Chromosome Suspensions Research Articles

High-Speed Chromosome Sorting

Dual-beam high-speed sorting has been developed to facilitate purification of chromosomes based on DNA staining with the fluorescent dyes Hoechst 33258 and chromomycin A3. Approximately 200 chromosomes per second of two types can be sorted from a suspension of chromosomes isolated from human lymphoblasts while fluorescent objects (chromosomes, debris fragments, chromosome clumps, and nuclei) are processed at the rate of about 20,000 per second. This sorting rate is approximately ten times that possible with conventional sorters. Chromosomes of a single type can be sorted with a purity of about 90 percent. DNA from the sorted chromosomes is suitable for construction of recombinant DNA libraries and for gene mapping.

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%.

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.

22] Gene mapping with sorted chromosomes

This chapter focuses on gene mapping with sorted chromosomes. Sorted chromosomal DNAs isolated by single laser chromosome sorting and analyzed by standard restriction enzyme analysis were used to map the β-, γ-, and δ-globin and insulin genes to the short arm of chromosome. Hybridization of radiolabeled probe to chromosomes sorted onto spots with a single laser sorter determined the Y-chromosome containing histogram peak 2 and further sublocalized the myc gene. Spot-blot analysis of chromosomes separated with a high-resolution dual-laser chromosome sorter provided a straightforward, rapid method to map genes to whole chromosomes. Several developments contributed to the efficacy of the spot-blot method: (1) development of spermine buffer and improved lymphocyte chromosome suspension preparation, (2) identification of chromosomes sorted in spermine buffer, (3) sorting chromosomes directly onto filters, (4) dual laser chromosome sorting, (5) improved sorter design and performance, and (6) characterization of an additional chromosome stain pair.

Flow-sorting analysis of normal and abnormal human genomes.

Flow analysis of human chromosomes is a powerful new approach to study the human genome. Human chromosomes can be resolved almost completely as a result of improvements in metaphase chromosome suspension preparations, new fluorescent dye combinations, and advances in sorter optical design. We have applied this technique to map genes, detect abnormal chromosome constitutions, and construct individual recombinant chromosomal DNA libraries.

Comparison of Transformation, Chromosome Aberrations, and Reproductive Death Induced in Cultured Mammalian Cells by Neutrons of Different Energies

Dose-effect relations for cell transformation and reproductive death induced in C3H/10T1/2 cells, NBCH-3 cells, and WAGR-2 cells by 300 kV X rays and monoenergetic fast neutrons of 0.5, 4.2, and 15 MeV energy have been analyzed on the basis of the relations F(D) = t1D + t2D2 and S(D)/S(0) = exp - (a1D + a2D2), respectively. Values of a1 are a factor of about 10(3) larger than corresponding values of t1, while the dependence on neutron energy is similar for both effects. RBE values and relations between the a1 and t1 values are compared with characteristics, derived from corresponding parameters for chromosome aberrations and reproductive death, induced in other cell lines after irradiation with the same neutron beams. A hypothesis is developed assuming that nonrandom structural changes at a restricted number of sites on one or more chromosomes are the primary causes of transformation of cultured mammalian cells by ionizing radiations, whereas similar but randomly distributed chromosomal changes, which are induced at many sites on all chromosomes, cause observable aberrations and reproductive death. Flow cytometry of chromosome suspensions has been used to demonstrate nonrandom changes of chromosomes in selected clones of transformed cells and of random changes of chromosomes in cells inactivated by X rays and neutrons.

Immunofluorescent detection of histone 2B on metaphase chromosomes using flow cytometry.

A monoclonal antibody against histone 2B (anti-H2B) was used as a reagent to stain isolated chromosomes for analysis using flow cytometry. Chromosome suspensions were treated with a mouse monoclonal antibody specific for the histone 2B (clone HBC-7) and then with a fluorescein-labeled goat anti-mouse-IgM antibody. The chromosomes were also stained for DNA content with either Hoechst 33258 or propidium iodide. The amount of antibody and the amount of DNA-specific stain bound to each chromosome were measured simultaneously using flow cytometry. The order of the steps in the staining protocol is important. Propidium iodide prevents anti-H2B from binding to chromosomes, and therefore must be added only after antibody labeling is completed. In contrast, the addition of Hoechst 33258 before antibody labeling reduces antibody binding by only 20%-30%. Binding of anti-H2B was proportional to the DNA content of both human and Chinese hamster chromosomes. Human chromosomes bind an average of three to four times more anti-H2B than do Chinese hamster or mouse chromosomes of the same DNA content. This was determined by analyzing mixtures of human and Chinese hamster chromosomes and human and mouse chromosomes. The results demonstrate that it is possible to label the proteins of chromosomes in suspension with fluorescent antibodies and to use these reagents for the analysis of chromosome structure by flow cytometry.

Flow Cytometric Determination of Radiation-Induced Chromosome Damage and Its Correlation with Cell Survival

Chinese hamster M3-1 cells were irradiated with several doses of X rays or alpha particles from 238Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and alpha particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.

Preparation of chromosome suspensions for flow cytometry

Certain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4 degrees C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.

Purifying human Y chromosomes by flow cytometry and sorting.

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.

Optimization of mammalian chromosome suspension preparations employed in a flow cytometric analysis.

Metaphase chromosomes from Chinese hamster V-79 cells have been stained with propidium iodide and their DNA content was measured by flow cytometry. The chromosomes in suspension must be stabilized during the preparation to preserve their morphological integrity. In order to obtain high resolution in the flow karyotype, several modifications were made to the method used to prepare chromosome suspensions. The influence of several ions, detergents and detergent mixtures was investigated. Magnesium (3-5 mM) together with sodium (10 mM), Tris (10 mM) and Triton X-100 (0.4%) gave the best results. In contrast, sodium and potassium exerted no effect, while calcium had a negative effect on the preparation as measured with flow cytometry. Compared with a solution of potassium chloride + propidium iodide, chromosomes prepared in Tris buffer resolved better with less background. A histogram quality factor which contains the variables peak height, depth of valley and coefficient of variation, was used to quantitate the histograms.

Design and operation of a dual laser chromosome sorter.

We describe the design and operation of a dual laser fluorescence activated chromosome sorter that can analyze and sort chromosome suspensions stained with two complementary DNA stains. We used two 18-watt argon-ion lasers to provide maximal excitation of the DNA-specific stains Hoechst 33258 and chromomycin A3. The lasers are focused onto the sample stream by independent optics permitting optimal focusing of any pair of laser wavelengths. We incorporated beam expanders to decrease the energy density on the optics and improve the focal spot uniformity. Adjustable focusing lens holders are used to set the optimal focal spot size. The redesigned sorter separates suspensions of normal human fibroblast chromosomes into 21 fractions and can reliably sort more pure chromosome fractions than was previously possible.

Construction and characterization of genomic libraries from specific human chromosomes.

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.

Application of the BrdU/thymidine method to flow cytogenetics: differential quenching/enhancement of Hoechst 33258 fluorescence of late-replicating chromosomes.

Discrimination between many types of isolated mammalian chromosomes can be accomplished by dual-beam flow cytometry following DNA staining with Hoechst 33258 (HO) and Chromomycin A3 (CA3). In this report, we show that the bivariate discrimination of selected late-replicating Chinese hamster M3-1 chromosomes can be improved by appropriate treatment of the cells with 5-bromo-2'-deoxyuridine (BrdU) prior to chromosome isolation and staining. Two labeling schemes are reported. In one scheme the chromosomes are collected from cells labeled with BrdU only during late S phase. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is substantially quenched by the incorporated BrdU, thus improving their discrimination. In the other scheme, chromosomes are collected from cells labeled with thymidine (dT) during late S phase following 20 h of growth in BrdU-containing medium. The Hoechst fluorescence of the 10, 11, M2, and Y chromosomes is quenched less than the other chromosomes, again improving their discrimination. Y chromosomes from chromosome suspensions of untreated controls, of cells labeled with BrdU during late S phase, and of cells labeled with dT during late S phase following 20 h growth in BrdU were separated by dual-parameter sorting. While the purity of the sorted Y chromosome was 15% in untreated controls, it was 70-75% using the BrdU/dT labeling protocols.

Assigning the polymorphic human insulin gene to the short arm of chromosome 11 by chromosome sorting.

We have determined the subchromosomal location of the human insulin gene by analyzing DNA isolated from sorted human metaphase chromosomes. Metaphase chromosome suspensions were sorted into fractions according to relative Hoechst fluorescence intensity by the fluorescence activated chromosome sorter. The chromosomal DNA in each fraction was characterized by restriction endonuclease analysis. Initial sorts indicated that the insulin gene-containing fragment resided in a fraction containing chromosomes 9, 10, 11, and 12. Studies of cell lines that contained chromosome translocations permitted the assignment of the insulin gene to a derivative chromosome that contains portions of the short arm of chromosome II. Simultaneous sorting of the normal homolog from this small derivative chromosome separated the two different sized insulin gene-containing restriction fragments in this individual. These data indicate that the two restriction fragments represent insulin gene polymorphism and not duplicate gene loci.

Immunoelectronmicroscopy of metaphase chromosomes.

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.

A reliable preparation of mono-dispersed chromosome suspensions for flow cytometry

A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4 degrees C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.

Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.

Reutilization of Presumably Degraded Radioactive Label from a Chromosomal Fraction of Homologous and Heterologous Cells

Summary(1) A method for producing a suspension of pure mitotic chromosomes of Chinese hamster cells with insignificant contamination by interphase nuclei or their fragments is described. The yield obtained is high and the chromosome morphology is well preserved. (2) H3 thymidine label from such chromosomes became specifically incorporated into the chromosomes of fresh Chinese hamster cells and of human leucocyte cultures. No gross alteration in the chromosomes of the receptor cells occurred, and uptake of label was inhibited by the presence of added carrier thymidine in the medium, in an amount insufficient to inhibit DNA synthesis. Therefore, most of the incorporation appears to have involved previous degradation of the DNA at least to the level of free nucleotide.The authors gratefully acknowledge the helpful suggestions of Dr. Theodore T. Puck, Dr. Sherman M. Weissman, and Dr. Gordon Allen, as well as the technical assistance of Mrs. Gloria E. Johnson.