Induction of chromosome banding in early stages of spermatogenesis by ethidium bromide

Distinct chromosome banding of early meiotic prophase (leptotene through early pachytene) can he induced in male Chinese hamsters by injecting ethidium bromide and Actinomycin D intratesticularly for 4 hours and making acetic orcein squash preparations from minced testicular tissue. Zygotene pairing apparently starts from chromosome ends.Fellow, China Medical Board of New York, Inc.     

Application of ethidium bromide for high-resolution banding analysis of chromosomes from human malignant cells

Ethidium bromide was added to cultured human leukemic bone marrow and solid tumor cells to evaluate its inhibitory effect on mitotic chromosome condensation and its possible application to high-resolution banding analysis. In most experiments ethidium bromide treatment resulted in a high proportion of mitotic cells having elongated chromosomes, without remarkable reduction in either the mitotic index or quality of metaphase chromosomes. Optimal effect on chromosome length was obtained by adding 10 micrograms/ml of ethidium bromide during the final 2 hr of culture. Because of the simplicity and reproducibility of the technique involved, ethidium bromide can be used routinely to extend the length of chromosomes for fine-banding analysis of malignant cells.     

Inhibitory effects of ethidium bromide on the growth kinetics ofKlebsiella aerogenes,adaptation to ethidium and cross-resistance to proflavine

The aerobic growth ofKlebsiella aerogenes adapted to a chemically defined glucose ammonium sulphate medium was studied in this medium containing ethidium bromide (EB). The viability of sensitive bacteria on solid medium decreased markedly with EB above 150 mg/liter, and only a fraction of the viable inhabitants of a colony grown with EB at 500 mg/liter survived after a second transfer on to this concentration. In liquid medium containing EB above 30 mg/liter, the logarithmic phase was composite, consisting of a slower first mode followed by a faster second mode, and with EB above 50 mg/ liter composite growth was preceded by a lag. This lag was preceded by a mass doubling and one cell division with EB below 100 mg/liter; at higher concentrations only a mass doubling occurred, and the bacteria became elongated.During continued subculture in liquid medium containing EB, the lag vanished, the second mode of growth replaced the first, its rate increased and cell size became normal. Bacteria trained to EB in liquid medium were cross-resistant to proflavine and vice versa. 1.25 Moles of EB were about as inhibitory as 1 mole of proflavine. The mechanism of training to EB is discussed and cross-resistance is related to the similar mode of action of EB and proflavine.The author is grateful to the late Professor Sir Cyril Hinshelwood, O.M., F. R. S., and to Dr. A. C. R. Dean of the Physical Chemistry Laboratory, South Parks Road, Oxford, for valuable advice. The author wishes also to thank Miss E. S. Smalley for technical assistance in the final stages.     

Chromosome shattering: a mitotic catastrophe due to chromosome condensation failure

Chromosome shattering has been described as a special form of mitotic catastrophe, which occurs in cells with unrepaired DNA damage. The shattered chromosome phenotype was detected after application of a methanol/acetic acid (MAA) fixation protocol routinely used for the preparation of metaphase spreads. The corresponding phenotype in the living cell and the mechanism leading to this mitotic catastrophe have remained speculative so far. In the present study, we used V79 Chinese hamster cells, stably transfected with histone H2BmRFP for live-cell observations, and induced generalized chromosome shattering (GCS) by the synergistic effect of UV irradiation and caffeine posttreatment. We demonstrate that GCS can be derived from abnormal mitotic cells with a parachute-like chromatin configuration (PALCC) consisting of a bulky chromatin mass and extended chromatin fibers that tether centromeres at a remote, yet normally shaped spindle apparatus. This result hints at a chromosome condensation failure, yielding a “shattered” chromosome complement after MAA fixation. Live mitotic cells with PALCCs proceeded to interphase within a period similar to normal mitotic cells but did not divide. Instead they formed cells with highly abnormal nuclear configurations subject to apoptosis after several hours. We propose a factor depletion model where a limited pool of proteins is involved both in DNA repair and chromatin condensation. Chromosome condensation failure occurs when this pool becomes depleted. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article has been submitted as a contribution to the festschrift entitled “Uncovering cellular sub-structures by light microscopy” in honour of Professor Cremer’s 65th birthday.     

Binding of ethidium bromide to fractionated chromatin

The binding of ethidium bromide (EB) to different chromatin preparations was tested. Scatchard plots showed that the slowly sedimenting fraction of sheared chromatin is enriched in dye-binding sites. Limited nuclease digestion of rat liver nuclei, which has been shown to preserve the subunit structure of chromatin, reduces the number of binding sites available for intercalation of the dye.     

Synchronization of human leukemic cells: Relevance for high-resolution chromosome banding

Summary The cell-cycle kinetics of synchronized K562 human leukemic cells and bone marrow cells from adults with acute leukemia were studied in order to develop more reliable methods for producing increased numbers of mitoses, particularly those with elongated chromosomes suitable for high-resolution banding. Parameters examined included DNA content, mitotic index (MI), and chromosome preparations. K562 cells synchronized with methotrexate (MTX), thymidine (Tdr), or hydroxyurea (HU) showed two-fold increases in peak MI. Optimal harvesting times after release from block were approximately 10.5, 12.5, and 14.5 h for MTX, HU, and Tdr, respectively. MTX was selected for studies with cells from patients. Cells from 7 of the 10 patients studied showed 4.4-fold increases in peak MI. The optimal harvesting time was 9.5 to 11.5 h after release from block, considerably later than the 6 h time previously assumed in studies using stimulated lymphocytes. Cells from the three remaining patients showed no increase in MI after synchronization: and the lack of response may have been related to the high proportion of cells in G₀+G₁ prior to MTX exposure. For both the K562 cell line and most patient specimens, the combination of synchronization with appropriate release times and short Colcemid exposure (10 min) resulted in substantially improved chromosome preparations.     

Individual and age-related characteristics of the mitotic condensation of human Y chromosome

Three types of contraction (steady, speeding and slowing) of fluorescent (f) and nonfluorescent (nf) parts of the human Y chromosome were revealed in the spiralization interval limited by reper chromosome 3 length from 16.6 to 2.9 mkm. On the basis of regression analysis it was shown that in the initial phase of the spiralization interval studied the f-block was condensed more rapidly than the nf-region; then the speed of contraction of the latter exceeded that of the former. A decline of the Y chromosome condensation in relation to ageing was revealed. A possible chromosome segregation disturbance in gametogenesis due to senescent changes of heterochromatic regions is discussed.     

Effects of ethidium bromide on mitosis and chromosomes: a possible material basis for chromosome stickiness

When two types of mammalian cells were treated with ethidium bromide for several hours, the mitotic figures showed no chromatid breaks or exchanges but a high incidence of sticky chromosomes. Electron microscopic examinations revealed that many chromosomes are connected by submicroscopic chromatin strands of various widths. Chromosome stickiness, therefore, is interpreted as entanglement of chromatin fibers between unrelated chromosomes, probably caused by abnormal condensation behaviors prior to mitosis. Presumably, chromatin breaks would occur when sticky chromosomes separate during anaphase. Such microscopically undetectable breaks expressed as various kinds of chromosomal aberrations in the next mitosis when the damaged cells were permitted to recover in the absence of ethidium bromide.     

Study of two cases of ring 13 chromosome using high-resolution banding.

The chromosomes of two patients with ring 13 (r13) were studied using high-resolution RBG banding of prometaphase cells. The rings of the two patients differ slightly in breakpoints. Cell with multiple single, double-sized rings, quadruple-sized rings, rod- and ring-shaped fragments, and fragments showing varied states of condensation were seen, as were cells monosomic for chromosome 13. The evolution of these cell lines as a result of sister chromatid exchange, nondisjunction, ring breakage, and premature chromosome condensation is discussed. Clinical features of these patients reflect the heterogeneity of phenotype for r13 patients. Each case includes a feature of trisomy 13. The significance of mosaicism of cell lines in patients bearing ring chromosomes is considered with respect to variation in clinical findings.     

Potentiometric characterization of ethidium bromide and of its reactions with nucleic acids

A liquid membrane electrode that allows the concentration of ethidium ion (Ed(+)) to be measured selectively and accurately in the range of 0.1 microM to 5 mM is made. For Ed(+) concentrations less than 1 microM or more than 0.1 mM, the trend is no longer linear, and the causes of this behavior are discussed. The mean activity coefficient of ethidium bromide exhibits deviations from the Debye-Huckel limiting law that are interpreted in terms of aggregate formation. The stability constants for Ed(2)(2+) and Ed(2)Br(+) are 230 kg mol(-1) and 3.0 x 10(4) kg(2) mol(-2), respectively. In NaCl solutions, clusters involving up to 4 Ed(+) units are detected and their stability constants are evaluated. The intercalation of ethidium into poly(A).poly(U) in 1M NaCl is investigated by the above electrode, and the results are compared with those obtained by spectrophotometry. The data are analyzed in terms of Scatchard plots. The potentiometric method is more accurate than the spectrophotometric one at low values of the binding degree (r) where negative deviations from linearity are observed. The deviations are ascribed to a cooperative behavior rather than to artifacts caused by minor systematic errors.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Effects of ethidium bromide and berenil on protein synthesis

The effects of ethidium bromide, an intercalating dye and berenil, a nonintercalating dye on the biological activities ofEscherichia coli ribosomes have been studied. Ethidium bromide treatment drastically reduced both enzymatic and nonenzymatic initiation complex formation, enzymatic as well as nonenzymatic binding of phenylalanyl tRNA, peptidyl transferase, GTPase as well as the overall protein synthesising activity as measured by the poly U-dependent polymerization of phenylalanine. On berenil treatment, however, only enzymatic formation of the initiation complex is marginally reduced. Other reactions are not markedly affected except the enzymatic phenylalanyl tRNA binding which is slightly decreased only at high Mg²⁺ concentration; the treated ribosome has lowered polymerizing activity at sub-optimal Mg²⁺ concentration (10 mM). Although it has already been shown in this laboratory that treatment with either dye leads to the unfolding of the structure of the ribosome, the present studies indicate that berenil treatment does not alter the structure of the ribosome drastically in contrast to ethidium bromide treatment.