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.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Stabilization of chromonema--one of the highest compactization levels--by visible light in the presence of ethidium bromide

This paper deals with the ultrastructure and behavior of interphase chromatin and metaphase chromosomes of L-197 culture cells under experimental conditions, which help to reveal the chromonemal level of chromosomal structure after the treatment of living cells with 0.1% Triton X-100 and 3 mM CaCl2. In these conditions, the chromonemata can be seen as dense chromatin fibers with thickness about 100 nm. Such chromosomes, whose chromonemal substructure after the treatment with hypotonic solution (10 mM Tris-HCl), look like loose chromosomal bodies composed of elementary 30 nm DNP fibrils. On the other hand, if chromosomes, in which chromonemal levels were revealed by 3 mM CaCl2, were treated with etidium bromide and then illuminated by light with length wave about 460 nm, no chromosomal decondensation in hypotonic conditions is observed. Chromonemata in chromosomes stabilized by light retain their density and dimensions. It is very important that chromonemata in stabilizated chromatin of metaphase chromosome keep specific connections between themselves and also general trend in their composition inside the chromosome. Thus, we have found conditions for observation of chromonemal elements in metaphase chromosome, providing the possibility for future three-dimensional investigation of chromonema packing in mitotic chromosomes.     

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.     

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.     

Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes

The mouse oocyte expresses a polypeptide of Mr 120,000 that cross-reacts with an antibody to the brain membrane skeletal protein adducin. Immunofluorescence localization showed a bright chromosomal staining reaction in metaphase I and metaphase II oocytes. Following in vitro fertilization the maternal chromosomes lost their immunoreactivity during pronuclear development. The fertilizing sperm chromatin and male pronucleus did not show any detectable staining reaction. Bright chromosomal fluorescence was again observed in the first mitotic metaphase when both maternal and paternal chromosomes gave a positive staining reaction. In contrast to the immunoreactivity of the maternal meiotic chromosomes, the meiotic chromosomes of male germ line cells failed to exhibit any detectable staining reaction and this difference was confirmed by immunolabeling of oocyte and spermatocyte karyotypes. Mitotic chromosomes in preimplantation embryos, fetal liver, adult intestinal epithelium, and MDCK cells also failed to show any detectable labeling reaction. The results suggest that expression of the immunoreactive chromosomal adducin may be a unique feature of oogenesis.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

Improved methods for the isolation of individual and clustered mitotic chromosomes

We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gei patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression

Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.     

Nondisjunction, disturbances in spindle structure, and characteristics of chromosome alignment in maturing oocytes of mice heterozygous for Robertsonian translocations

To correlate the chromosomal constitution of meiotic cells with possible disturbances in spindle function and the etiology of nondisjunction, we examined the spindle apparatus and chromosome behavior in maturing oocytes and analyzed the chromosomal constitution of metaphase II-arrested oocytes of CD/Cremona mice, which are heterozygous for a large number of Robertsonian translocation chromosomes (18 heterobrachial metacentrics in addition to two acrocentric chromosomes 19 and two X chromosomes). Spreading of oocytes during prometaphase 1 revealed that nearly all oocytes of the heterozygotes contained one large ring multivalent, apart from the bivalents of the two acrocentric chromosomes 19 and the X chromosomes, indicating that proper pairing and crossing-over between the homologous chromosome arms of all heterobrachial chromosomes took place during prophase. A large proportion of in vitro-matured oocytes arrested in metaphase II exhibited numerical chromosome aberrations (26.5% hyperploids, 40.8% hypoploids, and 6.1% diploids). In addition, some of the oocytes with euploid chromosome numbers (26.5% of the total examined) appeared to be nullisomic for one chromosome and disomic for another chromosome, so that aneuploidy levels may even be higher than expected on the basis of chromosome counts alone. Although oocytes of the complex heterozygous mice seemed able initially to form a bipolar spindle during first prometaphase, metaphase I spindles were frequently asymmetrical. Chromosomes in the multivalent did not align properly at the equator, centromeres of neighboring chromosomes in the multivalent remained maloriented, and pronounced lagging of chromosomes was observed at telophase I in oocytes obtained from the Robertsonian translocation heterozygotes. Therefore, disturbance in spindle structure and chromosome behavior appear to correlate with the chromosomal constitution in these oocytes and, ultimately, with failures in proper chromosome separation. In particular, reorientation appears to be a rare event, and malorientation of chromosomes may remain uncorrected throughout prometaphase, as we could not find many typical metaphase I stages in heterozygotes. This, in turn, could be the basis for malsegregation at anaphase and may ultimately induce a high rate of nondisjunction and aneuploidy in the oocytes of CD/Cremona mice, leading to total sterility in heterozygous females.     

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.     

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.     

Mouse satellite DNA, centromere structure, and sister chromatid pairing

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.     

Stabilization of chromosomes by DNA intercalators for flow karyotyping and identification by banding of isolated chromosomes

A number of structurally unrelated DNA intercalators have been studied as stabilizers of mitotic chromosomes during isolation from rodent and human metaphase cells. Seven out of the nine intercalators tested were found to be useful as chromosome stabilizing agents. Chromosome suspensions prepared in this way could be preserved for long periods of time. After isolation the chromosomal DNA was longer than 150 kb. With intercalated chromosomes high resolution flow karyotypes could be obtained as illustrated for the non-fluorescent intercalators 9-methylene-(1,3-dimethyl-2,4-dionepyrimidine-5-yl)-phenanthrid in iumchloride and 4'-aminomethyl-4,5', 8-trimethylpsoralen combined with DAPI and 33258 Hoeschst for fluorescent staining and for the fluorescent intercalator propidium iodide used as a stabilizer and as a fluorochrome. Passage of the intercalated chromosomes through the laser beam had no measurable effect on the length of the chromosomal DNA subsequently isolated. After flow analysis and collection on slides human chromosomes could easily be banded by Giemsa staining methods with the same resolution as obtained in conventional metaphase spreads. This allowed a ready identification of about 80 percent of all chromosomes in the unfractionated suspension collected after passage through the laser beam.     

水稻中期染色体和DNA纤维的高效制备技术

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25％。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。     

Isolation and fractionation of CHO chromosomes in aqueous two phase systems using charged polymers and base specific macroligands

Summary Chromosomes were isolated in a preparative scale by synchronisation of CHO cells with a double Thymidine block followed by an arrest in the metaphase by addition of Colcemid. Under proper cultivation conditions a mitotic index of 77% total cells could be routinely achieved. Bulk chromosome preparations free of nuclei and other subcellular particles have been obtained by low speed centrifugation followed by a 60 transfer countercurrent distribution using aqueous two phase systems composed of polyethylenglycol and dextran. The partition of CHO chromosomes previously purified in aqueous two phase systems were studied further to develop a protocol for the separation and isolation of individual chromosomes. Partition experiments with chromosomes changing the electrostatic phase potential by addition of charged PEG-derivatives suggest the existence of relatively highly charged chromosome groups. Most promising results with regard to separation were obtained using two PEG-derivatives, which interact specifically with the bases in DNA. For this affinity partitioning a GC- and AT-specific macroligand were employed. Comparing CCD's using each of these ligands information on the GC and AT content of exposed DNA in the chromosomes groups could be derived, demonstrating that specific sequences of DNA are accessible at the surface of metaphase chromosomes.     

Digital image analysis of chromatin fibre phenotype after "in situ" digestion with restriction endonucleases

Restriction Endonucleases (REs) may recognize, cleave and remove DNA from fixed chromatin producing specific chromosome banding patterns. However, the modifications produced in the chromatin fibre are not easy to evaluate and compare. The aim of the present investigation was to visualize differences resulting in the texture of the chromatin fibre from metaphase chromosomes after each digestion using digital image analysis (DIA) facilities. To this purpose, metaphase chromosomes derived from a L-929 mouse cell line were digested with different REs (AluI, HpaII and HaeIII). Since light microscopy does not permit the observation of the chromatin fibre, DIA was performed on digitalized images of metaphase chromosomes under electron microscopy. The application of a LUT (Look Up Table) within the DIA software assigns a colour to each grey level of a digital image. The results obtained using a particular LUT, which permits the discrimination of specific chromatin fibre phenotypes resulting from each digestion, are reported and compared with those obtained under the light microscope.     

Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes

Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.