scholarly journals A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

1998 ◽  
Vol 46 (10) ◽  
pp. 1203-1209 ◽  
Author(s):  
Françoise Jaunin ◽  
Astrid E. Visser ◽  
Dusan Cmarko ◽  
Jacob A. Aten ◽  
Stanislav Fakan

We describe a colloidal gold immunolabeling technique for electron microscopy which allows one to differentially visualize portions of DNA replicated during different periods of S-phase. This was performed by incorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chinese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate blocking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific staining of DNA with osmium ammine show that labeling corresponds well to replicated DNA. Different patterns of labeling distribution, reflecting different periods of DNA replication during S-phase, were characterized. Cells in early S-phase display a diffuse pattern of labeling with many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated with the nucleolus. The good correlation between our observations and previous double labeling results in immunofluorescence also proved the technique to be reliable.

1973 ◽  
Vol 58 (3) ◽  
pp. 564-573 ◽  
Author(s):  
Robert R. Klevecz ◽  
Leon N. Kapp

Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G1 between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.


1975 ◽  
Vol 66 (1) ◽  
pp. 95-101 ◽  
Author(s):  
K D Ley

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.


1964 ◽  
Vol 23 (1) ◽  
pp. 53-62 ◽  
Author(s):  
T. C. Hsu

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H3-thymidine labeling and the method of 5-fluorodeoxyuridine block with H3-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.


2004 ◽  
Vol 94 (1) ◽  
pp. 126-138 ◽  
Author(s):  
Karel Koberna ◽  
Anna Ligasová ◽  
Jan Malínský ◽  
Artem Pliss ◽  
Alan J. Siegel ◽  
...  

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