Incision assay

DNA repair knockout Inborn DNA repair defects Incision assay Base modifications... 

The BER capacity in mES cells is greater compared to MEF. It has been shown that proteins involved in BER, f.e. Apel, DNA ligase IE, Parpl, Pena, Ung2, Xrccl, are expressed to a higher extent in mES ceEs. Furthermore a BER incorporation assay and a DNA incision assay have shown a higher BER activity. The latter assay revealed a six-fold greater level of incision production in mES ceEs compared to MEF cells (Tichy et al. 2011). 

The test detects the ability of a chemical to enhance the exchange of DNA between two sister chromatids of a duplicating chromosome. SCEs are reciprocal interchanges of the two chromatid arms within a single chromosome. These exchanges are visuaUzed during the metaphase portion of the cell cycle and presumably require enzymatic incision, translocation, and ligation of at least two DNA helices. The most commonly used assays employ bone marrow or lymphocytes from mammalian species such as mice, rats, or hamsters. Human lymphocytes may also be used. 

A typical dermal irritation assay is conducted as follows. Six male albino rabbits are be clipped free of hair on the back. One area of skin is left intact, whereas another is abraded in a tic-tac-toe pattern with the point of a hypodermic needle so as to incise the superficial epidermis layer without causing bleeding. The test material, 0.5 ml of liquid or 0.5 g of solid or semisolid is applied to each site under a 1 x 1 in. gauze pad. The entire trunk of the animal is wrapped with an impervious material and held in place with tape for 24 h. The patches are then removed and excessive material wiped off. The skin reactions are scored at 24 and 72 h after the initial application according to a scheme such as that listed in Table 2. 

Figure 12.1 Outline of NER assay in human cell extracts. The hallmark of a successful mammalian NER dual-incision activity is the excision of internally 32P-labeled 24- to 32-nucleotide fragments containing the...

Experimental vitamin C deficiency in humans was assayed in the early forties by Crandon (1940), who underwent a skin incision after six months on a diet essentially free of ascorbate. Failure in the reparative process, together with deficient formation of intercellular matrix and vascular elements, were histologically observed in a biopsy of the wound taken ten days after a skin incision. Similar results were later confirmed by Wolfer and coworkers (1947). 

With our sensitive spectrophotometric methods (11,12,17) we have shown how the reaction velocity constants for peroxidase action can be measured directly from the kinetics of formation and disappearance of the enzyme-substrate complex. This is perhaps the most elegant and incisive method of assay and can now be successfully applied not only to hemo-proteins but also to diphosphopyridine nucleotide enzymes. The detaUs of the methods lie outside the scope of this review and are available in detail elsewhere (3,6). 

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