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     

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.     

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.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

Patterns of DNase I sensitivity in the chromosomes of the ant Tapinoma nigerrimum (Hymenoptera,Formicidae)

We have analysed the patterns of DNase I/nick translation in the chromosomes of Tapinoma nigerrimum. The results show a non-uniform DNase I sensitivity in different chromosome domains. The hypersensitivity appears to be specially concentrated at both the NOR and the distal regions. The resemblance to and differences from the situation in other animal species, in which active genes are DNase I hypersensitive, are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Mechanisms of in situ nick translation of chromosomes using restriction endonucleases

In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA-protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions.     

DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I

DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I.     

Developmental changes in the chromatin of the brain of the rat: analysis by nicktranslation

Nick translation of nuclei of the brain of 3- 14- and 30-day old rats was carried out following their digestion by DNase I. The incorporation of ³H-dTMP at 14- and 30-day is significantly lower than at 3-day. This may be due to a lower proportion of active chromatin (DNase I hypersensitive sites) and condensation of chromatin with progressive development. When nuclei were digested by EcoRI and then nick-translated, the incorporation of ³H-dTMP showed the same pattern. Since the EcoRI sites are believed to be randomly distributed, the overall conformation of chromatin including the DNase I sensitive sites seems to undergo increasing compaction with development.     

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.     

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     

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.     

Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.     

Selective digestion of mouse metaphase chromosomes

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000xg pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

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     

DNase I digestion as a tool for the quantitative evaluation of C-heterochromatic-DNA in situ.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of the pre-treatments C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic-DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse metaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in 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.     

Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.