Selective digestion of mouse chromosomes with restriction endonucleases. II. X-ray microanalysis of HaeIII-treated chromosomes

We used X-ray microanalysis to study the changes induced in mouse metaphase chromosomes as a result of digestion with the restriction endonuclease HaeIII. The phosphorus X-ray signal was used as a marker for DNA and the sulfur signal for protein. Calcium, iron, copper, and zinc were also detected. HaeIII induced a loss of phosphorus from both the centromeres and chromosome arms, but the losses in the arms were much greater. These changes were accompanied by an increase in the electron density of the centromeres and a reduction in that of the arms. No reduction in the sulfur signal in either arms or centromeres occurred as a result of HaeIII digestion. Except for calcium, which showed only a moderate reduction, the inorganic ions exhibited very large losses as a result of HaeIII digestion. The differentiation of chromosome arms and centromeres as a result of HaeIII digestion is therefore not simply due to differential loss of DNA but also involves structural reorganization of the chromatin, as shown by electron microscopy. This reorganization does not involve loss of proteins but may be correlated with changes in the amounts of inorganic ions known to be involved in chromatin condensation.     

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.     

Alterations induced in mouse chromosomes by restriction endonucleases

Fixed chromosomes of mouse have been treated with Alu I, Eco RII, Hind III or Bam HI restriction endonucleases and subsequently stained with either Giemsa, Ethidium Bromide or Acridine Orange. The results obtained have been discussed in the light of preferential or non-preferential extraction of DNA from specific chromosome areas following enzyme digestion. The possible involvement of a particular structural organization of some classes of heterochromatin has been hypothesized to account for the findings after Alu I or Eco RII treatment. The meaning of the Giemsa banding observed after Hind III or Bam HI digestion has also been considered, in comparison to the different stain responses obtained by using a DNA-specific dye such as Ethidium Bromide.     

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.     

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.     

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.     

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.     

Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy.

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.     

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.     

The selective digestion of polytene and mitotic chromosomes of Drosophila melanogaster by the Alu I and Hae III restriction endonucleases

Polytene and mitotic chromosomes of Drosophila melanogaster were treated with either Alu I or Hae III restriction endonucleases. Subsequent staining with the DNA-specific fluorochrome ethidium bromide showed that these enzymes are capable of selectively digesting chromosomal DNA in fixed cytological preparations, as previously shown in mammalian metaphase chromosomes. Alu I or Hae III digestion made possible the localization in situ of some highly repetitive DNAs in both polytene and mitotic chromosomes, while only Alu I permitted the localization of the 5S RNA genes on the polytene chromosomes of D. melanogaster.     

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.     

Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases

A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.     

Factors affecting the digestion of moss DNA by restriction endonucleases

Abstract

The age of gametophytic tissues, de-starching, inclusion of PVP in the extraction medium, and column purification of isolated DNA have little or no effect upon the restriction of total DNA of Physcomitrella patens (Hedw.) Bruch, Schimp. & W.Gümbel. The relative longevity of restriction enzymes also appears unimportant. However, the extent of digestion of moss DNA by a given restriction endonuclease appears to correlate inversely with the number of cytosine residues in its recognition sequence that are susceptible to methylation in plant cells. Inclusion of spermidine in the restriction buffer slightly enhances restriction by a few specific endonucleases. This knowledge has practical significance when designing experiments in which it is desirable that restriction of isolated DNA samples is taken to completion.     

The specific blocks of heterochromatin on metaphase chromosomes of horse and Prjewalski horse detected by in situ digestion with restriction endonucleases

Restriction endonuclease in situ digestion of metaphase chromosomes gives an opportunity to reveal strips with different structure within GC-rich pericentric heterochromatin of the domestic horse and the wild Przewalski horse. Blocks of heterochromatin, which are insensitive to HaeIII and brightly stained with chromomycin A3 after restriction enzyme digestion, are localized on the border with euchromatin in the majority of chromosomes of Equus caballus and E. przewalskii. In contrast to chromosome 5 of E. caballus, acrocentric chromosomes of E. prezewalskii which are homologous to this chromosome have RE-CMA-blocks. We discuss a possible nature of the specific heterochromatin, which is insensitive to restriction enzyme digestion, and its role in the karyotype evolution.     

Factors affecting the digestion of restriction endonucleases in situ.

The influence of incubation buffers and glycerol on the enzyme activity of naked DNA of lambda phage and mouse, and of mouse chromosomal DNA was investigated. The results obtained varied in part from previously known data, but confirmed the importance of these factors in determining the patterns of in situ restriction enzyme digestion so far attributed exclusively to endonuclease activity.     

Hinf I restriction endonuclease digestion on human fixed metaphase chromosomes

The authors report on the activity of Hinf I restriction endonuclease on human fixed metaphase chromosomes. Experiments performed by digesting chromosomes just after harvesting or after ageing in methanol-acetic acid displayed a different pattern of digestion on metaphases, since only aged preparations showed gaps on heterochromatic regions of chromosomes 1, 9 and 16 and C-like bands on other chromosomes. In this view, the authors suggest that structural modifications of the DNA, induced by acid fixation, can influence Hinf I activity on fixed metaphase chromosomes.     

Cryptic variants of acrocentric human chromosomes as analysed by restriction endonucleases

Ten phenotypically normal human individuals have been analysed by in situ treatments with restriction endonucleases in order to obtain a better characterization of some cryptic variants of acrocentric chromosomes. Treatments with AluI, NdeII and Sau 3AI confirm the existence of two cryptic amplified regions on the short arms of both one chromosome 15 and one chromosome 22, in one female. These amplifications seem to be of different origin involving the nucleolar organizer region of chromosome 15 and the satellite of chromosome 22.     

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.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.