[Relation between the intensity of transcription and the rRNA gene content of individual nucleolar-forming regions of human chromosomes]. / Zavisimost' mezhdu intensivnost'iu transkriptsii i soderzhaniem genov rRNK v individual'nykh iadryshkoobrazuiushchikh raionakh khromosom cheloveka.

A comparison has been carried out of satellite strand lengths, Ag-staining intensity and labelling intensity in in situ hybridization of 3H-thymidine-labelled 28S-rRNA gene fragment in nucleolar-forming regions (NFR's) of individual acrocentric chromosomes from blood lymphocytes of 2 karyotypically normal individuals. To identify chromosomes modified R-staining was performed (5-bromdesoxy-pyridine + Höchst fluorochrome 33258 + Giemsa dye). The data obtained demonstrate, firstly, the variability between 10 acrocentric human chromosomes both in the content of ribosome genes and in silvering intensity and NFR satellite strand length and, secondly, a positive correlation between three studied characteristics of individual chromosomes. In one case an exception has been noted in one homologue of chromosome 21 of an individual A: high intensity of hybridization labelling was accompanied by weak Ag-staining and short satellite strand. It was concluded that the variability of transcription activity of individual NFR's detected by Ag-staining is, as a rule, based on the variability in the content of ribosome gene DNA in them and, in some cases, the presence of rRNA gene copies in inactive state.

[Chromosome characteristics of X-linked mental retardation. II. Autosomes]. / Khromosomnaia kharakteristika X-stseplennoi retsessivnoi umstvennoiotstalosti. Soobshchenie II. Autosomy.

The frequencies of chromosome and chromatid breaks and gaps were studied in blood lymphocytes of three groups of individuals: 21 males with X-linked mental retardation characterized by fragile X chromosome; 52 males with non-differentiated X-linked mental retardation having no fra(X) chromosome in their cells; 15 intellectually normal males. The lymphocytes were cultured both in medium 199 and in Eagle's medium supplemented with fluoro-deoxyuridine. The significantly higher frequencies of various autosomal lesions were observed in the individuals with the fragile X chromosome syndrome and in those with mental retardations without fra(X) chromosome, in comparison with normal males. The significant difference in some autosome lesions was also found between both groups of the patients. The distribution of chromosome lesions in autosomes of different groups was significantly higher in chromosomes A and lower in groups B, E, F and G, than expected in accordance with their relative length in the haploid set. In all the groups of individuals studied, the predominant localization of chromosome and chromatid breaks and gaps was observed in fragile sites 1p31, 3p14, 6q26 and 16q23.

[Variability in the expression of the fragile site of the (fra)X chromosome in 2 consecutive cell cycles]. / Izmenchivost' ékspressii lomkogo uchastka khromosomy fra(X) v dvukh posledovatel'nykh kletochnykh tsiklakh.

The number and morphology of X chromosomes were analysed in tetraploid cells induced with colcemid in cultured blood lymphocytes obtained from a patient with fra(X) syndrome of mental retardation. In contrast to diploid cells containing fra(X) chromosome in 22.7% of cells, the marker X was found in 51.6% of tetraploids, each cell containing only one fragile X out of the two expected ones. The data obtained indicate an extreme lability of the expression of fragile site (X) (q 27) in consecutive lymphocyte generation.

[Chromosomal characteristics of X-linked recessive mental retardation. I. The X chromosome]. / Khromosomnaia kharakteristika X-stseplennoi retsessivnoi umstvennoi otstalosti. Soobshchenie I. X-khromosoma.

The X-chromosome was studied in blood lymphocytes of 68 males with aspecific mental retardation (MR), their 57 relatives and 15 intellectually normal males. The incidence of a fragile X-chromosome (fra(X)) was found to be 4.7% in an unselected group of 42 patients, 50% among 10 probands in which pedigree data were suggestive of X-linked MR diagnosis, and 75% in the group of 15 patients selected for phenotype characteristic of the fragile X syndrome. The fra(X) was present in 1-43% of metaphases in different individuals, no such marker being observed in cells of 15 normal individuals. No significant difference was found when the incidence of the fra(X) was compared in cells cultured in the medium 199 with low folic acid content and the Eagle's medium supplemented with 5-fluorodeoxyuridine (10.62 +/- 2.94 SEM and 13.53 +/- 2.85 SEM, respectively). The possibility of false-positive diagnosis of the fragile X syndrome was quantitatively appreciated. A half of the patients showing a fra(C) in conventionally stained chromosomes were found to have fragile 6 autosome as the only marker in these cells, and in patients with the evident fragile (X) syndrome the fra(6) constituted about one-third of the fra(X) frequency. Both culture media employed were similar in the fra(6) induction.

Intercellular NOR-Ag-variability in man. II. Search for determining factors, clonal analysis.

Intercellular, nonartifactual variability of nucleolar organizer region (NOR)-Ag-staining was studied in cultured human peripheral blood lymphocytes, skin and embryonic fibroblasts. No differences in number and character of variable NORs and intensity of their staining were observed between lymphocytes stimulated to proliferate with phytohemagglutinin and pokeweed mitogen, as well as lymphocytes of first- and second division. The number of NOR associations per cell and the number of associated chromosomes per association were also similar. In a given individual these criteria were similar in lymphocytes and fibroblasts. In all nine clones derived from three independent parental fibroblast cultures the intercellular NOR-Ag-variability was similar to that observed in a given parental cell line. A significant decrease in the number of metaphases containing NOR associations was observed in second-division lymphocytes compared with first-division ones, as well as in skin fibroblasts compared with lymphocytes.

Intercellular NOR-Ag variability in man. I. Technical improvements and marker acrocentric chromosomes.

A new modification of the Ag I technique has been developed using human cultured blood lymphocytes, which involves ultra-violet irradiation of chromosome preparations during incubation in AgNO3. This technique enables detection in a short incubation time all NORs capable of being stained with silver. A peculiar morphological change in Ag-stainable NORs during the incubation is described, which can be used as a criterion of the completion of Ag staining. With the refined Ag-staining procedure, acrocentric marker chromosomes were studied which showed one or two satellite stalks within the same individual. Ag staining was highly coincident with this variability.

[Morphological changes in isolated metaphase chromosomes depending on the pH and ionic strength of the solutions]. / Izmenenie morfologii vydelennykh metafaznykh khromosom v zavisimosti ot pH i ionnoi sily rastvorov.

The morphology of isolated unfixed Chinese chamster chromosomes is described in relation to the composition of solutions used for chromosome isolation. The chromosomal morphology was studied with the aid of a flow-chamber and phase-contrast microscopy. Changes in chromosome length and width in relation to pH values (ranging from 2.2 to 9.15) and ionic strength (ranging from 0 to 2.0 M) are detected.

Genetic determination of NOR activity in human lymphocytes from twins.

Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Ag-stained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag + NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acorocentric association frequency were not significantly higher in DZ compared with MZ twins.

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium.

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, Cd-, Ag-NOR-staining). Chromosomes decondensation could be reversed by the addition of calcium ions to the medium. Ca ++-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.

Polymorphisms of Ag-stained nucleolar organizer regions in man.

Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population. The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation. In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.

[Genetic determination of the activity of nucleolar-organizing regions of human chromosomes]. / Geneticheskaia obuslovlennost' aktivnosti iadryshkoobrazuiushchikh raionov khromosom cheloveka.

Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes of 8 monozygotic and 8 dizygotic twin pairs using Ag-staining of acrocentric chromosomes. These chromosomes were identified by G-banding. NOR activity was estimated by the number of Ag-stained regions, their distribution among individuals acrocentrics and the size of silver deposits. No statistically significant differences were found as regards these criteria between the monozygotic twins. The dizygotic twins were distinguished, as a rule, in most pairs by one or another chromosome. The data obtained indicate that NOR, activity in each chromosome is of a heritable character, at least in blood lymphocytes. Chromosome 21 was found to have a greater NOR activity as compared with other acrocentrics.

5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes.

When [3H]dC was added with a high dose (4 x 10(-1) mM) of dT to human blood lymphocyte cultures, much heavier labeling of interphase nuclei and metaphase chromosomes was observed compared with that in cultures treated with [3H]dC alone. This observation indicates that in the presence of excess dT, exogenous dC is included into cytosine bases of DNA, releasing the cells from the thymidine block. BrdC 5 x 10(-2) mM added with a high dose of dT (4 x 10(-1) to 1.0 mM) to the cultues did not relieve the thymidine block as determined from the percentage of metaphases of the first to third divisions. It is concluded that BrdC, in contrast to dC, is not utilized as a cytosine DNA precursor even in the presence of high concentrations of dT. The frequency of SCEs per cell was the same when studied with the aid of BrdC and BrdU used under similar conditions. The distribution of SCEs among chromosomes was also identical for both analogues: The number of SCEs was significantly higher than expected in chromosomes of group B and lower than expected in chromosomes of groups E, F, and G.

[Sister chromatid exchanges in bromine incorporation into DNA cytosine nucleotides. III. 5-bromodeoxycytidine as a DNA thymine precursor. The characteristics of the exchanges]. / Sestrinskie khromatidnye obmeny pri vkliuchenii broma v tsitozinovye nukleotidy DNK. III. 5-bromdezoksitsitidin kak predshestvennik timina DNK. Kharakteristika obmenov.

Cell movement through the mitotic cycle and sister chromatid exchanges (SCE) were studied in human blood lymphocytes cultured in the presence of 5-bromodeoxycytidine (BrdC, 0.05 mM) plus thymidine (dT 0.4, 0.8, and 1.0 mM). In controls, lymphocytes were cultivated in the presence of 5-bromodeoxyuridine (BrdU, 0.05 mM) and deoxycytidine (0.1 mM), or BrdC alone. All nucleosides were added to the cultures 28 hours prior to fixation and were maintained in the medium for 16 hours. As determined from percentage of metaphases of 1st to 3rd divisions, BrdC did not release from thymidine block. This fact leads us to conclude that BrdC in contrast to deoxycytidine does not serve as a cytosine precursor. No significant differences in the frequency of SCE and their distribution among chromosomes were found between cultures treated with BrdC and with BrdU.

[Sister chromatid exchanges under bromine incorporation into DNA cytosine nucleotides. II. Utilization of 3H-deoxycytidine as a DNA cytosine precursor]. / Sestrinskie khromatidnye obmeny pri vkliuchenii broma v tsitozinovye nukleotidy DNK. II. Ispol'zovanie 3H-dezoksitsitidina v kachestve predshestvennika tsitozina DNK.

The incorporation of 3H-deoxycytidine (3H-Cdr) in the presence of thymidine (Tdr) into cultured human blood lymphocytes has been studied. The analysis of the label in interphase nuclei as well as in chromosomes at metaphase was carried out. The labeling was much higher when 3H-Cdr (0.5 to 1.0 C/ml, 2--4 x 10(-5) mM) was added to the cultures simultaneously with Tdr (4 x 10(-1) mM). This observation is considered as an indication that in the presence of high doses of Tdr exogeneous Cdr is utilized to synthesize cytosine of DNA rather than thymidine. During the first hours after its addition, the bulk of 3H-Cdr is eliminated from the culture medium. At 12 hrs of the incubation, the medium seems to be free of the nucleoside as shown particularly from the single chromatid localization of the label in chromosomes of the second mitosis. The incorporation into lymphocytes of 3H-Tdr administered in the same dose under the same conditions was registered for the whole period of observation (24 hrs). The data obtained are discussed in relation to lymphocyte catabolism of exogeneous nucleosides.

[Sister chromatid exchanges following incorporation of bromine into cytosine DNA nucleotides. I. Combined use of 5-bromdeoxycytidine and thymidine for the purpose of incorporating bromine into DNA cytosine]. / Sestrinskie khromatidnye obmeny pri vkliuchenii broma v tsitozinovye nukleotidy DNK. I. Sovmestnoe primenenie 5-bromdezoksitsitidina i timidina s tseil'iu vkliucheniia broma v tsitozin DNK.

In the cultured in vitro human blood lymphocytes treated with 5-bromodeoxycytidine (BrdC) plus thymidine (dT) in concentrations of 0.05 mM and 0.4 mM, respectively, metaphases of the second division, containing chromosomes with differentially stained sister chromatids appear after a 30 hours treatment, attaining 85 per cent at 36 hours. Under the treatment with BrdC or BrdU alone, the similar percentage of metaphases is observed at 28 hours. Similar results are obtained if, at 16--18 hours, the culture medium, containing BrdC plus dT is either replaced with a medium free of both the precursors, or supplied once more vith (0.05 mM) or dC(0.05 mM). The The observed slowing movement of cells through out the cell cycle, due to the treatment with BrdC plus dT, is explained by the inhibitory effect of dT on DNA synthesis which is manifested as BrdC catabolism. It is concluded that BrdC administered together with the cell cycle-slowing doses of thymidine is predominantly incorporated into cytosine of DNA.

Isochromosome X in man: different DNA replication patterns in the long arms.

An X isochromosome for the long arm was studied in 3 patients with Turner's syndrome using the BrdUrd-Hoechst 33258-Giemsa method and C-staining. In all 3 patients studied, the long arms of the i(Xq) were asymmetrical with respect to chronology of DNA synthesis. The most striking asynchrony of DNA replication was observed in large early replicating segments adjacent to the centromeric region. Two C bands of similar appearance were observed localized symmetrically in both arms. The data are interpreted in accordance with two possible origins of an abnormal X which is known as i(Xq).