Question

An experiment was designed using tritiated thymidine (radioactive). Growing cells of the bean Vicia fava were labeled. Individual chromatids could be visualized microscopically and the radioactivity seen as dark grains deposited over individual chromatids.   The cells were fixed and observed at three times--after labeling all chromatids with radioactive thymidine, after one mitotic division, and after two mitotic divisions. Given that the Watson and Crick semiconservative model for DNA replication is correct, how would the chromosomes and chromatids appear at the three metaphase stages after labeling? Indicate your answer by drawing a representative chromosome for each of the metaphase figures. PLEASE ONLY ANSWER IF YOU ACTUALLY KNOW THE ANSWER

Answer 1

Hii guys,

This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.

The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid). DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical “daughter” cells from a single “parent” cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

Methods of detecting and assay of DNA synthesis:

In a cell, DNA replication must happen before cell division can occur and the biochemical pathway correlates well with DNA synthesis which is relatively specific for cell division. Measurement of new DNA synthesis is, therefore, essentially synonymous with measurement of cell proliferation. Direct measurement generally involves the incorporation of a labeled nucleoside into genomic DNA. Examples include the tritiated thymidine ([3H]dT) and BrdU (bromodeoxyuridine) methods 1, 2. Radioactive tagging of newly synthesized DNA with 3H-labeled Thymidine (3H-T) is most frequently applied technique. In recent days, usage of BrdU non radioactive labeling has increased. Gratzner in 1982 2 developed a monoclonal antibody to BrdU and for identifying BrdU-labeled S-phase cells with immunoperoxidase, immunofluorescence, or avidin-biotin complexes 3, 4, 5, 6. Monoclonal antibody (MAb) techniques for detection of BrdU have the advantages of simplicity and speed over standard autoradiography. Therefore, for a long period of time the cell biologists have been using the technique of thymidine labeling, whereby a group of chemicals such as BrdU are incorporated into the DNA of dividing cells. The advantages of non-radioactive immune techniques using BrdU or anti-BrdU are undisputed but the scope and limitations of the systems for detecting BrdU cannot always be clearly defined. Besides enabling microscopic detection at the cellular level, the enzyme immune assay technique represents a method for routine screening. Two different detection systems are available: 1) the conventional colorimetric test and 2) a chemiluminescence assay also based on the anti-BrdU technique. One of these test methods will be suitable for use depending on the problem and the available laboratory facilities.

Conclusion:

This is a small review which may be helpful for the beginners, to learn technical details.

Hope this helps u guys!