Regions of active chromatin conformation in ‘inactive’ male meiotic sex chromosomes of the mouse

The sensitivity to DNase I of the meiotic sex chromosomes of the male mouse was determined by in situ nick translation. At pachytene and diakinesis-metaphase I, six segments, four at the ends of the X and Y chromosomes and two at internal sites on the X chromosome, were found to be more sensitive than the other parts of these chromosomes. The sensitive segments presumably reflect an active or potentially active chromatin conformation which is maintained in the sex chromosomes despite the earlier reported, almost complete cessation of uridine incorporation. The distribution of regions which are sensitive to DNase I corresponds to that of early DNA replication bands. Active conformation patterns like those figured here, probably exist in the sex chromosomes of other mammals as well.     

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation

Summary A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.     

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     

Analysis of methylation and distribution of CpG sequences on human active and inactive X chromosomes by in situ nick translation

In situ nick translation of fixed mitotic chromosomes after HpaII or MspI digestion allows us to detect different DNA methylation levels along chromosomes. We used this technique to analyse the methylation levels of CCGG sites in the active and inactive X chromosomes of female human cells. In addition, we analysed the distribution of these sites with respect to the banding pattern. Our data show that the inactive X, as a whole, is more methylated than the active one and that CCGG sequences are preferentially located on R-positive bands.     

An improved method for in situ nick translation of human chromosomes with biotin 11-labelled dUTP detected by biotinylated alkaline phosphatase

Nick translation of the DNA of conventionally prepared human metaphase chromosomes using DNase I and biotin dUTP combined with streptavidin-phosphatase-detection assay produced a banding-like appearance. This pattern seems to be due to differences in DNase I sensitivity along the chromosomes. The Y chromosome could be clearly distinguished from the other chromosomes because of its intensely dark labelled heterochromatic region. In addition to DNase I concentration, hypotonic treatment seems to be an important methodological factor influencing band resolution. Together with recently published similar methods these results indicate that in situ nick translation using biotinylated nucleotides may develop into a useful technique to overcome several problems of human cytogenetics.     

Mapping of DNAase I sensitive regions on mitotic chromosomes

We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of 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. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

Restriction endonuclease/nick translation of fixed mouse chromosomes: A study of factors affecting digestion of chromosomal DNA in situ

We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.by D. Schweizer     

DNase I sensitivity of Microtus agrestis active,inactive and reactivated X chromosomes in mouse-Microtus cell hybrids

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.Professor Marcus passed away on 2 January 1987     

Different DNA methylation pattern in A and B chromosomes of Crepis capillaris detected by in situ nick-translation. Comparison with molecular methods

Experiments were performed on Crepis capillaris callus lines with 0, 1 and 2 B chromosomes and on hairy root lines without or with 1 and 2 B chromosomes. Comparison of HPLC results for DNA from calli differing in number of B chromosomes did not reveal any significant differences in methylation level (30.4 +/- 1.1%, 30.9 +/- 1.2%, 31.7 +/- 1.7% in lines without or with one or two B chromosomes respectively) which could be attributed to the number of B chromosomes. Restriction patterns obtained after DNA digestion with HhaI, HpaII, MspI or HaeIII (i.e. restriction enzymes sensitive to cytosine methylation) were similar in calli and apical root segments and also did not depend on the presence or number of B chromosomes. Methylation of B chromosomes higher than that of A chromosomes was demonstrated by fluorescent in situ nick translation driven by HpaII, MspI or HaeIII in metaphase chromosomes. After short digestion (I and 3 h), B chromosomes, in contrast to A chromosomes, were weakly labelled or not labelled at all, which indicates longer distances between target sequences containing unmethylated cytosine in the former.     

The distribution of genes on chromosomes: A cytological approach

Studies during the last 20 years have shown that the chromosomes of many organisms, especially those of higher vertebrates, consist of a series of segments having different properties. These can be recognized as, for example, G- and R-bands. Recent studies have indicated that genes tend to lie in the R-bands rather than in the G-bands, although the number of genes that has been mapped with high precision is, as yet, only a very small proportion of the total, probably much less than 1%. We have therefore sought to study the distribution of genes on chromosomes using a cytological approach in conjunction with “universal” markers for genes. Such markers include mRNA and the gene-rich, G + C-rich H3 fraction of DNA, both of which can be localized using in situ hybridization, and DNase I hypersensitivity, and digestion by restriction enzymes known to show selectivity for the CpG islands associated with active genes, both of which can be detected using in situ nick translation. We have chosen to use the approaches involving in situ nick translation and have shown that the patterns of DNase I hypersensitivity and of CpG islands on human chromosomes show a strict correspondence to R-banding patterns: Deviations from R-banding patterns reported by previous investigators who have made similar studies appear to be attributable to excessive digestion. On the other hand, we have not found the expected differentiation between the active and inactive X chromosomes; this may perhaps be attributable to such factors as the demethylation of some non-island CpGs in the inactive X and the possible alterations of chromatin structure caused by methanol-acetic-acid fixation affecting DNase I hypersensitivity. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Patterns of DNase sensitivity in the chromosomes of Rana perezi (Amphibia: Anura).

We have analyzed the patterns of DNase I/nick translation in the chromosomes of Rana perezi. The results show a nonuniform DNase sensitivity in different chromosome domains; the hypersensitivity appears to be concentrated at both the NOR and the distal regions. The resemblance to the situation in mammals, where active genes are DNase I hypersensitive, is discussed.     

DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome

Summary We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

In situ nick translation of human metaphase chromosomes with the restriction enzymes MspI and HpaII reveals an R-band pattern.

Several restriction enzymes (HindIII, HaeIII, MspI, HpaII, EcoRI, KpnI, and NotI) were evaluated for their ability to induce bands in human metaphase chromosomes during in situ nick translation. MspI and HpaII were able to induce a completely developed R-band pattern. Preferential cleavage of R-band chromatin is due to the presence of unmethylated CpG-residues present in CpG-rich islands, which are apparently unevenly distributed and mainly concentrated in R-bands.     

Sorting of chromosomes by magnetic separation

Summary Chromosomes were isolated from Chinese hamster x human hybrid cell lines containing four and nine human chromosomes. Human genomic DNA was biotinylated by nick translation and used to label the human chromosomes by in situ hybridization in suspension. Streptavidin was covalently coupled to the surface of magnetic beads and these were incubated with the hybridized chromosomes. The human chromosomes were bound to the magnetic beads through the strong biotin-streptavidin complex and then rapidly separated from nonlabeled Chinese hamster chromosomes by a simple permanent magnet. The hybridization was visualized by additional binding of avidin-FITC (fluorescein) to the unoccupied biotinylated human DNA bound to the human chromosomes. After magnetic separation, up to 98% of the individual chromosomes attached to magnetic beads were classified as human chromosomes by fluorescence microscopy.     

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.     

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations

Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.