Methods
Two main assays are used to detect immortal DNA strand segregation: label-retention and label-release pulse/chase assays.
In the label-retention assay, the goal is to mark 'immortal' or parental DNA strands with a DNA label such as tritiated thymidine or bromodeoxyuridine (BrdU). These types of DNA labels will incorporate into the newly-synthesized DNA of dividing cells during S phase. A pulse of DNA label is given to adult stem cells under conditions where they have not yet delineated an immortal DNA strand. During these conditions, the adult stem cells are either dividing symmetrically (thus with each division a new 'immortal' strand is determined and in at least one of the stem cells the immortal DNA strand will be marked with DNA label), or the adult stem cells have not yet been determined (thus their precursors are dividing symmetrically, and once they differentiate into adult stem cells and choose an 'immortal' strand, the 'immortal strand' will already have been marked). Experimentally, adult stem cells are undergoing symmetric divisions during growth and after wound healing, and are not yet determined at neonatal stages. Once the immortal DNA strand is labelled and the adult stem cell has begun or resumed asymmetric divisions, the DNA label is chased out. In symmetric divisions (most mitotic cells), DNA is segregating randomly and the DNA label will be diluted out to levels below detection after five divisions. If, however, cells are using an immortal DNA strand mechanism all the labeled DNA will continue to co-segregate to the adult stem cell, and after five (or more) divisions will still be detected within the adult stem cell. These cells are sometimes called label-retaining cells (LRCs).
In the label-release assay, the goal is to mark the newly synthesized DNA that is normally passed on to the daughter (non-stem) cell. A pulse of DNA label is given to adult stem cells under conditions where they are dividing asymmetrically. Under conditions of homeostasis, adult stem cells should be dividing asymmetrically so that the same number of adult stem cells is maintained in the tissue compartment. After pulsing for long enough to label all the newly replicated DNA, the DNA label is chased out (each DNA replication now incorporates unlabeled nucleotides) and the adult stem cells are assayed for loss of the DNA label after two cell divisions. If cells are using a random segregation mechanism, then enough DNA label should remain in the cell to be detected. If, however, the adult stem cells are using an immortal DNA strand mechanism, they are obligated to retain the unlabeled 'immortal' DNA, and will release the all the labeled newly synthesized DNA to their differentiating daughter cells in two divisions.
Some scientists have combined the two approaches, by first using one DNA label to label the immortal strands, allowing to adult stem cells to begin dividing asymmetrically, and then using a different DNA label to label the newly synthesized DNA. Thus, the adult stem cells will retain one DNA label and release the other within two divisions.
Read more about this topic: Immortal DNA Strand Hypothesis
Famous quotes containing the word methods:
“With a generous endowment of motherhood provided by legislation, with all laws against voluntary motherhood and education in its methods repealed, with the feminist ideal of education accepted in home and school, and with all special barriers removed in every field of human activity, there is no reason why woman should not become almost a human thing. It will be time enough then to consider whether she has a soul.”
—Crystal Eastman (1881–1928)
“Commerce is unexpectedly confident and serene, alert, adventurous, and unwearied. It is very natural in its methods withal, far more so than many fantastic enterprises and sentimental experiments, and hence its singular success.”
—Henry David Thoreau (1817–1862)
“How can you tell if you discipline effectively? Ask yourself if your disciplinary methods generally produce lasting results in a manner you find acceptable. Whether your philosophy is democratic or autocratic, whatever techniques you use—reasoning, a “star” chart, time-outs, or spanking—if it doesn’t work, it’s not effective.”
—Stanley Turecki (20th century)