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J B Rattner

Publications and source records attributed to J B Rattner.

At least 73 records · Page 4Linked to original sources
PubMed1990

Distribution of CT-rich tracts is conserved in vertebrate chromosomes.

The distribution of d(CT)-rich pyrimidine tracts in the karyotypes of a variety of vertebrates was studied by in situ hybridization. The probe for these studies was a 56bp homopyrimidine/homopurine sequence obtained from a mouse genomic library constructed with DNA prepared from a restriction enzyme digestion of metaphase chromosomes. Single-stranded DNA nuclease digestions and two-dimensional gel analysis of topoisomers of this sequence indicated that it is capable of adopting a triplex conformation in vitro. In situ hybridization with this probe to the karyotypes of ten different vertebrate species revealed a highly conserved chromosomal distribution of d(CT)-rich tracts. These tracts are found throughout the chromosomal arms and in some karyotypes they are clustered, producing a banding pattern. However, at the resolution of the light microscope these tracts appeared to be absent from the centromeric regions of all chromosomes examined except those of chicken. The non-random distribution of these tracts to the chromosomal arm regions implies an organizational or functional role for this repeat class. It is unlikely that the 56 bp sequence type contributed to the formation of the triplex DNA structure previously detected in centromeric domains of mouse.

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PubMed1990

Autoantibodies to chromosomal domains in rheumatic diseases.

Autoantibodies to chromosomal proteins are frequently found in the sera of certain patients with rheumatic diseases. In patients with scleroderma, especially in those with the CREST syndrome, autoantibodies to a specific chromosomal domain (centromere) have been found as a common feature. In this report we describe the results of a study that utilized chromosomes prepared from fibroblasts of an Asiatic deer, the Indian Muntjac (IM). This substrate is sensitive and allows a more precise localization of chromosomal antigens. Using IM chromosomes we provide evidence that antibodies directed against chromosomal components are present in patients with rheumatic diseases and in some normal controls. The titre of these antibodies is high in scleroderma patients and low in the normal controls. In a group of ankylosing spondylitis patients there is a greater frequency of moderately elevated tires of antibodies to the kinetochore domain than in patients with rheumatoid arthritis or systemic lupus erythematosus.

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PubMed1989

Kinetochore structure: electron spectroscopic imaging of the kinetochore.

The structure of the kinetochore in thin section has been studied in the Indian muntjac by an electron spectroscopic imaging technique. This procedures allows the analysis of the distribution of phosphorus within the layers of the kinetochore. The results indicate that this element is a major component of both the inner and outer plates whereas it is largely absent in the middle plate and fibrous corona. The majority of the phosphorus is localized to a 30-nm fiber(s) that is woven through the layers of the kinetochore. The presence of phosphorus within this fiber, along with its morphological and biochemical features, indicates that it contains DNA. The fiber(s) occupies a major portion of the inner and outer plate where it forms a series of rows. It is rarely observed in the middle layer except where it passes between the inner and outer layers. The absence of structure in the middle plate suggests that it may represent a space rather than a plate that in turn may be related to the function of this region. The distribution of phosphorus within the kinetochore is neither altered by treatment with colcemid nor by the presence of microtubules at the kinetochore. Analysis of conventional micrographs of the kinetochore together with structural information obtained by electron spectroscopic imaging suggests that most microtubules insert and terminate between the rows of kinetochore fibers in the outer plate. However, some microtubules continue through the middle layer and terminate at the lower plate. The insertion of microtubules at different levels of the kinetochore may reflect the existence of functionally distinct microtubule classes. Electron spectroscopic imaging indicates that the microtubules associated with the kinetochore are phosphorylated.

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PubMed1988

Sequence organization and cytological localization of the minor satellite of mouse.

A complete 120 bp genomic consensus sequence for the mouse minor satellite has been determined from enriched L929 centromeric sequences. The extensive sequence homology existing between the major and minor satellite suggests an evolutionary relationship. Some sequences flanking the minor satellite has also been identified and they provide insight into centromeric DNA organization. Isotopic in situ hybridization analysis of the minor satellite to mouse L929 and Mus musculus metaphase spreads showed that this repetitive DNA class is localized specifically to centromeres of all chromosomes of the karyotype. With the use of high resolution non-isotopic fluorescence in situ hybridization the minor satellite is further localized to the outer surface of the centromere in a discrete region at or immediately adjacent to the kinetochore. Our cytological data suggests that the minor satellite might play a role in the organization of the kinetochore region rather than, as previously suggested, sites for general anchoring of the genome to the nuclear matrix.

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PubMed1988

Erythroid-specific gene chromatin has an altered association with linker histones.

The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.

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PubMed1988

Detection of distinct structural domains within the primary constriction using autoantibodies.

We report the immunological differentiation of structures within the primary constriction. These include the kinetochore and the connecting strand, a structure which connects sister kinetochores. The location and temporal appearance of the connecting strand antigen suggest that it could play a role in the maintenance of sister chromatid pairing. In addition, we report the identification of a novel epitope that is localized to discrete patches along the entire length of the junction between sister chromatids at metaphase (the junction patch antigen). The patches on the inner surface of the euchromatic arms can be disrupted by Colcemid treatment while those found in the primary constriction remain intact. The apparent heterogeneity of the patches suggests that they may play different roles in the regulation of sister chromatid pairing. Because of their cytological localization and possible functional role, the junction patch and connecting strand antigens have provisionally been collectively termed CLiPs (Chromatid Linking Proteins). All of these antigenic sites are shown to be distinct from centromeric heterochromatin, which can itself be immunologically differentiated from the euchromatic arms. The relationship between the antigenicity of the primary constriction and the unique manner in which chromatin is organized in this region is discussed.

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PubMed1988

Electron spectroscopic imaging of DNA.

Electron spectroscopic imaging (ESI) was carried out using a fixed beam electron microscope equipped with a parallel electron energy filter to form micrographs of purified plasmid DNA without the use of heavy metal stains and shadows. Inelastically scattered electrons that have ionized the phosphorus LII,III shell electrons were used to form phosphorus distribution maps of DNA and deoxyribonucleoprotein complexes. Signal-to-noise values of the net phosphorus content over single DNA molecules compared to two and four interwound DNA strands directly reflect the known stoichiometric levels of phosphorus content, illustrating that ESI can be used to determine the relative levels of nucleic acid in nucleoprotein complexes. An initial attempt to characterize nucleosomal and transcriptionally active chromatin from Saccharomyces cerevisiae with this technique reveals three distinct ultrastructural classes of the basic chromatin fiber.

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PubMed1988

Electron spectroscopic imaging of the centrosome in cells of the Indian muntjac.

Specific antibody labelling indicates that phosphoproteins are present at microtubule-organizing centres, including the centrosome. We have employed electron spectroscopic imaging techniques that permit high-resolution elemental analysis of thin sections of intact cells to investigate the precise distribution of phosphorus and therefore phosphoproteins at the centrosome of Indian muntjac cells. We report that these proteins are localized to both the pericentriolar matrix and the centriole. The matrix contains an abundance of phosphorus and is associated with microtubule elements. Within the mature centriole, major structures including the nine triplet blades and linking elements that connect adjacent blades are composed of phosphorylated proteins. In addition, phosphoproteins are abundant at the ends of the centriole, at the interface between the centriole lumen and the pericentriolar environment. From these observations we suggest that phosphoproteins may play both a structural and a functional role within the centrosome region.

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PubMed1987

Identification and characterization of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis.

An autoantibody that binds an antigen localized to the stembody of dividing cells has been identified in a patient with systemic sclerosis. Initially, this antigen is associated with the surface of the metaphase chromosomes. At the onset of anaphase the antigen becomes preferentially associated with the forming stembodies. This association is maintained as furrowing progresses during telophase and continues after the intercellular bridge is released from the daughter cells during G-1. Immunoblots indicate that the epitope detected by immunofluorescence is present on a protein with an apparent molecular weight of 38 kD.

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PubMed1987

The organization of the mammalian kinetochore: a scanning electron microscope study.

A procedure has been developed for scanning electron microscopy that enables the visualization of kinetochores along the surface of isolated chromosomes of the Indian muntjac. Indirect immunofluorescence and thin section analysis of the kinetochores of those isolated chromosomes verified that these structures retained in vivo composition and morphology during the isolation procedure. In scanning electron micrographs the outer surface of the outer kinetochore plate can be visualized as a series of fibers 25-30 mn in diameter that are arranged across the plate. These images are comparable to those obtained by whole mount transmission electron microscopic procedures (Rattner 1986) and are compatible with a model of the kinetochore in which chromatin fiber from the body of the chromosome extend to the outer kinetochore plate.

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PubMed1987

Mammalian kinetochore/centromere composition: a 50 kDa antigen is present in the mammalian kinetochore/centromere.

The composition of the mammalian kinetochore/centromere was studied by indirect immunofluorescence and immunoblotting protocols using serum from a patient with the CREST variant of scleroderma. The results of these studies suggest that a protein with a molecular weight of 50 kDa is localized at the surface of the primary constrictions (the kinetochore region) of both human and Indian muntjac chromosomes. In addition, we were able to verify the presence of a 19.5 kDa antigen (CENP-A), previously detected in human centromeres, within the kinetochore region of the Indian muntjac. These data suggest that the composition of the kinetochore region of the primary constriction is complex and that there is conservation in composition within the mammals. These features may reflect the important role of this unique chromosomal domain in the maintenance of ploidy.

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PubMed1987

The higher order structure of the centromere.

The architecture of the centromere region of mouse chromosomes has been studied in cells grown in the presence of 5-azacytidine. This drug interferes with normal condensation producing elongated centromere regions. It has been found that this effect is reversible in the presence of the drug, allowing the observation of the repackaging of the extended centromere into a structure exhibiting native centromere morphology. Light microscopy as well as transmission and scanning electron microscopy of this condensation process suggests that the native centromere is formed by the helical folding of a subfiber with an approximate diameter of 100 nm. This fiber is in turn composed of loops of the 30-nm fiber class. The boundary between successive gyres of the subfiber are obscured at the completion of condensation resulting in the formation of a homogenous 250- to 300-nm fiber that is the native centromere. These observations provide evidence for an additional level of chromatin organization within the metaphase chromosome.

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PubMed1986

Organization within the mammalian kinetochore.

The organization within the mammalian kinetochore was examined using whole-mount electron microscopic techniques on chromosomes digested with restriction enzymes or micrococcal nuclease. These preparations revealed that a portion of the kinetochore is highly resistant to nuclease digestion and can be visualized as a discrete structure. The relationship of this structure to the remainder of the chromosome suggests that it represents the outer kinetochore plate. The plate is composed of a series of fibrillar loops that are arranged in a parallel array along the plane of the plate. These fibers are 25-30 nm in diameter. The morphology, particulate substructure, and ultimate susceptibility to nuclease digestion suggest that these fibers contain DNA. A model is presented that suggests that the outer plate contains the apexes of chromatin loops that originate within the body of the primary constriction.

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PubMed1986

Assembly of DNA onto the histone octamer facilitates the B-to-Z transition.

Nucleosomal core particles containing the right- and left-handed conformations of DNA were examined for their ability to support the B----Z or Z----B transition. Nucleosomes were assembled onto the B-and Z-conformations of poly[d(Gm5C)] and the B-conformation of poly[d(GC)] as previously described. Absorbance and circular dichroic spectroscopy indicated that the DNA on all three core particle populations could undergo the conformational B----Z transition. Further, the right- to left-handed transition for both poly[d(Gm5C)] and poly[d(GC)] appeared to be facilitated by the DNAs association with the histone octamer. The DNA remained associated with the protein core subsequent to the transition, and electron microscopy and sedimentation velocity analysis indicated that there were no gross changes in nucleosomal structure. However, a change in the sedimentation value of the poly[d(Gm5C)] core particles was detected when the conformation of the DNA was altered from B to Z, resulting in a lower S20,w value for the Z-form particles than for the corresponding B-form particles.

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PubMed1986

An investigation of human sperm pronuclear chromosome "gaps" using scanning electron microscopy.

A common cytogenetic finding in both Q-banded and solid Giemsa-stained preparations of pronuclear chromosomes obtained from cross-species fertilization of hamster oocytes by human sperm is the presence of a variable-length "gap" in the centromeric region. Scanning electron microscopy was used to investigate these altered chromosomal regions. The centromere in most eukaryotic organisms appears as a constricted region approximately 200-300 nm in diameter. In contrast, the gap portion of the centromeric region of pronuclear chromosomes was found to contain a chromatin fiber with a diameter of 80-150 nm. The detection of this fiber confirms that the chromosome arms are continuous, and the size of the fiber explains the gap appearance in the light photomicrographs. The morphology of the fiber is consistent with the concept that the normal chromatin packaging has been altered in varied regions within the centromere of these chromosomes.

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