A new approach in recognition of heterochromatic regions of human chromosomes by means of restriction endonucleases.

The heteromorphisms of C-band regions of human chromosomes are evaluated by means of restriction endonucleases AluI, DdeI, MboI, and RsaI. Every chromosome exhibits heteromorphic markers of the C-band regions except chromosome 8. Each enzyme was found to be highly characteristic in its staining profile, a result that clearly suggests the diversity of heterochromatin. The inherent C-band-region heterochromatin variability that is revealed by these enzymes provides a valuable tool in identifying markers as compared with other previously described techniques.     

Expression of heterochromatin by restriction endonuclease treatment and distamycin A/DAPI staining of Indian muntjac (Muntiacus muntjak) chromosomes

The constitutive heterochromatin of the Indian muntjac (Muntiacus muntjak) was examined following digestion with various restriction endonucleases (AluI, HaeIII, HinfI, and MboI), as well as by selective fluorescence staining with distamycin A plus 4'-6-diamidino-2-phenylindole. Distinct areas within the C-bands were found to have characteristic staining patterns which were more conspicuous in the sex chromosomes. Two dot-like structures resistant to AluI were found in the X and Y1 chromosomes in the same position as the nucleolus organizer regions.     

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.     

Prediction of mammalian meiotic synaptic and recombinational behavior of inversion heterozygotes based on mitotic breakpoint data and the possible evolutionary consequences

The karyotype of moose (2n=68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analysed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating.The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-T richness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.     

The molecular characterization of the genome of Muraena helena L. I. Isolation and hybridization of two MboI-restricted DNA fractions.

To investigate the genome of the anguilliform fish Muraena helena at the molecular level we characterized total DNA by agarose gel electrophoresis after cleavage with AluI, HaeIII, MboI, and DdeI restriction endonucleases. Subsequently, we isolated the DNA from two specific electrophoretic fractions to be used as probes for Southern and in situ hybridization experiments. One such fraction showed an electrophoretic pattern typical of highly repetitive DNA localized in the centromeres of many chromosomes. The other fraction was shown to be located in the nucleolar organizer region, partially coincident with 45S rDNA, and to be composed of highly repetitive sequences.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

The AluI-induced bands in great apes and man: implication for heterochromatin characterization and satellite DNA distribution

Restriction endonucleases have recently been proved to be active on fixed chromatin, producing differences in staining of metaphase chromosomes. In this paper we show the results obtained by treating the metaphase chromosomes of Pan troglodytes, Pan paniscus, and Gorilla gorilla with the restriction enzyme AluI. These results demonstrate qualitative differences in the telomeric heterochromatin between Pan and Gorilla despite the fact that these areas appear homogeneous in the two genera by the C-banding method. The results found with individual chromosomes in the different species also appear relevant, in the light of the evolutionary relationships between these nonhuman primates and man. Lastly, the results suggest the presence, in great apes, of some highly repetitive DNA sequences different from the human satellites I-IV.     

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.     

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.     

New distinctions between regions of centromeric heterochromatin in human chromosomes by treatments with the isoschizomers NdeII-Sau3AI

The isoschizomers NdeII-Sau3AI (decreases GATC) have been used to characterize heterochromatic regions in human chromosomes. The findings with NdeII are identical with those previously published with MboI, but the results with Sau3AI provide evidence for new distinctions of centromeric heterochromatin in chromosomes 5 and 6. The results are discussed in relation to the chromatin organization at these regions an the mechanisms of the action of restriction endonucleases.     

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 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.     

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. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.     

Some remarks on the use of TaqI to detect highly repetitive DNA sequences in human chromosomes

In the attempt to conclude investigation of the action of restriction endonucleases on eukaryote chromosomes, we carried out a series of experiments digesting in situ human metaphase chromosomes with AluI/TaqI followed by Giemsa staining. We focused on the centromeric regions of chromosomes1, 2 and 16 and noted that those areas appeared as intensely stained blocks after AluI digestion, but were dramatically reduced in size or completely destroyed after subsequent TaqI treatment. These results permitted us to draw some conclusions on the highly repetitive DNA composition of these regions, in terms of alphoid and classical satellite DNAs.     

Characterization of human chromosomal constitutive heterochromatin

The constitutive heterochromatin of human chromosomes is evaluated by various selective staining techniques, i.e., CBG, G-11, distamycin A plus 4,6-diamidino-2-phenylindole-2-HCl (DA/DAPI), the fluorochrome D287/170, and Giemsa staining following the treatments with restriction endonucleases AluI and HaeIII. It is suggested that the constitutive heterochromatin could be arbitrarily divided into at least seven types depending on the staining profiles expressed by different regions of C-bands. The pericentromeric C-bands of chromosomes 1, 5, 7, 9, 13-18, and 20-22 consist of more than one type of chromatin, of which chromosome 1 presents the highest degree of heterogeneity. Chromosomes 3 and 4 show relatively less consistent heterogeneous fractions in their C-bands. The C-bands of chromosomes 10, 19, and the Y do not have much heterogeneity but have characteristic patterns with other methods using restriction endonucleases. Chromosomes 2, 6, 8, 11, 12, and X have homogeneous bands stained by the CBG technique only. Among the chromosomes with smaller pericentric C-bands, chromosome 18 shows frequent heteromorphic variants for the size and position (inversions) of the AluI resistant fraction of C-band. The analysis of various types of heterochromatin with respect to specific satellite and nonsatellite DNA sequences suggest that the staining profiles are probably related to sequence diversity.     

The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [¹⁴C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number of short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding.It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.     

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 DNA was then transferred to a nylon filter and ³²P-labelled major satellite DNA was used as a probe for hybridization experiments. Other preparations were simply stained with Giemsa after digestion in situ with AluI and BstNI. Our results show that although restriction endonuclease cleavage primarily depends on DNA base sequence, this factor is not always sufficient to explain nuclease-induced cytological effects. In fact, the structural organization of peculiar regions such as the centromeres of mouse chromosomes might affect cleavage efficiency when restriction enzyme digestion is performed in situ.M.L. Pardue     

Distribution of satellite DNA fractions within major heterochromatic regions of human chromosomes as revealed by PleI and TfiI digestion.

Banding patterns induced by selective DNA extraction with the restriction endonucleases PleI and TfiI reveal the distribution of human satellite DNAs within the major heterochromatic blocks on human metaphase chromosomes. PleI and TfiI are able to discriminate HinfI target sites, depending on the nature of the central base. PleI digestion specifically reveals regions, within major C-bands, that include the major sites of satellite II DNA and permits more precise localization of satellite II domains than does radioactive in situ hybridization. The close correspondence between the cytogenetic results presented here and previously reported molecular data seems to support the idea that the frequency of enzyme target sequences is the main factor in determining the action produced by restriction endonucleases on fixed human chromosomes and that chromatin conformation is not an important factor in limiting enzyme accessibility.     

The banding pattern produced by restriction endonucleases in mouse chromosomes

The metaphase chromosomes of mouse have been treated with the restriction endonucleases Alu I, Mbo I, Hae III and Eco RII and subsequently stained with the DNA-specific dye Ethidium bromide. A striking correspondence between previous biochemical findings and our cytological results has been noticed with Alu I, Mbo I and Hae III, which were capable of digesting all but the DNA localized at the centric constitutive heterochromatic areas. Digestion with Eco RII, on the contrary, resulted in cytological data which apparently did not fit in with the biochemical results previously obtained by digesting the DNA of this species with the same enzyme. It is postulated that factors such as chromatin organization, in addition to DNA base sequence, can determine the results we report.     

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.     

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.