Cytological, flow cytometric, and molecular analysis of the rapid evolution of mammalian chromosomes containing highly amplified DNA sequences

Transfection of a mouse dihydrofolate reductase (DHFR) cDNA contained in a plasmid "expression vector" into DHFR deficient Chinese hamster cells, followed by progressive selection of cells in increasing concentrations of methotrexate (MTX), leads to marked amplification of the exogenous DHFR sequences in the recipient hamster cells. This gene amplification is evident at the cytological level, in the form of homogeneously staining chromosomal regions (HSRs), at a gene expression level, in the form of fluorescein-methotrexate binding, and at the DNA level. Flow sorting, based on variable fluorescein-MTX binding, or direct cellular cloning, followed by chromosome analysis, revealed intercellular heterogeneity of HSRs in size and distribution. This suggested that there was a rapid evolution of HSRs in MTX-resistant transfectants. Chromosomal analysis of HSR evolution in situ, by examining individual colonies presumably derived from one or a few cells, underscored this impression of chromosome structural fluidity. Rates of HSR change in excess of 0.01 per cell division, increased by low doses of the recombinogen, mitomycin C, were detected. The Chinese hamster DHFR transfectants described should be amenable to detailed, coordinate cytological and molecular characterization. Such an analysis should contribute to an understanding of processes such as homologous recombination in mediating HSR evolution in mammalian chromosomes.     

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".     

Cell-by-cell flow cytometric analysis of chromosomes

The authors discuss various aspects of a recently developed method permitting a detailed flow cytometric analysis of the individual cell karyotypes such as instrumentation, histochemistry, data proceeding algorithms. Possible drawbacks of the method and the ways of their overcoming are considered. Results of analysis of the Chinese hamster cells are presented that illustrate the possibilities of the method, including the metaphase chromosome distribution according to their fluorescence intensity, the analysed cell distribution according to their chromosomes number, the table in which the individual cell karyotypes are distributed according to their fluorescence. The results obtained show that the developed method may be successfully used for investigating chromosomal iNstability and heterogeneity of the mammalian cells.     

Preparation and flow cytometric analysis of metaphase chromosomes of tomato

Summary A procedure for the preparation of tomato chromosome suspensions suitable for flow cytometric analysis is described. Rapidly growing cell suspension cultures of Lycopersicon esculentum cv VFNT cherry and L. pennellii LA716 were treated with colchicine to enrich for metaphase chromosomes. Metaphase indices between 20 and 35% were routinely obtained when cultures were exposed to 0.1% colchicine for 15–18 h after 2 days of subculture. Mitotic cells were isolated by brief treatment with cell wall digesting enzymes in a medium with low osmolarity (325 mOsm/kg of H₅₂O). The low osmolarity medium was needed to avoid the chromosome clumping and decondensation seen in standard media. Suspensions of intact chromosomes were prepared by lysing swollen protoplasts in various buffers (MgSO₄, polyamines, hexylene glycol, or KCl-propidium iodide) similar in contents to the buffers used to isolate mammalian chromosomes. For univariate flow cytometric analysis, chromosome suspensions were stained with a fluorescent DNA-binding stain (propidium iodide, Hoechst 33258, mithramycin, or chromomycin A3) and analyzed using an EPICS flow cytometer (Profile Analyzer or 753). Peaks for the chromosomes, chromatids, clumps of chromosomes, nuclei, and fluorescent debris were seen on a histogram of log of fluorescence intensity, and were confirmed by microscopic examination of the objects collected by flow-sorting. Chromosome suspensions prepared in MgSO₄ buffer have the highest frequency of intact chromosomes and the least fluorescent cellular debris. Peaks similar to theoretical univariate flow karyotypes of tomato chromosomes were seen on the observed univariate flow karyotypes, but were not as well resolved. Bivariate flow analysis of tomato chromosome suspension using double-stain combination, Hoechst 33258 and chromomycin A3, and two laser beams showed better resolution of some chromosomes.     

Capture of DNA sequences at double-strand breaks in mammalian chromosomes

To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene. To introduce a genomic DSB, cells were electroporated with a plasmid expressing endonuclease I-SceI, and clones that had lost tk function were selected. Among 253 clones analyzed, 78% displayed small deletions or insertions of several nucleotides at the DSB site. Surprisingly, approximately 8% of recovered mutations involved the capture of one or more DNA fragments. Among 21 clones that had captured DNA, 10 harbored a specific segment of the I-SceI expression plasmid mapping between two replication origins on the plasmid. Four clones had captured a long terminal repeat sequence from an intracisternal A particle (an endogenous retrovirus-like sequence) and one had captured what appears to be a cDNA copy of a moderately repetitive B2 sequence. Additional clones displayed segments of the tk gene and/or microsatellite sequences copied into the DSB. This first systematic study of DNA capture at DSBs in a mammalian genome suggests that DSB repair may play a considerable role in the evolution of eukaryotic genomes.     

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

Satellite DNA sequences flank amplified DHFR domains in marker chromosomes of mouse fibrosarcoma cells

This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10^–3 M MTX-res H2 and the 5×10^–7 M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band ofm as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a³H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.Abbreviations DHFR dihydrofolate reductase - MTX Methotrexate - HSR Homogeneously Staining Region - ASR Abnormally Staining Region - DM Double Minute - ISH In Situ Hybridization - FISH FluorescenceIn Situ Hybridization     

Models of sequence evolution for DNA sequences containing gaps

Most evolutionary tree estimation methods for DNA sequences ignore or inefficiently use the phylogenetic information contained within shared patterns of gaps. This is largely due to the computational difficulties in implementing models for insertions and deletions. A simple way to incorporate this information is to treat a gap as a fifth character (with the four nucleotides being the other four) and to incorporate it within a Markov model of nucleotide substitution. This idea has been dismissed in the past, since it treats a multiple-site insertion or deletion as a sequence of independent events rather than a single event. While this is true, we have found that under many circumstances it is better to incorporate gap information inadequately than to ignore it, at least for topology estimation. We propose an extension to a class of nucleotide substitution models to incorporate the gap character and show that, for data sets (both real and simulated) with short and medium gaps, these models do lead to effective use of the information contained within insertions and deletions. We also implement an ad hoc method in which the likelihood at columns containing multiple-site gaps is downweighted in order to avoid giving them undue influence. The precision of the estimated tree, assessed using Markov chain Monte Carlo techniques to find the posterior distribution over tree space, improves under these five-state models compared with standard methods which effectively ignore gaps.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.     

Flow cytometric analysis of genetic FRET detectors containing variable substrate sequences

A genetic Fluorescence Resonance Energy Transfer (FRET) detector undergoes a post‐translational modification (PTM)‐induced conformational change that results in increased FRET. To test if the PTM‐dependent FRET change can be quantified by flow cytometry, we purified and immobilized a genetic detector on microbeads and used flow cytometry to measure its FRET efficiency before and after Erk‐2–mediated phosphorylation. The fluorescence ratio R between the acceptor and donor fluorescence, which was obtained by fitting a straight line through the data points in linear space, increases following phosphorylation, thus demonstrating that flow cytometry is capable of detecting a PTM‐dependent FRET response. Furthermore, when Erk‐2 and a genetic detector are coexpressed in bacteria, the measured R value changes with the substrate sequence with near single residue resolution. Similarly, the cells coexpressing the glycosylating enzyme O‐GlcNAc transferase (OGT) and a genetic detector specific for OGT exhibit a PTM‐induced change in FRET efficiency. Therefore, the combination of flow cytometry and a genetic detector may be useful to characterize the substrate specificity of a PTM enzyme and identify the sequences that are preferentially targeted for PTM in vivo. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Rapid isolation of metaphase chromosomes containing high molecular weight DNA

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.     

An evaluation of the molecular clock hypothesis using mammalian DNA sequences

A statistical analysis of extensive DNA sequence data from primates, rodents, and artiodactyls clearly indicates that no global molecular clock exists in mammals. Rates of nucleotide substitution in rodents are estimated to be four to eight times higher than those in higher primates and two to four times higher than those in artiodactyls. There is strong evidence for lower substitution rates in apes and humans than in monkeys, supporting the hominoid slowdown hypothesis. There is also evidence for lower rates in humans than in apes, suggesting a further rate slowdown in the human lineage after the separation of humans from apes. By contrast, substitution rates are nearly equal in mouse and rat. These results suggest that differences in generation time or, more precisely, in the number of germline DNA replications per year are the primary cause of rate differences in mammals. Further, these differences are more in line with the neutral mutation hypothesis than if the rates are the same for short- and long-living mammals.     

The distribution of two highly repeated DNA sequences within Drosophila melanogaster chromosomes

In situ hybridization using ³H-RNA probes has been used to localize the sequences found in two satellites of density 1.705 g/cc and 1.672 g/ cc to specific sites within the chromosomal complement. A detailed analysis of the sites on the X chromosome was carried out using the scute series of inversions to relate the heterochromatic breakpoint relative to the location of the sequence on this chromosome. It has also been possible to establish the order of arrangement of 1.705 and 1.672 DNA at the heterochromaticeuchromatic junction on chromosome 3(R). A mitotic map is provided. The T_m of hybrids formed in situ showed that the hybrids were representative of the sequences being analyzed. The two satellites also were traced through a number of purification procedures to show that a covalent linkage may be likely between the 1.705 g/cc and 1.672 g/cc satellite as predicted from in situ hybridization analyses.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Origin and evolution of mammalian sex chromosomes

Mammals present an XX/XY system of chromosomal sex determination, males being the heterogametic sex. Comparative studies of the gene content of sex chromosomes from the major groups of mammals reveal that most Y genes have X-linked homologues and that X and Y share homologous pseudoautosomal regions. These observations, together with the presence of the two homologous regions (pseudoautosomal regions) at the tips of the sex chromosomes, suggest that these chromosomes began as an ordinary pair of homologous autosomes. Birds present a ZW/ZZ system of chromosomal sex determination where females are the heterogametic sex. In this case, avian sex chromosomes are derived from different pairs of autosomes than mammals. The evolutionary pathway from the autosomal homomorphic departure to the present-day heteromorphic sex chromosomes in mammals includes suppression of X-Y recombination, differentiation of the nascent non-recombining regions, and progressive autosomal addition and attrition of the sex chromosomes. Recent results indicate that the event marking the beginning of the differentiation between the extant X and Y chromosomes occurred about 300 million years ago.     

Two-parameter data acquisition system for rapid slit-scan analysis of mammalian chromosomes

A data acquisition system is described for recording two independent signals simultaneously from a laser-based flow cytometer for rapid slit-scan chromosome analysis. High-aperture microscope optics allow recording of fluorescence distributions along the longest axis of metaphase chromosomes with a spatial resolution better than 1 micron. Fluorescence and small angle forward light scatter as well as dual-wavelength fluorescence signals from Indian muntjac chromosomes stained with propidium iodide (PI) or acridine orange (AO) have been recorded simultaneously. While maintaining the multi-user operation of the computer, photomultiplier signals are digitized at a rate of 400 signals per second, stored temporarily in high-speed cache memories, and transferred subsequently to a minicomputer for further storage. Extensive software packages for data acquisition, analysis, and display of the results are described. Data acquisition is generally done in list mode, allowing complete reconstruction of individual signals (profiles) at any time. The distribution of stained constituents along the chromosomes can be displayed. Furthermore, histograms of various parameters of the input signals may be generated.     

Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA-specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air-spaced achromat for the standard focusing lens. Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies. The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.     

DNA mapping of gastric cancers using flow cytometric analysis

Although numerous studies of gastric cancers on DNA ploidy have been reported, differences in the degree of aneuploidy (DNA index, DI) during progression have not been identified. We attempted to chart the differences in DIs during progression to clarify the role of aneuploidy in gastric cancers. We classified the gastric cancers examined into intestinal (n = 88) and diffuse (n = 48) types, and then analyzed 136 gastric cancers (intramucosal cancer, 42; submucosal cancer, 39; advanced cancer, 55) by flow cytometry using multiple sampling. In addition, we examined the DNA ploidy pattern of mucosal and submucosal lesions using the same submucosal cancers to study the tumor progression in individual cancers. Intratumoral DNA differences in DNA ploidy were observed in both types of gastric cancers. In intestinal-type cancers, multiple subclones indicated by a different DI occurred during the early stage of gastric cancers, whereas in diffuse-type cancers, multiple subclones were found primarily in advanced cancers. Although the DI varied widely in early intestinal-type cancers between 1.0 and 2.0, in early diffuse-type cancers, the DI tended to be less than 1.2. However, in advanced stage gastric cancers, the DI distribution was similar for both histological types. In intestinal-type cancers, high DI (>1.3) aneuploidy was frequently found in mucosal lesions. In contrast, only low DI (<1.2) aneuploid clones were observed in mucosal lesions of diffuse-type cancers. The present results suggest that high DI aneuploid tumor clones in intramucosal cancers acquire invasive ability when they progress to submucosal cancers, whereas DNA aneuploidy itself plays an important role in submucosal invasion of diffuse-type cancers.