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V Goyanes

Publications and source records attributed to V Goyanes.

At least 37 records · Page 2Linked to original sources
PubMed1994

Sister chromatid differentiation after in situ detection of ultraviolet-induced DNA breaks under electron microscopy.

Chinese hamster DON cells with 5-bromodeoxyuridine (BrdU)-substituted chromosomes were ultraviolet (UV)-exposed and processed for in situ detection of induced DNA breaks under electron microscopy. For this purpose, UV-induced breaks were amplified by an exonuclease III digestion to obtain single stranded DNA motifs which could hybridize with oligonucleotides of random sequences. These reannealed motifs could be used as primers which were extended by the Klenow polymerase, incorporating biotinylated-dUTP that was detected by a gold-tagged streptavidin. After processing, the chromatid whose DNA was BrdU-substituted in one strand showed a higher electron density than the chromatid substituted in both strands. In contrast, the unifilarly substituted chromatid showed about twice the labelling of DNA breaks as the bifilarly substituted one. This result could be the consequence of a greater loss of chromatin tracts in the bifilarly substituted chromatid, as implied by an X-ray microanalysis which showed that the amount of phosphorous lost by the bifilarly substituted chromatid was higher than that of the unifilarly substituted chromatid.

Animals↗
PubMed1994

Assessment of Yqh translocations.

Two initially presumed Yqh translocations, one to Xp and another to 21p, were assessed by conventional banding procedures, 5-azacytidine treatment, electron microscopy, and fluorescence in situ hybridisation. While the Yqh nature of the heterochromatic block from Xp was confirmed, this was not the case with the 21ph+ variant. In conclusion, conventional banding techniques are insufficient to diagnose Y;G translocations.

Azacitidine↗
PubMed1994

An extra band within the human 9qh+ region that behaves like the surrounding constitutive heterochromatin.

An extra variant G band in a human 9qh+ region was analysed in normally condensed and 5-azacytidine undercondensed chromosomes. Fluorescence in situ hybridisation showed that specific, classical, alphoid and beta satellite DNA was not present. Nevertheless, this extra band behaves like the surrounding heterochromatin because (1) its chromatin fibres showed condensation inhibition after 5-azacytidine treatment, as confirmed by electron microscopy, and (2) it was not affected by in situ digestion with the restriction endonucleases AluI and Sau3A. These results suggest that this variant band may correspond to euchromatin that has become inactivated by a position effect.

5-Methylcytosine↗
PubMed1994

Human alpha-satellite DNAs are not susceptible to undercondensation after 5-azacytidine treatment.

Localization of alphoid human satellite DNAs using fluorescence in situ hybridization (FISH) of metaphase chromosomes following treatment with 5-azacytidine to produce undercondensation showed that human alpha-satellite DNA is not sensitive to the condensation-inhibition effect of 5-azacytidine. The difference in heterochromatic DNA subsets was particularly evident in the constitutive heterochromatin of chromosomes 1 and 9. Comparison of the results obtained after FISH with those obtained from electron microscopy and G-banding enabled accurate localization of this DNA domain.

Azacitidine↗
PubMed1994

Variations in length and area in human Y chromosome: an ultrastructural analysis.

Variations in length and area of human Y heterochromatin and euchromatin were analysed by electron microscopy for chromosomes in which the heterochromatin was previously undercondensed with distamycin A. Quantitation of data showed that heterochromatin variation was greater than euchromatin variation in the long arm.

Chromatin↗
PubMed1994

Frequency of sister chromatid exchanges in humans at 14-16 week foetal stage and at birth.

In order to identify transplacental induced mutagenicity in humans the baseline frequency of sister chromatid exchanges (SCEs) in amniotic fluid cells (8.56 +/- 0.28 per cell) from 14 to 16-week-old foetuses as well as in peripheral blood lymphocytes (7.96 +/- 0.26 per cell) from newborns was determined. A group of twelve patients who were analysed both at 14-16 weeks gestation and at birth showed no SCE variations. These results suggest that foetal amniocytes and T lymphocytes from newborns present a similar sensitivity to the mutagenic activity of the thymidine analogue 5-bromodeoxyuridine employed in the sister chromatid differentiation.

Adult↗
PubMed1993

Turner's syndrome: a behavioral and cytogenetic study.

This study focused on the behavioral and cytogenetic aspects of Turner's syndrome. We evaluated the behavioral scores by ESPQ, HSPQ, and TAMAI tests. In the cytogenetic study, six different variables were defined by a chromosome image analysis computer system (Applied Imagine) that assessed changes in band organization and the measurement of mean optical density (MOD) in six G-bands (Xp21, Xq12, Xq21, Xq23, Xq25, Xq27). We studied 33 females with Turner's syndrome and compared them with 30 females individually matched for normal karyotype, short stature, age, and education. Significant differences were found between the two groups.

Adolescent↗
PubMed1993

Selective digestion of mouse chromosomes with restriction endonucleases. Oligonucleotide priming of single-stranded DNA produced with exonuclease III.

L-929 mouse chromosomes prepared for electron microscopy have been treated with MspI, EcoRI, and HaeIII restriction endonucleases (REs). RE-induced nicks were amplified with exonuclease III to obtain single-stranded DNA (ss-DNA) motifs. The ss-DNA produced was enough to permit hybridization of a series of random oligonucleotides. These can be used as primers, which are extended by the Klenow fragment using non-isotopic labelled dUTP. The incorporation of biotinylated dUTP is detected with a gold-tagged streptoavidin as the reporter molecule. This allows, in mouse chromosomes, the detection of different rates of sensitivity to the digestion with specific REs in distinct intraheterochromatic DNA subsets. In addition, these results show that enzymatic production of ss-DNA seems to be adequate for electron microscopy work since the chromatin fiber is preserved better than in denatured DNA produced with heat, NaOH, or formamide.

Animals↗
PubMed1991

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.

Analysis of Variance↗
PubMed1991

Selective digestion of mouse chromosomes with restriction endonucleases. II. X-ray microanalysis of HaeIII-treated chromosomes.

We used X-ray microanalysis to study the changes induced in mouse metaphase chromosomes as a result of digestion with the restriction endonuclease HaeIII. The phosphorus X-ray signal was used as a marker for DNA and the sulfur signal for protein. Calcium, iron, copper, and zinc were also detected. HaeIII induced a loss of phosphorus from both the centromeres and chromosome arms, but the losses in the arms were much greater. These changes were accompanied by an increase in the electron density of the centromeres and a reduction in that of the arms. No reduction in the sulfur signal in either arms or centromeres occurred as a result of HaeIII digestion. Except for calcium, which showed only a moderate reduction, the inorganic ions exhibited very large losses as a result of HaeIII digestion. The differentiation of chromosome arms and centromeres as a result of HaeIII digestion is therefore not simply due to differential loss of DNA but also involves structural reorganization of the chromatin, as shown by electron microscopy. This reorganization does not involve loss of proteins but may be correlated with changes in the amounts of inorganic ions known to be involved in chromatin condensation.

Animals↗
PubMed1990

Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases.

Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.

Animals↗
PubMed1990

Scaffold-like structures in mouse chromosomes revealed by restriction endonuclease digestion and electron microscopy.

A scaffold-like structure is observed under the electron microscope when mouse chromosomes are digested with the restriction endonuclease Hae III. This structure, located in the inner part of chromatids, may correspond to those fragments of chromatin loops anchored to the chromosome scaffold and is obtained when chromosomes are treated either in suspension or attached to grids. The width of the structure is correlated with the extent of digestion in chromosomes treated in suspension. Those treated on grids show this structure whenever chromatids do not collapse. These results agree with the model of chromosome organization based on a non-histone protein scaffold.

Animals↗
PubMed1990

Differential action of restriction endonucleases on chromosomes prepared for light and electron microscopy.

Whole mounted chromosomes from the L929 mouse cell line were digested on grids with HinfI and AluI restriction endonucleases and studied by electron microscopy. Results show differences in the pattern of bands obtained in a marker chromosome when compared with those previously reported by light microscopy with the same restriction endonucleases. These differences suggest that the accessibility of restriction sites on chromosomes may be modulated by preparatory methods for chromosome analysis.

Animals↗
PubMed1990

In situ random primer extension of metaphase chromosomes.

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.

Chromosome Banding↗
PubMed1990

Discontinuous undercondensation of centromeric heterochromatin in mouse chromosomes: evidence in Hoechst 33258-treated cells.

Mouse chromosomes from the L929 cell line have been treated with Hoechst 33258 to induce undercondensation of centromeric heterochromatin. The morphological changes induced by this fluorochrome were analyzed in electron micrographs of whole-mounted chromosomes. Results show that the condensation inhibition of centromeric heterochromatin caused by Hoechst 33258 is not produced homogeneously and suggest compositional differences within an individual centromere.

Animals↗
PubMed1990

Morphometric analysis of human chromosome satellites and NOR asymmetries by transmission electron microscopy.

Human chromosome satellites appear as roughly spherical telomeric structures of 0.215 +/- 0.013 microns mean diameter by electron microscopy. Morphometric evaluations showed that in the short arms of D and G chromosomes lacking secondary constrictions, the chromatin which constituted the satellites appeared virtually integrated within the short arms. Asymmetry was detected in sister satellites from almost identical dimensions to the near absence of one of them. When Ag-NOR staining was employed to locate active nucleolar organizer regions (NORs) these appeared associated with satellite short arms and active NORs were never found in non-satellite chromosomes. Asymmetry was also evident between sister NORs.

DNA↗
PubMed1989

Detection of cryptic bands by AluI in eukaryotic chromosomes.

Selective digestion of fixed chromatin with the restriction endonuclease AluI (which cuts the sequence AG CT) uncovers a specific and repeatable pattern of bands within the euchromatin of two species of grasshoppers and of the L929 mouse cell line, which are not detectable by means of other banding techniques such as C-bands, specific fluorochromes, or other restriction endonucleases. It is tentatively suggested that this chromatin represents a special class of repetitive DNA embedded in the euchromatin, not containing the AluI restriction site to the same extent as in euchromatin and not associated with C-banded heterochromatic material.

Animals↗