Zur Problematik der Chromosomenautoradiographie

The distribution of silver grains on the chromosomes of group G was studied in 216 mitoses from five patients with Down's syndrome after incubation with tritated thymidine in order to test the replication pattern of these autosomes. The different labelling densities over the chromosomes were determined by comparative estimations of the blackened areas and subdivided into four degrees: very strongly labelled, strongly labelled, weakly labelled and unlabelled. The 56 combinations for five G chromosomes and four labelling densitives were divided into three groups: group A, with three strong and two weak labelling types, group B, with two strong and three weak labelling types, and group C, with the remaining combinations. Thereby 51 (23.6%) mitoses were counted in six combinations of group A, 31 (14.4%) in six combinations of group B, and 134 (62.0%) in 44 combinations of group C. In order to test the possibility of identifying the G chromosomes based on a different DNA replication pattern the findings were subjected to a statistical analysis. The expected values were calculated for the different combinations of labelling degrees and were compared with the observed values. This method being used, the question whether the extra chromosome is replicating late or early compared with the remaining G chromosomes could not be answered with sufficient certainty, because P_A=1, P_B=0 could not be assumed on our figures. We conclude from these findings that the termination phases for the chromosomes 21 as well as 22 lie close together and that apparently the autoradiographic representation of the replication pattern can be influenced by various factors. The influencing factors can only be understood in connection with the problems of quantitative autoradiography. They are subdivided into three groups and discussed in this way: A. Factors due to the physiology of the genetic material: 1. Duration of the DNA-synthesis and the G₂-period. 2. Chromosomal replication pattern. 3. The relation of ³H-Thymidin and unlabelled thymidine in the newly synthesized DNA. B. Factors due to the autoradiographic and cytologic method: 1. Statistics of the radioactive disintegrations. 2. -self-absorption. 3. Coincidence. 4. Effectivity. 5. Back-ground. 6. Fading. 7. Condensation of the chromosomes. 8. Cytologic preparation. 9. Thickness of the emulsion layer. 10. Duration of exposition and developping. C. Factors due to the method of evaluation: 1. Accuracy of counting. 2. Deficient analysis of the results. After considering these factors it seems clear that the autoradiographic technique is not capable to demonstrate subtle differencies of the DNA replication. Thus the autoradiographic identification of chromosomes remains questionable if the termination phases of DNA replication lie close together as this seems the case for the chromosomes 21 and 22.

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Studie im Rahmen der Assoziation Hämatologie EURATOM — GSF.