Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

High resolution chromosome analysis: one and two parameter flow cytometry

Isolated mammalian chromosomes have been quantitatively classified by high resolution flow cytometry. Chinese hamster chromosomes stained with 33258 Hoechst and excited in the UV showed a fluorescence distribution in which the 14 types of Chinese hamster chromosomes were resolved into 16 groups seen as distinct peaks in the distributions. Chinese hamster chromosomes were also stained with both 33258 Hoechst (HO) and chromomycin A3 (CA3); the two dye contents were measured by selective excitation in the UV and at 458 nm in a dual beam flow cytometer. The resulting two parameter distribution (HO versus CA3) showed 10 chromosome groups¹. Human strain LLL 761 chromosomes stained with HO and excited in the UV showed a fluorescence distribution in which the 23 types of human chromosomes were resolved into 12 groups. Human chromosomes stained with both HO and CA3 and measured in the dual beam flow cytometer produced two parameter fluorescence distributions which showed 20 groups. The chromosomes associated with each group were determined by quinacrine banding analysis of sorted chromosomes and by DNA cytophotometry of preidentified metaphase chromosomes. The relative HO and CA3 stain content and frequency of occurrence of chromosomes in each group were determined from the fluorescence distributions and compared to the results from DNA cytophotometry. The chromosome to chromosome variations in HO and CA3 staining are attributed to variations in chromosomal base composition.     

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization.

The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.     

Denaturation behaviour of DNA-protein-complexes detected in situ in metaphase chromosomes in suspension by Hoechst 33258 fluorescence.

The denaturation behaviour of DNA-protein complexes in metaphase chromosomes in suspension was analysed in situ by Hoechst 33258 fluorescence. The results indicate that due to the stability of the dye molecule and the product of the molecular extinction coefficient and the quantum yield at different temperatures, Hoechst 33258 is a suitable probe for the detection of double-stranded DNA. Thus, it is possible to monitor the concentration of double-stranded DNA in a suspension by measuring the total fluorescence intensity. The fluorescence denaturation profiles of DNA (calf thymus) were found to be comparable to absorption measurements. The decrease in fluorescence of metaphase chromosomes in suspension with increasing temperature may therefore be used to detect conformational changes of DNA in situ.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 M trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Metaphase chromosome accumulation and flow karyotypes in rice (Oryza sativa L.) root tip meristem cells.

Highly efficient cell synchronization and metaphase chromosome accumulation in rice root tip cells were achieved. Flow cytometric analysis was performed for obtaining optimal parameters to synchronize the cell cycles. High mitotic indices (about 57.6% in root tip meristemic area) were obtained by treating seedlings with 0.5 cm length using 0.5 mM hydroxyurea at 30 degrees C for 4 h, incubating in a hydroxyurea-free solution for 30 min, and then treating with 0.3 microM trifluralin for 3 h. After trifluralin treatment, incubation in distilled water for 15 min reduced chromosome clumping on metaphase spread. Uniformity of seed germination at the time of treatment is a critical parameter for obtaining high metaphase index. Isolated rice chromosomes were suitable for flow cytometric analysis and chromosome sorting. The morphology of flow sorted metaphase chromosomes was intact.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Effects of acetic acid-alcohol,trypsin, histone 1 and histone fragments on Giemsa staining patterns in chromosomes

Summary In an effort to minimize subjective bias, a classification scheme was devised to assess Giemsa staining patterns obtained with experiments involving acetic acid-alcohol and exogenously applied histone 1 and polypeptides. A single rinse of metaphase preparations with acetic acid-alcohol quantitatively reduced Giemsa dye binding. Acid-alcohol irrversibly changed the conformation of HI and its ability to interfere with trypsin G-banding. Our results suggest that, in addition to protein extraction, acid-alcohol may alter the conformation of acid-insoluble components of metaphase chromosomes. The carboxy-terminal polypeptide (residues 73–212) from NBS cleavage of H1 was an effective inhibitor of Giemsa staining and trypsin G-banding. However, this polypeptide which is preferential for supercoiled DNA was much less efficient in inhibiting Giemsa staining of trypsinized metaphase chromosomes. The molecular consequences of these experiments are discussed.     

The DNA-based structure of human chromosome 5 in interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.     

A new method for the preparation of metaphase chromosomes for flow analysis

A new method for the preparation of metaphase chromosomes for flow analysis has been evaluated. It has been shown that this method, which involves detergent lysis of metaphase cells and polyamines to stabilize the DNA, yields lower coefficients of variation and background levels in the DNA histograms than is currently obtained by hexylene glycol based methods. A conventional flow cytometer (FACS-II) has been used to resolve the human karyotype into about 14 peaks after ethidium bromide staining and excitation with a relatively low level of illumination (0.4 W at 488 nm). Flow karyotypes have also been obtained from suspension cell lines, in particular from the mouse cell line, Friend 707/B10. The only disadvantage of this method is that the chromosomes are highly condensed and therefore banding studies on sorted chromosomes may not be possible.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

Applications of fluorescence spectroscopy to molecular cytogenetics

Phenomena studied by fluorescence spectroscopy, including differential perturbations of dye fluorescence by variations in DNA composition and interdye energy transfer, can be applied to cytological studies of chromosome structure, replication, and repair. When combined with fluorescence-activated flow sorting, fluorescent differentiation between metaphase chromosomes can be exploited to obtain recombinant DNA libraries enriched for all or parts of individual chromosomes. Cloned DNA segments from such libraries continue to find application in studies of human chromosome structure and function and in molecular linkage analyses, thus leading to molecular genetic investigations of human disease states.     

Automatic detection and localization of sister chromatid exchanges.

Sister chromatids of human metaphase chromsomes from cells which have replicated twice in medium containing 5-bromodeoxyuridine exhibit unequal fluorescence when stained with the dye 33258 Hoechst. Sister chromatid exchanges occurring in these chromosomes are apparent as interchanges of brightly and dully fluorescing chromatids. A technique for detecting such exchanges by computer analysis of chromsome images has been developed and found to campare favorably with manual methods. The exchanges have been localized in the context of quinacrine banding patterns.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Occurrence of G-banding in metaphase chromosomes of Encarsia berlesei (Hymenoptera: Aphelinidae).

Well-defined G-bands were obtained on somatic metaphase chromosomes of Encarsia berlesei using trypsin and warm 2x SCC in sequence. The G-banded pattern allowed rapid identification of all five metacentric chromosomes, which appeared uniformly lighted when stained with DAPI fluorochrome dye. It is stressed that ageing affects G-banding in this insect species; in fact, good banded chromosomes were obtained on 1-month air-stored chromosomes. Evidence for asynchronous condensation on the chromosomes of this species is also provided.     

Metaphase synchronization and chromosome preparation from the OK opossum cell line having a potentially isolatable X chromosome

As part of a study on X chromosomes, metaphase cell synchrony and chromosome isolation methods were developed for the opossum (Didelphis virginiana) kidney epithelial cell line (OK). The cell synchrony yielded large amounts of metaphase cells using a relatively simple method in which a key feature was a calcium- and magnesium-free balanced salt wash. A neutral pH chromosome isolation method was developed for the kidney epithelial cells, because they were somewhat difficult to disrupt fully by other methods. FACS IV flow microfluorometric analysis of OK chromosomes confirms a clear difference between the sizes of opossum X chromosomes and autosomes.     

Human chromosome-specific repetitive DNA sequences: novel markers for genetic analysis

Two recombinant DNA clones that are localized to single human chromosomes were isolated from a human repetitive DNA library. Clone pHuR 98, a variant satellite 3 sequence, specifically hybridizes to chromosome position 9qh. Clone pHuR 195, a variant satellite 2 sequence, specifically hybridizes to chromosome position 16qh. These locations were determined by fluorescent in situ hybridization to metaphase chromosomes, and confirmed by DNA hybridizations to human chromosomes sorted by flow cytometry. Pulsed field gel electrophoresis analysis indicated that both sequences exist in the genome as large DNA blocks. In situ hybridization to intact interphase nuclei showed a well-defined, localized organization for both DNA sequences. The ability to tag specific human autosomal chromosomes, both at metaphase and in interphase nuclei, allows novel molecular cytogenetic analyses in numerous basic research and clinical studies.