Staining of Satellite DNA in Metaphase Chromosomes

Nature ◽  
1971 ◽  
Vol 231 (5304) ◽  
pp. 532-533 ◽  
Author(s):  
JORGE J. YUNIS ◽  
LILIANA ROLDAN ◽  
WALID G. YASMINEH ◽  
J. C. LEE
Keyword(s):  
Satellite Dna ◽  
Metaphase Chromosomes

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 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.

Evidence for the existence of satellite DNA-containing connection between metaphase chromosomes

2007 ◽  
Vol 101 (4) ◽  
pp. 1046-1061 ◽  
Author(s):  
I.S. Kuznetsova ◽  
N.I. Enukashvily ◽  
E.M. Noniashvili ◽  
A.N. Shatrova ◽  
N.D. Aksenov ◽  
...  
Keyword(s):  
Satellite Dna ◽  
Metaphase Chromosomes

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.

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.

Satellite DNA and heterochromatin of the flour beetle Tribolium confusum

Genome ◽  
1993 ◽  
Vol 36 (3) ◽  
pp. 467-475 ◽  
Author(s):  
Miroslav Plohl ◽  
Vlatka Lucijanić-Justić ◽  
Durdica Ugarković ◽  
Eduard Petitpierre ◽  
Carlos Juan
Keyword(s):  
Satellite Dna ◽  
Tandem Repeats ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Tribolium Confusum ◽  
Inverted Repeats ◽  
Metaphase Chromosomes ◽  
Bent Dna ◽  
Distamycin A

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40–45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 × 105 per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.Key words: tandem repeats, DNA sequence, bent DNA, inverted repeats, Coleoptera.

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

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