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.     

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flow-sorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.     

The chromosomes of Puschkinia libanotica during mitosis

The chromosome complement of Puschkinia libanotica is described. In addition to five pairs of A chromosomes plants may possess up to 7 B chromosomes. Part of the long arm of the B chromosome gives rise to a heterochromatic mass in interphase nuclei and this can be seen to be a double structure in G₁ nuclei and a quadruple structure in G₂ nuclei. It is believed that these configurations represent the pre- and post-replication forms of subchromatids in the heterochromatic segment of the B chromosome. Microdensitometry of metaphase chromosomes shows that the segment of the B chromosome that is heterochromatic during interphase has no more DNA per unit volume than any of the euchromatic A chromosomes.     

Prometaphase banding of human chromosomes with basic fuchsin

Summary A technique is described for the production of detailed and richly contrasting G-band patterns in human prometaphase chromosomes with the aid of the triphenylmethane dye basic fuchsin. The usefulness of this method is illustrated by its application for the precise analysis of two chromosome 11 rearrangements. It is also demonstrated that high-resolution banding with basic fuchsin can reveal bands not present in the international standard idiogram of human prophase chromosomes (ISCN 1981). The technique described can also be used for easy recognition of the late replicating X chromosome, which stains darker than its early replicating homologue. A preliminary analysis of the late replicating X chromosomes in a 49,XXXXY individual suggests that the three supernumerary X chromosomes do not necessarily replicate synchronously.     

On-line sorting of human chromosomes by centromeric index,and identification of sorted populations by GTG-banding and fluorescent in situ hybridization

Summary Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9–12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the offline inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%–99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.     

Quantitative analysis of fluorescence profiles of chromosomes : Influence of DNA base composition on banding

Chromosome banding has been analysed in terms of DNA content and base composition distribution along five human chromosomes. Three intercalative dyes (quinacrine, proflavine and ethidium bromide) whose fluorescence quantum yield in the presence of DNAs of different base compositions has been determined, have been used to examine the influence of base composition on the chromosome patterns. Considering that the amount of DNA as determined by the Feulgen reaction is almost constant along the chromosome arms and assuming that base composition is the only factor influencing the fluorescence of these dyes, a distribution of the A-T base pair content along the chromosomes has been calculated from the fluorescence intensity profiles. From the ratio of the intensity profiles obtained with quinacrine and proflavine, patterns showing the variation of the DNA content and of the A-T base pair content could also be obtained independently. The validity of these different approaches is discussed.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine in relation to Q-banding on CHO chromosomes has been investigated using X-ray microanalysis. Technical problems involved in this type of experiment were studied in detail. It was necessary to use a solution of quinacrine acetate in acetic acid to ensure that the only chlorine detectable in quinacrine-stained chromosomes was in the quinacrine molecule. Electron irradiation during analysis rapidly destroys quinacrine fluorescence, but the chlorine is not lost from the chromosomes, and there are several reasons for supposing that a reliable distribution of quinacrine on the chromosome can be obtained by the method. — Small variations along the chromosome in the amounts of chlorine (representing quinacrine) and of phosphorus (mainly DNA) occur. The distribution patterns for chlorine and phosphorus show a good resemblance to each other for each homologous chromosome; quinacrine fluorescence patterns (Q-bands) do not resemble chlorine distribution patterns, however. The results of this study therefore support the view that Q-bands result from the differential quenching of fluorescence along chromosomes to which the quinacrine is essentially uniformly bound, and do not reflect differential binding of quinacrine along the chromosome.With an Appendix by A. D. Carothers and D. Rutovitz     

Background and peak evaluation of one parameter flow karyotypes on a PC/AT computer

Flow cytometry has become a fast, quantitative method for the classification of metaphase chromosomes in suspension (flow karyotyping) stained with fluorescent dyes. Such a flow karyotype (frequency distribution of the fluorescence signals) consists of several peaks. The peak pattern characterizes the analyzed chromosome complement. In many cases flow karyotypes contain a continuum of an unspecific background deriving from chromosome fragments or chromosome aggregates. For the quantitative evaluation of a flow karyotype this background has to be subtracted by a suitable background function. In this approach the application of chi 2-functions is described. The feasibility of this method to flow karyotypes has been concluded from a computer simulation of chromosome breaking under different conditions. In spite of the rather rough assumptions of the model compared to the complex reasons that influence chromosome breaking, the chi 2-function fits the background better than the exponential function in current use. The approximation of a Gaussian distribution function by the chi 2-function also makes it possible to use the same subtraction procedure for chromosome aggregates. The procedure was tested for isolated chromosomes of Chinese hamster cell lines under different states of breaking. For further evaluation of one parameter flow karyotypes a setup of computer routines has been developed for PC/AT and compatible computer systems. Different peak values of these flow karyotypes can be determined (e.g. peak mean, standard deviation, absolute and relative peak area etc.). The applied method is to fit Gaussian curves to each peak of an experimentally measured histogram by using an interactive program. Fluctuations depending on 'noise' may be suppressed by a 'k-nearest-neighbours' smoothing procedure.     

Inference of genetic architecture from chromosome partitioning analyses is sensitive to genome variation,sample size,heritability and effect size distribution

Genomewide association studies have contributed immensely to our understanding of the genetic basis of complex traits. One major conclusion arising from these studies is that most traits are controlled by many loci of small effect, confirming the infinitesimal model of quantitative genetics. A popular approach to test for polygenic architecture involves so‐called “chromosome partitioning” where phenotypic variance explained by each chromosome is regressed on the size of the chromosome. First developed for humans, this has now been repeatedly used in other species, but there has been no evaluation of the suitability of this method in species that can differ in their genome characteristics such as number and size of chromosomes. Nor has the influence of sample size, heritability of the trait, effect size distribution of loci controlling the trait or the physical distribution of the causal loci in the genome been examined. Using simulated data, we show that these characteristics have major influence on the inferences of the genetic architecture of traits we can infer using chromosome partitioning analyses. In particular, small variation in chromosome size, small sample size, low heritability, a skewed effect size distribution and clustering of loci can lead to a loss of power and consequently altered inference from chromosome partitioning analyses. Future studies employing this approach need to consider and derive an appropriate null model for their study system, taking these parameters into consideration. Our simulation results can provide some guidelines on these matters, but further studies examining a broader parameter space are needed.     

The use of fluorochromes in the cytogenetics of the small-grained cereals (Triticeae)

Summary This paper describes some of the major advances that have been made in the cytogenetics of the small-grained cereals (Triticeae) using fluorochromes to detect nucleic acids in situ. The method, widely known as fluorescence in situ hybridization, has made a contribution in several areas including (i) chromosome mapping programmes, and (ii) cereal breeding programmes. Flow cytometry of cereal chromosomes has now been developed for the generation of chromosome enriched libraries; these libraries will ultimately be of use in both the cereal mapping and breeding programmes. Fluorescence in situ hybridization has also made a major contribution to the understanding of cereal genome structure by elucidating the distribution of different classes of DNA sequence. By using suitable nucleic acid probes whole chromosomes can now be identified in interphase nuclei. The labelling patterns have revealed a structured arrangement of chromosomes at interphase. Not only are chromosomes organized but the ribosomal RNA genes also show structured patterns of condensation and expression. Progress in each of these areas has been rapid in recent years and this progress is described.     

Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss

A colony color assay that measures chromosome stability is described and is used to study several parameters affecting the mitotic maintenance of yeast chromosomes, including ARS function, CEN function, and chromosome size. A cloned ochre-suppressing form of a tRNA gene, SUP11, serves as a marker on natural and in vitro-constructed chromosomes. In diploid strains homozygous for an ochre mutation in ade2, cells carrying no copies of the SUP11 gene are red, those carrying one copy are pink, and those carrying two or more copies are white. Thus, the degree of red sectoring in colonies reflects the frequency of mitotic chromosome loss. The assay also distinguishes between chromosome loss (1:0 segregation) and nondisjunction (2:0 segregation). The most dramatic effect on improving mitotic stability is caused by increasing chromosome size. Circular chromosomes increase in stability through a size range up to approximately 100 kb, but do not continue to be stabilized above this value. However, linear chromosomes continue to increase in mitotic stability throughout the size range tested (up to 137 kb). It is possible that the mitotic stability of linear chromosomes is proportional to chromosome length, up to a plateau value that has not yet been reached in our synthetic constructions.     

Chromosomes in the flow to simplify genome analysis

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.     

Deletion Analysis of the Selfish B Chromosome, Paternal Sex Ratio (Psr), in the Parasitic Wasp Nasonia Vitripennis

Paternal Sex Ratio (PSR) is a ``selfish' B chromosome in the parasitoid wasp Nasonia vitripennis. It is transmitted via sperm, but causes supercondensation and destruction of the paternal chromosomes in early fertilized eggs. Because this wasp has haplodiploid sex determination, the effect of PSR is to convert diploid (female) eggs into haploid (male) eggs that carry PSR. Characterizing its genetic structure is a first step toward understanding mechanisms of PSR action. The chromosome is largely heterochromatic and contains several tandemly repeated DNA sequences that are not present on the autosomes. A deletion analysis of PSR was performed to investigate organization of repeats and location of functional domains causing paternal chromosome destruction. Deletion profiles using probes to PSR-specific repetitive DNA indicate that most repeats are organized in blocks on the chromosome. This study shows that the functional domains of PSR can be deleted, resulting in nonfunctional PSR chromosomes that are transmitted to daughters. A functional domain may be linked with the psr22 repeat, but function may also depend on abundance of PSR-specific repeats on the chromosome. It is hypothesized that the repeats act as a ``sink' for a product required for proper paternal chromosome processing. Almost all deletion chromosomes remained either functional of nonfunctional in subsequent generations following their creation. One chromosome was exceptional in that it reverted from nonfunctionality to functionality in one lineage. Transmission rates of nonfunctional deletion chromosomes were high through haploid males, but low through diploid females.     

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes: I. The test system and the effects of streptonigrin

An appropriate method for evaluating transmissible genetic damage in female germ cells is presented. Analysis of karyotype preparations from preimplantation (6-day) rabbit embryos for consistent structural chromosome abnormalities is described as a sensitive way in which these determinations can be made. A table prepared from summarized research reports shows that less than 1 in 2000 rabbit blastocysts karyotyped thus far has a consistent structural chromosome abnormality. In the new data presented in this study, none of the 90 control blastocysts karyotyped had consistent structural chromosome abnormalities while 13 of 278 from female rabbits treated with streptonigrin (NSC-45383) before conception did. These findings demonstrate that karyotype analysis for consistent chromosome lesions in preimplantation rabbit embryos from females exposed to chemical clastogens can serve as a sensitive and quantitative means of estimating effects on oocytes. The results of the study also show that the consistent structural chromosome lesions in the blastocyst karyotypes are what would be predicted from the mode of action of streptonigrin and the segregation of chromosomes during meiosis. The relative efficiency of this system based on the rabbit as compared to another recently described system based on the hamster is also discussed.     

Visualization of the chromosome scaffold and intermediates of loop domain compaction in extracted mitotic cells

A novel extraction protocol for cells cultured on coverslips is described. Observations of the extraction process in a perfusion chamber reveal that cells of all mitotic stages are not detached from coverslips during extraction, and all stages can be recognized using phase contrast images. We studied the extracted cell morphology and distribution of a major scaffold component - topoisomerase IIalpha, in extracted metaphase and anaphase cells. An extraction using 2M NaCl leads to destruction of chromosomes at the light microscope level. Immunogold studies demonstrate that the only residual structure observed is an axial chromosome scaffold that contains topoisomerase IIalpha. In contrast, mitotic chromosomes are swelled only partially after an extraction using dextran sulphate and heparin, and it appears that this treatment does not lead to total destruction of loop domains. In this case, the chromosome scaffold and numerous structures resembling small rosettes are revealed inside extracted cells. The rosettes observed condense after addition of Mg2+-ions and do not contain topoisomerase IIalpha suggesting that these structures correspond to intermediates of loop domain compaction. We propose a model of chromosome structure in which the loop domains are condensed into highly regular structures with rosette organization.     

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes.

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.     

Standardization of karyotyping plant chromosomes by a newly developed chromosome image analyzing system (CHIAS)

Summary A chromosome image analyzing system (CHIAS) has been developed especially for plant chromosomes. A standard karyotyping method using CHIAS is also described. The characteristics of the CHIAS are as follows: 1) the main objects of the analysis are plant chromosomes, 2) it constructs a man-machine interactive system to put researchers' decisions into the analytical process, 3) it can automate the routine part of an analysis as much as possible, and 4) it digitizes the image information of chromosomes and analyzes them. Software for karyotype analysis of plant chromosomes has been developed. Thus, in the case of rye chromosomes, it is possible to get quantitative data for all chromosomes and a karyogram within 25 min.     

Clustering of Genes Coding for DNA Binding Proteins in a Regionof Atypical Evolution of the Human Genome

Comparison of the human and mouse genomes has revealed that significant variations in evolutionary rates exist among genomic regions and that a large part of this variation is interchromosomal. We confirm in this work, using a large collection of introns, that human chromosome 19 is the one that shows the highest divergence with respect to mouse. To search for other differences among chromosomes, we examine the distribution of gene functions in human and mouse chromosomes using the Gene Ontology definitions. We found by correspondence analysis that among the strongest clusterings of gene functions in human chromosomes is a group of genes coding for DNA binding proteins in chromosome 19. Interestingly, chromosome 19 also has a very high GC content, a feature that has been proposed to promote an opening of the chromatin, thereby facilitating binding of proteins to the DNA helix. In the mouse genome, however, a similar aggregation of genes coding for DNA binding proteins and high GC content cannot be found. This suggests that the distribution of genes coding for DNA binding proteins and the variations of the chromatin accessibility to these proteins are different in the human and mouse genomes. It is likely that the overall high synonymous and intron rates in chromosome 19 are a by-product of the high GC content of this chromosome.Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CSIC, Jordi Girona 18, 08034 Barcelona, Spain