Biomedical subjects
Publications and source records attributed to V Goyanes.
A high resolution procedure for analyzing human centromeric heterochromatin is described. The combined use of decondensation agents of pericentromeric heterochromatin and electron microscopy of whole mounted chromosomes allows a more precise identification of centromere and pericentromeric regions.
Mouse chromosomes from the L929 cell line have been digested with the restriction endonuclease HaeIII and analyzed by electron microscopy. Results show a different effect of the enzyme depending on the conditions of the digestion. Thus, while chromosomes digested in suspended cells show a double scaffold-like structure per chromatid, a similar banding to that found in chromosomes treated for light microscopy is obtained when chromosomes are digested on grids. Some aspects concerning the capacity of the cleaved DNA to be removed from the chromatin are discussed.
Denaturation and posterior renaturation of metaphase chromosomes after different treatments were tested by acridine orange staining. A relationship between conservation of ultrastructure and the capacity to renature was discovered. Amyl acetate, commonly used in electron microscopy to fix chromosomes, avoids the action of denaturing agents and this effect is not reversed by chromosome rehydration.
Five morphological parameters of sister chromatids, viz radius, length, centromeric index, volume and centromere width, were studied by electron microscopy, from 140 human number 1 chromosomes, and 122 human number 2 chromosomes. The degree of variation obtained ranged from 0.277 +/- 0.003% and 0.286 +/- 0.005% for the centromeric index of chromosomes 1 and 2, respectively, to 9.835 +/- 0.933% and 13.472 +/- 1.461% for centromere width.
If cells are allowed to replicate in the presence of different concentrations of BrdUrd throughout three consecutive cell cycles, FPG stained third division chromosomes show three different levels of staining. This fact allows identification of four different types of SCE's, corresponding to the first-, second- and third-cycle exchanges. Finally, the relationship between the degree of BrdUrd-substitution and the frequency of SCE's per cell cycle supports that most of the SCE's detected in BrdUrd-substituted chromosomes are induced by this thymidine analogue incorporated into DNA.
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Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.
Since sister chromatid exchange (SCE) relates directly to DNA synthesis which is circadian periodic, this study examines whether a circadian rhythm also characterizes SCEs. In 4-hourly blood samples collected for 48 hours on three diurnally active, nocturnally resting subjects with no family history of chromosome aberrations or instability, a circadian rhythm is demonstrable with an acrophase occurring around 14:45. The circadian amplitude is only of the order of 10% of the MESOR in untreated cultures or in cultures exposed to cefodizime. It remains more or less unchanged in cultures exposed to mitomycin-C, while the MESOR increases by about a factor of 2. In studies of human chromosome aberrations, the circadian rhythmicity of SCE should be taken into consideration.