Adriana de Capoa

Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP‐ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP‐ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP‐ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3‐aminobenzamide, an inhibitor of poly(ADP‐ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti‐5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP‐ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the hetero‐chromatic regions colocalized with the extended methyl‐rich domains.—de Capoa, A., Febbo, F. R., Giovannelli, F., Niveleau, A., Zardo, G., Marenzi, S., Caiafa, P. Reduced levels of poly(ADP‐ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell‐by‐cell computer‐assisted quantitative analysis. FASEB J. 13, 89–93 (1999)

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.

Frequency of satellite association in individuals with structural abnormalities of nucleolus organiser region

SummaryThe frequency of involvement of acrocentric chromosomes into satellite association has been extensively studied in recent years. These studies have aimed to show the existence of a relationship between satellite association, centric fusion and non-disjunction. In the present paper, evidence is given that different D-group chromosomes all bearing structural abnormalities in the nucleolus organizer region are preferentially involved in satellite association. Some interpretations of this finding are discussed.ZusammenfassungDie Häufigkeit der Beteiligung akrozentrischer Chromosomen bei Satellitenassoziation ist in den letzten Jahren eingehend untersucht worden. Diese Untersuchungen hatten zum Ziel, die Beziehungen zwischen Satellitenassoziation, zentrischer Fusion und Non-disjunction aufzuzeigen. Hier könnte dargelegt werden, daß verschiedene D-Chromosomen, die einen Defekt in der Region des Nucleolus-Organisators haben, bevorzugt an der Satellitenassoziation beteiligt sind. Erklärungen dieser Befunde werden diskutiert.

Clonal inheritance of rRNA gene activity: Cytological evidence in human cells

Clone-specific silver staining patterns of the nucleolus organizers (NOs) were observed in clones deriving from a human diploid fibroblast culture. Each specific staining pattern corresponds to one of the nine patterns observed in the general population. Since silver stainability of the NOs is related to rRNA gene function, these data demonstrate the clonal inheritance of rRNA gene activity.

Double satellites: Autoradiographic study of a chromosomal marker observed in two generations

Summary2 cases of a familial balanced translocation of satellites and part of the short arm between two D-group chromosomes are described.One of these chromosomes shows double satellites on its short arm, and the other appears deprived of visible satellites and part of its short arm. These chromosomes have been identified by means of autoradiography as a number 15 and a number 14, respectively.The possible implications of this type of translocation are discussed in the text.ZusammenfassungEs wird bei Tochter und Mutter eine balancierte Translokation von Satelliten und einem Teil des kurzen Arms zwischen zwei D-Chromosomen beschrieben.Eines dieser Chromosomen zeigt doppelte Satelliten an seinem kurzen Arm, bei dem anderen ist ein Satellit nicht zu erkennen, und es scheint ein Teil des kurzen Arms zu fehlen. Mit Hilfe der Autoradiographie ist jenes als Nr. 15, dieses als Nr. 14 charakterisiert.Die mögliche Bedeutung dieses Typs von Translokation wird besprochen.

Unusual segregation of t(11;22) resulting from crossing-over followed by 3:1 disjunction at meiosis I

Reciprocal translocation t(11;22)(q23;q11) is of particular interest because the unbalanced offspring of the translocation carriers usually present with a supernumerary derivative chromosome 22. This common unbalanced karyotype is the result of 3:1 chromosome segregation during meiosis. We report the third case of a rare segregation pattern of a paternal 11; 22 translocation. The probands karyotype revealed the presence of a der(11) and two copies of a der(22), i.e. 47, XX, t(11; 22)(q23;q11), +der(22) t(11;22)pat. The karyotype is the result of paternal 3:1 segregation after crossing‐over involving the derived and the normal chromosome 22, as revealed by chromosome polymorphism analysis. Contrary to the preferential maternal transmission of this common unbalanced translocation, the data from the literature, including our case, may suggest preferential paternal transmission of this rare type of unbalanced translocation.

Persistence of increased levels of ribosomal gene activity in CHO-K1 cells treated in vitro with demethylating agents

The rate of ribosomal gene activity was evaluated by silver staining of the Nucleolus Organisers (NOs) in cultured CHO-K1 cells after a 12 h pulse with two demethylating agents (L-ethionine and 5-azacytidine). Silver staining of the NOs was measured every 24 h, from 24 up to 110 h after seeding. The purpose was to test the hypothesis that drug-induced demethylation is associated to heritable modifications of rDNA activity. Ribosomal gene activity was shown to be significantly increased by both agents. The increase persisted throughout the experiments, thereby suggesting the heritability of this epigenetic modification. The analysis of heritable DNA damage or modification is an important task in studying the risk of cancer onset and the mechanisms of cancer induction. In these studies two main results were obtained: (i) heritable DNA variations can be induced by both mutational and epigenetic changes; (ii) the modified end-point was not negatively selected.

Autoradiographic identification of a 13/21 translocation

We have studied by means of autoradiography a case of D/G translocation after terminal labelling of leukocyte cultures with 3H-thymidine. The D-group chromosome involved in the translocatio

Labelling of human chromosomes with 3H-AMD

SummaryThis paper presents data on the pattern obtained by labelling in vitro with 3H-AMD human chromosomes from peripheral blood cultures. Preparations were divided into two experimental groups: 1. preparations labelled with 3H-AMD; 2. preparations treated with H2SO4 and labelled with 3H-AMD.The results show evidence of non-random grain distribution for some chromosomes and of differential intensity of labelling of chromosomes within some chromosome groups. Treatment with H2SO4 before labelling with 3H-AMD resulted in random distribution of grains along the chromosomes with disappearance of any labelling pattern. The same result was observed when preparations were stained with quinacrine dihydrochloride before labelling with 3H-AMD. These data suggest the existence of a differential distribution of proteins along the chromosomes.ZusammenfassungIn der vorliegenden Arbeit wird die in vitro-Markierung von menschlichen Chromosomen mit 3H-Aktinomycin D (AMD) in Kulturen des peripheren Blutes beschrieben. Es handelt sich um zwei Versuchsgruppen: 1. Markierung der Präparationen mit 3H-AMD; 2. Markierung der Präparationen mit 3H-AMD nach Vorbehandlung mit Schwefelsäure.Bei diesen Versuchen ergaben sich Hinweise auf eine nichtzufallsmäßige Kornverteilung auf einigen Chromosomen und auf differentielle Markierungsmuster in den Chromosomen einiger Chromosomengruppen. Diese Markierungsmuster wurden durch Vorbehandlung mit Schwefelsäure zerstört. Das gleiche Resultat wurde erhalten, wenn die Präparate vor der Markierung mit Chinakrin-Dihydrochlorid gefärbt wurden. Diese Ergebnisse lassen vermuten, daß die Proteine auf den Chromosomen ungleichmäßig verteilt sind.

Autoradiographic study of DNA synthesis in homologous chromosomes distinguishable by morphology.

Autoradiographic terminal labeling studies performed on leukocyte cultures of a man (A. de J.) and his son (H. de J.), both carriers of an enlarged satellite on a D-group chromosome, demonstrated that the morphological marker was a No. 15. Counts of grains on the long arms of the two chromosomes 15 showed that the marker chromosome was less heavily labeled than its normal homolog. This difference was highly significant in both cases. The marker chromosome was also shorter than its homolog, though not significantly so in one case. Possible interpretations of these findings, i.e., chromosomal polymorphism or autosomal lyonization, were discussed.