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H Zentgraf

Publications and source records attributed to H Zentgraf.

At least 109 records · Page 6Linked to original sources
PubMed1980

Comparison of different rearranged immunoglobulin kappa genes of a myeloma by electronmicroscopy and restriction mapping of cloned DNA: implications for "allelic exclusion".

We have studied the organization and function of different rearranged kappa genes in a myeloma, MOPC-21. Two kappa genes were cloned into Charon 4A and compared with each other and with a cloned germline CK gene by restriction mapping and electron microscopy. One MOPC-21 clone corresponds to the gene coding for the MOPC-21 kappa chain polypeptide; it has the V21 gene joined with the CK gene at the J2 sequence. The other MOPC-21 clone corresponds to a nonfunctional rearranged MOPC-21 kappa gene, except for a lkb deletion, 3' of J4. A similar deletion is also found in a "new" kappa gene present in NS-1, a cellular subclone of MOPC-21. The clone of the "nonfunctional" kappa gene has a V gene which is distinct from V21 which is joined to CK in the vicinity of J2. The undeleted form of this gene codes for a KRNA having the size of mature KmRNA which, however, is not translated into kappa chains. Thus the defect of the "nonfunctional" gene manifests itself at a late step of gene expression. The basis for "allelic exclusion" of antibody genes may simply be the complexity of the processes between genes and gene products, resulting in the expression of only one gene.

Alleles↗
PubMed1980

Conversion of bacteriophage fd into an efficient single-stranded DNA vector system.

Single-stranded DNA vectors were constructed in vitro by insertion of various DNA fragments into the Intergenic Region of the single-stranded DNA phage fd. These inserts introduce into the phage genome unique cleavage sites for restriction nucleases which are suited for sticky joining in cloning experiments. Since these sites are usually located within genes coding for antibiotic resistance, inactivation of a resistance gene by insertion can be used as a marker for the successful cloning of a DNA fragment. Resistance genes also allow to select for recombinant DNA phages and to minimize the loss of DNA inserts which otherwise becomes significant above an insert size of about one kb. Cloning of several DNA fragments is described and strand separation of double-stranded DNA fragments by means of cloning into fd DNA is given as an example for application of single-stranded DNA vectors.

Bacteriophages↗
PubMed1980

Visualization of nucleosomes in thin sections by stereo electron microscopy.

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.

Animals↗
PubMed1980

Coexistence of nucleosomal and various non-nucleosomal chromatin configurations in cells infected with herpes simplex virus.

Coexistence of four different forms of chromatin was observed by electron microscopy in nuclear spread preparations of monkey kidney cells during late stages of infection with herpes simplex virus (HSV-1 AMG). Besides typical nucleosomal (i) chromatin, thin (3-5 nm) strands morphologically indistinguishable from protein-free DNA were frequent, without (ii) or with (iii) sparse 10-22 nm large granules different from nucleosomes. In addition, uniformly thick (mean 17 nm), heavily stained chromatin strands (iv) were seen. The non-nucleosomal character of types (iii) and (iv) chromatin was also demonstrated by their resistance to histone removal in Sarkosyl and heparin. All four forms were seen in capsid-associated HSV-DNA molecules, and various combinations of these forms occurred in adjacent regions of the same DNA molecule, including the vicinity of replication branch points. Especially frequent were regions of chromatin types (ii) or (iii) alternating with thickly coated intercepts of type (iv) chromatin, the latter often displaying "bubble"-like strand separations. The appearance of chromatin types (ii)-(iv) was dependent on viral replication. These chromatin arrays were compared with structures observed in purified HSV-DNA from these cells. Patterns of single-stranded regions were found in HSV-DNA that were similar to those observed in the thickly coated type (iv) chromatin. It is concluded that, in these nuclei, non-nucleosomal arrangements can be formed, at least on viral DNA, under conditions of continued DNA synthesis and inhibited protein synthesis, and that single-stranded DNA is packed into a characteristic thick strand of non-nucleosomal chromatin by association with a special, probably virus-coded protein.

Animals↗
PubMed1980

Supranucleosomal organization of sea urchin sperm chromatin in regularly arranged 40 to 50 nm large granular subunits.

The organization of the chromatin of sperm nuclei of sea urchin (Parcentrotus lividus and Sphaerechinus granularis) was studied by electron microscopy using thin sections of fixed cells and spread preparations of chromatin obtained after various procedures involving incubation in low salt buffers. In fixed cells and in moderately dispersed preparations chromatin appeared in the form of fibrillar chains formed by closely apposed, distinctly sized granules that were larger than chromatin subunit structures so far described in other kinds of chromatin (mean diameters of particle units determined were 36 nm, in thin sections, and 47 nm, in spread preparations). Upon prolonged incubation in low salt buffer an increasing number of these large granular units was transformed into extended nucleosomal chains (nucleofilaments). Estimations indicated that the large granules contained 20 to 26 nucleosomes, i.e. 4.8 to 6.2 kb DNA, resulting in a linear DNA contraction ratio of 33 to 55, which is in a similar range as values reported for the supranucleosomal chromatin particle of the SV40 minichromosome but greater than contraction ratios of "superbead" structures. The unravelling of the large supranucleosomal granule into the extended nucleofilament chain in low salt buffers seemed to be a gradual process since intermediate stages of variable particles sizes and shapes were observed. This novel type of chromatin particle is considered to represent an example of a naturally occurring globular supranucleosomal packing unit of chromatin which is predominating, if not the only forms of supranucleosomal levels of chromatin organization and in relation to the special DNA content and histone composition of sea urchin sperm nucleosomes.

Animals↗
PubMed1980

Reversible in vitro packing of nucleosomal filaments into globular supranucleosomal units in chromatin of whole chick erythrocyte nuclei.

Structural transitions between extended and condensed forms of chromatin have been studied by electron microscopy of whole chicken erythrocyte nuclei incubated in low buffer concentrations (0.5 mM sodium borate; 1 to 5 mM Tris-HCl) with and without additions of monovalent or divalent cations. Both spread preparations and ultrathin sections have been examined. It is shown that the condensed chromatin present in chicken erythrocytes is organized, at the supranucleosomal level, in granular particles (mean diameter 29 +/- 3 nM0 tightly apposed against each other in beaded chains (higher order fibrils). These chains of higher order granules are gradually unravelled in low salt buffers lacking divalent cations into extended nucleosomal filaments. When nuclear chromatin swollen in low salt buffer and extended uniformly into nucleosomal filaments is exposed to buffers containing higher concentrations of monovalent ions (e.g., 100 mM NaCl) or low concentrations of divalent cations (e.g., 0.05-0.5 mM MgCl2) rapid and almost complete recondensation of the entire chromatin of a nucleus into one compact spheroidal aggregate of chains of higher order granules (diameters 25-30 nm) is observed. This transition is reversible and whole nuclear chromatin can be induced, in vitro, to undergo several cycles of such conformational changes. The results show that reconstitution of globular forms of higher order packing of nucleosomes is not restricted to oligonucleosomal units but also takes place, in a rather uniform and regular mode, in long nucleosomal filaments as they are present in whole nuclei. The data are discussed in relation to other studies of chromatin ultrastructure and are interpreted to indicate that the predominant, if not exclusive form of supranucleosomal packing existing in condensed inactive chromatin of living cells is a chromatin fibril made up of serially arranged, close-packed granular particles ("suprabeads" sensu in [64]). In addition, the results suggest that higher order packing of chromatin into such supranucleosomal granules is not simply a function of ionic strength but that the type of ions present strongly influences the state of chromatin packing.

Animals↗
PubMed1978

Higher order structure of simian virus 40 chromatin.

Simian virus 40 nucleoprotein complexes undergo an ionic strength-dependent structural transition. At moderate ionic strength they contain histone H1 as well as the nucleosomal histones and have a compact conformation with globular subunits 190 angstroms in diameter. At high ionic strength histone H1 is released, and the structure unfolds into chains with an average of 24 nucleosomes. The extended viral chromatin converts to the compact form by the addition of histone H1. Transcriptionally active simian virus 40 chromatin undergoes the same structural transitions. The higher order structure of viral chromatin may be analogous to the compact state of cellular chromatin fibers observed at physiological ionic strength.

Chromatin↗
PubMed1978

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.

Centrifugation, Density Gradient↗
PubMed1978

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd.

Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transpoon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 mu to 1.7 mu, and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In non-defective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducing phage from different defective clones varied from 0.6 mu to 3.0 mu and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.

Coliphages↗
PubMed1978

Supranucleosomal structure of chromatin.

Rat liver chromatin was moderately digested by micrococcal nuclease and analysed by centrifugation in isokinetic sucrose gradients and electron microscopy. Two classes of particles sedimenting with about 33S and 60S were characterized. Kinetics of their appearance and disappearance during progressive digestion suggests that they represent monomers and dimers cleaved from a higher order (supranucleosomal) structure of chromatin. Biochemical and electron microscopical results suggest that the monomers and dimers contain eight and sixteen nucleosomes, respectively, which are densely packed into 23 nm (monomer) and 29 nm (dimer) globules.

Animals↗
PubMed1978

Transcription patterns of amplified Dytiscus genes coding for ribosomal RNA after injection into Xenopus oocyte nuclei.

Oocytes of the frog Xenopus were injected with purified circular DNA containing amplified rRNA genes of the water beetle Dytiscus. Nuclear contents of injected oocytes were spread and examined by electron microscopy. Most of the Dytiscus DNA seen in injected nuclei contained regions indensely packed with polymerases and nascent transcripts. Apparently normal, as well as abnormal, patterns of transcription were observed. By this type of experiment, it may become possible to recognize the transcribed regions and immediate transcripts of cloned DNA molecules whose activity cannot be seen by electron microscopy of normal nuclei.

Animals↗
PubMed1978

Biochemical and ultrastructural analysis of SV40 chromatin.

SV40 chromatin can be isolated in two forms: At moderate ionic strength (mu = 0.1-0.3) it contains histone H1 in addition to the four nucleosomal histones and has a highly condensed appearance in the electron microscope, being composed of a few closely connected large spheres [190 A (160, 220) diameter]. At high ionic strength (mu = 0.6-0.8) or after prolonged exposure to very low ionic strengths (mu less than 0.02), the compact form unfolds and the chromatin shows a typical nucleosomal morphology. Native SV40 DNA-protein complexes contain a median number of 24 nucleosomes. The number of superhelical turns does not differ in DNA obtained from the compact and the unfolded forms of chromatin. DNA-relaxing enzyme is found associated with SV40 chromatin and is capable of acting both on extraneously added circular DNA and on its own DNA in the nucleoprotein complex. Purified DNA-relaxing enzyme forms transiently nicked DNA intermediates where the enzyme can be found covalently attached to the site of the nick in the DNA. Transcriptionally active SV40 complexes undergo the same ionic-strength-dependent structural transition as that of bulk SV40 chromatin and may therefore also have a compact configuration at physiological salt concentrations.

Cell Line↗