Potentiation of Alu I-induced chromosome aberrations by high salt concentrations in Chinese hamster ovary cells

The restriction endonuclease Alu I induces chromosome-type aberrations in Chinese hamster ovary cells whose frequencies are considerably elevated in the presence of high concentrations of MgCl2, (NH4)2SO4, CaCl2 or NaCl. The most plausible explanation for these findings is that salt leads to partial dehistonization of the chromatin which makes more recognition sites available for Alu I.     

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.     

Effect of heat treatment on chromosomal aberrations induced by the alkylating agent trenimon or the restriction endonuclease Alu I in Chinese hamster ovary (CHO) cells

Heat treatment of CHO cells in the G1-phase of the cell cycle leads to chromatid-type aberrations in first posttreatment metaphases. Posttreatment of heat-treated cells with the alkylating agent trenimon leads to a synergistic effect on the production of chromatid-type exchanges. These results indicate that heat induces lesions which like the lesions produced by trenimon give rise to chromatid-type aberrations during the first posttreatment S-phase, and that these lesions can interact with each other to produce chromatid-type exchanges. Treatment of CHO cells in the G1-phase of the cell cycle with the restriction endonuclease Alu I induces chromosomal aberrations. Pretreatment of cells with heat leads to a reduction of Alu I induced chromosome-type aberrations. When cells are allowed to recover after heat treatment for 22 h, the aberration frequencies produced by Alu I are the same as in cells not treated with heat. These findings can be explained by assuming that heat-induced accumulation of accessory proteins in the chromatin protects the DNA from being cut by Alu I, and that the cells recovered from the heat-induced protein accumulation after 22 h.     

Endonuclease banding of isolated mammalian metaphase chromosomes

Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C-banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin.     

Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside

Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA-strand breaks by ara C.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro

The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid-type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone.     

The chromosome-breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S-phase of the cell cycle

The restriction endonuclease Alu I induces chromosomal aberrations in living Chinese hamster ovary (CHO) cells. Multiple fixation times reveal that the chromosome-breaking activity of Alu I is similar to that of ionizing radiation in that it is independent of the S-phase of the cell cycle. These results indicate that DNA double-strand breaks are the ultimate lesions for the production of chromosomal aberrations in all stages of the cell cycle.     

Effect of liver chromatin DNAse on closed circular DNA of PM-2 phage and simian virus 40]

Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.     

Cleavage map of bacteriophage phiX174 RF DNA by restriction enzymes.

phiX RF DNA was cleaved by restriction enzymes from Haemophilus influenzae Rf (Hinf I) and Haemophilus haemolyticus (Hha. I). Twenty one fragments of approximately 25 to 730 base pairs were produced by Hinf I and seventeen fragments of approximately 40 to 1560 base pairs by Hha I. The order of these fragments has been established by digestion on Haemophilus awgyptius (Hae III) and Arthrobacter luteus (Alu I) endonuclease fragments of phiX RF with Hinf I and Hha1. By this method of reciprocal digestion a detailed cleavage map of phiX RF DNA was constructed, which includes also the previously determined Hind II, Hae III and Alu I cleavage maps of phiX 174 RF DNA (1, 2). Moreover, 28 conditional lethal mutants of bacteriophage phiX174 were placed in this map using the genetic fragment assay (3).     

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.     

Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease

Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other forms of damage produced by 254-nm ultraviolet (UVC) radiation. We have isolated a DNA endonuclease, pI 7.6, from the chromatin of normal human lymphoblastoid cells which recognizes damage produced by UVC light, and have introduced this endonuclease into UVC-irradiated XPA cells in culture to determine whether it can restore their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS). Introduction of the normal endonuclease, which recognizes predominantly pyrimidine dimers, but not the corresponding XPA endonuclease into UVC-irradiated XPA cells restored their levels of UDS to approximately 80% of normal values. Electroporation of both the normal and the XPA endonuclease into normal human cells increases UDS in normal cells to higher than normal values. These results indicate that the normal endonuclease can restore UDS in UVC-irradiated XPA cells. They also indicate that XPA cells have an endonuclease capable of increasing the efficiency of repair of UVC damage in normal cells.     

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6-thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations.     

Ca/Mg-dependent endonuclease as a probe for detecting chromatin changes in lymphocytes in chronic lymphoid leukemia

Chromatin fragmentation of bovine peripheral blood lymphocytes from normal animals and the ones suffering from chronic lympholeucosis (CLL) by DNase I, micrococcal nuclease and purified Ca/Mg-dependent endonuclease from nuclei of human splenocytes was studied. The lymphocytes chromatin from CLL animals was shown to be more resistant to nucleases, than the one from normal animals. It was found that difference between fragmentation of chromatin samples from normal and CLL bovines was more dramatic when Ca/Mg- dependent endonuclease was used versus traditionally exploited DNase I and micrococcal nuclease. The data suggest that purified Ca/Mg-dependent endonuclease can be a useful enzymatic probe for detection of lymphocytes chromatin changes during CLL.     

Arthrobacter luteus restriction endonuclease recognition sequence and its cleavage map of SV40 DNA.

The nucleotide sequence at the cleavage site of the restriction endonuclease isolated from Arthrobacter luteus (Alu) has been determined. The endonuclease cleaves at the center of a palindromic tetranucleotide sequence to give even-ended duplex DNA fragments phosphorylated at the 5'-end. The endonuclease cleaves SV40 form I DNA into 32 fragments. The order and sizes of these fragments have been determined to provide an Alu cleavage map of the SV40 genome.     

Primary and secondary structure specificity of the cleavage of 'single-stranded' DNA by endonuclease Hinf I.

The interaction of endonuclease Hinf I with single-stranded fd DNA was examined. The sizes of the cleavage products indicate that the enzyme cuts this substrate at the same sequences as double-stranded DNA (GANTC). To determine whether or not the recognition sites in single-stranded DNA have to be present in double-stranded form in order to be cleaved, DNA fragments containing complementary or non-complementary Hinf I sequences were prepared and treated as substrates. The results suggest that completely base-paired recognition sites are necessary for cleavage. Sequences surrounding the Hinf I pentanucleotides significantly modulate the reaction rates.     

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 effects of DNA methylation by Hha I methylase on the cleavage reactions by Hae II, Aha II and Ban I endonucleases

The DNA methylated by Hha I methylase was resistant against cleavage of Hae II or Aha II endonuclease indicating that the methyl group of the C5 position of the inmost cytosine nucleotide interferes with the interaction between the enzyme and the hexameric recognition sequence. Considering that Hae II or Aha II methylase has not been isolated yet, the result explained above is a useful information for protecting a double stranded DNA from being cleaved by Hae II or Aha II endonuclease. In contrast to Hae II or Aha II endonuclease, Ban I endonuclease which also has Hha I sequence as its tetrameric core was able to cleave the same DNA normally. This result suggests that the C5 position of the inmost pyrimidine nucleotide is not an important contact point between Ban I endonuclease and its hexameric recognition sequence.     

Erythroid-specific gene chromatin has an altered association with linker histones.

The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.     

Excision repair of pyrimidine dimers from simian virus 40 minichromosomes in vitro

The ability of DNA repair enzymes to carry out excision repair of pyrimidine dimers in SV40 minichromosomes irradiated with 16 to 64 J/m2 of UV light was examined. Half of the dimers were substrate for the DNA glycosylase activity of phage T4 UV endonuclease immediately after irradiation, but this limit decreased to 27% after 2 h at 0 degrees C. Moreover, the apyrimidinic (AP) endonuclease activity of the enzyme did not incise all of the AP sites created by glycosylase activity, although all AP sites were substrate for HeLa AP endonuclease II. The initial rate of the glycosylase was 40% that upon DNA. After incision by the T4 enzyme, excision was mediated by HeLa DNase V (acting with an exonuclease present in the chromatin preparation). Under physiological salt conditions, excision did not proceed appreciably beyond the damaged nucleotides in DNA or chromatin. With chromatin, about 70% of the accessible dimers were removed, but at a rate slower than for DNA. Finally, HeLa DNA polymerase beta was able to fill the short gaps created after dimer excision, and these patches were sealed by T4 DNA ligase. Overall, roughly 30% of the sites incised by the endonuclease were ultimately sealed by the ligase. The resistance of some sites was due to interference with the ligase by the chromatin structure, as only 30-40% of the nicks created in chromatin by pancreatic DNase could be sealed by T4 or HeLa DNA ligases. The overall excision repair process did not detectably disrupt the chromatin structure, since the repair label was recovered in Form I DNA present in 75 S condensed minichromosomes. Although other factors might stimulate the rate of this repair process, it appears that the enzymes utilized could carry out excision repair of chromatin to a limit near that observed at the initial rate in mammalian cells in vivo.