RESUMEN
Genome size of Cyclops in cells at early stages of cleavage (up to the 5th division) and in somatic cells were estimated by a static digital Feulgen cytophotometry in order to study the quantitative changes in DNA content during chromatin diminution. Our realization of the cytophotometric method was approbeted on five different digital-imaging systems in blood cells of four vertebrate species. In all cases, we observed a direct correlation of the obtained and known from the literature data on the genome size and a high reproducibility, which allows to use these systems in future work. We also optimized the conditions for DNA hydrolysis of both blood smears, and for two species of Cyclops from the Moscow population, as 30 min in 5 N HCl at 24 degrees C. Here we first revealed chromatin diminution in two endemic Baikal species of Cyclopoida: Acanthocyclops incolotaenia and Diacyclops galbinus estimated the extent ofchromatin diminution in Diacyclops galbinus as 95.5-96.2 %. Cytometric analysis of the third species, Mesocyclops leuckarti, did not reveal obvious chromatin diminution. We also optimized the conditions for DNA hydrolysis of both blood smear preparations, and for two species of copepods from the Moscow population, as 30 min in 5N HCl at 24 degrees C.
Asunto(s)
Copépodos/citología , Copépodos/metabolismo , Citofotometría/métodos , ADN/metabolismo , Genoma/fisiología , Animales , Copépodos/genética , ADN/genética , SiberiaRESUMEN
We suggest a new theoretical method of the flow cytometry DNA histograms and apply it for Drosophila melanogaster imaginal discs cells. The model gives a possibility to determine the proportions of cells in G1, G2 (M) and S cell cycle phases. We show that the precision of G1 and G2 (M) DNA content measurements is limited by the precision of device zero signal arrangement. The usage of calculated device zero and dividing cells as the DNA content standards may improve the precision of DNA content measurements. We also compared the precisions of different DNA content methods and draw the conclusion that the current precisions of different methods are similar and lie within 2-6% interval.
Asunto(s)
Núcleo Celular/química , ADN , Citometría de Flujo/métodos , Discos Imaginales/química , Animales , Anuros , División Celular/genética , Pollos , ADN/análisis , Drosophila melanogaster , Fase G1/genética , Fase G2/genética , Modelos Teóricos , Reproducibilidad de los Resultados , Fase S/genética , Sensibilidad y Especificidad , Pez CebraRESUMEN
Quantitative nuclei DNA content measurement based on Feulgen reaction and the analysis of CCD images was tested. The measurements were performed in monochorome CCD option (650 X 514 pixels) with the wavelength 551 nm. The linear dependence of photomatrix elements signals on the falling light was shown with the use of multigraded light absorption filter. The optimal microscope and camera setting and an approach for elimination of the optic blur were found. We have shown that the proper Feulgen fluorescence does not affect our measurements. Densitometric DNA content measurements of blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens and Rana arvalis) showed good consistence to the literature data (http://www.genomesize.com). The precision of our approach is comparable to the other known methods. Current improvement of CCD technical parameters and wide usage of CCD cameras in biological applications gives perspectives for the suggested approach.