Excision assay

Figure 1. Diagrammatic representation of the P-element excision assay in embryonic soma.

The excision assay was tested also in two other Drosophila species to determine the functionality of the assay in other insects. Excision occurred in both D. simulans and D. grirashawii at nearly the same rate as observed in D. melanogaster (Table 1). The transposition activity of the P-element in the embryos of these species demonstrates that the excision assay can be employed as a reliable indicator of P-element behavior in other insects. 

The failure to observe P-element excision in A. suspensa and P. interpunctella embryos suggests that P-elements may not be functional in all insects. Although the reason for the lack of functionality has not been identified, the excision assay is designed with adequate latitude for modification so that it is amenable to experimental analysis. With sufficient effort, the biochemical basis for the lack of P-element activity can be identified and potentially be corrected. Preliminary results indicate that the transposase gene carried by the helper plasmid is being transcribed in A. suspensa, and efforts are now underway to determine if all of the posttranscriptional modifications necessary to generate a functional transposase raRNA occur (15). 

Parasorbic acid (Figure 2) was isolated from fruits of Sorbus aucuparia. Germination of mustard seed Sinapis alba) was affected adversely by parasorbic acid at 3.5 X 10-3 M and growth of excised tomato roots was inhibited at approximately 8.5 X 10 4 M (25). The acid also antagonized indoleacetic acid (IAA) in the Avena assay. Cornman 29,30) reported that parasorbic acid slowed down mitosis. Metaphase stages were observed to accumulate, but abnormalities were not detected. 

Figure 3. Influence at 500 (lM salicylic acid on ATP content of excised oat roots at four pH values. ATP determined by luciferin/ luciferase assay.

Table II. Sago Pondweed Excised Shoot Assay After One Week Exposure to Polar Spikerush Extracts...

In our laboratory, the viability of excised porcine buccal mucosa was assessed using histological evaluation and a 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) biochemical assay which has previously been used in assessing the viability of excised buccal mucosa and cornea [49, 50], Histological evaluation of tissue demonstrated that the buccal epithelium appeared viable up to 9 h postmortem, and this was supported by the MTT biochemical assay, which indicated that viability was maintained for up to 12 h [80], Therefore, we recommend that all permeation experiments using porcine buccal mucosa be performed within 9-12 h of animal death. 

Utilising a reversion assay in Salmonella enterica, Prieto et al reported an increased frequency of point mutations following bile-salt exposure. Mutations were predominantly nucleotide substitutions (GC to AT transitions) and -1 frameshift mutations.The frameshifts were dependent on SOS induction and linked to the activity of DinB polymerase (Pol IV). The authors proposed that the GC to AT transitions stimulated by bile, could have arisen from oxidative processes giving rise to oxidised cytosine residues. Consistent with this hypothesis, the authors demonstrated that strains of S. enterica-lacking enzymes required for base-excision repair (endonuclease III and exonuclease IV) and the removal of oxidised bases, demonstrated increased bile-acid sensitivity compared with competent strains. In another study using E. coli, resistance to the DNA-damaging effects of bile was associated with Dam-directed mismatch repair, a pathway also involved with the repair of oxidative DNA lesions. ... 

This assay is normally carried out only if positive effects have been obtained in earlier in vitro tests. The UDS test measures the DNA repair synthesis which occurs after excision and removal of a stretch of DNA containing the region of damage, induced in hepatocytes of animals treated with the test chemicals. UDS is measured by the uptake of radioactively labelled nucleotide, usually tritium-labelled th)unidine, into the DNA of the damaged hepatocytes. Animals, usually male rats, are treated with the test chemical, and... 

Metabolism and genetic toxicity have been reported to differ with the isomer of nitro-toluene. p-Nitrotoluene was not mutagenic in bacterial assays, but it did increase sister chromatid exchange frequencies and chromosomal aberrations in vitro-, in vivo it did not increase the frequency of micronuclei in bone marrow of treated rodents. Similar findings were reported for the ortho isomer, except that it did not induce chromosomal aberrations in vitro. Only the ortho isomer induces DNA excision repair in the in vivo-in vitro hepatocyte unscheduled DNA synthesis assay. Furthermore, ort/jo-nitrotoluene binds to hepatic DNA to a much greater extent than meta- or para-nitrotoluene, and investigators suggest that it may act similarly to the rodent hepatocarcino-gen 2,6-dinitrotoluene. ... 

Jones, J. D. G., Shlumukov, L., Garland, E., English, J., Scofield, S. R., Bishop, G. J. and Harrison, K. 1992. Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in plants. Transgenic Research, 1 285-297. 

Figure 19.5 Visualization of DNA damage induction in cultured human keratinocytes by photo-activated lomefloxacin using the comet assay. The presence of DNA breaks (induced either by ROS or by excision of DNA lesions) leads to fragmentation and electrophoretic migration to produce the comet tails, whereas bulky genomic DNA remains in the comets heads.

Tobacco accounts for about 90% of lung cancers. Some tobacco carcinogens induce DNA adducts that are repaired by the nucleotide excision repair (NER) pathway (3). The lowest DNA repair capacity (DRC), measured in peripheral blood lymphocytes by the host cell reactivation assay (HCR), was observed in lung cancer patients who were less than 60 years old, female, or lighter smokers, and in those with a family history of cancer (4,5). 

Later, Rupp et al. (1971) were able to demonstrate a physical exchange of material between molecules of DNA in irradiated cells defective in excision-repair. In RecB or Rec C mutants, this did not occur. The phenomenon could not be assayed in a rec-less mutant. 

Tg, 50H5,6H2T, 5,6H2T, 5HmU and 50H5MeHyd are readily detected by GC/ MS after excision from the damaged DNA and trimethylsilylation (Chap. 13.2). For the Tg lesion, an ultrasensitive assay is also available that even allows its detection in cells at a dose as low as 0.25 Gy and to follow its repair (Le et al. 1998). 

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