DNA changes involved in the formation of metaphase chromosomes, as observed in mouse duodenal crypt cells stained by osmium-ammine II. Tracing nascent dna by bromodeoxyuridine into structures arising during the S phase

1995 ◽  
Vol 242 (4) ◽  
pp. 449-461 ◽  
Author(s):  
Dong Feng Liu ◽  
Mohamed El-Alfy ◽  
Charles Philippe Leblond
Keyword(s):  
S Phase ◽  
Metaphase Chromosomes ◽  
Crypt Cells

scholarly journals Units of chromosome replication and packing

1983 ◽  
Vol 64 (1) ◽  
pp. 179-193
Author(s):  
A.M. Mullinger ◽  
R.T. Johnson
Keyword(s):  
Spatial Distribution ◽  
Light Microscope ◽  
Individual Cell ◽  
S Phase ◽  
Scanning Electron Micrographs ◽  
Metaphase Chromosomes ◽  
Impregnation Technique ◽  
Osmium Impregnation ◽  
The Relationship ◽  
Scanning Electron

Fusion between mitotic and S-phase cells induces the formation of prematurely condensed chromosomes (PCC) in the interphase partner. Viewed in the light microscope, S-phase PCC derived from the Indian muntjac appear to be fragmented and heterogeneous. In scanning electron micrographs prepared by an osmium impregnation technique, which avoids the need to sputter-coat the specimen, the S-phase fragments derived from an individual cell are resolved into about 1000 fibre aggregates, together with more dispersed fibres. Aggregates are roughly spherical and vary in diameter between about 0.25 and 1.6 micron. The spatial distribution of the aggregates shows some order: chains of single aggregates and, less commonly, duplicated chains occur. Regions of the PCC where the fibres are more dispersed are considered to be likely candidates for sites of replication at the time of fusion. The relationship between the condensed aggregate structure of the S-phase PCC and replication clusters is discussed, and also the assembly of aggregates to form metaphase chromosomes.

scholarly journals BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.

1990 ◽  
Vol 38 (2) ◽  
pp. 267-273 ◽  
Author(s):  
B Thoolen
Keyword(s):  
Small Intestine ◽  
Microwave Irradiation ◽  
Silver Staining ◽  
Leydig Cells ◽  
Protein A ◽  
S Phase ◽  
Immunocytochemical Study ◽  
Gold Solution ◽  
Proliferating Cells ◽  
Crypt Cells

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.

1976 ◽  
Vol 24 (1) ◽  
pp. 34-39 ◽  
Author(s):  
H G Gratzner ◽  
A Pollack ◽  
D J Ingram ◽  
R C Leif
Keyword(s):  
Deoxyribonucleic Acid ◽  
De Novo ◽  
Single Cells ◽  
Acid Synthesis ◽  
S Phase ◽  
Cho Cell ◽  
Metaphase Chromosomes ◽  
Time Intervals ◽  
Deoxyribonucleic Acid Synthesis ◽  
Sepharose 4B

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

scholarly journals Amplified dihydrofolate reductase genes are located in chromosome regions containing DNA that replicates during the first half of S-phase.

1982 ◽  
Vol 92 (2) ◽  
pp. 531-539 ◽  
Author(s):  
R E Kellems ◽  
M E Harper ◽  
L M Smith
Keyword(s):  
Dna Replication ◽  
Dihydrofolate Reductase ◽  
S Phase ◽  
Parental Line ◽  
Metaphase Chromosomes ◽  
Regional Patterns ◽  
Resistant Mouse ◽  
Karyotypic Analysis ◽  
Marker Chromosomes ◽  
Chromosome Regions

To obtain a better understanding of the relationship between metaphase chromosome banding patterns and genome organization, attention was focused on regions of metaphase chromosomes that were found to contain the genes for a specific cellular enzyme, dihydrofolate reductase (DHFR). These studies involved the use of highly methotrexate-resistant mouse lymphoblastoid cells (L5178YR), which contain approximately 300 times the number of DHFR genes present in parental cells (L5178YS). Karyotypic analysis revealed the presence of two very large, nonhomologous, marker chromosomes that were absent in the parental line. In situ hybridization of 3H-labeled cloned DHFR cDNA to metaphase chromosomes of L5178YR cells was used to localize the DHFR genes to a very large Giemsa (G)-negative region on each of the two large marker chromosomes. Regional patterns of DNA replication in metaphase chromosomes were studied by autoradiographic visualization of [3H]thymidine incorporation and by fluorescent microscopic visualization of bromodeoxyuridine incorporation. Because the amplified DHFR genes were present within two prominent cytogenetic regions on two easily identifiable chromosomes, it was possible to observe the following. The amplified DHFR genes were located in chromosome regions that replicated at the same time during the first half of a 9-h-S-phase. DNA replication began simultaneously and terminated simultaneously at many locations throughout each amplified region. We conclude that transcriptionally active DHFR genes are located within large G-negative regions of metaphase chromosomes and that the DNA within these regions replicates during the first half of S-phase.

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.

Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

1992 ◽  
Vol 50 (2) ◽  
pp. 1038-1039
Author(s):  
Shawn Williams ◽  
Xiaodong Zhang ◽  
Susan Lamm ◽  
Jack Van’t Hof
Keyword(s):  
Visible Light ◽  
Radiation Damage ◽  
Cross Section ◽  
Imaging Techniques ◽  
Dose Range ◽  
Specimen Preparation ◽  
X Rays ◽  
Metaphase Chromosomes ◽  
X Ray ◽  
Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Advances in imaging SIMS studies of stained and BrdU-labelled human metaphase chromosomes

1995 ◽  
Vol 53 ◽  
pp. 932-933
Author(s):  
R. Levi-Setti ◽  
J. M. Chabala ◽  
R. Espinosa ◽  
M. M. Le Beau
Keyword(s):  
High Performance ◽  
Secondary Ion Mass Spectrometry ◽  
Analytical Sensitivity ◽  
Metaphase Chromosomes ◽  
Banding Patterns ◽  
Sector Mass Spectrometer ◽  
Staining Methods ◽  
The University ◽  
Secondary Ion ◽  
Better Than

We have shown previously that isotope-labelled nucleotides in human metaphase chromosomes can be detected and mapped by imaging secondary ion mass spectrometry (SIMS), using the University of Chicago high resolution scanning ion microprobe (UC SIM). These early studies, conducted with BrdU- and 14C-thymidine-labelled chromosomes via detection of the Br and 28CN- (14C14N-> labelcarrying signals, provided some evidence for the condensation of the label into banding patterns along the chromatids (SIMS bands) reminiscent of the well known Q- and G-bands obtained by conventional staining methods for optical microscopy. The potential of this technique has been greatly enhanced by the recent upgrade of the UC SIM, now coupled to a high performance magnetic sector mass spectrometer in lieu of the previous RF quadrupole mass filter. The high transmission of the new spectrometer improves the SIMS analytical sensitivity of the microprobe better than a hundredfold, overcoming most of the previous imaging limitations resulting from low count statistics.

Sign in / Sign up

Export Citation Format

Share Document