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.     

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.     

Chromosome banding studies by replication and restriction enzyme treatment in vendace (Coregonus albula) (Salmonidae, Salmoniformes)

Chromosome banding studies were performed in vendace, Coregonus albula. Original data on distribution of early and late replication regions, restriction sites (AluI, DdeI, HinfI and HaeIII) on chromosomes in this coregonid fish have been used to analyse karyotype heterochromatin differentiation. Heterochromatic bands (C-positive and not digested by restriction enzymes) have been identified as late replicating regions. Extra bands produced by the applied methods have permitted the identification of several homologous pairs. The centromeres were differentially digested by the restriction enzymes. The studied population seems to be homogenic regarding karyotype characteristics.     

The effect of restriction enzyme digestion of human metaphase chromosomes on C-band variants of chromosomes 1 and 9

Human metaphase chromosomes, fixed on slides, have beent treated with 8 different restriction endonucleases and 29 combinations of 2 restriction enzymes prior to staining with Giemsa. The endonucleases AluI and DdeI and the combinations AluI + DdeI, AluI + HaeIII, AluI + HinfI, and AluI + MboI have then been used to digest metaphase chromosomes of nine individuals with C-band variants of chromosomes 1 or 9, obtained by the CBG technique. The restriction enzyme resistant chromatin of the paracentromeric regions of chromosomes 1 and 9 has been measured and compared with the corresponding CBG-bands. The size of the enzyme resistant chromatin regions depend upon the type of enzyme(s) used. Treatment with AluI + MboI was the only digestion that acted differently on different chromosome pairs. However, within one pair of homologous chromosomes, all digestions revealed the same variations as conventional C-banding.     

Comparative analysis of the fragments of highly repeated DNA of Homo, Pan, Macaca and Cercopithecus

In the present paper the restriction patterns of Homo, Pan troglodytes, Macaca fascicularis and Cercopithecus aethiops, obtained by digestion with Eco RI endonuclease, were compared. All these species showed two common bands at 0.3 and 0.6 Kb. In this region another band at 0.5 Kb was present in Macaca fascicularis and Cercopithecus aethiops. Furthermore Macaca fascicularis, Cercopithecus aethiops and Pan troglodytes showed a series of bands not present in humans. The distance matrix shows a strong similarity between Homo and Pan and a decreasing similarity with Macaca fascicularis and Cercopithecus aethiops. No intraspecific variability was observed.     

Qualitative analysis of constitutive heterochromatin and primate evolution

The results of qualitative heterochromatin analysis in 16 species of primates: Homo sapiens , Pan troglodytes and Gorilla gorilla (F. Hominidae), Hylobates syndactilus (F. Hylobatidae), Macaca fascicularis , M. tibetana , Mandrillus sphinx , M. leucophaeus , Cercopithecus aethiops , C. sabaeus and C. albogularis (F. Cercopithecidae), Cebus apella , Ateles belzebuth hybridus , Aotus azarae , Saimiri sciureus and Lagothrix lagothricha (F. Cebidae) are presented in this work. We characterized heterochromatin using: (a) in situ digestion with restriction enzymes AluI, HaeIII, RsaI and Sau3A, and (b) chromosome staining with DA/DAPI on unbanded chromosomes, on C-banded chromosomes and on sequentially G-C-banded chromosomes. The aim of this work was to relate the qualitative characteristics of constitutive heterochromatin observed with the cytogenetic evolutive processes in the primate group. Results obtained show that (1) in the family Cercopithecidae, Papionini species do not present chromosomal rearrangements when their karyotypes are compared and the heterochromatin characteristics are uniform, while Cercopithecini species show a high number of chromosomal reorganizations, but they have the same heterochromatic characteristics; (2) the Platyrrhini species analysed show variability in their karyological and heterochromatic characteristics; (3) the Hominoidea present two different situations: Pan , Gorilla and Homo with few chromosomal reorganizations among their karyotypes but with a high variability in their heterochromatin characteristics, and Hylobates with low heterochromatin variability and a highly derived karyotype. Speciation processes related to chromosome changes and heterochromatin variations in different groups of primates are discussed.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 107–124.     

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.     

In situ digestion of Drosophila virilis polytene chromosomes by AluI and HaeIII restriction endonucleases

Polytene chromosomes of Drosophila virilis were treated with AluI and HaeIII restriction endonucleases. Both enzymes were capable of extensively digesting chromosomal DNA, with the exception of some regions that contain repetitive DNAs. Moreover, a comparison was made between our data and the data already obtained with the same enzymes in D. melanogaster. On this basis, AluI digestion showed that the 5S RNA genes of D. virilis and D. melanogaster have different base composition, while digestion with HaeIII revealed resistance of the histone genes in D. virilis, contrary to what was previously found in D. melanogaster.     

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.     

Cytochemical heterogeneity of the C-band in human chromosome 1

Summary The heterogeneity of the C-band of human chromosome 1 has been evaluated using several selective staining methods: C-banding (CBG), distamycin A plus 4-6-diamidino-2-phenylindole (DA/DAPI) and Giemsa G-11 pattern following the treatment with the restriction endonucleases AluI and HaeIII. The bands produced by each method are characteristic but not identical. The total C-band is resistant to AluI treatment. The bands induced by HaeIII and the one stained by DA/DAPI are markedly similar but smaller than the C-band. The G-11 technique stains yet smaller regions than those of HaeIII and DA/DAPI. Depending on the expression of staining properties, the C-band of chromosome 1 usually consists of three subdivisions: the proximal, intermediate and distal regions, suggesting an extremely heterogeneous nature. The staining variations between different regions are further substantiated by studies of a reciprocal translocation where the proximal region and the remaining C-band of chromosome 1 are separate.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

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.     

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.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.     

Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.     

Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.     

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.     

Cytology of the Cercopithecidae: the banding pattern of Cercopithecus nigroviridis Pocock 1907

In the present note, some preliminary results on the chromosomes of Cercopithecus nigroviridis are shown. In the two subjects studied a diploid number of 48 was found. The chromosomes were also treated by enzymatic digestion (trypsin treatment) and the results are visualized. These results seem to confirm a certain affinity between the banding pattern of the chromosomes of C. nigroviridis and the banding pattern of the chromosomes of Macaca mulatta. During the present study a very high percentage of association between the marked chromosome pair was also noticed.