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J B Rattner

Publications and source records attributed to J B Rattner.

At least 91 records · Page 5Linked to original sources
PubMed1985

Tandem repeats of a specific alternating purine-pyrimidine DNA sequence adjacent to protamine genes in the rainbow trout that can exist in the Z form.

We have located an extensive (AC)n-rich but specific sequence downstream of three rainbow trout protamine genes. Although sharing considerable sequence homology, including a perfectly conserved 46 base pair repeat, the sequences exhibit a regular heterogeneity in the length of the (AC)n-rich tracts. Radioimmunoassay experiments, S1 nuclease sensitivity studies, two-dimensional electrophoretic analysis, and immunoelectron microscopy studies have been used to determine if the region could assume a Z DNA conformation. It was found that, in a supercoiled plasmid, the (AC)n-rich region has the ability to attain the Z DNA conformation under physiological conditions.

Animals↗
PubMed1985

Assembly and characterization of nucleosomal cores on B- vs. Z-form DNA.

The ability of right- vs. left-handed alternating purine/pyrimidine copolymers to support the formation of nucleosomes has been examined by using a trout testis assembly factor. The protein, which is thermostable, has a molecular weight of 29000 and will assemble nucleosomes onto both SV40 and calf thymus DNA. This assembly factor has been used to assemble nucleosomes onto the B and Z conformations of poly[d(Gm5C)] and the B conformation of poly[d(GC)]. The isolated B-form particles, which sediment at approximately 11 S in a sucrose density gradient, contain DNA of 140-200 bases in length and the four core histones. The isolated Z-form particles, which also sediment at approximately 11 S, contain the four core histones and DNA of 170-250 bases in length. Physical analysis of the particles by absorbance and circular dichroic spectroscopy indicates that the DNA remains in the original conformation throughout the isolation procedure. Further, the particles reconstituted onto left-handed DNA compete effectively for an anti-Z DNA antibody, while the corresponding right-handed particles do not. Analytical sedimentation velocity determinations indicate that the B-form poly[d(Gm5C)] and poly[d(GC)] particles sediment at 11.2 and 11.1 S, respectively. In contrast, the poly[d(Gm5C)] Z-form particles have an S20,w of 10.6 S. The differences in the sedimentation velocity and the density of the cores, and in the lengths of DNA associated with the particles, suggest that the conformation of the DNA affects the manner in which it associates with the histone octamer.

Animals↗
PubMed1985

Centromere organization in chromosomes of the mouse.

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.

Animals↗
PubMed1985

Radial loops and helical coils coexist in metaphase chromosomes.

Histone-depleted chromosomes have revealed a scaffold and loop architecture of metaphase chromosomes. In its simplest form this arrangement contradicts many classical observations suggesting chromosomes have a helical substructure. We have obtained preparations that allow the visualization of several levels of chromosome structure. These images suggest that metaphase packing is achieved by the compaction through helical coiling of a 200-300 nm fiber that is in turn composed of radial loops. These observations imply that any scaffold elements associated with radial loops are not distributed as previously proposed but must follow a complex and more extensive path within the metaphase chromatid.

Animals↗
PubMed1984

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout.

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.

Acetylation↗
PubMed1984

Sequence analysis of a PM2-DNA anti-Z-IgG-binding region.

An anti-Z-antibody-binding region between PM2-DNA map units 0.05 and 0.18, containing approx. 25% of the bound PM2 antibody molecules (1,2) has been sequenced. Analysis of this PM2 DNA sequence from map units 0.00 to 0.175 demonstrates that alternating purine/pyrimidine tracts capable of adopting the left-handed conformation are present within this antibody-binding region. Longer (GC)n-rich tracts are clustered together and comprise seven alternating purine/pyrimidine-rich areas (48%-84%) ranging from 19 to 142 nucleotides in length. The DNA located between these alternating purine/pyrimidine-rich areas exhibit a low level (0%-19%) of this sequence arrangement. There is a very strong correlation between the alternating purine/pyrimidine-rich areas and the anti-Z-DNA-IgG-binding sites. Nucleotides 1461-1583 of the PM2-DNA genome encode the bacteriophage capsid protein IV. One of the PM2 left-handed sites is located within this protein-coding sequence; a B-to-Z transition within this site may be involved in protein-IV gene regulation in vivo.

Bacteriophages↗
PubMed1984

The higher-order structure of chromatin: evidence for a helical ribbon arrangement.

Both intact and nuclease-isolated chromatin fibers have been examined at different degrees of salt-induced compaction, using a variety of preparation techniques. The results suggest that the initial folding step in nucleosome packing involves the formation of a zig-zag ribbon as has been proposed by others (Thoma F., T. Koller, and A. Klug, 1979, J. Cell Biol., 83:403-427; Worcel A., S. Strogartz, and D. Riley, 1981, Proc. Natl. Acad. Sci. USA, 78:1461-1465), and that subsequent compaction occurs by coiling of the ribbon to form a double helical structure. This type of folding generates a fiber in which the nucleosome-nucleosome contacts established in the zig-zag ribbon are maintained and in which the histone H1 molecules occupy equivalent sites. The diameter of the fiber is not dependent upon the nucleosome repeat length. Direct mass values for individual isolated fibers obtained from electron scattering measurements showed that the mass per length was dependent on ionic strength, and ranged from 6.0 X 10(4) daltons/nm at 10 mM NaCl to 27 X 10(4) daltons/nm at 150 mM salt. These values are equivalent to 2.5 nucleosomes/11 nm at 10 mM NaCl and to 11.6 nucleosomes/11 nm at 150 mM salt and are consistent with the range of packing ratios for the proposed helical ribbon.

Animals↗
PubMed1984

Ultrastructural organization of double minute chromosomes and HSR regions in human colon carcinoma cells.

The ultrastructural organization of double minute chromosomes (DMs) and marker chromosomes displaying homogeneously staining regions (HSRs) have been investigated in two cell lines, COLO-320 DM and COLO-320 HSR, derived from a human colon carcinoma. Double minute chromosomes in the COLO-320 DM cell line vary in number from several to several hundred and in diameter from 0.5 to 2 micron. These chromosomes lack kinetochore structures which appear as discrete electron dense plates on the centromeric chromosomes. The DMs are composed of regularly arranged nucleosome-containing fibers. These are folded to form 30 nm fibers which extend as loops from the central portion of the chromosome. In COLO-320 HSR, a majority of cells contain a single submetacentric HSR marker chromosome with HSR regions in both chromosome arms. The HSR chromosome appears structurally analogous in EM preparations to the other centromeric chromosomes of the karyotype.

Carcinoma↗
PubMed1983

Left-handed Z-DNA regions are present in negatively supercoiled bacteriophage PM2 DNA.

Bacteriophage PM2 DNA is a 10-kb covalently closed circular (ccc) molecule with a reported superhelical density of sigma = -0.12. Here we describe the binding of anti-Z-DNA antibodies to PM2 form I DNA under high and low salt conditions. The binding to PM2 DNA has been demonstrated by competitive radioimmunoassay (RIA), retardation of the DNA:antibody complexes in agarose gels and visualization by electron microscopy. The antibody binding is dependent on the degree of negative supercoiling. Thus, PM2 form II and form III did not bind the antibody. The low salt RIA results indicated the presence of 200-400 bp of left-handed DNA per PM2 molecule. This could reduce the effective superhelical density to sigma = -0.04 to -0.08, a range comparable with those found for other ccc DNAs in vivo. Electron microscopy revealed that a maximum of 22 antibody molecules bind to PM2. Single-site restriction with HpaII of the fixed DNA:antibody complex showed a cluster of four to five antibody molecules bound near one end of the linear DNA molecule. The evidence presented indicates that PM2 DNA contains regions of left-handed conformation under physiological conditions (low salt concentration) as well as at high salt concentrations. In addition, electrophoretic analyses of PM2 topoisomers indicate the presence of left-handed regions at superhelical densities less than that of isolated PM2 DNA.

Antigen-Antibody Complex↗
PubMed1983

Natural occurrence of left-handed (Z) regions in PM2 DNA.

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.

Antibodies, Viral↗
PubMed1982

Ultrastructural organization of yeast chromatin.

The ultrastructural organization of yeast chromatin was examined in Miller spread preparations of samples prepared from spheroplasts or isolated nuclei of Saccharomyces cerevisiae. Micrographs from preparations dispersed in 1 mM Tris (pH 7.2) illustrate that the basic chromatin fiber in yeast exists in two ultrastructurally distinct conformations. The majority (up to 95%) of the chromatin displays a beaded nucleosomal organization, although adjacent nucleosomes are separated by internucleosomal linkers of variable lengths. Ribonucleoprotein (RNP) fibrils are only occasionally associated with chromatin displaying the conformation. The remaining 5-10% of the chromatin appears to be devoid of discrete nucleosomes and has a smooth contour with a fiber diameter of 30-40 A. Transcriptional units, including putative ribosomal precursor RNA genes, defined by the presence of nascent RNP fibrils are restricted to chromatin displaying this smooth morphology. Chromatin released from nuclei in the presence of 5 mM Mg++ displays higher-order chromatin fibers, 200-300 A in diameter, these fibers appear to be arranged in a manner than reflects the two forms of the basic chromatin fiber.

Cell Nucleus↗
PubMed1981

Laser microsurgery in cell and developmental biology.

New applications of laser microbeam irradiation to cell and developmental biology include a new instrument with a tunable wavelength (217- to 800-nanometer) laser microbeam and a wide range of energies and exposure durations (down to 25 X 10(-12) second). Laser microbeams can be used for microirradiation of selected nucleolar genetic regions and for laser microdissection of mitotic and cytoplasmic organelles. They are also used to disrupt the developing neurosensory appendages of the cricket and the imaginal discs of Drosophila.

Animals↗
PubMed1981

Chromosome organization during male meiosis in Bombyx mori.

Chromatin organization during male meiosis in Bombyx mori has been investigated utilizing the Miller spreading procedure. During meiotic prophase, the linear 200-300 A chromatin fibers evident at interphase are folded into tandem arrays of approximately 7,000 loops per haploid genome. Adjacent loops visualized during early prophase are separated by 0.15-0.2 nm of nucleosomal DNA. Meiotic metaphase chromosomes display numerous loops which project radially from the central region of the chromosome suggesting that the loop conformation of prophase is maintained throughout meiosis. Spread preparations of spermatogenic stages through pachytene allow the visualization of actively transcribed ribosomal DNA. Throughout this period, these transcription units appear to be organized into loops in such a way that one active transcription unit exists on a single loop. Furthermore, there are various levels of transcription on different ribosomal loops, although the number of loops displaying active transcription remains constant throughout this period.

Animals↗
PubMed1980

Chromatin organization during meiotic prophase of Bombyx mori.

Chromatin organization during the early stages of male meiotic prophase in Bombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200-300 A in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5-25 mu of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.

Animals↗
PubMed1980

Folding up genes and chromosomes.

The basic chromatin fiber is composed of a tandem array of nucleosomes, adjacent nucleosomes being separated by a variable amount of DNA. In vivo, this fiber is folded to form a higher-order fiber with a diameter of 200 to 300 A. The distribution of nucleosomes within the higher-order fiber has been analyzed in negatively stained preparations of chromatin fibers released from metaphase cells or interphase nuclei by mechanical disruption. The fiber appears to be composed to discrete packing domains in each of which there is a high density of closely apposed nucleosomes; within these regions several discrete packing patterns are observed. Adjacent domains are connected by regions of the fiber which are thinner and contain a lower density of nucleosomes. From these observations we suggest that the 200 to 300 A fiber is mosaic and that domains differ from each other in composition so as to confer sequence specificity upon the structure. In addition, compositional differences may have relevance to the functional state of the underlying genes; thus, higher order structure of a region is related to its functional state. Above this level of organization, studies on the early stages of chromatin folding in meiotic prophase suggest that 200 to 300 A fibers are organized as loops which emanate from the longitudinal axis of the chromosome, in a manner consistent with a model for mitotic chromosome folding by means of radial loop formation. Active ribosomal RNA genes are identifiable in these preparations and appear to be organized in a similar fashion; each active gene appears to comprise a loop of chromatin.

Animals↗
PubMed1979

Nucleosome packing in interphase chromatin.

Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.

Animals↗