A third common allele in the transferrin system,Tf C3 , detected by isoelectric focusing

Summary The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.     

Condensation of all human chromosomes in phase G2 and early mitosis can be drastically inhibited by 33258-Hoechst treatment

Summary Condensation of human chromosomes in phase G₂ and early mitosis is inhibited by the fluorochrome 33258-Hoechst. This inhibitory effect is most apparent in primary diploid fibroblasts and lymphoblasts and least pronounced in peripheral blood lymphocytes. Condensation of the human Y chromosome, which contains a large heterochromatic region rich in A-T base pairs, is drastically inhibited by 33258-Hoechst treatment of fibroblasts and lymphoblasts. The difference in sensitivity of human chromosomes in different cell types to 33258-Hoechst probably reflects differences in the cell-membrane permeabilities to 33258-Hoechst.     

Condensation-inhibition by 33258-Hoechst of centromeric heterochromatin in prematurely condensed mouse chromosomes

The phenomenon of premature chromosome condensation has been applied to study the kinetics of condensation-inhibition exerted by the fluorochrome 33258-Hoechst (33258-H) on the centromeric heterochromatic regions of mouse chromosomes. Asynchronous mouse A-9 cells in culture were fused with mitotic HeLa cells in the presence of 33258-H. Pronounced condensation-inhibition of the c-heterochromatin was observed in prematurely condensed early G2, S and late G1 chromosomes in the 33258-H-treated cells. It is concluded that the c-heterochromatic regions begin to condense quite early in G2, decondense again late in G1 and remain decondensed in the S phase.     

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.     

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.     

Chromosome condensation and Hoechst 33258 fluorescence in meiotic chromosomes of the grasshopper Spathosternum prasiniferum (Walker)

Patterns of Hoechst 33258 fluorescence have been studied in grasshopper chromosomes. At metaphase of mitotic as well as meiotic divisions — when chromosomes were maximally compact — all the chromosomes fluoresced brightly but no differentially fluorescing regions were detected. However, when all the chromosomes, except the X, were highly extended at pachytene and diplotene stages a distinct differential fluorescence was observed: only the centromeres of the autosomal bivalents fluoresced brightly whereas the entire X univalent showed bright fluorescence. Restriction of differentially bright fluorescence to the more condensed regions of chromosomes suggests a modulatory role for chromosome condensation in the production of Hoechst fluorescence. This suggestion was further strengthened by the substantial quenching of fluorescence caused by removal of chromosomal proteins following treatment with H₂SO₄. Similarly, post-C-band-treatment staining with Hoechst also led to quenching, though now the centromeres of the chromosomes, including the X, retained their differential fluorescence. It is proposed, therefore, that in grasshopper chromosomes, H-fluorescence is modulated by chromosome condensation brought about by differential ratios of DNA/protein at different chromosome regions and at different division stages.     

The pleiotropic effects of 33258-Hoechst on the cell cycle in Chinese hamster cells in vitro

The fluorochrome 33258-Hoechst which binds to double-stranded DNA (dsDNA) has been previously shown to inhibit in several mammalian cell cultures the condensation of chromosomes in phase G2 and early mitosis. We have now found that this drug affects the cell cycle of Chinese hamster cells grown in vitro in several other ways. In cells treated with the drug, phase G2 is prolonged, the rate of DNA replication is drastically reduced and the cells are arrested most probably at very late S phase.     

Effects of Hoechst 33258 on human leukocytes in vitro.

The benzimidazole derivative Hoechst 33258 was added at various concentrations to human leukocyte cultures. After 16 or 24 h of treatment, with concentrations equal to or greater than 100 mug/ml of Hoechst 33258, a number of chromosomes showed regions in which the chromatin was undercontracted. The centromeric regions of chromosome 1 and, more rarely, of chromosomes 3 and 9 appeared to be decondensed. Short decondensed regions were also present on the long arms of chromosomes 1 and 2. The possible nature of these regions is discussed.     

Kinetochore formation in experimentally undercondensed chromosomes

Summary Treatment of human and mouse cell cultures with the cytidine analogue 5-azadeoxycytidine and the AT-specific DNA ligand Hoechst 33258 dramatically inhibited condensation of the pericentromeric heterochromatin in several chromosomes. When stained with antikinetochore autoimmune sera, these experimentally undercondensed chromosomes showed kinetochores with preserved antigenicity. The undercondensed and normally condensed chromosomes share the major antigenic determinants of the kinetochore.     

Hoechst 33258 induced uncondensed sites in marsupial chromosomes

The fluorochrome Hoechst 33258 induces pronounced uncondensed regions at mitosis at one or more specific sites on the X chromosomes of all eighteen species of marsupials belonging to the family Macropodidae which have been examined. The Y chromosomes of nearly all of these species also show sensitive sites. Autosomal regions which respond to this chemical were observed in only five species and there is evidence of polymorphism for two of these. The regions which respond usually show C-banding, but not all C-banding regions are affected. No specific effect was found in the chromosomes of eleven other species examined which are representative of 5 different Australian marsupial families. The implications of the apparent restriction of sex chromosome sensitive sites to macropods are discussed.     

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.     

Effects of DAPI on human leukocytes in vitro.

DAPI (4'-6-diamidino-2-phenylindole), a fluorochrome specific for AT-rich DNA, was supplied for 24 h at various concentrations to human leukocytes in culture. This treatment caused the appearance on the chromosomes of specific areas lacking spiralization. In particular, the centromeric regions of chromosomes 1,9, and 16, a short region on the long arm of chromosomes 1 and 2, and the distal heterochromatic part of the long arm of the Y chromosome were despiralized. The despiralization pattern of DAPI is compared with those previously obtained with Hoechst 33258 and Distamycin A.     

Identification of the microtubule-associated protein map2 and its binding sites on metaphase chromosomes from cultured cells

The microtubule-associated protein MAP₂, which binds preferentially to AT sequences of DNA, can bind to metaphase chromosomes. The binding pattern of MAP₂ to chromosomes is similar to that found for the binding of the bisbenzimidazole derivative 33258 Hoechst, which also binds preferentially to AT-rich regions.     

Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei.

We used intensity and fluorescence lifetime microscopy (FLIM) of 3T3 nuclei to investigate the existence of AT-rich and GC-rich regions of the nuclear DNA. Hoechst 33258 (Ho) and 7-aminoactinomycin D (7-AAD) were used as fluorescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo) was used as a dye that displays distinct fluorescence lifetimes when bound to AT or GC base pairs. We combined fluorescence imaging of Ho and 7-AAD with time-resolved measurements of Yo and took advantage of an additional information content of the time-resolved fluorescence. Because a single nucleus could not be stained and measured with all three dyes, we used texture analysis to compare the spatial distribution of AT-rich and GC-rich DNA in 100 nuclei in different phases of the cell cycle. The fluorescence intensity-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G2/M-phases compared to G0/1-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G2/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of AT- and GC-rich DNA during the cell cycle.     

Effects of Hoechst 33258 on condensation patterns of hetero- and euchromatin in mitotic and interphase nuclei of Drosophila nasuta

Embryonic and third instar larval brain cells of D. nasuta were cultured in vitro in the presence of Hoechst 33258 (H) and H + 5-bromodeoxyuridine (BUdR) for periods varying from 2 to 24 h at 24 °C. Air-dried chromosome preparations were made with and without hypotonic pretreatment and stained with Giemsa. Metaphase chromosomes from H-treated (2 h) embryonic preparations show typical inhibition of condensation of the A-T-rich heterochromatin as in mouse. Presence of BUdR with H causes inhibition of condensation in fewer embryonic metaphase cells, but in the affected metaphases the degree of inhibition is more severe. In larval brains, however, even a 24 h H or H + BUdR treatment does not cause any significant inhibition of heterochromatin condensation. It is suggested that the differences in H effect on metaphase chromosomes of embryos and larval brains is related to differences in chromosome organization in the two cell types. Exposure of H-treated embryonic as well as larval brain cells to a hypotonic salt solution prior to fixation causes a ‘supercondensation’ of the heterochromatic chromocentre in most interphase nuclei. Presence of BUdR along with H reduces the frequency of cells showing such ‘supercondensed’ chromocentre. The euchromatin region in H-treated interphase nuclei is, on the other hand, slightly more diffuse than in control nuclei. Apparently, H-binding to DNA affects the nucleoprotein organization in hetero- and euchromatic regions of interphase nuclei in specific ways.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Fluorometric properties of the bibenzimidazole derivative hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA

A new fluorescent probe of chromosomal DNA structure in situ, the bibenzimidazole derivative Hoechst 33258, shows enhanced fluorescence with both AT- and GC-rich DNA; however, enhancement by AT-rich DNA is greater than enhancement with GC-rich DNA. When this compound is used as a probe, it produces localized fluorescence which can be correlated with AT concentration in specific chromosome regions. By the use of 33258, Hilwig and Gropp (1972) were able to demonstrate the relatively AT-rich DNA present in centric regions of mouse chromosomes; these regions do not fluoresce brightly when treated with quinacrine because of the presence of guanine residues which are spaced with high periodicity and which therefore efficiently quench quinacrine fluorescence. The data obtained in this study with DNA polymers of defined structure or composition, as test model compounds, suggest that 33258 is a useful cytochemical reagent for generally identifying all types of AT-rich regions in chromosomes, including those which are not demonstrable with quinacrine.     

Chromosome condensation and sister chromatid pairing in budding yeast

We have developed a fluorescent in situ hybridization (FISH) method to examine the structure of both natural chromosomes and small artificial chromosomes during the mitotic cycle of budding yeast. Our results suggest that the pairing of sister chromatids: (a) occurs near the centromere and at multiple places along the chromosome arm as has been observed in other eukaryotic cells; (b) is maintained in the absence of catenation between sister DNA molecules; and (c) is independent of large blocks of repetitive DNA commonly associated with heterochromatin. Condensation of a unique region of chromosome XVI and the highly repetitive ribosomal DNA (rDNA) cluster from chromosome XII were also examined in budding yeast. Interphase chromosomes were condensed 80-fold relative to B form DNA, similar to what has been observed in other eukaryotes, suggesting that the structure of interphase chromosomes may be conserved among eukaryotes. While additional condensation of budding yeast chromosomes were observed during mitosis, the level of condensation was less than that observed for human mitotic chromosomes. At most stages of the cell cycle, both unique and repetitive sequences were either condensed or decondensed. However, in cells arrested in late mitosis (M) by a cdc15 mutation, the unique DNA appeared decondensed while the repetitive rDNA region appeared condensed, suggesting that the condensation state of separate regions of the genome may be regulated differently. The ability to monitor the pairing and condensation of sister chromatids in budding yeast should facilitate the molecular analysis of these processes as well as provide two new landmarks for evaluating the function of important cell cycle regulators like p34 kinases and cyclins. Finally our FISH method provides a new tool to analyze centromeres, telomeres, and gene expression in budding yeast.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.