Difference in constitutive heterochromatin behaviour between human amniocytes and lymphocytes detected by a sequential in situ exonuclease III digestion-random primer extension procedure.

Fixed chromosomes from human amniotic fluid cells and peripheral blood lymphocytes were digested in situ with exonuclease III and the single stranded DNA obtained was used as template for an in situ random primer extension. Under these conditions an R banding pattern, more evident in lymphocytes than in amniocytes, was obtained. Nevertheless, constitutive heterochromatin of chromosomes 1, 16, Yq, and mainly the pericentromeric region of chromosome 9 was far more intensely labelled in amniocytes than in lymphocytes.

Sister chromatid differentiation in 5-bromo-2'-deoxyuridine-substituted chromosomes: a study with DNA-specific ligands and monoclonal antibody to histone H2B.

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B.

Nuclear DNA introgression across a Pyrenean hybrid zone between parapatric subspecies of the grasshopper Chorthippus parallelus.

Two parapatric subspecies of the European grasshopper Chorthippus parallelus meet along the Pyrenees and form a hybrid zone. A nuclear DNA sequence marker (cpnl-1), involving the presence or absence of a 5 bp insertion, was found to differentiate between the two subspecies along either side of the High Pyrenees but further electrophoretic and sequence analyses revealed that considerable mixing of the subspecific genomes had occurred towards the western and eastern ends of the Pyrenees. The cline for this marker was relatively narrow in two adjoining western central high cols (Peyrelue: 9.

Banding human chromosomes using a combined C-banding-fluorochrome staining technique.

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pretreatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands.

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.

Direct incorporation of fluorescein-12-dUTP to insect fixed chromosomes by random primed extension.

The use of an in situ random primed system allows the direct incorporation of fluorescein-12-dUTP into fixed insect chromosomes, resulting in a strong fluorescent labelling. While in an orthopteran species (Eyprepocnemis plorans) a relatively uniform labelling of meiotic and mitotic chromosomes is produced, in Tenebrio molitor (Coleoptera) only the chromosomal arms, but not the pericentromeric heterochromatic areas of mitotic chromosomes, show positive labelling by this method. This indicates that the organization of DNA in heterochromatin is distinct from that in the euchromatin in distantly related species, and in such a way that, in some species, the random hexanucleotides are prevented from annealing with the chromosomal denatured DNA or (and) that primer extension by Klenow enzyme is impeded.

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.

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.

5-azacytidine produces differential undercondensation of alpha, beta and classical human satellite DNAs.

Fluorescence in situ hybridization employing human alphoid, beta and classical satellite DNA probes was performed on 5-azacytidine treated and untreated chromosomes obtained from human lymphocytes. The individual used in this study presented a polymorphism of constitutive heterochromatin of chromosomes 1 and 9 as revealed by in situ digestion with the restriction endonuclease Alul. Neither the alphoid nor the beta satellite DNA domains were susceptible to condensation-inhibition by 5-azacytidine.

Heterochromatin heterogeneity and rapid divergence of the sex chromosomes in Chorthippus parallelus parallelus and C. p. erythropus (Orthoptera).

Mitotic chromosomes from individuals of a Pyrenean hybrid zone between two subspecies of the grasshopper Chorthippus parallelus have been pretreated as for C-banding and subsequently stained with 4,6-diamidino-2-phenylindole (DAPI) and (or) Chromomycin A3 (CMA). The results, which are different from those obtained with Giemsa C-banding or standard DAPI - CMA treatments show (i) hidden heterochromatic heterogeneity that may be correlated with the existence of distinct families of repetitive DNAs, (ii) information about the possible independent origin of the three detected types of heterochromatin, and (iii) a further marker difference between the sex chromosomes of these two subspecies. This last result leads us to discuss the possible differential rates of evolution of sex chromosomes and autosomes in these subspecies and provides us with a new tool for the study of the structure and dynamics of this hybrid zone.

In situ nick translation of meiotic chromosomes to demonstrate homologous heterochromatin heterogeneity.

The in situ nick translation procedure performed on fixed meiotic chromosomes partially cleaved with restriction endonucleases shows a different staining of homologous heterochromatic regions, which could be explained through a differential restriction endonuclease cleavage. Mutations occurring before massive tandem duplication and involving those DNA motifs that produce these heterochromatic blocks, together with the absence of DNA recombination that characterizes these particular regions, could explain the observed results. This method for chromosome labelling is most useful to demonstrate a certain level of heterochromatin heterogeneity that is present in the genome of living species but remained cryptic to other techniques that are also able to induce longitudinal differentiation of the chromosomes.

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.

Detection of DNA strand breaks induced by hydroxyl radicals in nuclear and chromosomal chromatin by electron microscopy.

Chinese hamster Don cells were treated with 10 mM hydrogen peroxide. DNA strand breaks induced by hydroxyl radicals were amplified in 3' termini by an exonuclease III digestion, resulting in single stranded DNA motifs. In situ detection of these motifs was performed on chromatin fibres of isolated whole-mounted nuclei and chromosomes by a random priming procedure, using biotinylated-dUTP which bound a gold-tagged streptavidin.

The DNA fragments produced by AluI and BstNI digestion of fixed mouse chromosomes.

AluI and BstNI restriction endonucleases were used to study cytological and biochemical effects on centromere DNA in fixed mouse chromosomes. These enzymes were employed, as it is known that AluI is incapable of attacking major satellite DNA, contrary to BstNI that is known to cut this DNA fraction into monomers of 234 bp. After digestion in situ, electrophoretic analysis was carried out to characterize the DNA purified (1) from the material remaining on the chromosomes and (2) from the material solubilized from chromosomes.

The distribution of genes on human chromosomes as studied by in situ nick translation.

We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme--nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns.

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.

Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins.

Fluram (Fluorescamine; 4-phenylspiro(furan-2(3H),1'-phthalan)-3,3'-dione) is a fluorogenic reagent, which permits the detection of primary amines by forming highly fluorescent pyrrolinone derivatives. This reagent has been used on methanol-acetic acid fixed metaphase chromosomes of mouse and man and proved to be very effective in differentiating chromosome regions in both genomes. Mouse centromeric heterochromatin is highly reactive, showing intense fluorescence in all centromeric regions, whereas human chromosomes show no fluorescence in such regions.

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin.

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes.

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.

C-banding with specific fluorescent DNA-ligands: a new approach to constitutive heterochromatin heterogeneity.

The employment of certain DNA-specific fluorescent stains on unbanded and C-banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed.

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.

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.