The structure of SV 40 chromatin.

Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.     

Regulation of potassium uptake in the sodium-resistant (NaCl(r)) and thalium-resistant (TlCl(r)) mutant strain of diazotrophic cyanobacterium Anabaena variabilis

A thalium chloride-resistant (TlCl(r)) mutant strain and a sodium chloride-resistant (NaCl(r)) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K(+)) analog, and nitrosoguanidine (NTG), respectively. The TlCl(r) mutant strain was found to be defective in K(+) transport and showed resistance against 10 microM TlCl. However, it also showed sensitivity against NaCl (LD(50), 50 m M). In contrast, neither wild-type A. variabilis nor its NaCl(r) mutant strain could survive in the presence of 10 microM TlCl and died even at 1 microM TlCl. The TlCl(r) mutant strain exhibited almost negligible K(+) uptake, indicating the lack of a K(+) uptake system. High K(+) uptake was, however, observed in the NaCl(r) mutant strain, reflecting the presence of an active K(+) uptake system in this strain.DCMU, an inhibitor of PS II, inhibited the K(+) uptake in wild-type A. variabilis and its TlCl(r) and NaCl(r) mutant strains, suggesting that K(+) uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention is further supported by the inhibition of K(+) uptake under dark conditions. Furthermore, the inhibition of K(+) uptake by KCN, DNP, and NaN(3) also suggests the involvement of oxidative phosphorylation in the regulation of an active K(+) uptake system.The whole-cell protein profile of wild-type A. variabilis and its TlCl(r) and NaCl(r) mutant strains growing in the presence of 50 m M KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlCl(r) mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K(+) and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaCl(r) mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt tolerance of the NaCl(r) mutant strain was owing to increased K(+) uptake and accumulation, whereas the salt sensitivity of the TlCl(r) mutant strain was owing to the lack of K(+) uptake and accumulation.     

The structure of nucleoprotein cores released from adenovirions.

The morphology, protein composition and DNA organization of nucleoprotein core complexes isolated from type 5 adenovirions have been examined by electron microscopy and biochemical techniques. The morphology of such core structures is in some ways strikingly similar to that exhibited by cellular chromatin. 'Native' core preparations contain compact and less highly-folded forms: the latter appear as thick fibres, 150-300A in diameter. Upon exposure to 0.4M NaCl, adenovirus cores undergo a transition to a beaded string form, reminiscent of nucleosomes. Of the three arginine-rich proteins, polypeptides V, VII and mu present in 'native' cores, only polypeptide VII remains associated with viral DNA in the presence of 0.4M NaCl. We therefore conclude that the nucleosome-like beads are constructed solely of polypeptide VII. The results of micrococcal nuclease digestion experiments suggest that polypeptide VII is sufficient to protect some 100-300bp of adenoviral DNA.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

Characterization of DNA-Binding Proteins Involved in the Assembly of Mitochondrial Nucleoids in the Yeast Saccharomyces cerevisiae

Mitochondrial nucleoids (mt-nucleoids) isolated from the yeastSaccharomyces cerevisiae were analyzed to identify the proteincomponents that are involved in the compact packaging of mtDNA.The isolated mt-nucleoids were disassembled by the additionof 2 M NaCl and the disassembled mt-nucleoids were reassembledonce again into compact structures by dialysis against a bufferthat contained NaCl at concentrations below 0.1 M, as monitoredby staining of the DNA with 4',6-diamidino-2-phenylindole. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa,50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolatedmt-nucleoids by column chromatography on DNA cellulose afterdigestion of mt-nucleoids by DNase I in the presence or absenceof 2 M NaCl. Purified mtDNA was compactly packaged into nucleoid-likestructures upon the addition of fractions that contained DNA-bindingproteins and subsequent dialysis to reduce the concentrationof NaCl. Five proteins, with molecular masses of 67 kDa, 52kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinityfor double-stranded DNA than that of the 20-kDa protein. Thefraction that contained the five DNA-binding proteins otherthan the 20-kDa protein was also able to fold mtDNA compactlyinto nucleoid-like structures. By contrast, the combinationof the 20-kDa protein and mtDNA resulted in formation of lesstightly packed, string-of-bead structures. These results suggestthat at least six different DNA-binding proteins are involvedin the organization of the mt-nucleoids. (Received April 7, 1995; Accepted July 10, 1995)     

Some properties of tobacco protoplast chromatin.

Chromatin was prepared from tobacco-leaf protoplasts. Its solubility in increasing molarities of NaCl was studied and the structure of the soluble fraction observed by electron microscopy. We demonstrate that in plants, the DNA and histones are associated in beaded structures similar to those called omicron-bodies or nucleosomes in animal chromatin. The nucleosomes were associated with DNA in either compact or extended forms. The compact arrangement was predominant in the fraction solubilized between 0.1 and 0.4 M NaCl. The extended form, present at 0.5 and 0.6 M NaCl. showed DNA filaments of various lengths interspacing the nucleosomes. At these ionic strengths ring structures were present, associated with the DNA. At 0.7 M NaCl and above, only DNA filaments were present, occasionally associated with big rings, and nucleosomes were compoetely dissociated. Free DNA molecules were present at all ionic strengths used. The possible origin and significance of the rings are discussed.     

Composition of the folded chromosome from Anabaena variabilis

A "heavy" nucleoid (folded chromosome) from A. variabilis has been isolated in preparative amounts. The composition of the folded chromosome and that of a more simple DNA--protein complex isolated from the "heavy" nucleoid of A. variabilis by chromatography on a column with methylated albumin (MAK) were studied. It was shown that the "heavy" nucleoids contain total cell DNA in a complex with the definite membrane fragment, which can be discovered by a large number of membrane proteins, phospholipids, lipopolysaccharides and amino sugars. After MAK chromatography the DNA--protein complex also contains total cellular DNA, a negligible amount of membrane polypeptides and noticeable amounts of phospholipids and lipopolysaccharides.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

Simple isolation of DNA hydrophobically complexed with presumed gene regulatory proteins (M3).

Chromatin from chicken reticulocytes and mouse Ehrlich ascites tumor cells has been extracted with 2 M NaCl, leaving a portion of the DNA still complexed with a fraction of nonhistones (designated M3, since it can be dissociated from DNA in solutions of 3 M NaCl containing 5 M urea). The DNA complexed with M3, separated from the bulk DNA by centrifugation, was found to contain sequences poorly represented in bulk DNA. Specifically we found that DNA--M3 complexes isolated from chicken reticulocyte chromatin were enriched in globin gene sequences by 20-fold relative to unfractionated DNA and by over 1000-fold relative to DNA rendered free of protein following the extraction of chromatin with 2 M NaCl. We have therefore isolated DNA fractions complexed with M3 which are enriched in specific sequences as may be determined by M3.     

The chromatographic properties of the DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]

The DNA-dependent DNA-polymerase (DNA polymerase I which is not sorbed on the column with DEAE-cellulose, and DNA-polymerase II, which is absorbed by this column and is eluted from it by 0.3 M of NaCl), have been isolated from Acholeplasma laidlawii PG-8. DNA-polymerase I in homogeneous state was obtained as a result of the stepwise treatment by heparin-sepharose (elution at 0.35 M of NaCl) and poly-U-sepharose (elution at 0.3 M of NaCl). It was presented on the electrophoregram by one polypeptide with molecular weight of 72 kDalton. The second form of DNA polymerase was also obtained in homogeneous state as a result of sequential treatment on heparin-sepharose (elution at 0.3 M of NaCl) and on poly-A-sepharose (elution at 0.25 M of NaCl): the protein which had manifested polymerase activity was a polypeptide with molecular weight of 45 kDalton.     

The type of DNA attachment sites recovered from nuclear matrix depends on isolation procedure used

A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change.     

Molecular cloning of the phosphoenolpyruvate carboxylase gene of Anabaena variabilis

Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322. Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A. variabilis genomic library. A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A. variabilis PEPCase. E. coli HB101 cells transformed with plasmid pTRH1 have approx. 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A. variabilis PEPCase.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.     

Multiple restraints to the unfolding of spermidine nucleoids from Escherichia coli

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged.     

Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase

A L-methionine- D, L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N(2) reduction. In order to determine the biochemical basis for the NH(4)(+)-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn(+2) for optimum activity, while SA0-GS was Mg(+2) dependent. SA0-GS had the following apparent K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH(4)(+)-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K(m) values for NH(4)(+) and glutamate, and thus limited the assimilation of NH(4)(+) generated by N(2) reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH(4)(+) binding and metal ion coordination.     

The mechanism of action of the deoxyribonuclease of rat liver chromatin on DNA]

A mechanism of action of DNAase, isolated by chromatin extraction with 0.4 M NaCl, on DNA is described. The enzyme is an endonuclease, it does not require the presence of double-stranded regions in the DNA molecule, does not distinct single-stranded breaks in DNA, and it preferably attacks single-stranded DNA. It hydrolyses DNA for 3'-phosphodiester bonds to octane nucleotides which are resistant to the enzyme activity. The action of the enzyme on DNA does not depend on the position of terminal phosphates. Chromatin DNAase is not specific to DNAs from different origins.     

Purification and comparative study of the properties of glucocorticoid- and estrogen-receptor complexes in the rat liver

Parallel purification of glucocorticoid- and estrogen-receptor complexes from rat liver cytosol has been accomplished. Some properties of purified steroid-receptor complexes (SRC) were determined. The procedure developed earlier when the two-step treatment of cytosol with DNA-cellulose alternated with SRC ammonium sulphate precipitation, was shown to be universally applicable for purification of various SRC. Certain modifications have been devised allowing some increase in the degree of receptor purification. The amount of estrogen-receptor complexes (ERC) isolated from male liver cytosol was 20-40 times less than that of glucocorticoid-receptor complexes (GRC) isolated simultaneously from the equal volume of the same cytosol. Both SRC types bind intensively to homologous DNA but not to poly(A). The elution of GRC from DNA cellulose was mainly achieved at 0.4 M NaCl. With this, GRC and ERC showed small, but reliable differences in the salt resistance of their associates with DNA: the ERC-DNA link was stable toward NaCl up to 0.1 M, whereas an appreciable amount of GRC was eluted from DNA-cellulose at 0.1 M NaCl. The stability of purified ERC exceeded that of purified GRC, which apparently reflects the differences in the hormone-receptor binding constants. The receptor stability under various environmental conditions is discussed and some recommendations on the improvement of the SRC stability and its control are given.     

Delayed methylation and the matrix bound DNA methylase

It is shown that the methylation of DNA that occurs in isolated nuclei is "delayed methylation". This methylation is not reduced in nuclei which have been pretreated with 0.2M NaCl to extract the soluble methylase suggesting that this methylation is the product of a firmly bound matrix associated DNA methylase. Evidence is provided that, like the methylase, the DNA substrate is associated with the nuclear matrix.