Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion. Of these, some nonhistones were primarily detected in the extracted chromatin while others were apparently resistant to extraction and located principally in the residual chromatin. (7) The chromatin in constitutive heterochromatin is transiently resistant to cleavage by micrococcal nuclease.     

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.by E.R. Schmidt     

DIGITAL IMAGE ANALYSIS OF CHROMATIN FIBRE PHENOTYPE AFTER ‘IN SITU’ DIGESTION WITH RESTRICTION ENDONUCLEASES

Restriction Endonucleases (REs) may recognize, cleave and remove DNA from fixed chromatin producing specific chromosome banding patterns. However, the modifications produced in the chromatin fibre are not easy to evaluate and compare. The aim of the present investigation was to visualize differences resulting in the texture of the chromatin fibre from metaphase chromosomes after each digestion using digital image analysis (DIA) facilities. To this purpose, metaphase chromosomes derived from a L-929 mouse cell line were digested with different REs (AluI,HpaII andHaeIII). Since light microscopy does not permit the observation of the chromatin fibre, DIA was performed on digitalized images of metaphase chromosomes under electron microscopy. The application of a LUT (Look Up Table) within the DIA software assigns a colour to each grey level of a digital image. The results obtained using a particular LUT, which permits the discrimination of specific chromatin fibre phenotypes resulting from each digestion, are reported and compared with those obtained under the light microscope.     

In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.by D. Schweizer     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Cytological and electrophoretic analysis of DNA methylation in the holocentric chromosomes of Megoura viciae (Homoptera, Aphididae).

Chromosomal and purified DNA methylation patterns were determined in the holocentric chromosomes of Megoura viciae by treatment with MspI and HpaII. Both enzymes produced a clear C-like banding pattern but widely digested one telomere of the X chromosome, which appeared as heterochromatic after C-banding treatment and brightly fluorescent after chromomycin A3 staining. Quantitative microfluorometric evaluations of DNA extraction performed on cytological preparations showed that both isoschizomers resulted in the same DNA extraction (about 30%). Contrary to what was found by in situ endonuclease treatment, the electrophoretic patterns of purified and digested DNA showed that digestion with MspI was slightly more extensive than that with HpaII in a zone of fragments ranging from 23 to 9 kb. This result indicates that aphid chromatin is not wholly unmethylated. The discrepancy between electrophoretic and cytological data has been explained by taking into consideration that DNA fragments with high molecular weights could be cleaved in situ by the enzymes but not extracted from the chromatin.     

Some factors affecting the action of restriction endonucleases on human metaphase chromosomes

We have investigated whether restriction endonucleases produce bands on human chromosomes by extracting DNA, using staining methods which are stoichiometric for DNA. Restriction enzymes that produce C-band patterns appear to remove DNA extensively from chromosome arms. In general, however, those restriction enzymes that produce G-bands do not extract DNA from chromosomes, and their effects are believed to be due to conformational change in the chromosomal DNA; in these cases, the chromosomal regions affected appear to be determined by the chromosome structure and not by the specificity of the enzyme. DNA loss from chromosomes due to digestion by restriction enzymes may in some cases be uniform, although a G-banding pattern is visible after Giemsa staining.     

The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee,gorilla, and orangutan and its evolutionary significance

Summary The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid geneaalogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human.     

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands,C-bands,and in situ hybridization

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase chromosomes with three restriction endonucleases (EcoRI, MboI and PstI). Banding patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.     

The Alu I-induced bands in metaphase chromosomes of orangutan (Pongo pygmaeus)

Summary Restriction endonucleases have been recently proved to be active on fixed chromosomes, thus they are useful in chromatin structure studies. Within this class of enzymes, Alu I is able to detect the presence and localization of highly repetitive DNA sequences in human and in other mammalian and dipteran species. In this paper the pattern obtained on fixed metaphase chromosomes of orangutan (Pongo pygmaeus) by Alu I digestion and Giemsa staining is shown. The results are discussed in the light of the distribution, in this species, of the I–IV human satellite DNAs. It is also suggested that in Pongo some highly repetitive sequences, different from the major human satellites, are present.     

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m² there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). At 70–300 J/m² the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. — BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800–15000 J/m² did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.     

Analysis of DNase 1 sensitivity and methylation of active and inactive X chromosomes of kangaroos (Macropus robustus) by in situ nick translation

The overall nuclease sensitivity and methylation of active and inactive X chromosomes of kangaroos were examined by in situ nick translation. Cultured fibroblasts of subspecies wallaroo-euro (Macropus robustus robustus; Macropus robustus erubescens) hybrids were used, enabling the paternally and maternally derived X chromosomes to be distinguished. No difference was found between the active and inactive X chromosomes with DNase I or MspI digestion. When chromosomes were digested with the methylation sensitive restriction enzymes HpaII and HhaI, the inactive X chromosome was labelled to a greater extent. These results indicate no overall difference in chromatin condensation between the active and inactive X chromosomes and greater overall methylation of the active X chromosome. This relative undermethylation of the inactive X chromosome may be important in X chromosome inactivation, but its function, if any, remains to be determined.by A. Bird     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

The biochemical and cytological characterization of Vicia faba DNA by means of MboI,AluI and Bam HI restriction endonucleases

Summary Restriction endonucleases were employed to characterize both cytologically and electrophoretically the DNA of Vicia faba. The electrophoretic pattern of total DNA digested with AluI and MboI shows a continuous smear. Bam HI also shows a continuous smear for the bigger polynucleotide fragments and several bands in the lower part of the lane. Digestion of fixed chromosomal DNA produces metaphase longitudinal differentiation when MboI and AluI are used, while no appreciable banding pattern is present when Bam HI is employed. These results are discussed in relation to the organization of chromosomal DNA, to other data in the literature on chromosome banding and on the digestion of total DNA of other species.     

Relationship of ultraviolet light-induced DNA-protein cross-linkage to chromatin structure

The production of banding patterns in metaphase chromosomes by restriction enzymes is inhibited by ultraviolet (UV) irradiation. Irradiation of fixed chromatin produces a 15-fold decrease in DNA extraction by restriction enzymes in comparison with that observed by irradiation before fixation. Alcohol-acid fixation of chromatin produces two major changes, the extraction of histones and dehydration. The effect of UV light is probably the result of a net increase in the yield of DNA-protein cross-links at comparable fluences of UV light and of the stabilization of the structural changes in the fixed chromatin fibril induced by the photoadducts. The X-irradiation of cells before fixation, as well as the rehydration of fixed chromatin, increases the extraction of DNA from fixed chromatin irradiated with UV light to levels similar to or even higher than those obtained with living cells. The effect of UV light before and after fixation on the extraction of DNA by restriction enzymes and proteinase K can be related to changes in chromatin structure and DNA conformation.     

Restriction endonuclease digestion of meiotic chromosomes of Allium subvillosum L.

Meiotic chromosomes of the liliaceous plant Allium subvillosum were characterized by means of digestion with the restriction endonucleases (REs) Hae III and Msp I followed in some cases by treatments with proteinase K or nuclease S1. Both REs are capable of digesting euchromatin, giving a C-like banding pattern. Something similar can be observed when chromosomes are digested with the two restriction endonucleases followed by treatments with proteinase K. By contrast, heterochromatic regions can be digested only after sequential treatments using Hae III plus nuclease S1. These results are discussed in relation to the structural organization of plant meiotic chromosomes as well as the special conformation of plant heterochromatin.     

CpG islands detected by self-primed in situ labeling (SPRINS)

We have studied the distribution and methylation of CpG islands on human chromosomes, using the novel technique of self-primed in situ labeling (SPRINS). The SPRINS technique is a hybrid of the two techniques primed in situ labeling (PRINS) and nick translation in situ. SPRINS detects chromosomal DNA breaks, as in nick translation in situ, and not annealed primers, as is the case in PRINS. We analyzed in situ-generated DNA breaks induced by the restriction enzymes HpaII and MspI. These restriction enzymes enable the detection of chromosomal CpG islands. Both HpaII- and MspI-SPRINS produce a banding pattern resembling R-banding, indicating a higher level of CpG islands in R-positive bands than in R-negative bands. Our SPRINS banding observations also indicate differences in sequence copy number in the satellites of homologous acrocentric chromosomes. Furthermore, a comparison of homologous HpaII-SPRINS-banded X chromosomes of females from lymphocyte cultures grown without methotrexate or bromodeoxyuridine revealed methylation difference between them. The same comparison of homologous X chromosomes from the cell line GM01202D, which has four X chromosomes, one active and three inactive, revealed the active X chromosome to be hypermethylated. Received: 5 February 1998; in revised form: 8 May 1998 / Accepted: 11 May 1998     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.