECTHAM-cellulose . More slowly solubilised chromatin, containing fewer hnRNP particles, binds much more strongly to ECTHAM-cellulose . In confirmation of results with mechanically sheared chromatin, the affinity of particular chromatin fractions is not dependent on the size of chromatin particles, rather it reflects the differing composition, and in particular the non-histone protein and hnRNP content, which, we propose, determines the conformation adopted by different chromatin fractions in the cation conditions used for elution from ECTHAM-cellulose.

Biochim Biophys Acta, 1982 May 31, 697(2), 178 - 84
A sequence specific endonuclease from Micrococcus radiodurans; Wani AA et al.; A new sequence specific endonuclease, Mra I has been purified from Micrococcus radiodurans . This enzyme cleaves bacteriophage lambda DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on phi X174 DNA . It has no sites on SV40, PM2 and pBR322 DNA . The three sites on phage lambda DNA are different from those cleaved by Sma I, Xma I and Xor II . The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of lambda DNA . Mra I is shown to be an isoschizomer of Sac II and Sst II recognizing the palindromic nucleotide sequence '5-CCGC reduced GG-3' . The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol . The enzyme shows activity at a broad range of temperature and pH with an optimum at 45 degrees C and pH 7.0 . Mra I represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.

J Exp Zool, 1982 May 20, 221(1), 61 - 79
Transmission and scanning electron microscopic studies of the human sperm chromatin decondensed by micrococcal nuclease and salt; Sobhon P et al.; The human sperm chromatin was gently decondensed by treating the sperm head sequentially with micrococcal nuclease and 2 M NaCl . All histones, about 10% of DNA, and a small amount of degraded protamines were released into the soluble fraction, leaving mainly nucleoprotamines in the pellet fraction . Transmission and scanning electron microscopic studies revealed that the nucleoprotamine pellet consisted of chromatin cords of two dimensions, viz., 330- to 420-A and 650- to 1200-A thick cords laced together by very fine strands of 60- to 80-A fibers; both types of cord appeared knobby and had zig-zag patterns throughout their length . It appeared that these cords were derived from two types of sperm heads of approximately equal population; one type contained chiefly the thick cords and the other chiefly the thin cords . Further treatment of the pellet nuclease-NaCl with urea and mercaptoethanol resulted in the dissociation of the thick into the thin cords and unravelling of the thin cords into smaller sized fibers; whereas the treatment of the pellet nuclease-NaCl with DNAase I resulted in the disappearance of the 60- to 80-A fibers, and the remaining cords were chiefly of thick type together with the sperm head exoskeletons . From these results the packing order of the chromatin in human sperm was proposed.

Biochemistry, 1982 May 11, 21(10), 2349 - 56
On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus; Marlier JF et al.; The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus . The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP . The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A) . Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident . During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation . Thus there are two stereochemically distinct subsites for initiating polymerization . ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete . The average chain length of the polymeric product is considerably smaller for ADP alpha S vs . ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.

Biochem Pharmacol, 1982 May 1, 31(9), 1671 - 9
Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites; Green D et al.; All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity . A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, GANU) of glucose . Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia . To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated . With the use of the endonuclease micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined . Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM {14C}chloroethyl drug . The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells . This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin {L . C . Panasci, D . Green and P . S . Schein, J . clin . Invest . 64, 1103 (1979)} . In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU . Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU . The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.

Mutat Res, 1982 May, 94(1), 213 - 28
Enhanced radiosensitivity and defective DNA repair in cultured fibroblasts derived from Rothmund Thomson syndrome patients; Smith PJ et al.; Rothmund Thomson syndrome (RTS) is an oculocutaneous and cancer-prone disorder in which enhanced carcinogen sensitivity, mediated through abnormal DNa metabolism, may be an associated factor . Cultured fibroblasts from 5 RTS patients have been examined for their colony-forming abilities and DNa repair capacities following gamma-irradiation . 2 of the 4 RTS strains showed enhanced sensitivity following hypoxic gamma-irradiation, and 1 of these 2 strains also showed enhanced sensitivity under toxic conditions . Defective DNA repair was implicated in the above abnormal responses to gamma-radiation since both strains displayed reduced levels of repair synthesis and slow removal of radiogenic DNA lesions (assayed by their sensitivity to strand-incising activities present in protein extracts of Micrococcus luteus cells) . A hypothesis is presented to rationalize the origin and heterogeneity of these laboratory phenotypes of RTS.

Mol Biol (Mosk), 1982 May-Jun, 16(3), 541 - 50
{Splitting of mononucleosomes into histone-containing subnucleosomes}; Domanskii NN et al.; Histone-containing subnucleosomal particles SN7 and SN4 revealed initially in micrococcal nuclease digest of mouse chromatin were studied . It was found that production of SN7 and SN4 did not depend on the preservation of supranucleosomal levels of chromatin organization and was the result of intranucleosomal splitting . Micrococcal nuclease that preferentially attacks internucleosomal linker DNA can cut mononucleosomes with the formation of 40 b . p . DNA fragment complexed with H2a and H2b and SN7 particle containing six histones (H2a, H2b, 2H3 and 2H4) and 108 b . p . DNA . It is proposed that such a splitting of nucleosomes reflects their intimate organization, particularly a histone-DNA interaction within core particles . These results are interpreted in terms of a nucleosomal model in which H2a-H2b pairs are localized in the peripheral parts of the nucleosomal DNA superhelix.

J Gen Virol, 1982 May, 60(Pt 1), 77 - 85
Cell-free coupling of Newcastle disease virus RNA transcription, translation and Co-translational processing; Content J et al.; A cell-free coupled system for transcription, translation and glycoprotein processing of the Newcastle disease virus genome is described . The system consists of a rabbit reticulocyte lysate preincubated with micrococcal nuclease and of detergent-disrupted purified Newcastle disease virions . {35S}methionine incorporation was linear for 2 h . Polypeptides NP and M, the presumably unglycosylated analogues of glycoproteins HN and possibly F, were identified as translation products . When in vitro synthesis was carried out in the presence of dog pancreas microsomes the HN analogue (pre-HN) was converted to an 80K (approx.) protein which comigrated on polyacrylamide gels with HN synthesized in vivo and which, except for a small fragment, was protected from proteolytic degradation . In immunoprecipitation studies, antiserum against HN purified from virions reacted with both the processed and the unprocessed form of HN synthesized in vitro.

Biull Eksp Biol Med, 1982 May, 93(5), 60 - 3
{Proteins and the functional properties of the chromatin fractions from the nuclei of the normal liver and of experimental hepatomas}; Lobanenkov VV et al.; Chromatin fragments generated in normal liver and solid hepatoma nuclei due to the action of endogenous nucleases and in ascites hepatomas nuclei treated with micrococcal nuclease differ in the ability to be released from the nuclei into a medium of low ionic strength . It is suggested that such a fractionation is based on different solubility of DNP fragments attached to the nuclear skeleton and of those that are not bound with it . DNP fragments extracted in a low-salt buffer contain all five histones with a negligible admixture of nonhistone proteins having the protein/DNA ratio about 1.1 . No endogenous RNA-polymerase activity could be detected in these DNP fragments . The bulk of the RNA-polymerase activity is found in the matrix-associated DNP fragments that appear to be enriched in nonhistone proteins (their protein/DNA ratio amounted to 2.5) . The possibility that transcribable DNP fragments are associated with the matrix through low-salt-stable linkages like those in the DNA-(RNA-polymerase-RNA or RNP)-matrix seems to be confirmed by the data obtained.

J Bacteriol, 1982 May, 150(2), 649 - 56
Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus; Traxler CI et al.; A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of {14C}glucose from UDP-{14C}glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA) . Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate . The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin . Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin . Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan . There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA . In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.

Proc Natl Acad Sci U S A, 1982 May, 79(10), 3143 - 7
Assembly of new histones into nucleosomes and their distribution in replicating chromatin; Russev G et al.; We studied the assembly of new histones into nucleosomes and their distribution in replicating chromatin in growing P815 mouse cells . New histones and new DNA were density-labeled with 13C, 15N, 2H-substituted amino acids together with {3H}arginine or with 5-iododeoxyuridine and {3H}thymidine, respectively, for 1 hr (approximately 20% of S phase) . Mono- di-, tri-, tetra- and larger oligonucleosomes were isolated by sucrose gradient centrifugation of micrococcal nuclease-digested chromatin, and their density distribution was analyzed, without fixation, in metrizamide/triethanolamine density gradients {Russev, G . and Tsanev, R . (1976) Nucleic Acids Res . 3, 697-707} in which mono- and oligonucleosomes containing dense amino acids or 5-iododeoxyuridine separate from the corresponding normal nucleosomes . Under these conditions, approximately 74% of the new histones are found in nucleosomes on newly replicated DNA, and the remainder are on unreplicated DNA . The majority of new histones form entirely new nucleosomes; a minor fraction may form hybrid nucleosomes that also contain preexisting histones . New nucleosomes are distributed to both new daughter DNA molecules with approximately equal probability, and our evidence suggests, but does not prove, that they are distributed in a random manner along new DNA.

Appl Environ Microbiol, 1982 May, 43(5), 1041 - 50
Antimicrobial characteristic of insoluble alkylpyridinium iodide; Nakagawa Y et al.; Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized . The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers . The insoluble agent {C12(50)} that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent . C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol . This washing became ineffective after a few cycles of antibacterial treatment and refreshment . Such C12(50) recovered the activity upon 1.0 N NaOH treatment . The antibacterial activity of C12(50) depended on its surface area . It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts . But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes . The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions . The activity of C12(50) was higher at a higher temperature or at a lower cell concentration . The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer . C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave . The mode of action of the laurylpyridinium iodide solution against E . coli and M . luteus was similar to that of C12(50) except for the influence of E . coli cell concentration.

J Cell Biol, 1982 May, 93(2), 278 - 84
Analysis of DNA attached to the chromosome scaffold; Kuo MT; Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold . The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp . Quant . Biol . 42:351--360) . Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation . Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl . Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA . Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences . The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.

Hautarzt, 1982 May, 33(5), 257 - 65
{Clinico-experimental studies on the effect of benzoylperoxide}; Puschmann M; Clinical and experimental investigations to characterize therapeutic effects of topically applied benzoyl peroxide (5 and 10% in alcohol-free gel formulation) were performed with: quantitative determination of bacteria in the follicular filaments with the cyanoacrylate technique (P . acnes and micrococcaceae); agar diffusion method for bacteriostatic effects; semi-quantitative determination of skin surface lipids (ground glass method); lipid solvent and thin-layer chromatography (free fatty acids vs . triglycerides); scanning electron microscopy of the skin surface; exfoliative cytology with corneocyte counts in a Fuchs-Rosenthal chamber (corneocytes/cm2 skin surface); determination of corneocyte surface are in micrometer2; and a clinical trial concerning efficacy and tolerance of the gel formulation . Topically applied benzoyl peroxide acts antibacterially and keratolytically, has anti-lipolytic activity, reduces bacteria in the follicular infundibula, but does not inhibit sebum production as measured by skin surface lipids . Benzoyl peroxide stimulates the epidermopoiesis with reduction of corneocytes/cm2 from 87,400 +/- 29,000 to 36,000 +/- 19,000 (day 15 of treatment) with diminution in size of corneocytes from 1,018 micrometers2 +/- 74 to 865 micrometers2 +/- 65 vs . 832 micrometers2 +/- 85 (5% vs . 10% benzoyl peroxide) . Alcohol-free gels of benzoyl peroxide are better tolerated by acne patients than those containing alcohol, in particular when combined with topical tretinoin (vitamin A acid) treatment.

Chem Biol Interact, 1982 May, 40(1), 85 - 96
Effects of 1,1'-hexamethylene-bis-{(5-p-chlorophenyl)-biguanide} on the genome and on the synthesis of nucleic acids and proteins in the bacterial cells; Ackermann-Schmidt B et al.; The strongly effective bactericidal compound 1,1'-hexamethylene-bis-{(5-p-chlorophenyl)-biguanide} (HCG), which is used as a disinfectant alterates the DNA of B . subtilis as shown in the rec assay, induced auxotrophic mutants in E . coli B and causes prophage induction in Micrococcus lysodeikticus 53-40 (N5) . In vivo experiments with E . coli B have demonstrated that HCG extensively breaks down bacterial DNA and interacts with the synthesis of cellular DNA to the similar extent as found for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The structural integrity of ribosomes and of ribosomal subunits remains intact in the presence of HCG.

Biochim Biophys Acta, 1982 Apr 26, 697(1), 60 - 70
The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction; Kitzis A et al.; It was shown with the use of specific probes that mild micrococcal nuclease digestion released from chromatin actively-transcribed genes as small nucleosome oligomers . In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease . It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA . The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A+) RNAs, however larger released fragments include some transcribed sequences, while the nuclease resistant chromatin is considerably impoverished in coding sites . These evidences are the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease . We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5-2% of the total nuclear DNA, formed of single-stranded DNA . In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes . Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.

J Biol Chem, 1982 Apr 25, 257(8), 4655 - 60
The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin; Saffer JD et al.; The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells . HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells . Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated . However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells . Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively . The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I . 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA . Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.

J Biol Chem, 1982 Apr 10, 257(7), 3477 - 83
Uracil-DNA glycosylase . Purification and properties of uracil-DNA glycosylase from Micrococcus luteus; Leblanc JP et al.; A uracil-DNA-glycosylase from Micrococcus luteus has been purified more than 3,000-fold . The enzyme preparation appears homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It is devoid of nonspecific endonucleases, specific endonucleases for apurinic and apyrimidinic sites, 3-methyladenine or 7-methylguanine-DNA-glycosylases . It behaves as a monomer protein of 19,400 daltons . It has an isoelectric point of 7.0 +/- 0.1 . It has an optimal activity between pH 5.0 and 7.0 . It has no cofactor requirement and is not inhibited by EDTA . Uracil-DNA-glycosylase is highly specific for DNA containing dUMP residues, releasing uracil as product of the reaction . It is 2-fold more active on single-stranded DNA than on double-stranded DNA . If it releases uracil dimers from ultraviolet-irradiated PBS1 DNA, it is at the threshold of the detection . The apparent Km is 7 X 10(-8) M, and uracil acts as a noncompetitive inhibitor with a Ki of 3.2 X 10(-4) M . Cis-syn cyclogbutadiuracil also is a potent inhibitor, while some analogs, produced by x-irradiation of uracil and thymine, are weak inhibitors . Spermine, between 10 and 400 microM, increases the enzymatic activity by 50% and is not inhibitory at other concentrations . Spermidine activates the enzyme at concentrations of 40 to 120 microM, but becomes inhibitory at 200 and 400 microM . A new finding is that drugs which intercalate in DNA, such as ethidium bromide and ellipticine, cause a 2- to 2.5-fold activation of this enzyme activity . The concentrations giving maximal activation depend on the drug . The enzyme does not behave as a processive enzyme during uracil excision.

Nucleic Acids Res, 1982 Apr 10, 10(7), 2275 - 93
Nucleosome spacing in rat liver chromatin . A study with exonuclease III; Strauss F et al.; Exonuclease III was used to uniformly trim DNA ends of micrococcal nuclease-prepared chromatin fragments down to the first major impediment encountered by the enzyme, which arises from the interaction of H1 with the nucleosome . This trimming, when performed on nucleosome dimers, allowed one to quantitatively determine the center-to-center distance of nucleosomes . This distance, of mean 198 base pairs, was found to essentially vary between about 180 and 215 base pairs, with extremes of 165 and 230 base pairs . Trimming of trimers further revealed that the overall arrangement of nucleosome center-to-center distances along the chromatin fiber is that expected on a statistical basis.

Nucleic Acids Res, 1982 Apr 10, 10(7), 2323 - 35
Nucleosomes containing histones H1 or H5 are closely interspersed in chromatin; Torres-Martinez S et al.; The distribution of histones H1 and H5 along chromatin fibers has been examined in the nucleated hen erythrocyte . Nucleosome oligomers, produced by micrococcal nuclease digestion of nuclei, were sequentially reacted with affinity-chromatography purified rabbit anti-H5 and sheep anti-rabbit antibodies . Quantitation of the relative amounts of H1 and H5 in the precipitated and supernatant fractions as a function of the oligomer number was consistent with a close interspersion of both types of histones, probably a random one . This conclusion was supported by the immunoprecipitation of longer chromatin fibers . This pattern of distribution appears to apply both to bulk chromatin and to chromatin inactivated during the maturation of the erythrocyte.

J Steroid Biochem, 1982 Apr, 16(4), 495 - 501
Regulation of the peptide elongation reaction on uterine ribosomes by estrogens; Whelly SM et al.; Administration of 17 beta-estradiol to ovariectomized mature rats for 1 h induces an increased capacity of subsequently isolated uterine ribosomes to synthesize protein in a cell-free protein synthesis system . The increased rate of protein synthesis can be ascribed to an effect of estrogen on the rate of peptide elongation rather than synthesis of additional new peptides . The increased rate of peptide elongation is dependent upon the dose of estradiol over the range of 0.1 to 10 micrograms/animal, and exhibits hormone specificity; 17 beta-estradiol, diethylstilbesterol, estrone and estriol but not 17 alpha-estradiol, progesterone, dihydrotestosterone or corticosterone will induce the response . Removal of ribosome associated proteins by extraction with 0.5 M KCl results in activation of protein synthesis by uterine ribosomes from control rats to rates that are equal to that of ribosomes from estrogen-stimulated rats suggesting that ribosomes from control animals are in an inhibited state . The KCI extracted ribosomal factors from control animals inhibit the synthesis of protein by salt-washed uterine ribosomes when added back to the ribosomes prior to assay and the inhibitory properties of these factors are greater if derived from ribosomes of control rather than 1 h estradiol-treated rats . The extracted inhibitor is inactivated by heat, is insensitive to treatment with N-ethylmaleimide, is insensitive to micrococcal nuclease and is reversible . The early activation of uterine ribosomes by estrogen appears to result from either the removal or inactivation of a ribosome associated-peptide elongation reaction, inhibitory factor.

Mol Cell Endocrinol, 1982 Apr, 26(1-2), 201 - 16
The intranuclear distribution of rat uterine estrogen receptors determined after nuclease treatment and chromatin fractionation; Pavlik EJ et al.; The intranuclear locations at which rat uterine estrogen receptors interact with chromatin have been probed using digestions performed with DNAase I and micrococcal nuclease . Exposure to nuclease has been controlled to effect limited to extensive digestion of nuclear DNA under conditions which maintain the integrity of the {3H} estradiol-receptor complex . The effect of divalent cation concentration on the release of estrogen receptors fron nuclease-treated chromatin was examined and found to be of consequence above 2 mM . Exposure to nuclease released nuclear estrogen receptors from chromatin, with DNAase I being more efficient than micrococcal nuclease in mediating this release . The release of the bulk of nuclear estrogen receptors closely paralleled the nuclease-mediated digestion of chromatin DNA . At 1 h after exposure to estrogen, substantial quantities of uterine estrogen receptors (80-90%) were distributed in chromatin fractions which, on the basis of fractionation terminology, have been termed 'transcriptionally inactive' by convention . Enrichment of estrogen receptors in chromatin which has been termed ' transcriptionally active' only occurred with 10-20% of the estrogen receptors . Hence, our findings support a model where, at early times after estrogen exposure, receptors from the rat uterus are enriched to only a minor extent in chromatin to which 'transcriptional activity' is generally assigned while the bulk of receptors are localized in chromatin which is generally considered 'transcriptionally inactive'.

J Biochem (Tokyo), 1982 Apr, 91(4), 1411 - 7
Affinities of various nucleases to DNA-Sepharose under non-digestive conditions: survey for productive affinity chromatography; Tanaka H et al.; 1 . It has been reported that DNase I can be highly purified from pancreas extract by affinity chromatography on a dDNA-Sepharose column under non-digestive conditions . In the present study, the adsorption-elution of other nucleases on the column under non-digestive conditions was studied . 2 . All the seven kinds of nucleases tested were adsorbed when applied on a dDNA-Sepharose column under conditions which did not allow the enzymes to hydrolyze the DNA . The non-digestive conditions were as follows . i) For DNase II (pI=10.2), pH 3.0 in the presence of 50 mM sodium sulfate (inhibitor), ii) for micrococcal nuclease (pI=9.6), pH 4.0 in the absence of Ca2+ (activator), iii) for restriction endonucleases Eco RI (pI=5+1), Hind III (pI=5+1), and Bam HI (pI=5+1), pH 4.0 in the presence of 20% glycerol and 0.1% Neopeptone (stabilizers), and iv) for nucleases S1 (pI=5+1) and nuclease P1 (pI=4.5), pH 7.0 . At the respective pH's, the enzymes other than nucleases S1 and P1 were cationic so as to exhibit electrostatic attraction to the anionic dDNA-Sepharose . Although S1 and P1 were anionic, they still adsorbed to the column . 3 . All the adsorbed nucleases described above were eluted by a concentration gradient of KCl without changing pH . The ionic strengths required for elution were 0.19 for DNase II, 0.53 for micrococcal nuclease, 0.73 for Eco RI, 0.72 for Hind III, 0.37 for Bam HI, 0.17 for P1, and 0.13 for S1 . The fact that the ionic strength required for the elution of DNase I (pI=5.0) was 0.39 at pH 4.0 indicates that the former five enzymes except DNase II can be chromatographed with almost the same or higher efficiency than DNase I, because the proteins adsorbed with no-specific affinity could be mostly eluted at lower ionic strength . On the other hand, the fact that nucleases P1 and S1 were adsorbed in spite of electrostatic repulsion suggests that these two enzymes can also be effectively chromatographed, especially when other cationic proteins are previously removed by an appropriate method such as adsorption to a typical cation exchanger.

Cell Differ, 1982 Apr, 11(2), 91 - 8
Changes in endonuclease activity and in chromatin structure of rat hepatocytes during fetal and neonatal life; Rossi R et al.; A developmental study of rat hepatic endonuclease has been performed . Nuclei, from different stages of hepatocyte maturation, were analyzed for endogenous endonuclease activity . The chromatin extracted from these nuclei does not show any fragmentation during the first 17 days of fetal development . On the 18th day of fetal life there is a massive increase in specific endonuclease activity . At birth this activity reaches a maximum level (3.5 units/mg DNA); thereafter it undergoes a gradual decrease . The size of the basic DNA repeats produced by the endonuclease action is 218.9 +/- 1.6 in 18-day-old fetuses and decreases to 204.9 +/- 2.5 in 19-day-old fetuses, a value which remains constant in the following fetal and postnatal life . This difference in monomer size is due to changes in the chromatin structure . Micrococcal nuclease digests show that the "nucleosome core" does not change during hepatocyte development . Therefore, the difference in size of the endonuclease DNA fragments must be due to the linker regions.

Biochim Biophys Acta, 1982 Mar 29, 696(3), 267 - 74
Effect of histone composition on the stability of chromatin structure; Puigdomenech P et al.; The mode of fragmentation of chromatin by micrococcal nuclease has been studied in nuclei from different sources at physiological ionic strength and low temperature . During digestion, the size of chromatin was reduced until an average S value of 95-100 (hen erythrocyte) or 60-65 (rat liver) was attained . The accumulation of these structures correlated with the period of maximum solubility (80%), indicating that the bulk of chromatin behaved in this manner . Further digestion did not result in a corresponding decrease in S value but in a bimodal sedimentation pattern . As opposed to this behavior, chromatin containing actively acetylated core histones showed a continuous variation in size during the digestion . Indirect immunoprecipitation of chromatin by anti-H5 antibody and sheep anti-rabbit antibody revealed that the acetylated chromatin is partially depleted of H5.

Nucleic Acids Res, 1982 Mar 11, 10(5), 1481 - 94
Analysis of highly purified satellite DNA containing chromatin from the mouse; Zhang XY et al.; A purification scheme for satellite DNA containing chromatin from mouse liver has been developed . It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases . Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions . Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively . In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA . This material has the same histone composition as bulk chromatin . No significant differences were detected in an analysis of minor histone variants . Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.

Mol Cell Biochem, 1982 Mar 5, 43(1), 27 - 34
Partial characterization of membrane-associated proteinases from Micrococcus lysodeikticus; Rivas L et al.; We have identified cytoplasmic and membrane-associated proteinases from Micrococcus lysodeikticus (M . luteus) by the use of 125I-labeled casein and insulin as substrates . The membrane-associated activities were released by shock washing . Proteolytic activities showed pH optima at slightly alkaline values and we have concentrated on the activities at pH 8.0 . The total units of both proteolytic activities were higher in the cytoplasmic than in any other fractions but the situation was different when the results were expressed in terms of specific activity . The activities against casein and insulin were differentiated by the action of inhibitors, divalent metal ions, Arrhenius plots and dependence on ionic strength . On these grounds, it is proposed that the membrane-associated enzyme acting on insulin is a single thiol proteinase while the proteolysis of casein reflects the action of, at least, two enzymes (thiol proteinase and serine proteinase) . The distinction between the casein and insulin degrading activities was confirmed by crossed-inhibition experiments and by their behaviour on gel chromatography and concentration-dependence experiments . The aggregating properties have hampered the purification of the enzymes . The present results raise doubts about the significance of preventing membrane damage and degradation of membrane proteins by the addition of indiscriminated proteinase inhibitors during membrane isolation and manipulation.

Biochemistry, 1982 Mar 2, 21(5), 985 - 92
Higher order folding of two different classes of chromatin isolated from chicken erythrocyte nuclei . A light scattering study; Fulmer AW et al.; Bulk chromatin fragments were excised from chicken erythrocyte nuclei by digestion with micrococcal nuclease . Fractionation into S chromatin (soluble at physiological ionic strengths) and I chromatin (insoluble at physiological ionic strengths) was achieved by dialysis against buffers containing 0.15 M NaCl . The effects of NaCl concentration on the molecular dimensions of S and I chromatins were determined by dynamic and static light scattering . Series of fragment lengths were obtained by gel filtration of S and I chromatins under ionic conditions which lead to maximal intramolecular compaction . Hydrodynamic radii and radii of gyration were determined for fragment lengths ranging from 8 to 53 nucleosomes . These data are in excellent agreement with calculations for extended helical structures . Close-packed solenoidal or superbead structures are not compatible with these data . Comparisons of molecular dimensions derived from light scattering and electron microscopy indicate that considerable shrinkage of chromatin fragments can occur when common methods of sample preparation are used for microscopy.

J Virol, 1982 Mar, 41(3), 1044 - 54
Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure; Ben-Asher E et al.; Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer . Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S . In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin . They were found to be composed of replicative forms of MVM DNA and cellular histones . After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified . These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively . We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.

J Biochem (Tokyo), 1982 Mar, 91(3), 967 - 73
Studies on histone oligomers . IV . Reassociation of chromatin from histones of various conformations; Kawashima S et al.; Chicken erythrocyte chromatin, whole or (H1,H5)-depleted, was dissociated in 2 M NaCl in the presence or absence of 5 M urea at pH 8 or pH 5, and reconstituted by decreasing the concentration of NaCl and urea . The reassociated products were characterized by their solubilities in 0.1 mM EDTA, micrococcal nuclease digestion, cross-linking of histones, and fractionation of histone oligomers by solubility in ammonium sulfate solution . In the absence of urea, the nucleosome structure and the histone octamer were reconstituted perfectly at both pH 5 and pH 8 . When chromatin was exposed to urea, no nucleosome structure or histone octamer was obtained at pH 5 either decreasing the concentration of salt first or that of urea first . At pH 8, the chromatin structure was regained fairly well by decreasing the concentration of urea first, but only partially by decreasing the concentration of salt first . Solubility in 0.1 mM EDTA was found to be a good criterion for monitoring the proper reassociation of chromatin.

Can J Biochem, 1982 Mar, 60(3), 379 - 88
Structural studies on yeast nucleosomes; Lee KP et al.; Mononucleosomes isolated from micrococcal nuclease digests of stationary phase chromatin of the yeast Saccharomyces cerevisiae were compared both compositionally and physiochemically with those from chicken and bovine calf . It was found that while yeast mononucleosomes are similar in composition, their thermal denaturation profiles and circular dichroism spectra indicate a less constrained structure . Furthermore, yeast nucleosomes were discovered to be labile in solutions of low ionic strength and could not be reconstituted by methods applicable to calf and chicken nucleosomes . On the basis of the reconstitution of a hybrid nucleosome containing calf histones H2A, H2B, and H3 and yeast histone H4, it was concluded that variations in the yeast H4 sequence are unlikely to be responsible for the apparent decrease in the stability of yeast nucleosomes . Examinations of histone-histone interactions in free solution revealed a change in the H3-H4 interaction and together with the previously published results of other researchers it was inferred that changes in the H3 sequence might be responsible for this structural variation.

Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1839 - 43
Hemin-independent control of globin synthesis in Friend erythroleukemia cells induced to differentiate; Stringer EA et al.; A hemin-independent translational inhibitor that prevents synthesis of rabbit globin when uninduced Friend leukemia (FL) cell and rabbit reticulocyte lysates are mixed {Cimadevilla, J . M . & Hardesty, B . (1975) Biochem . Biophys . Res . Commun . 63, 931-937} cannot be detected in FL cells induced to differentiate . Mixing of lysates of FL cells induced with hexamethylene bisacetamide or aminonucleoside of puromycin and rabbit reticulocytes does not cause inhibition of rabbit globin synthesis . Induction also results in the cells acquiring sensitivity to the inhibitor from uninduced FL cells . A reduction in total protein synthesis is observed when uninduced and induced FL cell lysates are mixed . Inhibition does not result from competition by an excess of uninduced FL cell mRNA species for the translational machinery because uninduced FL cell lysates retain their inhibitory activity after treatment with micrococcal nuclease . Rabbit globin mRNA recovered from rabbit reticulocyte lysates that have been incubated with lysates of uninduced FL cells can still be translated effectively, indicating that inhibition does not result from modification of other species of mRNA by uninduced FL cell lysates . A switch to hemin-dependent translational control does not follow induction of differentiation . The rate of amino acid incorporation in induced FL cell lysates--like that in uninduced FL cell lysates--is unaffected by omission of exogenous hemin from the system . Its presence is not required to prevent activation of heme-regulated inhibitor . From these data, it is clear that the control of protein synthesis in FL cells--whether or not they are induced--is different from that regulated by hemin in normal erythroid cells.

Proc Natl Acad Sci U S A, 1982 Mar, 79(6), 1771 - 5
Effect of the B--Z transition in poly(dG-m5dC) . poly(dG-m5dC) on nucleosome formation; Nickol J et al.; We have studied the properties of complexes formed between histones and the methylated synthetic polydeoxynucleotide poly(dG-m5dC) . poly(dG-m5dC) . This polymer undergoes the transition from B DNA to left-handed Z DNA at moderate ionic strength . When the polymer is in the Z form it will bind histones, but nucleosomes are not detected . When the polymer in the B form is combined with equimolar quantities of the four core histones and digested with micrococcal nuclease, particles are formed which behave in all respects as normal nucleosome cores . When these core particles are placed in solvents that would result in conversion of the protein-free polymer to the Z form, no transition is observed . The formation of a nucleosome core particle thus stabilizes the B form, whereas the presence of the Z form prevents nucleosome formation . The results suggest that if Z DNA is present in eukaryotic nuclei, it will serve to disrupt the normal chromatin structure.

J Virol, 1982 Mar, 41(3), 781 - 91
Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity; Centrella M et al.; Infection of mouse L-cell spinner cultures by vesicular stomatitis virus (VSV) effected the selective translation of viral mRNA by 4h after viral adsorption . Cell-free systems prepared from mock- and VSV-infected cells reflected this phenomenon; protein synthesis was reduced in the virus-infected cell lysate by approximately 75% compared with the mock-infected (control) lysate . This effect appeared to be specific to protein synthesis initiation since (i) methionine incorporation into protein from an exogenous preparation of initiator methionyl-tRNA gave completely analogous results and (ii) the addition of a ribosomal salt wash (containing protein synthesis initiation factors) stimulated protein synthesis by the infected cell lysate but had no effect on protein synthesis by the control . Micrococcal nuclease-treated (initiation-dependent) VSV-infected cell lysates were not able to translate L-cell mRNA unless they were supplemented with a ribosomal salt wash; a salt wash from ribosomes from uninfected cells effected a quicker recovery than a salt wash from ribosomes from infected cells . When salt wash preparations from ribosomes from uninfected and infected cells were tested for initiation factor 2 (eIF-2)-dependent ternary complex capacity with added GTP and initiator methionyl-tRNA, we found that the two preparations contained equivalent levels of eIF-2 . However, initiation complex formation by the factor from virus-infected cells proceeded at a reduced initial rate compared with the control . When the lysates were supplemented with a partially purified eIF-2 preparation, recovery of activity by the infected cell lysate was observed . Mechanisms by which downward regulation of eIF-2 activity might direct the selective translation of viral mRNA in VSV-infected cells are proposed.

Mol Biol (Mosk), 1982 Mar-Apr, 16(2), 392 - 7
{Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion}; Georgieva E et al.; Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker . Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined . The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA . None of the fractions displayed the properties characteristic of active chromatin.

J Biochem (Tokyo), 1982 Feb, 91(2), 571 - 87
Chemical and immunological properties and amino acid sequences of three lysozymes from Peking-duck egg white; Kondo K et al.; Three lysozymes (DLs-1, -2, and -3) were purified from Peking-duck egg white by adsorption on CM-Sephadex C-25 resin, followed by CM-Sephadex C-25 and Sephadex G-50 column chromatographies . The three enzymes each moved as a single band, but showed different electrophoretic mobilities, on disc-polyacrylamide gel electrophoresis at pH 4.1 . Final yields of DL-1, DL-2, DL-3 were 18.2%, 22.0%, and 6.0%, respectively, from the crude material adsorbed on CM-Sephadex resin . The enzymatic activities of DL-1, DL-2, and DL-3 were 1.53, 1.52, and 1.34 times that of hen egg white lysozyme, respectively, using Micrococcus lysodeikticus cell wall as a substrate at pH 6.2 and 37 degrees C . All the DLs lacked histidine and their amino acid compositions differed from each other by a few amino acid exchanges . The amino acid sequences of DL-2 and DL-3 differed from that of DL-1 by two displacements (Ser-37 to Gly and Gly-71 to Arg) and three displacements (Pro-79 to Arg in addition to the same substitutions), respectively . In comparison with Duck II and Duck III lysozymes from Kaki-duck (Hermann and Jolles (1970) Biochim . Biophys . Acta 200, 178-179; Hermann et al . (1971) Eur . J . Biochem . 24, 12-17) as regards amino acid sequences, Duck II is identical to DL-1 except for one displacement of Gln-57 in DL-1 to Glu in Duck II, while Duck III is rather different from our three lysozymes . All DLs gave single precipitin lines with any rabbit antisera against DLs and each precipitin line fused completely with that of any of the DLs in Ouchterlony double diffusion tests . However, the radiobinding inhibition assay showed that some anti-DL antisera clearly discriminated fine differences among the three DLs . The displacement at residue 79 (Pro in equilibrium Arg) gave the clearest immunological difference among the three kinds of displacements found in DLs.

Eur J Biochem, 1982 Feb, 122(1), 199 - 203
Preparation of a mRNA-dependent cell-free translation system from whole cells of Saccharomyces cerevisiae; Hofbauer R et al.; A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by mechanical breakage of cells, isolation of a 30000 x g supernatant fraction and removal of endogenous mRNA by treatment with micrococcal nuclease . The system thus isolated is dependent on added mRNA and translates yeast mRNA to discrete products, many of then identical with yeast proteins synthesized in vivo . Activity and properties of this system are comparable to those of other eukaryotic cell-free translation systems . It offers the following advantages, compared to yeast translation systems described previously . (a) Its isolation is simple and fast . (b) Since it is not isolated from spheroplasts there is no danger of its inactivation by contaminants in enzymes used for spheroplast preparation . (c) Isolation appears to be less strain-dependent and can be carried out starting from cells in various physiological states.

Chem Biol Interact, 1982 Feb, 38(3), 261 - 74
Excision of benzo{a}pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts; Kaneko M et al.; The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF) . For this purpose NF were prelabeled in their DNA with {14C}-thymidine and treated with {3H}BPDE I . The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo{a}pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine . The adduct composition did not change significantly in 24-h post treatment incubation . Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis . The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p . chromatosomal DNA and in total nuclear DNA . From these data the concentration in nucleosomal linker-DNA was calculated . The initial adduct distribution was non-random and 6.3 times higher in 47 b.p . linker-DNA relative to 145 b.p . core-DNA and 9.2 times higher in 27 b.p . linker-DNA relative to 165 b.p . chromatosomal DNA . Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA . After this early phase the adducts located in 145 b.p . core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period . In contrast, further adduct removal was observed from nucleosomal linker-DNA.

Biokhimiia, 1982 Feb, 47(2), 296 - 304
{Existence of boundary lipids in reconstituted hydrophobic protein-lipid system}; Dergunov AD et al.; A fraction of hydrophobic proteins (9, 14, 18 kD) soluble in a chloroform-methanol mixture (2:1) has been isolated from Micrococcus lysodeikticus bacterial membranes . The proteins obtained were introduced into proteoliposomes at a protein/lipid weight ratio ranging from 0.1 to 0.25 in combination with the fluorescent probe pyrene or the spin probe 2-(14-carboxytetradecyl)-2-ethyl-4.4-dimethyl-3-oxasolidinyloxyl . The excimertization of pyrene upon direct excitation of its molecules (gamma excit.=338nm) and under conditions of energy transfer from the excited protein chromophores to pyrene (gamma excit.=286nm) and the spin-spin exchange between the spin probe molecules was investigated . The experimental results suggest that the hydrophobic protein molecules are surrounded by a structurally heterogenous lipid area containing up to 3.3 mg of lipid per l mg of protein . The maximal expression of structural heterogeneity was observed at the minimal content of protein in the proteoliposomes . Treatment with the membranotropic antibiotic gramicidin S resulted in disappearance of lateral heterogeneity of lipids in the constituted system and in lipid aggregation in bacterial membranes . It is assumed that the aggregability of membrane proteins depends on the structural rearrangement of some part of lipid bilayer around them.

Biochim Biophys Acta, 1982 Jan 26, 696(1), 76 - 86
Structure of adenovirus chromatin; Mirza MA et al.; The structure of adenovirus type 2 chromatin isolated from wild-type and ts1 virions was investigated by micrococcal nuclease digestion and electron microscopy . Partial digestion of wild-type and ts1 chromatin with micrococcal nuclease generated a multimeric DNA smear devoid of the 200 basepair nucleosome repeating pattern characteristic of cellular chromatin . However, 11 S monomer cores of 150 basepairs were detectable . The chromatin of ts1 (39 degrees C) was more resistant to digestion by micrococcal nuclease . Two-dimensional electrophoresis of the monomer core showed that wild-type core contained protein VII while ts1 (39 degrees C) core contained PVI and PVII . Protein V appears to be located on the variable-length intermonomer region . Crosslinking studies suggest that proteins PVII and VII exist in dimeric form within the monomer core . Electron microscopy revealed a 5.5-fold-condensed two-micron-long beaded structure with about 200 monomer particles spaced irregularly . Based on these observations, a model for adenovirus prochromatin and chromatin is proposed that differs in important aspects from the model proposed previously (Corden, J., Engelking, H.M . and Pearson, G.D . (1976) Proc . Natl . Acad . Sci . USA 73, 401-404).

Biochim Biophys Acta, 1982 Jan 26, 696(1), 7 - 14
Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore; McHugh MM et al.; Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug . The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here . Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei . Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels . Chromatin breakdown measured under these conditions is double-stranded in nature . In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks . Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay . When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin . No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol . For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin) . Such degradation also occurs more rapidly with chromophore than with neocarzinostatin . Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min . Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei . The released chromatin can serve as a substrate for micrococcal nuclease digestion . Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.

J Biol Chem, 1982 Jan 25, 257(2), 1007 - 16
Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction; Barbehenn EK et al.; A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid . The physicochemical properties of M . luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition . The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell . Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr . Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids . Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration . The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels . It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis . This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

J Biol Chem, 1982 Jan 25, 257(2), 946 - 52
Translational control by messenger RNA competition for eukaryotic initiation factor 2; Rosen H et al.; Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl{35S}methionyl-tRNAf into protein . The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products . On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation . The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2) . Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis . No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent . These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2 . In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor . The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity . These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2 . They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species . Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.

Biochemistry, 1982 Jan 19, 21(2), 248 - 56
Histone-dependent reconstitution and nucleosomal localization of a nonhistone chromosomal protein: the H2A-specific protease; Watson DK et al.; We have described earlier a chromatin-bound protease with unique specificity for histone H2A {Eickbush, T . H., Watson, D . K., & Moudrianakis, E . N . (1976) Cell (Cambridge, Mass.) 9, 785--792} . In the present study, we explore the nature of interactions that form and stabilize the enzyme-chromatin system by using the activity of the protease to monitor its binding to DNA and DNA-histone complexes . During salt extraction of chromatin, the protease is released at an ionic strength between that required for the extraction of the slightly lysine-rich histones (H2A and H2B) and the arginine-rich histones (H3 and H4) . The reassociation of this nonhistone protein to DNA has an absolute requirement for the H3--H4 tetramer and is only enhanced by the H2A--H2B dimer in the presence of the tetramer . We believe that the binding of the enzyme onto DNA requires some histone-elicited compaction of the helix . We have also examined the distribution of this enzyme within the chromatin fiber by isolating pools of monomer nucleosomes from micrococcal nuclease digests of 0.6 M NaCl extracted chromatin and from reconstituted DNA-protein complexes . The H2A-protease is found with these monomer nucleosome pools, and no activity can be detected in the low molecular weight products released during the digestion . Thus, by virtue of its extraction characteristics from chromatin and its association with isolated nucleosomes, this nonhistone protein exhibits properties hitherto assigned only to the inner histones.

EMBO J, 1982, 1(7), 811 - 6
The chromatin repeat length of brain cortex and cerebellar neurons changes concomitant with terminal differentiation; Jaeger AW et al.; Chromatin repeat lengths in neuronal, glial, and liver nuclei of the rat were determined by micrococcal nuclease digestion followed by gel electrophoresis . The repeat length of cortex neurons decreased from 200 base pairs (bp) before birth to 170 bp at 14 days and all subsequent stages . Administration of {3H}thymidine to pregnant rats during the period of fetal neurogenesis allowed neurons differing in their time of origin to be labeled individually . This revealed that the shortening of the chromatin repeat length affected only neurons generated early during development, i.e., between gestational days 13/14 and 18/19, whereas neurons continuing to proliferate beyond gestational day 19 and up to birth (day 22) did not undergo shortening of their repeat length . In contrast to the cortex neurons, cerebellar neurons (granule cells) underwent lengthening of the repeat length from 165 bp at fetal and early post-natal stages (up to day 4) to 218 bp after day 30 . Thus, in both cortex and cerebellar neurons the changes occurred temporally coincident with major developmental processes . No changes were detected in liver nuclei during the same period . Non-astrocytic glia cells of the adult cortex had 200 bp repeats.

EMBO J, 1982, 1(12), 1487 - 92
The structural role of histone H1: properties of reconstituted chromatin with various H1 subfractions (H1-1, H1-2, and H1o); Biard-Roche J et al.; A previous study on the distribution of histone H1 subfractions in chromatin suggested that these proteins differ in the protection they confer to DNA . To elucidate further this suggestion, reconstitution experiments were carried out with purified H1 subfractions (H1-1, H1-2, H1o) and H1-depleted chromatin . We have studied the structural properties of H1o as compared to those of other H1 fractions by electrophoretic analysis of DNA and mononucleosomes obtained after micrococcal nuclease digestion, thermal denaturation, and electron microscopy . The three fractions studied reassociate to H1-depleted chromatin . However, differences in the extent of DNA protection are observed between H1o and the other fractions: H1o induces a more rapid degradation of long oligomers into mononucleosomes; these mononucleosomes bearing H1o only, have a greater electrophoretic mobility; furthermore, thermal denaturation shows that a small fraction of DNA is less efficiently protected by H1o than by the other fractions . Electron microscopy, on the other hand, shows that these differences are not due to areas of chromatin devoid of H1o in the reconstitute and that the reconstituted samples are able, under proper ionic conditions, to refold in a higher-order structure.

Arch Dermatol Res, 1982, 272(3-4), 321 - 9
Antimicrobial effects of an antiperspirant formulation containing aqueous aluminum chloride hexahydrate; Holzle E et al.; To document deodorant efficacy the antimicrobial activity of a gelatinous antiperspirant formulation of aqueous aluminum chloride hexahydrate was investigated . In vitro assays demonstrated highly bactericidal activity on microorganisms comprising the resident axillary skin flora, including micrococcaceae and aerobic diphtheroid bacteria . Gram-negative bacteria and yeast were partially inhibited . In vivo experiments utilizing occlusive patches on forearm skin and bacterial sampling of the axilla showed pronounced bacteriostasis and persistence of aluminum chloride on the skin . Inhibition of microbial growth lasted more than 3 days after a single treatment of the axilla . Following repeated open applications to the volar aspect of the forearm, the skin remained virtually sterile for 3 days.

Cancer Biochem Biophys, 1982, 6(2), 95 - 9
Euchromatin protein analysis in preneoplastic and neoplastic cell lines: effects of endogenous chromatin solubilization; Smith GJ; No differences were detected in the micrococcal nuclease or deoxyribonuclease I-solubilized (euchromatin) proteins from several preneoplastic and neoplastic mouse epithelial cell lines . Significant levels of endogenous chromatin solubilization were detected in these cell lines prior to the addition of the exogenous nucleases . The endogenous solubilization markedly reduced the susceptibility of the remaining chromatin to solubilization by micrococcal nuclease or deoxyribonuclease I . Evidence is presented which suggest that the endogenous solubilization may obscure differences in the protein components of the euchromatin fraction from these preneoplastic and neoplastic cell lines.

Prostate, 1982, 3(5), 439 - 57
Responses to androgens of rat ventral prostate nuclear androgen-binding sites sensitive and resistant to micrococcal nuclease; Davies P et al.; Rat ventral prostate nuclei were separated into three major fractions by mild digestion with micrococcal nuclease and two fractions by extensive digestion . All fractions contained androgen-binding sites . Almost 50% of nuclear binding sites were resistant to enzymic digestion when only 5-15% of total DNA was resistant . Under milder digestion conditions, 21% of nuclear binding sites were associated with an intermediate fraction, representing 16% of total nuclear DNA, which was enriched in specific androgen-regulated gene sequences . This fraction was rapidly degraded by more extensive digestion . The nuclease sensitivity of these particular genes was markedly influenced by castration and the administration of dihydrotestosterone to castrated animals . The nuclear content of both nuclease-resistant and -sensitive androgen-binding sites was decreased by castration . Whereas the administration of androgen to animals castrated 1 day previously preferentially replenished nuclease-resistant sites, nuclease-sensitive sites, including those associated with transcriptionally active regions, had apparent priority when androgen was supplied to animals castrated 7 days previously . The significance of these observations to the regulation of nuclear processes and the possible interrelationships of nuclease-sensitive and -insensitive sites are discussed.

Folia Microbiol (Praha), 1982, 27(4), 228 - 36
Valine production from hydrocarbon by Micrococcus varians; Chatterjee M et al.; A bacterium isolated from Assam (India) soil was found to accumulate L-valine in the growth medium and was identified as Micrococcus varians . The strain grew and accumulated valine in a purely synthetic medium, but supplementation with either casamino acids or yeast extract or with both, improved the yield . The entire fermentation period could be divided into a growth phase and a production (phase which could be prolonged by adjustment of pH to neutral range . Among the different hydrocarbon and nitrogen sources tested straight run gas-oil and ammonium sulphate, respectively, were found most suitable . Antibiotics inhibited growth but stimulated extracellular valine accumulation . Vitamins stimulated growth and valine yield and an inoculum level of 10% was found to be optimal . The yield of L-valine under optimal conditions was 2.95 g/L.

Exp Gerontol, 1982, 17(3), 173 - 7
Age changes in the H1 group of histones from rat liver; Wagner AP et al.; Histones were obtained from young and old rat livers by extracting them in 0.25 N HCl . They were fractionated on 15% acid urea polyacrylamide gels containing 6.25 M urea and the changes in the ratio of the major histone fractions as a function of age were calculated . Data presented show a significant increase in the amount of H1 degree fraction in the liver of old rats as compared to young rats . This data is discussed and the possible involvement of H1 degree fraction in an increased resistance of old rat liver chromatin to micrococcal nuclease digestion of linker DNA is suggested . Finally, in connection with this increased resistance, some possible consequences in chromatin structure are discussed.

Basic Life Sci, 1982, 20, 315 - 22
New approaches to DNA damage and repair: the ultraviolet light example; Haseltine WA et al.; DNA fragments of defined sequence are used as probes to study DNA damage and repair . The case of ultraviolet light is presented and includes the following: (a) Description of the distribution of cyclobutane pyrimidine dimers within defined DNA sequences . Considerations of the effect of neighboring base composition, dose rate, and double- or single-stranded property of the DNA are discussed . (b) Dissection of the anatomy of the incision event and subsequent repair steps . A three-step incision model for repair of cyclobutane dimers by the Micrococcus luteus repair enzymes will be presented . The steps are (1) recognition of the lesion and N-glycosylase scission, (2) cleavage of the phosphodiester bond 3' to the newly created apyrimidinic site, and (3) scission of the apyrimidinic sugar on the 5' side . (c) Use of human alphoid sequences as indicators of DNA damage in intact human cells . (d) Biological significance of a novel ultraviolet light-induced photoproduct . This photoproduct occurs at pyrimidine-cytosine sequences and may have a significant biological role.

J Virol, 1982 Jan, 41(1), 334 - 9
Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis; Matsuo T et al.; The mRNA species encoded by early region 4 (E4) (map position {mp} 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100) . The mRNA's were translated in vitro using {35S}methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates . Five major (35,000-molecular-weight {35K}, 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used . The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed . Assignment of these polypeptides to E4 was further established by hybrid arrested translation . Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K . The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J . Biol . Chem, 255:6772--6779, 1980) . Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related . The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them . These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.

Eur J Biochem, 1982 Jan, 121(2), 401 - 5
Effect of cations on the acetylation of chromatin in vitro; Dod B et al.; The effect of the differently charged cations Na+, Mg2+, spermidine and spermine on the acetylation of histones in vitro in soluble chromatin and in core particles was investigated . Up to a given concentration, which depends on the charge of the cation, all four cations activate the acetylation of these fractions . Above this critical concentration a gradual inhibition of the acetyltransferase activity occurs . Spermine, spermidine and Mg2+, but not Na+, affect the relative accessibilities of the acetyltransferase to the individual histones . As the concentration of these three polyvalent cations increases there is a gradual increase in the relative acetyl incorporation in histones H3 accompanied by a corresponding decrease in H4 . Kinetic aspects of the system were also studied . A minor fraction of the soluble chromatin (mononucleosomes and dinucleosomes) which is preferentially digested by micrococcal nuclease is also acetylated preferentially . The role chromatin structure plays in determining the specificity of histone acetylation is discussed.

Carcinogenesis, 1982, 3(2), 151 - 3
Caffeine inhibits DNA polymerase I from Escherichia coli: studies in vitro; Balachandran R et al.; Caffeine inhibits the activity of DNA polymerase I (E . coli) and its proteolytic large fragment in in vitro DNA replication system . DNA polymerase from Micrococcus luteus is also equally inhibited by caffeine . The extent of inhibition was more with the activated adenovirus, T4 and calf thymus DNA than with synthetic DNA template-primers . Results obtained from time-course studies indicated that caffeine inhibition reached maximum by 30 min of incubation . Enzyme kinetic studies showed that inhibition was competitive with respect to DNA template.

Invest Ophthalmol Vis Sci, 1982 Jan, 22(1), 103 - 10
Investigation of the accuracy of tear lysozyme determination by the Quantiplate method; Copeland JR et al.; The accuracy of lysozyme concentration determination by the method of measuring lysis diameter in agarose gel slabs containing Micrococcus lysodeikticus bacteria uniformly suspended throughout the gel was determined for various methods of tear sample collection . The effects of storage of lysozyme solution samples at subzero temperatures for several days was also examined . It was found that a power rather than the suggested exponential dependence between the lysozyme concentration and lysis diameter provides the most accurate fit and thus should be used for interpolation . Storing samples frozen in glass capillaries lowered the lysozyme concentration in a predictable manner . When Weck-Cel sponges were used to collect the samples the lysozyme concentration was greatly diminished in a nonlinear manner because of internal adsorption . The relative loss (cause by adsorption) depended on the actual lysozyme concentration as well as on the sample volume/sponge weight ratio . Storing samples absorbed by such sponges in a frozen state further altered the results in an unpredictable way . The observation that smaller tear samples for a given sponge size yielded lower apparent values for lysozyme concentration casts doubt on findings that have reported lower lysozyme concentration in the tears of keratoconjunctivitis sicca patients, where either cellulose sponges or filter paper discs were used for tear collection.

DNA, 1982, 1(4), 345 - 53
Evolutionary conservation of vitellogenin genes; James TC et al.; Homologous and heterologous hybridizations in solution were performed between sheared genomic DNA and DNA complementary to vitellogenin mRNA of Xenopus, chicken, and migratory locust . The kinetics of hybridization and the thermal stability of the hybrids formed suggested a high degree of conservation of coding sequences of insect, amphibian, and avian vitellogenin genes . These cDNA probes hybridized to calf thymus DNA to a slight, but significant, extent, and not at all to Micrococcus lysodektikus DNA . DNA complementary to Xenopus albumin mRNA did not cross-hybridize significantly with locust or chicken DNA . Further evidence for the evolutionary conservation of vitellogenin genes was obtained from Southern blot analysis of restriction endonuclease-digested genomic DNA from a variety of vertebrate and invertebrate oviparous animals (Xenopus, chicken, migratory and desert locusts, yellow meal worm, carab moth, and Mediterranean fruitfly) . When probed with cloned vitellogenin cDNAs from Xenopus and migratory locust, the DNA of these organisms showed varying degrees of homology of parts of the vitellogenin coding sequences . Southern blot analysis also showed that a part of the sequence specified in the cloned Xenopus vitellogenin cDNA was represented as repetitive DNA in the locust genome . However, cloned locust vitellogenin cDNA hybridized to discrete fragments of the restricted vertebrate DNA . These studies demonstrate a remarkably high degree of conservation of insect, amphibian, and avian vitellogenin genes.

Chromosoma, 1982, 87(3), 345 - 57
Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B; Turner BM; A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types . Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern . Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads . It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained . Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment . It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions . Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay . This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B . As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.

EMBO J, 1982, 1(2), 223 - 30
Disruption of the typical chromatin structure in a 2500 base-pair region at the 5' end of the actively transcribed ovalbumin gene; Bellard M et al.; We examined the chromatin organizations of approximately 3 kb of DNA in the 5'-end flanking region of the ovalbumin gene in chicken erythrocyte and oviduct cell nuclei . With specific DNA probes and an indirect end-labeling technique, we analysed the pattern of the DNA fragments obtained after micrococcal nuclease digestion and generated comparative maps of the nuclease cuts . This region of the chicken genome displays a "typical" chromatin arrangement in erythrocyte nuclei, with nucleosomes apparently positioned at random . In contrast, in oviduct nuclei, the same region has an "altered" chromatin structure, and lacks a typical nucleosomal array . The existence of specifically positioned proteins and of alterations in the DNA secondary structure in this region of the oviduct chromatin is suggested by comparison of the nuclease cleavage maps which reveals specific changes: disappearance of nuclease cuts present in "naked" and erythrocyte chromatin DNAs, and appearance of new cuts absent from these DNAs.

Can J Biochem, 1982, 60(10), 1001 - 5
Adenoassociated virus has a unique chromatin structure; Brown M et al.; The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2) . Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600-2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers . This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.

Basic Life Sci, 1982, 20, 147 - 55
Single-stranded gaps as localized targets for in vitro mutagenesis; Shortle D et al.; Short single-stranded gaps in circular DNA molecules can be generated enzymatically, often at predetermined sites . These can serve as targets for in vitro mutagenesis procedures that result in alterations in nucleotide sequence within or very near the gap . Deamination of unpaired cytosine residues with sodium bisulfite has been used to induce mutations in the BglI restriction site of SV40 DNA and within defined regions of the beta-lactamase gene on pBR322 . A new method of induction of mutations at gaps, called "gap misrepair," has been developed; it was used to cause changes at the HindIII and C1aI restriction sites on pBR322 DNA . Gap misrepair reactions using DNA polymerase I of Micrococcus luteus in the presence of T4 DNA ligase and three of the four deoxynucleoside triphosphates yielded all three possible substitutions for adenine and cytosine residues in the DNA.

Carcinogenesis, 1982, 3(4), 435 - 8
In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide; Galiegue S et al.; Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO) . It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed . For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides . This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released . The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used . The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.

J Virol, 1982 Jan, 41(1), 309 - 12
Analysis of human cytomegalovirus nucleoprotein complexes; St Jeor S et al.; When chromatin was isolated from cells infected with human cytomegalovirus, the virus DNA remained with the chromatin fraction . If deproteinized virus DNA was added to either isolated nuclei or chromatin, the DNA was lost during the chromatin isolation . When isolated chromatin from cytomegalovirus-infected cells was banded in isopycnic metrizamide gradients, a single peak with a density of 1.18 g/cm3 was present . Analysis of this peak in isopycnic neutral CsCl gradients indicated that it contained both human cytomegalovirus and human embryonic lung cell DNAs . When infected nuclei were treated with micrococcal nuclease, 11S subunit particles which cosedimented with cell nucleosomes and contained virus DNA were isolated.

Carcinogenesis, 1982, 3(3), 341 - 4
Mechanism for the loss of preferential benzo {a} pyrene binding to the linker DNA of chromatin; Jack PL et al.; We have examined the fate of the asymmetric chromosomal distribution of DNA adducts generated by the chemical carcinogen r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo{a}pyrene (BPDE) . Treatment of mouse embryo cells with BPDE results in 3.5 times more binding to the linker DNA regions between nucleosome cores than to the nucleosome core DNA itself, but 24 h post-treatment incubation of these cells leads to a loss of this non-random binding . A similar result was obtained when post-treatment incubation was carried out in the presence of hydroxyurea indicating that factors other than DNA replication are responsible for this changes in adduct distribution . However in the case of excision repair deficient xeroderma pigmentosum (XP12/BE) cells the non-random adduct distribution was stable over a period of 48 h, whereas with excision repair proficient XP variant (XP4/BE cells, loss of preferential binding did occur . There results indicate that the loss of non-random nucleosomal DNA modification with time can be accounted for by the preferential removal of adducts from micrococcal nuclease sensitive linker DNA and further, demonstrates that in certain cells at least, the relative position of nucleosome core structures on DNA remains unchanged over a period of at least 48 h.

Chromosoma, 1982, 84(5), 717 - 27
Localization in mouse-L-Cell chromosomal sites of transferred immunoglobulin genes; Fittler F et al.; Mouse thymidine kinase negative (tk-) L-cells were cotransformed with two different kappa light chain genes (cloned from mouse myeloma) and the tk gene from Herpes simplex virus I . (Transformation is defined as change in the genotype by introduction of foreign DNA.) About 80% of the tk+ -transformants contained varying amounts of transferred kappa light chain sequences, one transformant about 150 copies per genome . The transferred immunoglobulin genes appear to be organized in a nucleosomal substructure, as deduced from digestion experiments with micrococcal nuclease . In situ hybridization experiments revealed, that the transferred genes are not distributed randomly across the chromosomes of the recipient cell . Instead they are clustered at one or a few chromosomal locations.

Proc Natl Acad Sci U S A, 1982 Jan, 79(1), 118 - 22
Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA; Musich PR et al.; The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis . When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease . A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease . The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide . In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence . This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.

Allerg Immunol (Leipz), 1982, 28(4), 251 - 9
{Analysis of methods for optimizing lysozyme determination}; Stelzner A et al.; In order to optimize the detection of lysozyme in the clinic, studies on photometric and agar diffusion methods were performed with different preparations of micrococcus luteus (lysodeikticus) . Living or living lyophilized preparations of micrococcus were suitable . The influence of temperature, test period and lysozyme concentration on the result was demonstrated . The lysoplate technique has several advantages in clinical routine tests, however an exact adjustment of the reagents and the test is necessary (living micrococcus, 3 hours incubation, 50 degrees C test temperature) . The lysozyme measurements of different papers are transferable not without reserve and require a careful interpretation in respect of reproducibility and clinical relevance.

Mol Gen Genet, 1982, 186(1), 127 - 34
The role of pyrimidine dimers in postreplication repair in Neurospora; Calza RE et al.; Using the Micrococcus luteus dimer specific endonuclease assay of Wilkins (1973), and photoreactivation we have examined the induction and fate of ultraviolet induced pyrimidine dimers in the excision defective strain, uvs-2, of Neurospora crassa . Dimer induction was fluence dependent from 0 to 800 ergs/mm2 UV . An interdimer distance of 19.6 x 10(6) DNA molecular weight was found after a fluence of 220 ergs/mm2 . We confirm the earlier report that this mutant is completely excision defective (Worthy and Epler 1972) . Photoreactivation (PR), which greatly enhanced survival (by 10 fold after 440 ergs/mm2 UV), reduced significantly (40-44%) the number of UV-endonuclease sensitive sites found in irradiated DNA . This treatment also alleviated immediately some of the temporary blocks to high molecular weight DNA synthesis (elongation or ligation) seen in irradiated cells . We have also attempted to elucidate the mechanism of cellular postreplication repair used to overcome the UV inhibition to DNA synthesis . It was determined that during postreplication repair, Neurospora does not use recombination to bypass dimers and that single stranded DNA gaps opposite dimers do not appear to be present during the time when DNA being synthesized is made only in short pieces.

J Immunol, 1982 Jan, 128(1), 314 - 22
Mapping the antigenic epitope for a monoclonal antibody against lysozyme; Smith-Gill SJ et al.; A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of Kohler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds . The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody . Duck lysozyme did not react with the antibody at all . Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes . Arg 68 was identified as a determining amino acid . Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope . The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme . Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114 . The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site . Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.

J Biol Chem, 1981 Dec 25, 256(24), 13188 - 92
Translation of vesicular stomatitis and Sindbis virus mRNAs in cell-free extracts of Aedes albopictus cells; Gillies S et al.; We have developed a cell-free system from Aedes albopictus (mosquito) cells which is able to carry out endogenous protein synthesis and is stable to freezing and thawing . Successful preparation of extracts was found to depend on the presence of purified placental RNase inhibitor during cell breakage . Micrococcal nuclease-treated extracts translated exogenously added Sindbis 26S or vesicular stomatitis virus mRNA with a high degree of fidelity, demonstrating that initiation of protein synthesis had occurred . Evidence is presented showing that when cell fractions containing intracellular membranes were used to translate vesicular stomatitis virus mRNA, the G protein was glycosylated and inserted into microsomal vesicles . Additional studies indicate that initiation of protein synthesis in this system is dependent on a capped and methylated 5'-terminal structure in the mRNA.

Biochim Biophys Acta, 1981 Dec 14, 638(2), 275 - 81
F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein; Lim SH et al.; It has been claimed (Andreu, JM, Warth, R . and Munoz, E . (1978) FEBS Letter, 86, 1-5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars . Even after extensive purification of M . lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found . Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A . Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase . Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography . No qualitative or quantitative changes in the subunit (alpha, beta, gamma, delta and epsilon) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography . We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein . The presence of carbohydrate is attributable to contamination with lipomannan.

J Biol Chem, 1981 Dec 10, 256(23), 12574 - 80
Chemical composition of nucleosomes among domains of calf thymus chromatin differing in micrococcal nuclease accessibility and solubility properties; Davie JR et al.; Calf thymus chromatin was fractionated by the Sanders' procedure ((1978) J . Cell Biol . 79, 97-109) . this procedure involves sequential elutions of micrococcal nuclease-digested nuclei with buffers of increasing ionic strength . Through the use of the nuclei nick translation technique of Levitt et al . (Levitt, A., Axel, R., and Cedar, H . (1979) Dev . Biol . 69, 496-505) which specifically labels the transcriptionally competent regions of the chromosome, the lowest salt eluted, micrococcal nuclease-sensitive chromatin fraction, was found to be enriched in transcriptionally competent chromatin . This chromatin fraction contained approximately equimolar amounts of the core histones and low amounts of histone H1 . In addition, this fraction was enriched both in the acetylated species of histone H4 and in the high mobility group (HMG) proteins 14 and 17, but it was depleted in 5-methylcytosine . As the ionic strength of the elution buffers increased, chromatin fractions from less micrococcal nuclease-sensitive chromatin domains were eluted . The nuclease-insensitive fractions were enriched in the unacetylated species of histone H4, 5-methylcytosine, and histone H1 . Although these fractions had a smaller proportion of nucleosomes containing HMG-14 and HMG-17, they contained about 50% of the total HMG-14 and HMG-17 population.

Biochemistry, 1981 Dec 8, 20(25), 7284 - 90
Characterization of rat liver oligonucleosomes enriched in transcriptionally active genes: evidence for altered base composition and a shortened nucleosome repeat; Berkowitz EM et al.; A transcriptionally active chromatin fraction of oligonucleosome size has been separated and isolated by a modified micrococcal nuclease fractionation procedure . After mild enzymatic digestion, rat liver nuclei were lysed, and the chromatin was separated by centrifugation on linear sucrose gradients . Fractions from four regions of the gradient were pooled and labeled, from the top to the bottom, A, B, C, and D, respectively . Fraction A, which contained 20% or less of the total DNA, was determined to have a mean size of a hexanucleosome . By hybridization with {3H}cDNA transcribed from total cytoplasmic poly(A) mRNA, DNA from fraction A was shown to be 10-15-fold enriched in transcribing genes when compared with total DNA . This fraction also has a somewhat higher concentration of AT base sequences . Significant differences were observed in nucleosome phasing . Fraction A has the shortest repeat length, fractions B and C are intermediate, and fraction D, which is depleted in transcribing DNA sequences, has the longest . Thus, we have isolated a chromatin fraction of oligonucleosome size enriched in transcribing genes and organized with reduced nucleosome spacing.

Mol Cell Biol, 1981 Dec, 1(12), 1094 - 105
Adenovirus chromatin structure at different stages of infection; Daniell E et al.; We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease . Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin . This pattern was maintained in parenteral DNA throughout infection . Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate . Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches . Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected . This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers . All regions of the genome were represented in the protected DNA . To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with {3H}thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis . With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat . We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting . High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Eur J Biochem, 1981 Dec, 121(1), 15 - 9
Tightly bound non-histone proteins in nucleosomes from pig-liver chromatin; Caiafa P et al.; Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine . By this method non-histone proteins have been found to be associated with the core particles . Proteins tightly bound to the core particle DNA (i.e . dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71000 to 20000 . They show, furthermore, a peculiar amino acid composition . Other tightly bound proteins have been shown to be present only in the spacer regions . The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are compatible or incompatible, respectively, with the simultaneous binding of the histone octamer.

Gann, 1981 Dec, 72(6), 965 - 8
A simple and convenient assay method for phosphorolysis of 5'-deoxy-5-fluorouridine; Miwa M et al.; 5'-Deoxy-5-fluorouridine (5'-DFUR) exhibits antitumor activity through its conversion to 5-fluorouracil by uridine phosphorylase (UPase) in mice . This mode of activation was confirmed by the fact that a mutant strain of Micrococcus flavus resistant to 5'-DFUR but sensitive to 5-fluorouracil was isolated and found to be deficient in UPase activity . Furthermore, since the enzyme level in tumor tissue should be useful as an indicator for therapy with 5'-DFUR, a simple assay method for the enzyme activity with use of the above bacterium was developed, in which the amount of 5-fluorouracil cleaved from 5'-DFUR by the enzyme was estimated through its antibacterial activity without separation from 5'-DFUR . This method is simple and efficient for handling a large number of samples.

Mol Biol Rep, 1981 Nov 30, 8(1), 51 - 6
Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle; Mukherjee AK et al.; A low molecular weight RNA species, in the 70-90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography . This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash . The iRNA at the concentration of 20-80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e . chick muscle poly(A)+mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature . The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA . The iRNA does not appear to be double-stranded RNA . It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.

Biochemistry, 1981 Nov 24, 20(24), 6921 - 9
Hybridization of nuclear matrix attached deoxyribonucleic acid fragments; Basler J et al.; Annealing studies were performed on DNA fragments associated with rat and mouse liver interphase nuclear matrix and the metaphase scaffold of Chinese hamster DON cells . Matrix and scaffold bound DNA fragments, reassociated with an excess of total genomic DNA, displayed kinetics virtually identical with total nuclear DNA probes . Moreover, both the extent and kinetics of these hybridizations were independent of the matrix DNA fragment size (less than 350--5000 base pairs) and the method of nuclease digestion used in their preparation (DNase I, micrococcal nuclease or endogenous digestion) . The repetitive DNA component of the matrix DNA was examined by reacting discrete sizes of matrix DNA fragments (less than 350--5000 base pairs) from mouse liver with a library of cloned repetitive sequence DNA fragments which included mouse major satellite sequences . Our results demonstrate that short DNA fragments anchored to the nuclear matrix contain these cloned sequences is similar proportion of total nuclear DNA and, when viewed in light of the annealing results, indicate that matrix DNA is not enriched in either repetitive or unique sequences . Furthermore, the matrix DNA fragments appear to contain the entire sequence complexity of the genome . Finally, we hybridized both matrix and total nuclear DNA fragments with cDNA to total nuclear polyadenylated RNA . The kinetics and extent of hybridization indicate that most, if not all, of the actively transcribed DNA sequences are present in similar concentrations . We conclude that in the overall organization of eukaryotic DNA within the nucleus, the repeating domains or loops which have been demonstrated by a number of investigators are not anchored at specific attachment sequences in interphase cells or during mitosis . These findings are discussed with regard to current concepts of eukaryotic DNA loop organization.

Biochemistry, 1981 Nov 24, 20(24), 6781 - 9
Association of the thyroid hormone receptor with rat liver chromatin; Jump DB et al.; We have investigated the association of the triiodothyronine (T3) nuclear receptor with rat liver chromatin by the use of selective endonuclease digestion and differential solubilization . The T3 receptor was found in a fraction of chromatin having some of the characteristics of active chromatin: (a) It is highly sensitive to DNase I and micrococcal nuclease digestion; (b) it is enriched in nonhistone proteins and depleted of histone (H-1) . Micrococcal nuclease and pancreatic DNase I excised two receptor-containing fragments from chromatin, a minor (12--14 S) form and a major (5.5--6.0 S) form . The latter structure has a Stokes radius of 42 +/- 2 A and an estimated molecular weight of 95400 when a partial specific volume of 0.725 cm3/g for protein is used . It contains DNA but no histones . Similar receptor-containing fragments were excised from chromatin of other rat tissues, including brain, kidney, and heart . Both the 5.5--6 S and the 12--14 S receptor-containing chromatin structures are converted by 0.5 M KCl to the 3.5 S form (R0 35 A molecular weight 50500) . Titration with micrococcal nuclease and pancreatic DNase I revealed that the 5.5--6 S form is preferentially excised by endonuclease . Neither receptor occupancy nor thyroidal status had an apparent effect on the susceptibility of chromatin to endonucleolytic digestion nor did they influence the distribution of T3 receptors in chromatin . Our results suggest that T3 receptors are not tightly associated with nucleosomes, the basic subunit of chromatin, but are associated with the DNA-linking nucleosomes in structurally modified regions of chromatin in rat liver nuclei . The T3 receptor-containing fragment may well represent a higher order of structural complexity necessary for T3 action at the cellular level.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5811 - 23
Partitioning of zinc and copper within subnuclear nucleoprotein particles; Bryan SE et al.; Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing . After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin . This level of metal was essentially unchanged with subsequent washings . Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper . Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA . Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used . In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents . Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure . A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5609 - 21
Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin; Cognetti G et al.; The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease . In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates . The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases . The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5533 - 52
The distribution of benzo(a)pyrene DNA adducts in mammalian chromatin; Jack PL et al.; This paper describes the distribution of DNA-lesions generated by the potent carcinogen benzo(a)pyrene (BP) or its ultimate metabolic derivative 7 alpha, 8 8 beta, di-hydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) within mammalian chromatin using the enzymic probe micrococcal nuclease . We have shown that the progress of the nuclease on naked DNA is unaffected by the presence of the hydrocarbon lesion at moderate extents of digestion . Digestion of nuclei isolated from murine erythroleukaemic cells immediately following BPDE treatment, and analysis of micrococcal nuclease resistant DNA by TCA precipitation, hydroxyapatite chromatography and gel electrophoresis demonstrates a non-random distribution of lesions . Approximately three times more binding occurs on the linker DNA regions between nucleosome cores than on the nucleosome core DNA itself . A similar result was obtained with BPDE treated primary mouse embryo cells; however nuclei isolated from these cells after prolonged treatment with BP (to allow metabolic activation) showed no such preferential binding . Post-treatment incubation of BPDE-treated cells shows that this difference can be accounted for by the loss of preferential localisation with time.

Biochemistry, 1981 Nov 10, 20(23), 6648 - 58
Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease; Cusick ME et al.; Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin . MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction . In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released . On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers . The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments . Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA . This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks . Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template . The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels . In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA . These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.

Vopr Med Khim, 1981 Nov-Dec, 27(6), 815 - 20
{Effect of histidine analogs and histidyl-containing dipeptides on the decarboxylase activity of Micrococcus sp . n . histidine decarboxylase}; Gonchar NA et al.; L-Histidinol and L-histidine hydroxamate were shown to inhibit competitively histidine decarboxylase from Micrococcus sp . n responsible for histamine biosynthesis . Constants of their inhibitory effect were found . Histidyl-containing dipeptides did not exhibit the pronounced inhibitory effect . The rate of inhibition was increased after esterification of histidyl dipeptide . Kinetics of the histidine decarboxylase inhibition by L-histidyl-L-leucine methyl ester was studied.

Mutat Res, 1981 Nov, 84(1), 73 - 82
Repair of 3-methyladenine and 7-methylguanine in nuclear DNA of Chlamydomonas: requirement for protein synthesis; Sweet JM et al.; The removal of 3-methyladenine and 7-methylguanine from nuclear DNA was determined following exposure of Chlamydomonas reinhardi to methyl methanesulfonate (MMS) . The amount of 3-methyladenine in DNA was determined using an extract from Micrococcus luteus that has a 3-methyladenine-DNA glycosylase . The amount of 7-methylguanine was estimated by heating the DNA for 30 min at 70 degrees followed by alkaline hydrolysis of the resulting apurinic sites . The molecular weight of the DNA was determined using alkaline sucrose gradients . The 3-methyladenine is removed with a half-life of 2--3 h whereas the 7-methylguanine is removed with a half-life of 10--12 h . The rate of removal of the 7-methylguanine is more than an order of magnitude faster than the estimated non-enzymatic hydrolysis rate indicating the probability of enzymatic repair . Addition of cycloheximide immediately after MMS treatment inhibits the removal of 3-methyladenine and 7-methylguanine from DNA . If cycloheximide is added 1.5 h after treatment with MMS, there is much less inhibition of the removal of 3-methyladenine . These results are interpreted to mean that MMS induces the synthesis of 1 or more proteins that are required for the repair of 3-methyladenine from Chlamydomonas DNA.

Eur J Biochem, 1981 Nov, 120(2), 399 - 405
Partial purification of nuclear androgen receptor by micrococcal nuclease digestion of chromatin and hydrophobic interaction chromatography; Bruchovsky N et al.; Extensive (20%) digestion of linker DNA of prostatic chromatin with micrococcal nuclease resulted in the precipitation of 95% of the nuclear androgen receptors . The receptor-enriched precipitate was dissolved in Tes buffer, pH 7.0, containing 0.6--1.2 M NaCl and analysed by hydrophobic interaction chromatography . The adsorption of receptor to omega-amino-alkyl derivatives of agarose increased with the length of the alkyl substituent indicating the presence of hydrophobic regions on the surface of the receptor molecule . Digestion of linker DNA followed by chromatography of precipitated chromatin proteins using 5-aminohexyl-agarose gave rise to a mean 93-fold purificaton of receptor with a recovery of 45% . This approach to the partial separation of nuclear androgen receptor may prove useful in conjunction with more selective purification techniques such as affinity chromatography.

Biokhimiia, 1981 Nov, 46(11), 1970 - 80
{Role of tryptophan in the enzymatic activity of histidine decarboxylase from Micrococcus sp . n.}; Gonchar NA et al.; The effect of N-bromosuccinimide (BSI) on micrococcal histidine decarboxylase in 0.07 M phosphate buffer, pH 5.6 was studied . Data from spectral and amino acid analyses suggest that at 20-fold molar excess of BSI three of 12 tryptophane residues undergo selective modification, resulting in 80-85% loss of the enzyme activity . Using fluorescent method and polyacrylamide gel electrophoresis, it was shown that modification of these reactive tryptophane residues does not cause structural changes of the enzyme . Presumably tryptophane residue responsible for enzymatic activity are either located in the enzyme active site of close to it . At 40-50-fold molar excess of BSI 6 to 9 tryptophane and 2 to 3 cysteine residues are subjected to modification; the other 3 tryptophane residues are unaffected by BSI . These are probably located deep inside the histidine decarboxylase molecule . The maximum of the protein fluorescent spectrum during modification of the 40-50-fold molar excess of BSI is shifted towards higher wavelength values, thus suggesting conformational changes of the enzyme . It can be therefore assumed that the enzyme molecule contains at least 2 groups of structurally and catalytically essential tryptophane residues which significantly differ in reactivity . Some diazonium salts were shown to inhibit micrococcal histidine decarboxylase . The kinetics of the inhibiting effect of these compounds were investigated.

Arch Androl, 1981 Nov, 7(3), 251 - 7
Human sperm chromatin has a nucleosomal structure; Wagner TE et al.; Digestion of human sperm chromatin with micrococcal nuclease reveals an 160 base pair (b.p.) DNA fragment that is further degraded to a series of DNA fragments remarkably similar to the micrococcal nuclease digestion products of eukaryote somatic cellular chromatin . DNase I digestion of human sperm chromatin also yields an identical pattern of DNA fragments to that observed upon DNase I digestion of somatic chromatin . These data, together with earlier electron microscopic observations, suggest a nucleosomal structure for human sperm chromatin.

J Neurochem, 1981 Nov, 37(5), 1193 - 202
Changes in chromatin structure associated with Alzheimer's disease; Lewis PN et al.; The enzyme micrococcal nuclease was used to examine the accessibility of chromatin extracted from brains of 13 patients with senile and presenile dementia of the Alzheimer type . Compared with chromatin extracted from brains of 8 patients without neurological signs or brain pathology and brains of 7 patients with nonAlzheimer dementia, Alzheimer chromatin was less accessible to this enzyme . Reduced accessibility was reflected by a reduced yield of mononucleosomes in comparison with dinucleosomes and larger oligomers . Both neuronal and glial chromatin were found to be similarly affected . The reduced yield of mononucleosomes from Alzheimer chromatin is not due to their increased breakdown, but is probably related to protein associated with the internucleosomal linker region that retards nuclease action . Dinucleosomes isolated from control and Alzheimer nuclease digests were examined for their protein complement . Three perchloric acid-soluble proteins situated in the histone H1 region of sodium dodecyl sulfate (SDS) gels were present in elevated levels in Alzheimer dinucleosomes . These results represent the first example of altered chromosomal proteins associated with a diseased state of the brain.

Biophys J, 1981 Nov, 36(2), 429 - 41
Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus; Sedliakova M et al.; In Escherichia coli B/r Her+ irradiated with two separate fluences, dimer excision is prematurely interrupted . The present study was designed to follow tha fate of dimers remaining unexcised . The results imply that these dimers (or distortions containing dimers) are transformed on replication from the state of sensitivity to the state of insensitivity to endonuclease from Micrococcus luteus . This conclusion is based on the following findings: (a) dimers were radiochromatographically detectable in DNA replicated after UV, which indicated that they were tolerated on replication . (b) Similar amounts of dimers were detected radiochromatographically both in DNA remaining unreplicated and DNA twice replicated after UV, This along with the low transfer of parental label into daughter DNA, indicated that dimers remained in situ in parental chains . (c) Immediately after UV, all parental DNA contained numerous sites sensitive to the extract from M . luteus . 2 h after UV, a portion of parental DNA still contained a number of endonuclease-sensitive (Es) sites, while another portion of parental DNA and all daughter DNA were free of Es sites . (d) The occurrence of parental DNA free of Es sites was not temporally correlated with dimer excision, but with the first round of DNA replication . (e) The amount of DNA free of Es sites corresponded to the amount of replicated DNA . (f) Separation of replicated and unreplicated DNA, and detection of Es sites in both portions separately showed that the replicated DNA was almost free of Es sites, whereas unreplicated DNA contained a number of such sites.

J Virol, 1981 Nov, 40(2), 465 - 71
Excision repair and patch size in UV-irradiated bacteriophage T4; Yarosh DB et al.; We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair . The average patch was small, approximately four nucleotides long . In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.

Cell, 1981 Nov, 27(1 Pt 2), 57 - 64
Micrococcal nuclease as a probe of DNA sequence organization and chromatin structure; Keene MA et al.; We have investigated micrococcal nuclease digestion of chromatin and purified DNA at the heat-shock locus 67B in Drosophila melanogaster . At early stages of the reaction a distinct set of fragments is generated, indicating the presence of preferential cleavage sites . These sites are also observed when purified recombinant plasmid DNA is used as the substrate, demonstrating that the sites are specified by the DNA sequence . At Drosophila locus 67B, prominent sites occur frequently, spaced approximately 200 bp apart, within the nontranscribed portions of the locus, but are generally not observed within the regions that are transcribed . In contrast, such sites are randomly distributed along the procaryotic plasmid pBR322 . The results indicate that specific patterns of digestion of eucaryotic chromatin by micrococcal nuclease cannot be simply interpreted as the consequence of the nucleosome array . However, it is possible that the organization of eucaryotic DNA sequences detected by micrococcal nuclease bears a functional relationship to the organization of DNA by nucleosomes and, in fact, was so selected through evolution.

Cancer Lett, 1981 Nov, 14(2), 205 - 15
Distribution of DNA-bound carcinogen 4-nitroquinoline 1-oxide and of repair-synthesized DNA in chromatin of WI-38 cells; Nose K; Distribution of DNA-bound 4-nitroquinoline 1-oxide (4NQO) and repair-synthesized DNA after treatment with 4NQO in chromatin was investigated with human diploid fibroblast WI-38 . Cells were incubated with {3H} 4NQO, and sites of binding on chromatin DNA were analyzed either by sensitivity to micrococcal nuclease or DNase I, or by fractionation of chromatin on sucrose gradient centrifugation . The results showed that 4NQO preferentially binds to DNA of linker region of chromatin, but the binding was random with respect to transcriptional activity of chromatin . The distribution of repair-synthesized DNA in chromatin damaged by 4NQO was also studied by similar experiments . {3H} Thymidine incorporated into repaired DNA was more sensitive to nucleases, but distributed almost equally among DNAs of chromatin subfractions with different transcriptional activity.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 7088 - 90
Organization of the diversity--joining region in rabbit immunoglobulin heavy chains as revealed by cleavage of a specific methionine residue in a100 allotype; Tonnelle C et al.; Three anti-micrococcus antibodies of restricted heterogeneity have been isolated from the antisera of homozygous a100/a100 rabbits . Heavy chains contained an unusual methionine residue at position 87 that may correlate with the a100 specificity . From this position, the sequence of a stretch of 35-50 amino acid residues was determined, permitting the definition of variable (V), diversity(D), and joining (J) segments in rabbit Ig heavy chains, with their most probable boundaries . Rabbit D regions so defined appear to be highly variable, both in sequence and in length, which varies, in the heavy chains analyzed, between 6 and 11 residues . The J regions are highly homologous to the mouse J2 segment.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6704 - 8
Carbohydrate modifications of the high mobility group proteins; Reeves R et al.; This paper reports the results of numerous biochemical analyses which indicate that the "high mobility group" proteins (HMGs) of mouse and bovine cells are bona fide glycoproteins and can, in addition, be modified by poly(ADP-ribose) addition in vitro . The sugars N-acetylglucosamine, mannose, galactose, glucose, fucose, and one unknown sugar (possibly xylose) have been identified in purified preparations of HMGs 14 and 17 . Furthermore, the fucose-specific lectin Ulex europeus agglutinin I bound both to the isolated HMGs and to monomer nucleosomes containing HMGs released from "active chromatin" by micrococcal nuclease digestion . Selective alkaline borohydride reductive cleavages of the HMGs suggested that the oligosaccharide prosthetic groups are primarily bound to these proteins by N-glycosidic linkages . The unexpected finding that the HMGs contain covalently bound complex carbohydrate moieties allows for a potentially great amount of variability and specificity in these proteins that may have important biological implications.

Nucleic Acids Res, 1981 Oct 24, 9(20), 5359 - 81
Localization of DNA methyltransferase in the chromatin of Friend erythroleukemia cells; Creusot F et al.; Chromatin fragments released from intact Friend erythroleukemia cell nuclei during limited incubation with micrococcal nuclease, DNase II or DNase I were analyzed to determine the distribution of DNA methyltransferase in chromatin . The enzyme was released in a free form when internucleosomal DNA was digested with micrococcal nuclease but was found associated with Mg++-precipitable polynucleosomes after DNase II digestion . Less than 25% of the enzyme was released from nuclei incubated with DNase I under conditions where transcriptionally active chromatin should have been completely digested . These results indicated that the bulk of DNA methyltransferase was bound to "linker" DNA in condensed regions of chromatin . Preferential rebinding of free enzyme to linker DNA was also demonstrated in vitro . The possibility that chromatin proteins play a role in regulating access of DNA methyltransferase to specific sites in DNA is discussed in light of these findings.

J Biol Chem, 1981 Oct 10, 256(19), 10124 - 8
Condensation of dinucleosomes by individual subfractions of H1 histone; Liao LW et al.; Dinucleosomes purified from micrococcal nuclease digests of steer kidney nuclei were stripped of H1 histone by exposure to 0.50 M NaCl . They were then formed in a complex with individual subfractions of calf thymus H1 histone by dialysis of histone-dinucleosome mixtures from 0.50 M NaCl to concentrations of NaCl between 0 M and 0.08 M; between 0.30 M and 0.10 M the complexes precipitated, and so were not included in the study . The presence of H1 in the complexes was shown to cause an asymmetrical, ordered condensation as revealed by distortions of the circular dichroic spectrum of the DNA . The distortions were negligible at 0.04 M NaCl and below, and increased as a function of ionic strength between 0.05 M and 0.08 M . The degree of distortion of the spectrum, and therefore the nature of dinucleosome condensation, differed greatly from one H1 subfraction to the next . One of the three subfractions tested had almost no effect on the circular dichroism in comparing its dinucleosome complex to H1-depleted dinucleosomes . The other subfractions to different degrees produced large distortions that resulted in spectra that were of the psi type at the higher salt concentration.

Nucleic Acids Res, 1981 Oct 10, 9(19), 5093 - 108
DNA fragments associated with chromosome scaffolds; Bowen BC; Following extensive digestion of HeLa metaphase chromosomes with either Hae III endonuclease or micrococcal nuclease, nonhistone protein scaffolds may be isolated . Scaffolds isolated after Hae III digestion have about 1.5% of the chromosomal DNA attached to them . This DNA is heterogeneous in size, ranging from about 0.2 to 20 kbp . It can be cleaved with either Eco RI or Hae III - Eco RI, producing a series of repeated fragments, of which the most abundant is 1.7 kbp in length . The 1.7-kdp fragment is tandemly repeated and is enriched (about 50-fold) in the scaffold-associated DNA . It is located primarily on human chromosome 1 and is probably a component of human satellites II and III . Scaffolds isolated after micrococcal nuclease digestion have about 0.1% of chromosomal DNA attached . This DNA is present in two size classes - fragments larger than 10 kbp and fragments approximately 0.2 kbp long . Restriction enzyme digestion of this DNA gives no prominent repeated fragments . Its reassociation kinetics are similar to those of total DNA, indicating that it is not enriched in either highly repetitive or middle repetitive sequences.

Anat Rec, 1981 Oct, 201(2), 225 - 35
Electron microscopic studies of rat sperm heads treated with urea, dithiothreitol, and micrococcal nuclease; Sobhon P et al.; Urea and dithiothreitol can decondense the chromatin in some of rat sperm heads . By this treatment, we have observed that in the nuclei of rat sperm the chromatin is organized into two morphologically distinct portions, namely: the compact chromatin rods of about 450 to 1,000 A thick, and the interlacing fibers about 250-290 A in thickness . When these treated sperm are further digested with micrococcal nuclease, the small fibers disappear, whereas the chromatin rods are still present in the "urea-nuclease pellet." From the available evidence, we suggest that the chromatin rods represent the highly packed nucleoprotamine, whereas the small fibers represent the more loosely organized nucleohistone.

Proc Natl Acad Sci U S A, 1981 Oct, 78(10), 6126 - 9
32P-labeling test for DNA damage; Randerath K et al.; Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test . DNA preparations exposed to chemicals known to bind covalently to DNA {N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, beta-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea} were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1) . The digests were treated with {gamma-32P}ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis-phosphates . These compounds were then separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions . Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents . Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical . This method detected a single adduct in 10(5) DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.

Proc Natl Acad Sci U S A, 1981 Oct, 78(10), 5968 - 72
Chicken erythrocyte nucleus contains two classes of chromatin that differ in micrococcal nuclease susceptibility and solubility at physiological ionic strength; Fulmer AW et al.; Inactive chromatin of the chicken erythrocyte nucleus is shown to consist of two distinct classes (I and S) . I chromatin (approximately 60% of the total genome) is insoluble at greater than 0.1 M ionic strength whereas S chromatin (approximately 40% of the total genome) is soluble at all ionic strengths studied (0.01--0.3 M) . These chromatins are released from nuclei upon digestion with micrococcal nuclease by two separate parallel processes that do not have a precursor--product relationship to each other . Isolated I-chromatin fragments show a progressive reduction in size from 250 to approximately 50 nucleosome equivalents with increasing digestion times at 0-2 degrees C . Prolonged digestion of nuclei at 37 degrees C results in conversion of I chromatin to mononucleosomes that are insoluble at greater than 30 mM NaCl . Isolated S-chromatin fragments show a constant size distribution, independent of digestion time, that peaks at approximately 35 nucleosome equivalents . Prolonged digestion of nuclei at 37 degrees C results in the conversion of S chromatin to mononucleosomes that are soluble at physiological ionic strength . Both I and S chromatins contain a full complement of histones with no nonhistone proteins.

Biokhimiia, 1981 Oct, 46(10), 1880 - 6
{Interaction of SV40 T-antigen with tumor cell chromatin}; Zheshovska-Vol'ny I et al.; The T-antigen of SV40 virus can be found in purified chromatin prepared from virus-induced tumour cells of the Syrian hamster . After treatment of chromatin or isolated nuclei with micrococcal nuclease this protein is detected in the high molecular weight and oligonucleosomal fractions . Data from sedimentation analysis and gel electrophoresis suggest that the T-antigen is predominantly linked with the oligonucleosomal fraction and in a lesser degree with mononucleasomes containing linker DNA and histone H1 . A small amount of the T-antigen is found in the mononucleosome complex devoid of histone H1; however, the ratio of the T-antigen to DNA in this case is about 30 times less than that in the oligonucleosomal fraction . In order to investigate the nature of T-antigen binding to nucleosomes, the interaction between the T-antigen and nucleosomes from normal rat liver was studied under restricted binding of the antigen to DNA (pH 8.0) . The T-antigen was effectively bound to the nucleosomes and coprecipitated with them in 5 mM MgCl2 . It was shown that the T-antigen was adsorbed on columns packed with immobilized histones H1 and nucleosomal histones without H1; the former eluted at 0.15 - 0.25 M NaCl, the latter - at 0.35 - 0.5 M NaCl . The possibility of T-antigen interaction with cellular DNA and protein components of chromatin (primarily to H1) is discussed.

J Biochem (Tokyo), 1981 Sep, 90(3), 877 - 80
Genome multiplicity and radiation resistance in Micrococcus radiodurans; Harsojo et al.; The number of genome equivalents of DNA per cell of M . radiodurans changed from approximately 5 to 10 depending on the media used . The sensitivity to ultraviolet light or gamma-rays was not different between the cells with different genome multiplicity . This suggests that the efficient repair process for DNA damage expressed in M . radiodurans is not influenced by the multiplicity of genomes in a cell.

Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5802 - 6
Nucleotide sequence of constant and 3' untranslated regions of a kappa immunoglobulin light chain mRNA of a homozygous b4 rabbit; Heidmann O et al.; A homozygous a2/a2 and b4/b4 rabbit has been hyperimmunized with Micrococcus lysodeikticus . Poly(A)-containing RNA has been isolated from the spleen and translated in vitro, and translation products have been analyzed by NaDodSO4/polyacrylamide gel electrophoresis . Double-stranded cDNA has been synthesized from poly(A)-containing RNA template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC).oligo(dG) tailing procedure . Tetracycline-resistant ampicillin-sensitive clones containing cDNA complementary to a kappa light chain mRNA have been selected by differential screening and their ability to hybridize to a spleen mRNA having the same size as a mouse kappa light chain mRNA . Two clones, pRk-15 and pRk-32, have been selected to determine the nucleotide sequence of the constant and 3' untranslated regions of kappa light chain mRNA, by the Maxam and Gilbert partial degradation method . Comparison of homologous regions of mouse kappa chain mRNA and b4 rabbit kappa chain mRNA reveals 61% homology in the constant region and 59% homology in the 3' untranslated region.

Biokhimiia, 1981 Sep, 46(9), 1712 - 6
{Interaction of the non-histone protein PS1 with some chromatin components}; Karavanov AA et al.; The binding of non-histone protein from mouse spleen chromatin located in the sites highly sensitive to micrococcal nuclease and DNA-ase I, to DNA and histones was studied . The binding of the DNA-protein complexes to nitrocellulose filters demonstrated the absence of protein binding to DNA . A highly selective binding of protein PS1 to histones H1 and H2A and to one of the non-histone proteins (presumably HMG 14) was revealed . It is concluded that protein PS1 is incorporated into chromatin by the protein-protein interactions.

J Invest Dermatol, 1981 Sep, 77(3), 311 - 3
Excision repair of UV-induced pyrimidine dimers in human skin in vivo; D'Ambrosio SM et al.; The induction and loss of pyrimidine dimers in human skin in vivo was determined using UV endonuclease, alkaline sucrose sedimentations, and the fluorescent detection of nonradiolabeled DNA . The number of dimers induced following exposure of the skin to radiation emitted from a Burdick UV-800 sunlamp was quantitated by reacting the extracted DNA with Micrococcus luteus endonuclease specific for pyrimidine dimers . Exposure to 15 and 30 seconds of radiation emitted from this lamp produced the formation of 12.8 and 23.6 dimers per 10(8) daltons DNA, respectively . Approximately 50% of the dimers induced were lost 58 min after irradiation . Only a small percentage (less than 10) remained 24 hr postirradiation . These data partially characterize the process by which pyrimidine dimers are excised from human skin DNA in vivo.

Eur J Biochem, 1981 Sep, 119(1), 183 - 8
Identification of a bacterial energy-transducing ATPase as a metallo (Zn2+) protein . Effect of chelating agents and divalent metal ions on ATPase activity; Mollinedo F et al.; Purified F1-ATPase from Micrococcus lysodeikticus contains zinc in the amount of 1 mol/mol of enzyme . This zinc content correlates with standard values of ATPase activity (assayed with Ca2+-ATP as substrate) of the protein, i.e . 5--6 mumol substrate hydrolysed . min-1 . mg-1 . Prolonged dialysis against EDTA results in a zinc-free protein which concomitantly loses its ATPase activity . Chelators such as Zincon, EDTA and L-cysteine inhibit the ATPase activity in concentration and/or time dependence related to their affinity for the metal ion involved . Reconstitution of the metallo (Zn2+) protein is demonstrated by the incorporation to the zinc-free protein of 65Zn2+ in amount near the 1 mol/mol of enzyme . This incorporation was concomitant with the regain of ATPase activity . The inhibition by EDTA and Zincon is reversed specifically by Zn2+ while the inhibition by EDTA is prevented by Zn2+ and Mn2+ and to, a minor extent, by Cd2+ . Zn2+ and Ca2+ ions are involved and are probably mandatory in the ATPase activity of M . lysodeikticus F1 but their roles appear to be different and not exchangeable . Other divalent metal ions inhibit the Ca2+-ATPase activity of the Zn2+ protein by the following decreasing order; Hg2+, Fe2+, Co2+, Cd2+, Mn2+, Mg2+ . M . lysodeikticus F1-ATPase is thus identified as a metallo (zinc) protein, which requires additional divalent metal ions for ATP hydrolysis.

J Biol Chem, 1981 Aug 25, 256(16), 8249 - 51
The reassociation with chromatin of H1 fragments bisected with thrombin; Ishimi Y et al.; When fragments of H1 histone formed by bisection of H1 histone with thrombin were allowed to reassociate with H1 histone-depleted chromatin, the carboxyl-terminal segment reassociated in such a manner as to protect the micrococcal nuclease-sensitive site(s) of the nucleosome core . On the other hand, the NH2-terminal segment of H1 histone protected 20 base pairs of linker DNA adjacent to the nucleosome core particle . These data provide strong evidence that the NH2-terminal portion and the carboxyl-terminal portion of H1 histone interact with 20 base pairs of linker DNA and the core DNA of the nucleosome, respectively.

Nucleic Acids Res, 1981 Aug 25, 9(16), 3991 - 4005
Changes in chromatin structure at the replication fork . II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I; Galili G et al.; Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels . One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978 . The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA . DNAase I was used to probe whether any nascent DNA is in nucleosomes . Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure . However, a striking background of nascent DNA between nascent DNA peaks was observed . This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I . One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region . Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.

Nucleic Acids Res, 1981 Aug 11, 9(15), 3671 - 80
Protection of discrete DNA fragments by the complex H1-octamerhistones or H5-octamerhistones after micrococcal nuclease digestion; Muyldermans S et al.; Several authors, including ourselves, have reported the existence of chromatosomes with DNA size larger than 166 bp in bird erythrocyte chromatin . It was tempting to correlate this increased DNA size with the presence of histone H5 . In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on: chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted . The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions: - denaturing gels clearly reveal a first discrete pause at 178 nucleotides in H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at the next intermediate 166 nucleotide chromatosome pause . - the generation of all discrete chromatosome bands is critically dependent on low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sites by histone H5 or H1, C or N terminal domains.

Biochemistry, 1981 Aug 4, 20(16), 4781 - 5
Carbon-13 nuclear magnetic resonance spectroscopic study of teichuronic acid from Micrococcus luteus cell walls . Comparison of the polysaccharide isolated from cells with that synthesized in vitro; Johnson SD et al.; Teichuronic acid isolated from the cell walls of Micrococcus luteus has been examined by natural-abundance (13)C NMR spectroscopy . Proton-decoupled and proton-coupled spectra were obtained for native teichuronic acid and also after the teichuronic acid had been oxidized with periodate and reduced with borohydride . The spectra are consistent with the structure {ManNAcUAp-( beta -1,6)-G1cp-( alpha -1,4)}n . Teichuronic acid synthesized in vitro from suitable substrates by the particulate enzyme fraction obtained from M . luteus yielded a (13)C NMR spectrum which is indistinguishable from that of the native teichuronic acid, indicating a structural identity of the teichuronic acid synthesized in vitro with that isolated from cell walls.

J Cell Biol, 1981 Aug, 90(2), 279 - 88
Regulation of the higher-order structure of chromatin by histones H1 and H5; Allan J et al.; Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously . The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA . The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes . The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests . The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.

J Cell Sci, 1981 Aug, 50, 209 - 24
Reconstruction of complexes of histone and superhelical nuclear DNA; Levin JM et al.; When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei . These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin . Their DNA is supercoiled and so intact . Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the 'core' histones without breaking the DNA . We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling . The superhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones . Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures . These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.

J Cell Biol, 1981 Aug, 90(2), 427 - 34
In vitro uptake and processing of prezein and other maize preproteins by maize membranes; Burr FA et al.; A cell-free, mRNA-dependent system has been developed for the translation and processing of zein preproteins . A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages . When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place . The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently . The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent . This small uptake, or absorption, or unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein . Finally a sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system has been developed which is particularly suited for the separation of low molecular weight proteins (less than 10,000 daltons) . Using this method, we examined the products of in vitro zein processing and detected no presequence polypeptides . This suggests that the zein cleavage proteinase is probably an exopeptidase.

J Virol, 1981 Aug, 39(2), 603 - 11
Simian virus 40 maturation: chromatin modifications increase the accessibility of viral DNA to nuclease and RNA polymerase; Brady J et al.; The accessibility of extracellular and nuclear simian virus 40 (SV40-M and SV40-I, respectively) virion chromatin DNAs to micrococcal nuclease, DNase I, BglI, EcoRI, and RNA polymerase was examined . Our results support the following conclusions: (i) the intranucleosomal DNA of SV40-I chromatin, similar to the precursor 75S chromatin complex, is resistant to enzymatic activity; and (ii) SV40-M virion chromatin is modified in a manner which increases the accessibility of viral DNA to enzymes, and the distinction between nucleosomal DNA and linker DNA is absent . Micrococcal nuclease digestion of SV40-I virion chromatin gave a typical nucleosomal DNA ladder pattern with a repeat unit of 205 base pairs of DNA . SV40-I chromatin was sensitive to cleavage with endonuclease BglI, but not with EcoRI . When SV40-I virion chromatin was used as a template, the rate of incorporation of ribonucleoside triphosphates into RNA was 5% of that obtained with naked form SV40 form I DNA . Micrococcal nuclease digestion of SV40-M virion chromatin resulted in submonomeric DNA fragments of approximately 55 base pairs, but no larger repeating unit of DNA was observed . SV40-M virion chromatin was sensitive to cleavage with either BglI or EcoRI and was approximately 20% more susceptible to digestion with DNase I than was SV40-I virion chromatin . The transcriptional efficiency of the extracellular virion chromatin was almost equivalent to that of naked SV40 form I DNA and was 16-fold higher than the rate observed with nuclear virion chromatin . The increased transcriptional activity was dependent upon the presence of nonhistone viral protein VP1 or VP2 or both.

J Cell Biol, 1981 Aug, 90(2), 289 - 99
A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis; Franke WW et al.; The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents . This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting . The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex . DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase . Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins . The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells . It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Eur J Biochem, 1981 Aug, 118(1), 47 - 52
Concerning the mode of action of micrococcin upon bacterial protein synthesis; Cundliffe E et al.; The antibiotic, micrococcin, binds to complexes formed between bacterial 23-S ribosomal RNA and ribosomal protein L11 and, in doing so, inhibits of thiostrepton . In assay systems simulating partial reaction of protein synthesis, micrococcin inhibits a number of processes believed to involve the ribosomal A site while stimulating GTP hydrolysis dependent upon ribosomes and elongation factor EF-G . The latter effect is not observed upon ribosomes lacking a protein homologous with protein L11 . Nor is it apparent upon those containing 23-S RNA previously subjected to the action of a specific methylase known to render ribosomes resistant to thiostrepton . It is concluded that stimulation by micrococcin of factor-dependent GTP hydrolysis results from the binding of the drug to its normal target site which involves 23-S RNA and protein L11.

J Biol Chem, 1981 Jul 25, 256(14), 7549 - 56
Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver; Christensen ME et al.; The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence . Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin . The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines . Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe . In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis . Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific . Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine . Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.

Nucleic Acids Res, 1981 Jul 24, 9(14), 3389 - 401
Effect of thyrotropin on 32P-labelled histones H1 and H3 in specific populations of nucleosomes in the thyroid; Cooper E et al.; Thyrotropin (TSH) increases the labeling of histones of H1 and H3 in thyroid slices incubated with 32Pi . We have prepared nuclei from control and TSH-treated thyroid slices, digested them with micrococcal nuclease, and extracted specific populations of nucleosomes by salt fractionation . Mononucleosomes, derived from the most nuclease-sensitive regions of chromatin, appeared to be selectively enriched in 32P-labeled H1 and H3 . However, we were able to detect TSH enhancement of H1 and H3 labeling only in nucleosome multimers derived from less nuclease-sensitive chromatin . Recent studies have indicated that transcriptionally competent regions of chromatin may be more susceptible to micrococcal nuclease digestion than inactive regions . Our results therefore suggest that H1 and H3 may be actively phosphorylated in transcriptionally competent chromatin; however, they suggest either that hormone-dependent phosphorylation of H1 and/or H3 does not confer transcriptional competence, or that not all transcriptionally competent chromatin is nuclease sensitive.

J Biol Chem, 1981 Jul 10, 256(13), 8608 - 16
Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease . A three-step incision model; Gordon LK et al.; The early steps of excision repair of cyclobutane pyrimidine dimers are investigated . It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus . This nick is not a substrate for T4 polynucleotide ligase . The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I . However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease . A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.

Eur J Biochem, 1981 Jul, 117(2), 375 - 82
Compositional heterogeneity of the chloroplast DNAs from Euglena gracilis and Spinacia oleracea; Schmitt JM et al.; The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using different experimental approaches . Euglena chloroplast DNA has a dG + dC content of 28% . Preparations averaging 20 x 10(6) in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content . Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity . From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC . Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT-rich sequences must occur in short stretches interspersed with dG + dC-rich stretches . Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile . But the higher resolution methods of melting, bouyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition . About 30% of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.

Eur J Biochem, 1981 Jul, 117(2), 245 - 50
Nuclease-sensitive regions on the extrachromosomal r-chromatin from Tetrahymena pyriformis; Borchsenius S et al.; The extrachromosomal DNA coding for the ribosomal precursor in Tetrahymena contains a transcribed region with a size of 6 x 10(3) base pairs plus non-transcribed central and distal spacers . In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared . Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures . The specific DNA fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P-labelled RNA probes . Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region . Specific regions on the r-chromatin are hypersensitive to DNase I . The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3' end . The hypersensitivity of the central part of r-chromatin is also found by autodigestion in isolated nucleoli.

Antibiotiki, 1981 Jul, 26(7), 537 - 45
{Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture}; Sokolova TM et al.; A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon . The optimal conditions for translation of its mRNA were developed . The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells . The higher the purity level of RNA, the higher the translation level . With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA . By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon . The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates . It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system . Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.

J Nutr, 1981 Jul, 111(7), 1258 - 64
Reduced transcription activity of rat liver chromatin after protein restriction and selective digestion of nuclei with micrococcus nuclease; Astrom S et al.; Restriction of protein intake in the diet has been shown to produce a change in transcription activity as determined in vitro . Conditions for a reduced transcription activity were investigated with selective digestion of nuclei with micrococcus nuclease (EC 3.1.4.7) . Liver nuclei from young male rats fed a diet containing either 20 or 3% casein for 6 days were digested with 1.3 microgram of micrococcus nuclease protein/mg of nuclear DNA (specific enzyme activity 15,000 or 150 units/mg of protein) . Part of the chromatin-bound RNA polymerase activity was transferred from the 2,000 x g to the 102,000 x g pellet . Independent of the type of endonuclease use, the specific activity of chromatin-bound and soluble RNA polymerase I plus III was similar in the two groups of rats . After protein restriction RNA polymerase II activity was significantly diminished in the 2,000 x g pellet, and was unchanged in the 102,000 x g pellet . Heparin-stimulated and soluble RNA polymerase II activities were significantly reduced . Number and length of RNA chains synthetized by chromatin-bound RNA polymerase I plus III remained unchanged by dietary treatment . After a low protein diet, RNA polymerase II in the absence and presence of heparin synthesized an unchanged number of RNA chains with reduced length . A selective digestion of chromatin with micrococcus nuclease is needed to show the reduction in RNA polymerase II activity after protein restriction.

J Nutr, 1981 Jul, 111(7), 1249 - 57
Increased transcription activity of rat liver chromatin after protein restriction and limited digestion of nuclei with micrococcus nuclease; von der Decken A et al.; Protein restriction has been shown to produce either an enhancement or a reduction of transcription activity in vitro . Conditions for an enhanced transcription activity were investigated . Young male rats were fed a complete diet containing either 20% or 3% casein for 6 days . Body weight changed +7.4 g/day and -0.5 g/day, respectively . Liver wet weights were 6.8 and 3.7 g and the DNA amounts/g were 1.31 and 1.67 mg . Liver nuclei were incubated without or with 1 unit micrococcus nuclease (EC 3.1.4.7) per milligram of nuclear DNA, and chromatin was fractionated into a 2,000 x g, a 102,000 x g pellet and a supernatant fraction . In the livers of rats fed a low protein diet, chromatin-bound RNA polymerase I plus III and II activity/mg of fractional and nuclear DNA and soluble RNA polymerase activity were increased, while heparin-stimulated RNA polymerase II activity remained unchanged . An increased number of chains was synthesized by RNA polymerase I plus III without change in chain length and incorporation rate per chain . The length and incorporation rate per chain increased, while the number of chains synthesized by RNA polymerase II did not . After stimulation by heparin, an increased number of short chains was synthesized at a lower rate of incorporation per chain . In the complex chromating structures the capacity for RNA synthesis was determined by specific enzyme activity, RNA chain number and length.

Nucleic Acids Res, 1981 Jun 25, 9(12), 2643 - 58
Sequence specific cleavage of DNA by micrococcal nuclease; Horz W et al.; Micrococcal nuclease is shown to cleave DNA under conditions of partial digestion in a specific manner . Sequences of the type 5'CATA and 5'CTA are attacked preferentially, followed by exonucleolytic degradation at the newly generated DNA termini . GC-rich flanking sequences further increase the probability of initial attack . Unexpectedly, long stretches containing only A and T are spared by the nuclease . These results, which were obtained with spared by the nuclease . These results, which were obtained with mouse satellite DNA and two fragments from the plasmid pBR22, do not support the previous contention that it is the regions of high At-content which are initially cleaved by micrococcal nuclease . This specificity of micrococcal nuclease complicates its use in experiments intended to monitor the nucleoprotein structure of a DNA sequence in chromatin.

Nucleic Acids Res, 1981 Jun 25, 9(12), 2761 - 75
The characterisation of 1SF monomer nucleosomes from hen oviduct and the partial characterisation of a third HMG14/17-like in such nucleosomes; Goodwin GH et al.; Nucleosomes released from oviduct nuclei during brief micrococcal nuclease digestions are enriched in transcribed sequences (bloom K.S . and Anderson, J.N . (1978) Cell, 15, 141-150) . Such nucleosomes released into this 1Sf supernatant fraction are enriched in proteins HMG14, 17 and a third lower molecular weight protein which we show in this paper to be related to HMG14 and 17 . This protein, which we call HMGY, runs as a doublet on polyacrylamide gels . A similar doublet is present in smaller quantities in chicken erythrocyte nuclei . Monomer nucleosomes in the 1SF supernatant have been separated by polyacrylamide gel electrophoresis into two main bands . The slower moving band contains the three HMG proteins HMG14, 17 and Y but lacks histone H1.

Nucleic Acids Res, 1981 Jun 25, 9(12), 2659 - 73
High sequence specificity of micrococcal nuclease; Dingwall C et al.; The substrate specificity of micrococcal nuclease (EC 3.1.4.7.) has been studied . The enzyme recognises features of nucleotide composition, nucleotide sequence and tertiary structure of DNA . Kinetic analysis indicates that the rate of cleavage is 30 times greater at the 5' side of A or T than at G or C . Digestion of end-labelled linear DNA molecules of known sequence revealed that only a limited number of sites are cut, generating a highly specific pattern of fragments . The frequency of cleavage at each site has been determined and it may reflect the poor base overlap in the 5' T-A 3' stack as well as the length of contiguous A and T residues . The same sequence preferences are found when DNA is assembled into nucleosomes . Deoxyribonuclease 1 (EC 3.1.4.5.) recognises many of the same sequence features . Micrococcal nuclease also mimics nuclease S1 selectively cleaving an inverted repeat in supercoiled pBR322 . The value of micrococcal nuclease as a "non-specific" enzymatic probe for studying nucleosome phasing is questioned.

Biochemistry, 1981 Jun 23, 20(13), 3695 - 702
Structure of transcriptionally active and inactive nucleosomes from butyrate-treated and control HeLa cells; Egan PA et al.; Nuclei from butyrate-treated or control HeLa cells were separated into micrococcal nuclease sensitive and resistant chromatin . Those regions most sensitive to the nuclease, amounting to some 10% of the chromatin, consisted mainly of mononucleosomes with equimolar amounts of the inner histones H2A, H2B, H3, and H4, very little H1, and equimolar amounts of the two small high-mobility group (HMG) proteins, HMG-14 and -17 . Both in butyrate-treated and in control cells, these nuclease sensitive monomers were some 5--7-fold enriched in DNA sequences which are transcribed into cytoplasmic polyadenylated RNA, while resistant monomers are depleted in the same sequences . Electrophoretic analyses of the transcriptionally active mononucleosomes revealed heterogeneity . Several subcomponents were resolved when monomers of butyrate-treated or control cells were electrophoresed at low ionic strength . Active monomer subcomponents differ in their molar content of HMG-14 and -17, in their content of H1 and A24, and in the length of their DNA . Some minor differences between nucleosomes of butyrate-treated and control cells were observed.

Biochim Biophys Acta, 1981 Jun 22, 644(2), 284 - 94
Phase behaviour in monolayers and in water dispersions of mixtures of dimannosyl diacylglycerol with phosphatidylglycerol; Lakhdar-Ghazal F et al.; Mixtures of dimannosyl diacylglycerol, extracted from the membrane of Micrococcus luteus, with synthetic dipalmitoyl phosphatidylglycerol or with samples of phosphatidylglycerol and phosphatidylinositol, extracted from the same bacterium, have been studied . Through a monolayer (pi, delta V) study and from fluorescence polarization data relative to diphenylhexatriene embedded in vesicles of the mixed lipids, it is shown that the glycolipid interacts with the phospholipids . These interactions are independent of the structure and physical state of the phospholipid acyl chains, of the lipid molecular packing and of the nature of the cations (monovalent, bivalent) present in the aqueous phase . No phase separation was detected, either in monolayers or in water dispersions . Furthermore, the data presented demonstrated a marked influence of the glycolipid on the phase behaviour of phosphatidylglycerol, both in the presence of monovalent (Na+, K+) and bivalent (Ca2+, Mg2+) cations . This point is of particular interest with regard to the highly rigid phase this phospholipid is known to assume in the presence of bivalent cations . It is then suggested that the glycolipid could act as a regulator of the membrane fluidity by preventing a too high rigidity of the lipid phase when bivalent cations are present at the membrane surface.

J Biol Chem, 1981 Jun 10, 256(11), 5802 - 9
Structural integrity of DNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes; Kennedy TD et al.; After treatment at a microsomal nuclease concentration too low to reduce the endogenous amino acid-incorporating activity of freshly prepared reticulocyte lysate, there is little, if any, intact 26 S RNA left in the ribosomes of either wheat germ or rabbit reticulocyte cell-free protein synthesizing extracts . The primary scissions, probably at highly exposed sites in the rRNA of plant and animal ribosomes, produce two fragments which remain complexed until thermal denaturation reveals "hidden breaks." Molecular weights of the fragments are approximately 0.5 x 10(6) and 0.8 x 10(6) in the case of wheat, and 0.4 x 10(6) and 1.3 x 10(6) in the case of rabbit . There is little perceptible degradation of 5 S, 5.8 S, and 18 S rRNA, or of tRNA in the same extracts . Even though limited degradation of 26 S rRNA by a reticulocyte nuclease has been reported to severely impair the translational mechanism in reticulocyte ribosomes, micrococcal nuclease-induced degradation of rRNA, whether limited or extensive, does not seriously impair the ability of reticulocyte lysates to discriminate, by selective polypeptide synthesis, between complex populations of cellular mRNA . In an allied study, it is shown that under conditions well suited to recovery of the 5.8 S/26 S rRNA complex, with its naturally occurring hidden break, 5 S/18 S rRNA complexing is not detectable in the RNA or metabolizing embryos, nor in the RNA from untreated or nuclease-treated protein synthesizing extracts from wheat and rabbit . The significance of this finding is briefly elaborated in relation to the suggestion that 5 S rRNA may interact with the M2(6)A-m2(6)A hairpin near the 3'-end of 18 S rRNA.

Biochemistry, 1981 Jun 9, 20(12), 3610 - 4
Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease; Berger NA et al.; Synthesis of DNA and poly(adenosine diphosphoribose) {poly(ADPR)} was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease . M . luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells . UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis . In UV-irradiated cells treated with M . luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold . Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis . Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis . Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis . These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA . The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).

J Biochem (Tokyo), 1981 Jun, 89(6), 1881 - 8
The interaction of H1 histone with nucleosome core; Ishimi Y et al.; The structural changes caused by H1 histone depletion of calf thymus chromatin or nucleosome monomer were studied by using the criterion of accessibility to micrococcal nuclease . When H1 histone was depleted from monomer, the DNS structure unfolded and became sensitive to the nuclease at a particular site on the nucleosome core, 105 base pairs from a chain terminus of core DNA . In reconstitution experiments with chromatin, this sensitive site on the core could be protected from the attack of the nuclease only when H1 histone was present . These results indicate that H1 histone interacts not only with the nucleosome linker region, but also with the core particle.

Arch Toxicol, 1981 Jun, 47(3), 169 - 77
Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration; Naslund B et al.; The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied . After a single oral dose of dimethylnitrosamine (2-75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease . Nucleosomes were separated on sucrose density gradients . There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals . The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 gav) and nucleosomal peak fractions were used for isolation of DNA . DNA was heat denatured in 7 M urea or formamide . After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area . The increase in DNA fragmentation was dimethylnitrosamine dose response dependent . When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight . A good correlation between mobility and log of chain length of phi chi 174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea . DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides . Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin . It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.

Mutat Res, 1981 Jun, 82(1), 11 - 22
Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli; Seeberg E; Partially purified extracts of Escherichia coli containing either uvrA+ or a mixture of uvrB+ and uvrC+ gene products were tested for an endonuclease activity on DNA treated with 8-methoxypsoralen plus 360-nm light . Neither of these fractions was active alone . The combined fractions, however, caused extensive strand cleavage of the psoralen-treated DNA . The endonuclease activity was dependent upon addition of ATP and Mg2+ to the reaction mixtures, and hence appeared similar to the UV-endonuclease activity previously shown to be reconstituted from the same fractions . It is concluded that the uvr+ gene products in these fractions interact to cause breakage of both psoralen-treated and UV-irradiated DNA . An examination of the dose-dependence relationship of the break formation in psoralen-treated DNA revealed that the enzyme acts upon psoralen mono-adducts . By varying the experimental conditions to increase the ratio of interstrand cross-links to mono-adducts it was found that the enzyme also acts upon cross-links, but with lower efficiency than for mono-adducts . Further studies of break formation in UV-irradiated DNA showed that elimination of pyrimidine dimers by treatment with photoreactivating enzyme in the light resulted in a loss of endonuclease-sensitive sites . This shows directly that pyrimidine dimers are the lesions recognized by the complemented uvr+ gene products in UV-irradiated DNA . For comparison, another endonuclease acting at pyrimidine dimers in DNA, the Micrococcus luteus UV-endonuclease, was also tested with psoralen-treated DNA, but no activity was observed . This and other data indicate that the repair endonuclease encoded by the uvr+ genes in E . coli is basically different from the other dimer-specific endonucleases previously characterized.

Cancer Res, 1981 Jun, 41(6), 2433 - 8
Altered gel electrophoretic mobility of bleomycin-mediated release of nucleosomal DNA; Kuo MT; Nucleosomal DNA isolated from bleomycin-treated nuclei shows retarded electrophoretic mobility in neutral gels as compared with the sample isolated from micrococcal (or endogenous) nuclease-digested nuclei . The retardation in electrophoretic mobility is probably due to the presence of single-strand regions in the DNA duplex of bleomycin-treated samples as determined by single-strand-specific S1 nuclease digestion and buoyant density measurements . This observation argues against the possibility that the generation of nucleosomal DNA following drug treatment of nuclei is due to an activation of endogenous nuclease by bleomycin and strongly suggests that the drug has a unique feature of action on chromatin.

Biochim Biophys Acta, 1981 May 29, 653(3), 391 - 407
Role of cytosol proteins in DNA chain growth and chromatin replication in Friend erythroleukemia cell nuclei; Muller MT et al.; The influence of cytosol proteins on the replication of DNA and chromatin in isolated nuclei from Friend erythroleukemia cells has been investigated . The overall process has been clearly shown to proceed stepwise . In the absence of cytosol proteins DNA chain growth tends to stop after the addition of approximately 200 nucleotides to the ends of growing chains . In the presence of cytosol proteins these sections grow to approximately 250 nucleotides, and participate in the stepwise extension of the replication process through adjacent nucleosomal sections of the template . Immediately following pulse labeling, the newly replicated DNA resides in a chromatin form which appears to be relatively resistant to digestion by micrococcal nuclease . During a chase interval, the association of the pulse-labeled DNA with nuclear proteins matures to a form which yields lengths of DNA upon digestion with micrococcal nuclease that correspond to mono-, di-, tri- and polynucleosomal units of chromatin . In the absence of cytosol proteins the nuclease resistant state of the labeled DNA tends to predominate and persist . The data support the view that DNA replication in a chromosomal setting proceeds stepwise over successive nucleosomal sections of template made accessible by the interaction of the cytosol proteins at or near the replication fork.

J Biol Chem, 1981 May 25, 256(10), 5077 - 86
A ribonuclease-resistant cytoplasmic 10 S ribonucleoprotein of chick embryonic muscle . A potent inhibitor of cell-free protein synthesis; Sarkar S et al.; A ribonucleoprotein (RNP) particle sedimenting at 10 S in sucrose gradients had been isolated from the post-polysomal fraction of homogenates of 14-day-old chick embryonic leg and breast muscle by sucrose gradient fractionation and gel filtration . The 10 S RNP contains a 4 S RNA species (base composition: AMP, .3%; GMP, 22.2%; CMP, 24.2%; and UMP, 23.2%), and shows three major bands in the 70-90-nucleotide size range by polyacrylamide gel electrophoresis in 99% formamide . The 4 S RNA does not contain oligo(U)- and oligo(A)-rich tracts . The RNP has a characteristic buoyant density of 1.410 g/ml, which corresponds to an RNA/protein ratio of about 1:4 . The UV absorption spectra of the RNP is very distinct from that of its RNA component . Both 4 S RNA and the 10 S RNP are potent inhibitors of translation of a variety of mRNAs such as chick muscle poly(A)+ mRNA, rabbit globin mRNA, EMC virus RNA, and poly(A)- and mRNA of rat liver in micrococcal nuclease-treated rabbit reticulocyte lysate . The inhibitory action of the RNA and the RNP on mRNA translation appears to involve the initiation process . The RNA and RNP do not have a nuclease activity associated with them . The hyperchromicity profile of the inhibitory RNA with increasing temperature indicates that it does not contain a significant amount of double-stranded structure . This is also supported by the complete loss of biological activity of the RNA by treatment with pancreatic RNase . In contrast, the inhibitory activity of the RNP was resistant to RNase . Electrophoresis of the protein moieties of the inhibitory RNP using both one- and two-dimensional gel techniques in the presence of sodium dodecyl sulfate shows a complex pattern of polypeptides of Mr = 12,000-150,000 . The protein pattern of the 10 S particle is quite different from those of free and polysomal mRNP and poly(A)-protein complexes of chick embryonic muscles, indicating that most, if not all of the mRNA-associated proteins, are absent in the 19 S RNP . The properties of the inhibitory RNA indicate that it is different from the various low molecular weight RNA species which are involved in the modulation of protein synthesis in cell-free systems . It is concluded that the 10 S particle represents a novel class of RNP, which may be involved in posttranscriptional regulation of protein synthesis in embryonic muscles.

Cancer Lett, 1981 May, 12(4), 311 - 21
Methylation of chromosomal DNA by two alkylating agents differing in carcinogenic potential; Berkowitz EM et al.; The affinity of 2 methylating agents, N-methyl-N-nitrosourea (MNU), a potent carcinogen, and dimethyl sulfate (DMS), a very weak or non-carcinogen, for specific structural or functional regions of DNA has been studied in an in vitro system using rat liver nuclei . The release of alkylated nucleotides from nuclei following limited nuclease digestion was measured . Under restrictive conditions, pancreatic DNase I preferentially digests DNA sequences active in RNA transcription while micrococcal nuclease digests spacer DNA between nucleosome cores . Nucleotides methylated by methylnitrosourea were preferentially released early during the digestions, suggesting a localization in both actively transcribing regions and spacer DNA . DMS alkylation, on the other hand, showed a random distribution in chromosomal DNA as measured by micrococcal nuclease and only limited accumulation in transcribing DNA.

J Biochem (Tokyo), 1981 May, 89(5), 1573 - 80
Geranylgeranyl pyrophosphate synthetase lacking geranyl-transferring activity from Micrococcus luteus; Sagami H et al.; Geranyl pyrophosphate synthetase, which catalyzes the condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate to give geranyl pyrophosphate, was purified 490-fold from Micrococcus luteus extracts by DEAE-Sephadex, hydroxylapatite, and Sephadex G-100 column chromatography . The enzyme has a pH optimum at 7.7 and the molecular weight was estimated to be 70,000 by Sephadex gel filtration . The Km values for isopentenyl pyrophosphate and dimethylallyl pyrophosphate were 8 microM and 62 microM, respectively . The enzyme required Mg2+ for maximum activity . Tween 80 showed a stimulative effect whereas Triton X-100 was rather inhibitory on the enzyme activity . Inorganic pyrophosphate and iodoacetamide were both potent inhibitors of the enzyme . The purified enzyme fraction was also capable of catalyzing the synthesis of geranylgeranyl pyrophosphate from isopentenyl pyrophosphate and farnesyl pyrophosphate, but lacked geranyl-transferring activity . The catalytic activities of geranylgeranyl pyrophosphate synthesis and geranyl pyrophosphate synthesis were affected differently by iodoacetamide and Triton X-100 . This enzyme fraction may be a mixture of two enzymes, geranyl pyrophosphate synthetase and geranylgeranyl pyrophosphate synthetase catalyzing the reactions of C5 greater than C10 and C15 greater than C20, respectively, or a single enzyme with two independent catalytic sites responsible for the C5 greater than C10 and C15 greater than C20 reactions . In any case, the existence of a new geranylgeranyl pyrophosphate synthetase different from the known geranylgeranyl pyrophosphate synthetase catalyzing the continuous condensation reaction of C5 greater than C10 greater than C15 greater than C20 was demonstrated.

J Biochem (Tokyo), 1981 May, 89(5), 1445 - 52
Biosynthesis of menaquinones . Enzymatic prenylation of 1,4-dihydroxy-2-naphthoate by Micrococcus luteus membrane fractions; Saito Y et al.; 1,4-Dihydroxy-2-naphthoate:polyprenyltransferase was detected in the membrane fraction from Micrococcus luteus . The specificity of the enzyme ws so tolerant as regards the prenyl-donating substrate that prenyl pyrophosphates ranging in chain length from C15 to C45 were active as substrates . The monophosphate esters were also active, though the reactivities were much lower than those of the corresponding pyrophosphates . The enzyme showed rigorous specificity with respect to the aromatic substrate . Neither 1,4-dihydroxynaphthalene nor its 2-methyl derivative was active at all . 1,4-Dihydroxy-3-methyl-2-naphthoate could be prenylated to afford menaquinone, but the reactivity was much less than that of its demethyl derivative . These results support the view that menaquinone biosynthesis involves the prenylation of 1,4-dihydroxy-2-naphthoate prior to decarboxylation or methylation.

Mol Cell Endocrinol, 1981 May, 22(2), 195 - 210
Isopycnic banding in metrizamide of the uterine cytosol and nuclear estradiol receptor; Baskevitch PP et al.; We have characterized the cytosol and the nuclear estrogen receptors (RE) of immature lamb uterus and their complexes formed with DNA and chromatin by using metrizamide isopycnic gradients . In low salt, the cytosol RE had a density of 1.238 +/- 0.002 g/cm3 . This density was increased by Ca2+-activated proteolysis (1.275 g/cm3) and heat transformation of the receptor (1.257 g/cm3) and lowered by trypsin treatment (1.20 g/cm3), DNA binding (1.20 g/cm3) or molybdate treatment . In high salt (0.5 M KCl) both the native and the heat-"transformed" cytosol RE banded at the same density of 1.26 g/cm3 . The 8S RE had the same density when bound to E2 or to 4-hydroxy-tamoxifen . Endometrial nuclei purified after nuclear translocation of RE were digested by micrococcal nuclease to solubilize the nuclear RE under low salt conditions . The majority of the extracted nuclear RE had a density similar to that of the 8S cytosol RE (1.239 +/- 0.002) and thus was different from the proteolyzed and heat "transformed" forms . Conversely, after a slight digestion of the nuclei, RE banded with chromatin at 1.208 +/- 0.001 g/cm3 . In low salt, both forms of the nuclear RE, but not the trypsin proteolyzed nuclear RE, were displaced by naked DNA added in vitro . We conclude that the cytosol RE and the free nuclear RE have, in low salt, the same density and DNA-binding ability and that metrizamide isopycnic analysis is a good method for quantifying the interactions of the receptor with DNA and chromatin.

Cancer Res, 1981 May, 41(5), 1829 - 33
Photoreactivation of ultraviolet radiation-induced pyrimidine dimers in neonatal BALB/c mouse skin; Ananthaswamy HN et al.; The numbers of ultraviolet light (UV)-induced pyrimidine dimers in the DNA of neonatal BALB/c mouse skin were measured by assessing the sensitivity of the DNA to Micrococcus luteus UV endonuclease . Irradiation of neonatal BALB/c mice with FS40 sunlamps caused a dose-dependent induction of endonuclease-sensitive sites (pyrimidine dimers) in DNA extracted from back skin . Exposure of these UV-irradiated neonatal mice to photoreactivating (PR) light ("cool white" fluorescent lamp and incandescent lamp) caused a reduction in the number of pyrimidine dimers in the DNA, as revealed by a shift in low-molecular-weight DNA to high-molecular-weight DNA . In contrast, DNA profiles of the skin of either UV-irradiated mice or UV-irradiated mice kept in the dark for the same duration as those exposed to PR light did not show a loss of UV-induced endonuclease-sensitive sites . Furthermore, reversing the order of treatment, i.e., administering PR light first and then UV, did not produce a reduction in pyrimidine dimers . These results demonstrate that PR or UV-induced pyrimidine dimers occurs in neonatal BALB/c mouse skin . The optimal wavelength range for in vivo PR appears to be in the visible region of the spectrum (greater than 400 nm) . Although dimer formation could be detected in both dermis and epidermis, PR occurred only in the dermis . Furthermore, the PR phenomenon could not be detected in the skin of adult mice from the same inbred strain.

Proc Natl Acad Sci U S A, 1981 May, 78(5), 3118 - 22
Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi; Sano H et al.; Two molecular weight forms of DNA (cytosine-5-)-methyltransferase {S-adenosyl-L-methionine:DNA (cytosine-5-)- methyltransferase, EC 2.1.1.37}, both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle . The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mt-) and female (mt+) mating types . The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo . Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites and the Mr 200,000 enzyme was shown to methylate both unmethylated and hemimethylated sites, the latter at an elevated rate . Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component . The data suggest strongly that the Mr 200,000 enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase . It is proposed that methylation of chloroplast DNA in female gametes and zygotes is regulated by assembly of the multimeric Mr 200,000 active enzyme, which in turm determines the maternal inheritance of chloroplast DNA.

J Biochem (Tokyo), 1981 May, 89(5), 1375 - 89
Phasing of nucleosomes in SV40 chromatin reconstituted in vitro; Hiwasa T et al.; Phasing of nucleosomes on SV40 DNA was studied by the reconstitution of chromatin from SV40 DNA form I and core histones . The reconstituted chromatin sedimented with increasing S values as the histone : DNA ratios increased, and the buoyant densities in CsCl decreased concomitantly . The average repeat lengths of nucleosomes in the chromatin reconstituted at ratios of 1.0 and 1.5 were estimated to be 168 and 143 base pairs, respectively, by electrophoretic analysis of DNA fragments generated by micrococcal nuclease digestion . The chromatin generated a series of DNA bands that differed in size by about 10 nucleotides upon DNase I digestion followed by heat-denaturation . Phasing of nucleosomes was probed by the use of single-site restriction endonucleases, EcoRI, BamH1, BglI, and HpaII: the latter two cleave DNA at and near the origin of DNA replication and transcription . The form I DNA in the chromatin reconstituted at ratios of 0.5, 1.0, and 1.5 was cleaved up to 60 to 80%, 20 to 60%, and 0 to 10%, respectively . Although the frequency of cleavage by these enzymes was not very different at the ratio 0.5, the BglI site became relatively more susceptible than the other sites at the ratio 1.0 . At the ratio 1.5, the DNA was almost resistant to these enzymes, though a significant amount (10%) was cleaved by BglI . These results suggest that the origin is the site unfavorable for nucleosome phasing although the region can be almost completely covered with nucleosomes at higher histone : DNA ratios . The fraction of chromatin immunoprecipitated with anti-T serum after in vitro T antigen binding also decreased with increase in the histone : DNA ratios . The results suggest that T antigen binds preferentially to the internucleosomal region . T antigen preferentially bound to the chromatin reconstituted with the DNA fragment containing the origin . Inefficient phasing of nucleosomes at the origin of DNA replication may facilitate the binding of T antigen to the origin.

Biochim Biophys Acta, 1981 Apr 17, 674(1), 144 - 54
Effects of deoxyribonuclease I and micrococcal nuclease on the release of dexamethasone-receptor complex from nuclei of sensitive and insensitive hepatoma cell lines; Taira M et al.; (1) Fu5 cells were sensitive to the glucocorticoid inhibition of cell growth and the hormonal induction of tyrosine aminotransferase (but not fructose-1,6-bisphosphatase and glycogen synthase) . AH-130 and AH-7974 cells were insensitive to both effects . (2) The release of {3H}dexamethasone radioactivity from the nuclei of Fu5 and AH-130 cells preincubated with {3H}dexamethasone increased as the KCl concentration increased from 0 to 0.4 M, with no significant difference between the two cell lines . (3) The radioactivity was more sensitively released in Fu5 nuclei than in AH-130 nuclei upon treatment with DNAase I . The release of radioactivity was always larger than the release of DNA in both cell nuclei . In contrast to DNAase I, micrococcal nuclease treatment did not show any difference between the two cell lines in the release of radioactivity from nuclei, always showing a release of radioactivity equal to that of DNA.

Biochem J, 1981 Apr 15, 196(1), 115 - 9
Chromatin organization in the rat hypothalamus during early development; Whatley SA et al.; The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease . A short chromatin repeat length (approx . 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx . 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx . 174 base-pairs) . Whereas in cortical neurons the chromatin repeat length shortened from approx . 200 base-pairs in the foetus to approx . 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.

Biochemistry, 1981 Apr 14, 20(8), 2212 - 9
Stereochemical and kinetic investigation of 32P-labeled inorganic phosphate exchange reaction catalyzed by primer-independent and primer-dependent polynucleotide phosphorylase from Micrococcus luteus; Marlier JF et al.; The SP diastereomer of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) is a substrate for the 32P-labeled inorganic phosphate exchange reaction catalyzed by the T and I forms of polynucleotide phosphorylase . The exchange reaction occurs with retention of configuration . This exchange reaction is very slow when only ADP alpha S(SP) is presented but is greatly activated by dinucleotide primers and ADP alpha S(RP), although the latter is not a substrate for the exchange reaction . Ap(S)A(RP) is an approximately 50% better activator of the exchange than the SP diastereomer . Furthermore, high levels of the ADP alpha S(SP) eliminate the activation by primers and by ADP alpha S(RP) . A phosphatase activity is present with the I form of the enzyme which converts ADP alpha S(RP) to AMPS . This activity may be responsible for the formation of the 5'-phosphate end group for de novo polymerization or for the processivity of this reaction.

Biochemistry, 1981 Apr 14, 20(8), 2127 - 32
Micrococcal nuclease cleavage of chromatin displays nonrandom properties; LaFond RE et al.; A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process . DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated . In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage . The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical . Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5 . The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed . Some possible mechanisms to account for this property are discussed.

J Biol Chem, 1981 Apr 10, 256(7), 3313 - 8
The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin; Perry M et al.; We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2 . DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths . Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei . This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones . These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.

J Cell Sci, 1981 Apr, 48, 171 - 9
Ultrastructural studies on chromatin digestion by micrococcal nuclease in the presence of polyamines; Rowlatt C et al.; Nuclei purified from C57BL mouse submandibular salivary gland were treated with a range of micrococcal nuclease concentrations and times of treatment (from 0.5 unit for 2.5 min to 50 units for 30 min) in the presence of polyamines . About 50% of the chromatin was solubilized initially but with prolonged digestion this chromatin became insoluble again . Electron microscopy showed destruction of the finely dispersed chromatin with mild digestion, followed by aggregation of chromatin with more vigorous digestion . The early disappearance of finely dispersed chromatin filaments was not accompanied by preferential solubilization of chromatin associated with RNA polymerase II (euchromatin) . These data suggest that the polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.

Eur J Biochem, 1981 Apr, 115(3), 545 - 50
Studies on the thermal denaturation of histone-H1-depleted chromatin; Dimitrov SI et al.; The multiphasic thermal denaturation profile of histone-H1-depleted chromatin was studied by using a nucleoprotein preparation which lacked the first high temperature transition at about 72 degrees C . Such a preparation was obtained by heating at 72 degrees C H1-depleted chromatin, the DNA of which was cross-linked with psoralen to ensure a complete renaturation of DNA upon cooling . When this nucleoprotein was redenatured, its melting profile was found to be significantly altered: only one high temperature peak centered at about 82 degrees C was observed in addition to the low temperature transition at about 53 degrees C . The kinetics of digestion of this material with micrococcal nuclease showed a limit digest equal to that found for the 'native' H1-depleted chromatin but the rate of hydrolysis was higher . The monomer particles prepared from this nucleoprotein were found similar to the 'native' monosomes in respect to protein:DNA ratio and size of DNA but showed an altered melting profile: the premelt area was broader, bigger, and centered at lower temperature; the main peak was reduced in size with no change in its melting temperature . On the basis of these data, the following conclusions were drawn: (a) the last two thermal transitions in H1-depleted chromatin most likely reflect the presence within each nucleosome of two regions with different stability of DNA; (b) DNA involved in the first high thermal transition of H1-depleted chromatin belongs to nuclease-accessible DNA, and (c) the main peak in the biphasic melting profile of the monomer particles reflects the denaturation of DNA regions which in the polymer nucleoprotein are involved in the two high temperature transitions.

Can J Microbiol, 1981 Apr, 27(4), 421 - 5
Inhibition of carotenoid synthesis in Micrococcus roseus; Cooney JJ et al.; Micrococcus roseus forms bicyclic keto-carotenoids . The effects of nicotine, piperonyl butoxide, and 2-(4-chlorophenylthio)-triethylamine hydrochloride (CPTA) were studied with regard to their ability to selectively inhibit carotenogenesis in the organism . Nicotine caused accumulation of beta-zeacarotene; piperonyl butoxide caused accumulation of phytoene and traces of phytofluene, zeta-carotene, and beta-zeacarotene . In both cases canthaxanthin biosynthesis was inhibited . CPTA inhibited canthaxanthin synthesis and caused accumulation of beta-zeacarotene and gamma-carotene and their mono- and di-hydroxy derivatives . Regardless of the inhibitor used, canthaxanthin was the major colored carotenoid biosynthesized . The expected precursors of carotenoid cyclization, neurosporene and (or) lycopene, were not detected in CPTA- or nicotine-inhibited cultures . Therefore, carotenoid cyclization in M . roseus does not involve neurosporene or lycopene and must occur early in carotene biosynthesis, prior to the formation of beta-zeacarotene, zeta-Carotene is proposed as the cyclization substrate and beta-zeacarotene as the substrate for oxygen insertion.

J Bacteriol, 1981 Apr, 146(1), 285 - 90
Postreplication repair in Saccharomyces cerevisiae; Resnick MA et al.; Postreplication events in logarithmically growing excision-defective mutants of Saccharomyces cerevisiae were examined after low doses of ultraviolet light (2 to 4 J/m2) . Pulse-labeled deoxyribonucleic acid had interruptions, and when the cells were "chased," the interruptions were no longer detected . Since the loss of interruptions was not associated with an exchange of pyrimidine dimers at a detection level of 10 to 20% of the induced dimers, we concluded that postreplication repair in excision-defective mutants (or leaky mutants) does not involve molecular recombination . Pyrimidine dimers were assayed by utilizing the ultraviolet-endonuclease activity in extracts of Micrococcus luteus and newly developed alkaline sucrose gradient techniques, which yielded chromosomal-size deoxyribonucleic acid after treatment of irradiated cells.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2263 - 7
Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena; Blackburn EH et al.; A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes . This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules . Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length . The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion . Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion . The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

Biokhimiia, 1981 Apr, 46(4), 642 - 51
{Structure of the Micrococcus lysodeikticus respiratory chain using low concentrations of Triton X-100 and glutaric aldehyde}; Turgenbaeva DA et al.; Using low (0.0025 -- 0.025%) concentrations of Triton X-100, the correlation between the decrease of NADN- and malate oxidase activities and NADH- and malate dehydrogenase release in large fragments of Micrococcus lysodeikticus membranes was established . This was accompanied by membrane suspension clearance and a decrease of microviscosity of the membrane lipid component . Using NADH-dehydrogenase, it was shown that the attachment of NADH-dehydrogenase to the membrane treated with glutaric aldehyde occurs in two steps, this being indicative of different environment of this enzyme in the membrane . The data obtained are discussed in terms of laterally heterogenous structure of the bacterial membrane with respect to the electron transport enzymes, in particular in favour of an existence of individual sites of the membrane containing dehydrogenases rather than other respiratory chain components.

Proc Natl Acad Sci U S A, 1981 Apr, 78(4), 2189 - 93
Enhanced phosphorylation of high-mobility-group proteins in nuclease-sensitive mononucleosomes from butyrate-treated HeLa cells; Levy-Wilson B; The protein composition of nucleosome fractions differing in their sensitivity to micrococcal nuclease and derived from butyrate-treated or untreated HeLa cells has been compared . Most of the high-mobility-group-14 (HMG-14) and HMG-17 content of HeLa cell chromatin is associated with those nucleosomes that are preferentially sensitive to micrococcal nuclease . Furthermore, electrophoresis of these two HMG proteins from the transcriptionally active chromatin fraction MN1 of butyrate-treated cells resolves them into a series of bands . The multiple band pattern of HMG-14 and -17 from butyrate-treated cells results from hyperphosphorylation rather than hyperacetylation . Phosphorylation of these two small nonhistone proteins may play some role in the modulation of the structure of transcriptionally active chromatin.

J Biol Chem, 1981 Mar 25, 256(6), 2656 - 61
Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites; Ehrenfeld E et al.; The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined . Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation . Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system . Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action . Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates . On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation . These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation . Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA . Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable . We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.

Biochem J, 1981 Mar 15, 194(3), 963 - 74
The variation with age of nuclear phosphoproteins released during micrococcal-nuclease digestion and nucleosomal phosphoproteins in three cell types from rat liver; Zongza V et al.; The nucleosomal non-histone phosphoproteins, and phosphoproteins released during the digestion of nuclei by micrococcal nuclease, were studied in three rat liver nuclear populations, namely diploid stromal, diploid parenchymal, and tetraploid parenchymal nuclei, which were separated by zonal centrifugation, in 3-week-old rats in which the parenchymal cells contain diploid nuclei and in 2- and 4-month-old rats with increasing proportions of parenchymal tetraploid nuclei . Qualitative and quantitative differences in nucleosomal phosphoprotein band patterns were found among different types of nuclei and ages . More phosphoprotein bands were found in nucleosomes derived from parenchymal than stromal nuclei . The number of phosphoproteins released during micrococcal-nuclease digestion increased with age for parenchymal nuclei . The significance of these results, considered in conjunction with the increase of DNA repeat length and decrease of nuclease accessibility with age, is discussed.

Vopr Virusol, 1981 Mar-Apr, (2), 210 - 5
{Animal and viral mRNA translation in a mRNA-dependent cell-free system and rabbit reticulocytes}; Kisling U et al.; A system of cell-free synthesis from rabbit reticulocytes is described . A method of repeated bloodlettings for induction of reticulocytosis was used . The lysate of washed reticulocytes was treated with micrococcus nuclease enabling effective inhibition of synthesis in endogenous templates . The translating activity is manifested most completely in the presence of the following compounds: ATP, GTP, ATP-regenerating system, SH-compounds, polyamines, tRNA . This system must be optimized in K+ and Mg2+ concentrations for different templates . The system is tolerant to high concentrations of rRNA which allows the utilization of total RNA preparations in the experiments . This system may be satisfactorily used for translation of mRNA of different origins.

Mol Cell Biol, 1981 Mar, 1(3), 237 - 44
Permeabilization of ultraviolet-irradiated Chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus; Yarosh DB et al.; Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity . This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid . The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected . In addition, there was no increase in ultraviolet resistance in treated cells . This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity.

Biochem J, 1981 Mar 1, 193(3), 729 - 35
Influence of the alpha-, beta- and gamma-subunits of the energy-transducing adenosine triphosphates from Micrococcus lysodeikticus in the immunochemical properties of the protein and in their reconstitution studied by a radioimmunoassay method; Larraga V et al.; We developed a sensitive and specific radioimmunoassay of the energy-transducing adenosine triphosphatase (F1-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and extended the assay to the alpha-, beta- and gamma-subunits of the enzyme . We isolated these subunits and studied cross-reactions . We found the immunochemical properties of alpha- and beta-subunits to differ, and gamma-subunits showed an intermediate behaviour between that of alpha- and beta-subunits . Our findings indicate that each subunit of M . lysodeikticus F1-ATPase has its own identity and that conformational antigenic determinants and/or co-operative antigenic sites-arise from subunit assembly . Equimolecular amounts of alpha- and beta-subunits (up to three copies of each) reconstituted partially the immunochemical properties of the ATPase molecule, and addition of 2 mol of gamma-subunit per mol of alpha 3 beta 3 complex improved reconstitution . Our findings describe the first reconstitution of biological activity of this ATPase by assembly of the isolated subunits, and provide support for earlier proposals on the stoicheiometry of the alpha 3 beta 3 gamma 2 type for M . lysodeikticus F1-ATPase . The radioimmunoassay method affords opportunities to study the homologies between different energy-transducing ATPases and their constituent polypeptides before the primary structure of these complex proteins has been determined.

What is Food Microbiology?, What Is Botulism?, What Is Growth Medium?, What Is Dna?, What Is Biofilm?, i, Bacteria, c, Microorganisms, c, Microbe, i, Bacteriology, r, Microorganism, o, Antimicrobials, o, Bacteria, e, Meningococcus, i, Bacillus, r, Pathogenic bacterium, a, Thermophiles, i, Microorganism, r, Pathogenic bacterium, i, Staphylococcus, r, Culture medium, c, Antimicrobial, i, S. cerevisiae, s, Microbial, s, Microorganism, e, Halophilic bacterium, e, Escherichia coli, n, Salmonella typhimurium, e, Yeasts, e, Growth media, n, Yeasts, r, Growth media

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005