Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.     

Modified method of differential staining of sister chromatids]

A modified method of obtaining differential staining of sister chromatids is described. It is simple, rapid, and effective, and at the same time inexpensive and accessible, since it allows one to use available reagents. When 5-bromdeoxyuridine was administered 24 hours before fixation into the Chinese hamster cell culture the percentage of metaphases with a differential chromatid staining constituted 95--98, and when this substance was administered 28 hours before fixation into the human lymphocyte culture this percentage varied from 75 to 92, depending on the individual. The mean number of sister chromatid exchanges in human lymphocytes failed to depend on the time of fixation.     

Dependence of the specific activity of antistaphylococcal immunoglobulin on the degree of its fragmentation

In vitro experiments have revealed no perceptible decrease in the content of antitoxic antibodies to alphastaphylolysin in the preparations of antistaphylococcal immunoglobulin, containing 13-50% of Fab-fragments. These data confirm the necessity of controlling the molecular composition of the preparations of antistaphylococcal IgG by physico-chemical methods with the aim to evaluate their quality, as the fragmented preparations are rapidly eliminated from a human body.     

Dependence of the specific activity of antitetanus immunoglobulin on the degree of its fragmentation

Antitetanus immunoglobulin preparations with the increasing content of Fab-fragments (15, 30, 53%) have been obtained under specific experimental conditions. Tests for specific activity have revealed an insignificant decrease (13%) in this activity in the preparation containing 15% of Fab-fragments and its sharp drop in the preparations containing 30-50% of Fab-fragments. The specific activity of antitetanus immunoglobulin has been found to be related to the degree of its fragmentation.     

The effect of distamycin A on heterochromatin condensation of Drosophila chromosomes

Larval neuroblasts of four species of Drosophila (melanogaster, hydei, virilis and funebris) were treated with distamycin A, a DNA ligand which induces distinct undercondensation in AT-rich heterochromatin. For each species the patterns of undercondensation were correlated with distribution of quinacrine-bright regions and of satellite DNAs. An overlapping of distamycin A-sensitive and quinacrine-bright heterochromatic regions was demonstrated for D. melanogaster and D. virilis, but not for D. hydei and D. funebris. Distamycin A undercondensation is thus a further criterion for resolving heterochromatin into different parts and enables identification of the steps of the condensation process within heterochromatic regions.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

Summary The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25±0.27 mU/10⁷ cells and 6.18±0.87 mU/10⁷ cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10⁷ cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10⁷ cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10⁷ cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10⁷ cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.     

Dependence of DNA condensation on the correlation distance between condensed counterions

Based on the ground state of counterions condensed on a DNA molecule, a model has been developed to successfully detect the process of DNA condensation. Through further investigation, the process of DNA condensation strongly depends on the correlation distance between condensed counterions on DNA molecules. Generally, there are two routes. The process of DNA condensation with the correlation distance between condensed counterions being 2 nm or 4 nm is different from the one with the correlation distance between condensed counterions being 3 nm or 5 nm. Effects of ionic strength on the diameter of toroidal condensates originate from the increase of correlation distance between condensed counterions.     

Relationship between the proliferation waves of the hepatocytes after hepatectomy and the waves of diurnal rhythm of mitotic activity. 1. Dependence of the form of the mitotic wave and the time of attaining its maximal degree on the time of operation]

The diurnal rhythm of mitotic activity (MA) of intact animal hepatocytes and the proliferative wave of hepatocytes after partial hepatactomy at time t0 are thought to appear as a result of formation of an initial proliferative wave, Pk-wave, within the G0-phase at constant moments of the day--time tk+1=tk+TMA(TMA=24hrs/K, k=1, or 2, . . ., or K) under the influence of the regulating system of the organism. Cells of the Pk-wave pass during a short time deltat (deltat less than TMA) from the G0-phase into the transformation phase, and then into the G1-phase. The 1st stimulated proliferative wave is formed at time tk, if tk--TMA less than t0 less than or equal to tk; its intensity depends most likely on the intensity of the corresponding Pk-wave of the intact liver. It was noted that time t0 of partial hepatectomy was necessary to coordinate with tk, but not with the time of the maximal mitotic activity, and that it was necessary to hepatectomize all animals within the interval from time tk--TMA to time tk. The model was shown to compare well with data by Post et al. (1963), Barbason (1970), and Van Cantfort and Barbason (1972) for hepatocytes of Wistar rats with TMA=8hrs, and tk (k=1,2,3) within intervals (2 a.m.; 4 a.m), (10 p.m.; noon), and (6 p.m. 8 p.m.). The maximal rate of liver generation was observed for all the hepactomized animals with the time of operation being between 8 p. m. and 2 a.m.     

Dependence of the specific activity of an immunoglobulin against tick-borne encephalitis on the degree of its fragmentation

Immunoglobulin preparations against tick-borne encephalitis with the increasing content of Fab-fragments (from 16% to 45%) have been experimentally obtained. As revealed by testing these samples in vivo for specific activity (in the biological neutralization test), the preparations containing 16% of Fab-fragments show no perceptible decrease of specific activity; its sharp decrease (2-16 times) can be observed in preparations with a high degree of fragmentation (the content of Fab-fragments being equal to 30-45%).     

Dependence of epidermal growth factor activation on its affinity and oligomerization]

Epidermal growth factor receptor (EGF-R) oligomerization has been followed on A-431 cells using covalent labeling by 125I-EGF and EGF-dependent autophosphorylation of receptor-kinase. High molecular weight complexes corresponding to monomeric, dimeric, and trimeric forms of EGF-R are detected. The process of oligomerization occurs effectively at 37 degrees C while at 4 degrees C no oligomer formation is detected. PMA or ATP treatment reduces the number of high-affinity EGF-binding sites but has no influence on dimer formation. Dimerisation of the EGF-R in the absence of the ligand has been established on formalin-fixed A-431 cells.     

Study of constitutive heterochromatin with a new and simplified fluorescence staining technique

A new fluorochrome that preferentially binds itself to the centromeric or the constitutive heterochromatin is described. This stain allows an easy assay, through fluorescence, of the repetitive DNA or bands, supposedly composed of constitutive heterochromatin, in insectivores, rodents and man, without following the in situ hybridization of Pardue & Gall (1970) or the DNA denaturation-renaturation processes of Arrighi & Hsu (1971). The staining patterns with this derivative of a Benzimidazole compound (Hoechst 33258) are induced in the chromosomes without incubation or pretreatment with SSC and are identical to those produced by other techniques. This stain may eventually contribute to elucidating the hitherto unknown molecular mechanisms involved in the relationship between the repetitious DNA sequences and the banding patterns, and to interpreting the mechanisms responsible for the chromosomal rearrangements and aberrations involving the peri-and non-pericentric regions of the chromosomes.Paper read by P.K.S. at the NATO Advanced Study Institute on Comparative Biology of Primates Turin 7–19 June 1972. Supported by the Alexander von Humboldt-Stiftung, Bonn-Bad Godesberg.     

Patterns of heterochromatin replication and condensation correlate in rat kangaroo PtK2 cells

Chromosome replication in mammalian cells in an ordered phenomenon. This is true also for the condensation in G2 of the heterochromatic chromosomal regions in mouse cells. The generality of this phenomenon and its mechanism are not known, nor is it known whether the order of condensation of the heterochromatic chromosomal segments in G2 reflects the order of replication or is independent of it. We determined the order of replication during the S phase and of condensation in G2 of the short heterochromatic chromosomal regions in the rat kangaroo cell line PtK2. The kinetics of condensation of these regions in G2 was studied in cells treated with Hoechst 33258. Their order of replication was established with the use of a sensitive technique based on the treatment of living cells with 5-bromodeoxyuridine and Hoechst 33258. Our results show that these regions exhibit a similar pattern of replication in S and condensation in G2.     

A study of heterochromatin in Drosophila nasuta by the 5-bromodeoxyuridine-giemsa staining technique

Larval brain ganglia of Drosophila nasuta were cultured in vitro in the presence of 5-bromodeoxyuridine for 1 or 5 h at 24° C and the air-dried chromosome preparations stained by the Hoechst 33258-Giemsa technique to reveal bromodeoxyuridine induced sister chromatid differentiation. In 1 h as well as 5 h preparations, 10–15% of well spread metaphase plates show a sister chromatid differentiation in only C-band heterochromatin regions of different chromosomes. We infer that this sister chromatid differentiation in all heterochromatic regions is seen after bromodeoxyuridine incorporation for only one replication cycle and is related to the presence of asymmetric A-T rich satellite sequences in all the C-band regions of D. nasuta karyotype.