scholarly journals Mouse satellite DNA, centromere structure, and sister chromatid pairing.

1986 ◽  
Vol 103 (4) ◽  
pp. 1145-1151 ◽  
Author(s):  
L M Lica ◽  
S Narayanswami ◽  
B A Hamkalo
Keyword(s):  
Electron Microscopy ◽  
Sister Chromatid ◽  
Satellite Dna ◽  
Marker Chromosome ◽  
Restriction Enzymes ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Mouse Satellite Dna

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.

scholarly journals Morphology and behaviour of silver-stained chromatid cores in mitotic chromosomes analysed by whole mount electron microscopy

1996 ◽  
Vol 68 (1) ◽  
pp. 1-7
Author(s):  
Jian Zhao ◽  
Shaobo Jin ◽  
Shui Hao ◽  
Ruiyang Chen
Keyword(s):  
Electron Microscopy ◽  
Sister Chromatid ◽  
Entire Length ◽  
Central Position ◽  
Core Network ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
The Core ◽  
Whole Mount ◽  
Segregation Of Chromosomes

SummaryUsing silver staining and the whole mount electron microscopy technique of squashed chromosomes, we studied the substructural organization and behaviour of chromatid cores in mitotic chromosomes of spermatogonia of the grasshopper Oedaleus infernalis during mitosis. It was found that the formation of mitotic chromatid cores takes place during the transition from prophase to prometaphase. Each chromosome contains two compact chromatid cores which are surrounded by a halo of dispersed argyrophilic material emanating radially from the cores. In early metaphase the chromatid core usually appears as an extended, slender network running longitudinally through the entire length of the chromatid, while in late metaphase the core frequently has a spiral appearance. In addition, our results revealed the existence of interconnections between sister chromatid cores along their entire length, as a result of which sister chromatid cores appear as a single interconnected core network in mitotic metaphase chromosomes. At this stage the core occupies a lateral position in each chromatid. However, during the transition from metaphase to anaphase, the interconnections are gradually released to allow the individualization of sister chromatid cores and the segregation of chromosomes. The core comes to occupy a central position in each segregated chromatid. These findings demonstrate the presence of an intrinsic interconnected core network within metaphase chromosomes which could be involved in the maintenance and segregation of chromosomes during mitosis.

Molecular Hybridization of Mouse Satellite DNA-Complementary RNA in Ultrathin Sections Prepared for Electron Microscopy

1974 ◽  
Vol 14 (2) ◽  
pp. 253-261
Author(s):  
J. JACOB ◽  
KATHERINE GILLIES ◽  
D. MACLEOD ◽  
K. W. JONES
Keyword(s):  
Electron Microscopy ◽  
Satellite Dna ◽  
Similar Distribution ◽  
Tissue Sections ◽  
Satellite Sequences ◽  
Interphase Cells ◽  
Nucleolar Chromatin ◽  
Ultrathin Sections ◽  
Mouse Satellite Dna

The feasibility of in situ hybridization in tissue sections prepared for electron microscopy has been examined using mouse satellite DNA-complementary RNA and mouse L cells. The results obtained are encouraging, although certain technical aspects require further clarification. In interphase cells, hybrid-forming sites occur in chromatin patches positioned along the nuclear envelope. It is also confirmed that satellite DNA occurs in nucleolus-associated chromatin. The results suggest that satellite sequences are present in intranucleolar and peri-nucleolar chromatin. A similar distribution is indicated for ribosomal cistrons.

scholarly journals Electron microscopy of gold-labeled human and equine chromosomes.

1989 ◽  
Vol 37 (9) ◽  
pp. 1443-1447 ◽  
Author(s):  
P E Messier ◽  
R Drouin ◽  
C L Richer
Keyword(s):  
Electron Microscopy ◽  
Chromosome Banding ◽  
Protein A ◽  
Gold Complex ◽  
Chromosome Identification ◽  
Chromosomal Dna ◽  
Metaphase Chromosomes ◽  
Sister Chromatids ◽  
Antigen Localization ◽  
Chromatin Reorganization

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.

A Scanning Electron Microscopy Study of the Structure of Human Mitotic Chromosomes

1981 ◽  
Vol 39 ◽  
pp. 442-443
Author(s):  
Kenneth W. Adolph
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Divalent Cations ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Thin Sectioning ◽  
Reproducible Manner ◽  
Scanning Electron

The organization of the fundamental 200-300A chromatin fibers in metaphase chromosomes has been investigated by various techniques of electron microscopy. Thin sectioning studies have suggested that a primary mode of fiber packaging is a radial distribution of loops. In these sectioning studies, the arrangement of fibers was observed by adjusting the concentration of divalent cations (Mg2+, Ca2+) in resuspension buffer to slightly expand the chromosomes and separate the fibers. Scanning electron microscopy has now been applied to obtain information about the surface organization of isolated chromosomes. As with thin sectioning, the degree of expansion of the chromosomes was controlled in a defined and reproducible manner by adjusting the concentration of divalent cations.Chromosomes were prepared from metaphase-arrested suspension cultures of HeLa cells. The isolation buffer contained 50 mM NaCl, 5 mM HEPES pH 7.4, 5 mM MgCl2, 0.5 mM CaCl2, 0.1 mM PMSF, and the mitotic cells were disrupted in a Dounce homogenizer with the aid of detergents.

scholarly journals In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

1982 ◽  
Vol 95 (2) ◽  
pp. 609-618 ◽  
Author(s):  
NJ Hutchison ◽  
PR Langer-Safer ◽  
DC Ward ◽  
BA Hamkalo
Keyword(s):  
In Situ Hybridization ◽  
Electron Microscope ◽  
Satellite Dna ◽  
Light Microscope ◽  
Test System ◽  
Microscope Level ◽  
Metaphase Chromosomes ◽  
Light Microscope Level ◽  
Mouse Satellite Dna

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

Molecular features of heterochromatin condensation

1993 ◽  
Vol 51 ◽  
pp. 72-73
Author(s):  
B.A. Hamkalo ◽  
K. Lundgren ◽  
M.H. Parseghian ◽  
M.Z. Radic ◽  
M. Saghbini
Keyword(s):  
Protein Interactions ◽  
Chromatin Condensation ◽  
Higher Order ◽  
Centromeric Heterochromatin ◽  
Order Structure ◽  
Sequence Motif ◽  
Dna Curvature ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Molecular Features

Eukaryotic chromosomes are nonuniformly condensed in both interphase and metaphase. This difference is most apparent in mitotic chromosomes in which centromeric heterochromatin is distinguishable from euchromatic arms because of a higher degree of condensation. This difference prevails in interphase and has led to the designation of this type of heterochromatin as “constitutive”. Differences in condensation presumably are a consequence of differential stability of higher order chromatin structure. Since higher order structure is likely to be due to distinctive DNA-protein and protein-protein interactions, our goal is to identify components of such interactions as a first step in understanding the molecular basis for differential chromatin condensation.Analysis of the structure of the mouse major centromeric satellite DNA revealed the presence of stable DNA curvature which could be reversed if certain small ligands which recognize a sequence motif common to this highly repeated DNA were bound to the DNA. In addition, growth of cells in the presence of these ligands prevented complete condensation of centromere regions of metaphase chromosomes (Fig.1).

Phosphorylation of histone H3 is correlated with changes in the maintenance of sister chromatid cohesion during meiosis in maize, rather than the condensation of the chromatin

2000 ◽  
Vol 113 (18) ◽  
pp. 3217-3226 ◽  
Author(s):  
E. Kaszas ◽  
W.Z. Cande
Keyword(s):  
Sister Chromatid ◽  
Meiotic Chromosome ◽  
Histone H3 ◽  
Sister Chromatid Cohesion ◽  
Chromosome Condensation ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
H3 Phosphorylation ◽  
Chromatid Cohesion ◽  
Metaphase I

Meiotic chromosome condensation is a unique process, characterized by dramatic changes in chromosome morphology that are required for the correct progression of pairing, synapsis, recombination and segregation of sister chromatids. We used an antibody that recognizes a ser 10 phosphoepitope on histone H3 to monitor H3 phosphorylation during meiosis in maize meiocytes. H3 phosphorylation has been reported to be an excellent marker for chromosome condensation during mitotic prophase in animal cells. In this study, we find that on maize mitotic chromosomes only pericentromeric regions are stained; there is little staining on the arms. During meiosis, chromosome condensation from leptotene through diplotene occurs in the absence of H3 phosphorylation. Instead, the changes in H3 phosphorylation at different stages of meiosis correlate with the differences in requirements for sister chromatid cohesion at different stages. Just before nuclear envelope breakdown, histone H3 phosphorylation is seen first in the pericentromeric regions and then extends through the arms at metaphase I; at metaphase II only the pericentromeric regions are stained. In afd1 (absence of first division), a mutant that is defective in many aspects of meiosis including sister chromatid cohesion and has equational separation at metaphase I, staining is restricted to the pericentromeric regions during metaphase I and anaphase I; there is no staining at metaphase II or anaphase II. We conclude that changes in the level of phosphorylation of ser10 in H3 correspond to changes in the cohesion of sister chromatids rather than the extent of chromosome condensation at different stages of meiosis.

scholarly journals Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.

1988 ◽  
Vol 8 (3) ◽  
pp. 1336-1344 ◽  
Author(s):  
D Talarico ◽  
A F Peverali ◽  
E Ginelli ◽  
R Meneveri ◽  
C Mondello ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Sequences ◽  
Satellite Dna ◽  
Repetitive Sequences ◽  
Metaphase Chromosomes ◽  
Highly Repetitive Dna ◽  
Cellular Dna ◽  
Mouse Satellite Dna ◽  
Simplex Virus

To study the influence of clustered highly repetitive DNA sequences on the expression of adjacent genes, LTK- cells were cotransfected with the herpes simplex virus thymidine kinase (tk) gene and mouse satellite DNA. TK+ transformants containing a few copies of the tk genes flanked by satellite DNA were isolated. In situ hybridization on the metaphase chromosomes indicated that in each cell line the TK sequences resided at a single chromosomal site and that integration occurred preferentially into regions of the cellular DNA rich in highly repetitive sequences. The prominent feature of these cell lines was their phenotypic instability. Suppression and reexpression of the tk gene occurred at high frequency (greater than 3%) and did not correlate with any significant change in the organization of foreign DNA or with the presence of selective agents. These results indicate that satellite DNA, the major component of constitutive heterochromatin, may influence the expression of adjacent genes by affecting the chromatin structure.

Distribution and function of non-histone proteins in human chromosomes

1992 ◽  
Vol 50 (1) ◽  
pp. 556-557
Author(s):  
W.C. Earnshaw ◽  
C.A. Cooke
Keyword(s):  
Mitotic Spindle ◽  
Centromeric Heterochromatin ◽  
Mitotic Chromosomes ◽  
Histone Proteins ◽  
Sister Chromatids ◽  
Structural Specialization ◽  
And Function ◽  
Passenger Proteins

The role of non-histone proteins in the structure and movements of mitotic chromosomes remains poorly understood. We describe here experiments aimed at characterization of the distribution of two very different classes of these proteins. The first is composed of integral components of the centromere (or primary constriction). The second class consists of proteins that we have termed “chromosome passenger proteins”. These proteins are chromosomal during most of the cell cycle, but appear to be associated with the cytoskeleton during anaphase and telophase.The centromere regions of chromosomes perform three essential functions in mitosis. (1) They form the site of attachment of the chromosomes to the mitotic spindle. (2) They contain the mechanochemical motor molecules that are responsible for the movements of the chromosomes along microtubules. (3) They regulate the pairing of sister chromatids during mitosis. The first two of these mitotic functions are properties of a disk-shaped structural specialization, the kinetochore, which is located at the surface of the centromeric heterochromatin.

Cohesin dependent compaction of mitotic chromosomes

2016 ◽  
Author(s):  
Stephanie A Schalbetter ◽  
Anton Goloborodko ◽  
Geoffrey Fudenberg ◽  
Jon M Belton ◽  
Catrina Miles ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Mitotic Chromosome ◽  
Protein Complexes ◽  
Budding Yeast ◽  
Mitotic Chromosomes ◽  
Chromosome Conformation ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Structural Maintenance Of Chromosomes

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply-conserved SMC complexes, organize chromosomes in budding yeast. The canonical role of cohesins is to co-align sister chromatids whilst condensins generally compact mitotic chromosomes. We find strikingly different roles in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosomes arms, independent of and in addition to its role in sister-chromatid cohesion. Cohesin dependent mitotic chromosome compaction can be fully accounted for through cis-looping of chromatin by loop extrusion. Second, condensin is dispensable for compaction along chromosomal arms and instead plays a specialized role, structuring rDNA and peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that SMC-dependent looping is readily deployed in a range of contexts to functionally organize chromosomes.

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