R. Mezzanotte

Chromatin organization and restriction endonuclease activity on human metaphase chromosomes

Human metaphase chromosomes were treated with HaeIII, HindIII, EcoRI, and AluI restriction endonucleases and subsequently stained with either Giemsa or ethidium bromide. The results obtained seemed to suggest that the structural organization of specific chromosome regions can play a primary role in determining the cytological effect after digestion with specific restriction endonucleases.

CYTOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF THE IN SITU ENDONUCLEASE DIGESTION OF FIXED TENEBRIO MOLITOR CHROMOSOMES

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.

The characterization of Muraena Helena L. mitotic chromosomes: karyotype, C-banding, nucleolar organizer regions, and in situ digestion with restriction endonucleases

We investigated the chromosome complement of Muraena Helena L. using C-banding, nucleolar organizer region silver staining, and restriction endonuclease digestion. We found a diploid number identical to that previously found in other Muraenidae (2n = 42). C-banding revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes as well as in most telomeres of acrocentric chromosomes. Nucleolar organizer regions were detected only on the short arm of chromosome 7. Digestion with either HaeIII, MboI, or DdeI restriction endonucleases produced a clearcut, specific banding pattern for each enzyme and indicated the existence of at least two different classes of highly repetitive DNAs. The short arm of chromosome 7 varied in size and staining characteristics.

DNA base sequence is not the only factor for restriction endonuclease activity on metaphase chromosomes: evidence using isoschizomers

Human and mouse fixed metaphase chromosomes were treated with the isoschizomer sets MboI/Sau3A and EcoRII/BstNI. In both cases we found that each member of the isoschizomer pairs produced different results, indicating that factors other than DNA base composition may affect in situ digestion by restriction endonucleases and that the structure of the enzymes is one factor. We also found that MboI and Sau3A isoschizomers produced the same effect on the chromosomes of the grasshopper Oedipoda germanica. This indicated that differences in the chromatin structure of different species may be important in determining restriction endonuclease activity on eukaryotic chromosomes.

Correlation between constitutive heterochromatin and restriction enzyme resistant chromatin in Arcyptera tornosi (Orthoptera)

Fixed mitotic chromosomes of A. tornosi have been analysed by means of C-banding, DA-DAPI and Chromomicin A3 fluorescence, as well as by digestion in situ with Alu I, Hae III, Hinf I and Hind III restriction endonucleases. From the results obtained at least nine types of chromatin can be distinguished in A. tornosi, Some C-band positive areas (constitutive heterochromatin) which show a characteristic fluorescence pattern are digested by specific endonucleases, whilst others are undigested. C-band negative areas (euchromatin) are digested by some restriction endonucleases but not by others. Regions digested are supposed to contain highly repetitive DNAs. It is noteworthy, however, that the heterochromatin associated with NORs is not attacked by any of the enzymes we used, while regions believed to contain AT-rich DNA (DA-DAPI positive) are digested by Hae III that cleaves the GG↓CC base sequence target.

Alterations induced in mouse chromosomes by restriction endonucleases

Fixed chromosomes of mouse have been treated with Alu I, Eco RII, Hind III or Bam HI restriction endonucleases and subsequently stained with either Giemsa, Ethidium Bromide or Acridine Orange. The results obtained have been discussed in the light of preferential or non-preferential extraction of DNA from specific chromosome areas following enzyme digestion. The possible involvement of a particular structural organization of some classes of heterochromatin has been hypothesized to account for the findings after Alu I or Eco RII treatment. The meaning of the Giemsa banding observed after Hind III or Bam HI digestion has also been considered, in comparison to the different stain responses obtained by using a DNA-specific dye such as Ethidium Bromide.

Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex

The sibling species Anopheles atroparvus and Anopheles labranchiae are cytogenetically almost indistinguishable. The chromosome complement (2n = 6) consists of two pairs of autosomes and two heteromorphic sex chromosomes with largely homologous heterochromatic long arms. Treatment of chromosome preparations with the restriction endonucleases, Alu I, Hae III, Mbo I, Hpa II, revealed species-specific differences of the sex chromosome banding pattern. These differences involved both amount and location of digested heterochromatin. Heterochromatin heterogeneity and a high level of intraspecific polymorphism, undetected with standard banding techniques, were observed in both species. Quantitative heterochromatin differences between the sex chromosomes did not inhibit their pairing and chiasmata formation. The endonuclease Msp I, which cleaves the same target sequence as Hpa II, did not digest heterochromatic as well as euchromatic regions in both species: inhibition of cleavage by methylation of the target sequence or limited access of the enzyme to the target could be involved in this response.

Heterochromatin heterogeneity in Oedipoda germanica (Orthoptera) detected by in situ digestion with restriction endonucleases

Metaphase chromosomes of the grasshopper Oedipoda germanica were treated with AluI, DdeI, HaeIII, HinfI, HpaII, MboI, MspI, Sau3A and TaqI restriction endonucleases (REs) and subsequently stained with Giemsa. C-banding and flurochromes of different base pair specificities (DA-DAPI or CMA3) were also used to localize and characterize the heterochromatin. Results indicate that (a) the large blocks of paracentromeric heterochromatin are heterogeneous within each chromosome. This heterogeneity is homogeneously distributed among almost all chromosomes of the genome. (b) The use of REs on fixed chromatin gives rise to a banding pattern not completely correlated with that obtained with C-banding; (c) heterochromatin associated with active NORs displays distinct characteristics: GC-rich (positive CMA3), high frequency of the restriction site CC GG, specific for MspI and HpaII, and low levels of methylation in the internal cytosine.

Ageing of fixed cytological preparations produces degradation of chromosomal DNA

When fixed chromosome preparations were allowed to age for 1-72 h, they became progressively more susceptible to digestion by exonuclease III and by S1 nuclease. Analysis of DNA from these aged preparations on agarose gels showed that the molecular weight of the DNA decreased as ageing progressed. We conclude that DNA in fixed chromosome preparations becomes progressively degraded as the preparations age.

Autosomal, sex and B chromosomes in Eyprepocnemis plorans (Orthoptera) viewed with restriction endonuclease in situ digestion

Similarities and differences between different subsets of heterochromatin localized on the autosomes, sex chromosomes and a B-chromosome in the genome of the grasshopper Eyprepocnemis plorans were analysed by in-situ digestion with 23 different restriction endonucleases, three un-specific nucleases, C-banding and specific fluorescent DNA-ligands. Results show an equilocal distribution of the different heterochromatic subsets in this species and provide a possible evolutionary origin for the B chromosome.