Slit-scan flow cytometry of mammalian chromosomes.

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.     

Determination of the DNA content of human chromosomes by flow cytometry

The mean relative DNA content of each human chromosome was calculated from flow karyotypes of ethidium bromide-stained chromosomes obtained from healthy, normal individuals. These values were found to correlate closely with previously published data obtained by photometric scanning of stained, fixed chromosomes. Calculations of the normal variation in DNA content of each human chromosome indicated that chromosomes 1, 9, 16, and Y (chromosomes with large centric heterochromatic regions) were the most variable, followed by the acrocentrics, 13, 14, 15, 21, and 22. Chromosomes 2, 3, 18, and 19 were also found to vary significantly in DNA content. Chromosomes from a number of subjects with extreme heteromorphisms were flow karyotyped to obtain an estimate of the extent of variation in DNA content of each chromosome. The greatest difference between extreme variants was found for chromosome 1 (which differed by 0.82% of the total genomic DNA), followed by 16 and 9. The largest Y-chromosome variant was 85.9% bigger than the smallest. The precise karyotype analysis produced by flow cytometry resolved many differences between chromosome homologs, including some that cannot be readily distinguished cytogenetically. The implications of these findings for detection of chromosome abnormalities by flow karyotype analysis are discussed.     

Chromosome heteromorphisms in the gorilla karyotype. Analyses with distamycin A/DAPI, quinacrine and 5-azacytidine

The chromosomes of one male and three female gorillas were extensively studied with various regional banding methods. The chromosomes were stained with the fluorescent dyes quinacrine mustard and distamycin A/DAPI (DA/DAPI), which label different subsets of heterochromatin in the chromosome complement. Furthermore, lymphocyte cultures were treated with the cytidine analog 5-azacytidine (5-azaC). The 5-azaC-induced undercondensations were found in most of the DA/DAPI-bands as well as in many telomeric C-bands. The karyotype of the gorilla exhibits a considerable number of heterochromatin variants. Of the different types of heteromorphisms noted, the most striking is that involving the short arm regions of chromosomes 12 to 16 and 23 (satellite stalk regions) and the paracentromeric heterochromatin of chromosomes 17 and 18. There also are numerous heteromorphic C-bands localized in the telomeric regions of homologous chromosome arms. In comparison, only few heteromorphisms occur between C-bands in the centromeric and pericentromeric regions of homologs. Finally, a variability in the fluorescence intensity of quinacrine-bright satellites in the short arms of chromosomes 12 to 16, 22, and 23 is observed.     

Co-occurrences of polymorphic heterochromatin regions of chromosomes and effect on reproductive failure

Although the polymorphic heterochromatin regions of chromosomes (heteromorphisms) have been extensively studied for their phenotypic effects on humans, co-occurrences of chromosome 1, 9, 16 and Y heteromorphisms and of acrocentric variants have never been studied on humans with an objective scoring system. Here we compared the frequencies of individual heteromorphisms on a total of 602, 768 and 224 patients with the indications of infertility, recurrent miscarriage and in vitro fertilization (IVF) failure, respectively and on 272 controls. Then we examined whether there were significant co-occurrences between heteromorphisms within and between the groups. There were no statistically significant differences in the frequencies of heteromorphisms between the groups. Both statistically significant and non-significant correlations were observed within the non-acrocentric and certain acrocentric heteromorphisms in each group. When these co-occurrences were examined between the groups, a 2.2 fold increased risk of IVF failure in males in the presence of either chromosome 13 or chromosome 21 variants was observed (95 %CI:1.1–4.2). We conclude that the simultaneous manifestations of heteromorphisms have no effect on reproductive failure. There seems to be a correlation between the non-acrocentric heteromorphisms (1qh+, 9qh+, 16qh + and Yqh+/-), which might be the result of complex interactions of formation of these heterochromatin regions. The correlations observed between certain acrocentric chromosomes might be related to satellite association and nucleolus formation. The increased risk observed in males with IVF failure in the presence of either chromosome 13 or 21 variants should be interpreted cautiously due to the heterogeneity of the group.     

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

Summary The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.     

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.     

Human chromosome variation: the discriminatory power of Q-band heteromorphism (variant) analysis in distinguishing between individuals, with specific application to cases of questionable paternity.

The chromosomes from 57 persons were analyzed by means of quinacrine fluorescent staining in order to assess the amount of variation and the discriminatory power of Q-band heteromorphism analysis. Chromosomes 3, 4, 13, 14, 15, 21, 22, and Y of each person were visually compared to those of 56 others, for a total of 1,596 comparisons. No two persons were found to have the same set of variants. The number of differences between chromosomes for each comparison ranged from 2 to 12 out of a possible total of 14 for females and 15 for males. Relatives were also distinguishable, and differences ranged from two to seven. We used the frequency with which each chromosome was useful for telling two people apart, and estimated the probability of finding two persons with the same set of quinacrine variants as .0003. Distinctly different heteromorphisms were found in the 39 unrelated persons for each of the chromosomes examined. In this small population, the number of different sets of variants observed for chromosomes 3, 4, 13, 14, 15, 21, 22, and Y were six, seven, 27, 16, 20, 15, 24, and five, respectively, for a total number of possible combinations of 1.14 X 10(15). As a test of the usefulness of chromosome heteromorphisms in paternity cases, 12 father-mother-child trios of virtually certain paternity, owing to the father-child segregation of a rare structural rearrangement, were coded and recombined at random to produce 120 cases of uncertain paternity. When the code was broken, 108 "alleged fathers" had been excluded correctly and the 12 biological fathers had been included correctly.     

Uneven extraction of protein in Chinese hamster chromosomes during G-staining procedures

To elucidate the role of chromosomal protein in G-band production, changes of protein distribution in chromosomes were studied in situ at each step of G-staining procedures. As a highly specific stain for protein, dansyl Cl was used, which conjugated with amino groups in polypeptide to emit bright fluorescence under UV irradiation, so that the pattern of fluorescence of dansyl-stained chromosomes was expected to reflect the distribution of protein. Uniform fluorescence pattern observed in untreated, dansyl-stained chromosomes indicated even distribution of protein in the ordinary air-dried chromosomes. The pattern of fluorescence representing the distribution of chromosomal protein after pretreatments of G-staining showed brighter outlines of chromatids, reduced fluorescence of chromosome body, and a slight difference in intensity along chromosome arms which corresponded to G-bands. This correspondence was confirmed when Giemsa stain was removed from G-banded chromosomes and the chromosomes were stained with dansyl Cl. The resulting dim fluorescence pattern conformed to G-bands previously observed in the same chromosomes. Similar events were observed in HCl-extracted chromosome slides, although the fluorescence was considerably reduced in this case. Our results inferred that chromosomal protein was partially lost during pretreatments of G-staining, that acid-soluble protein assumed less significant role in G-staining mechanism, and that uneven deprivation of acid-insoluble protein may occur during G-staining procedures.     

Centromeric index measurement by slit-scan flow cytometry

We report here the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.     

The karyotype of the mouse

A denaturating and renaturating technique, applied to mouse chromosomes, makes visible characteristic banding patterns by which all elements of the karyotype can be individually distinguished. The Y chromosome as a whole appears darkly stained. The X chromosome comprises 6.33% of the homogametic haploid set. The banding pattern of the chromosomes is compared with that obtained by aid of the quinacrine dihydrochloride fluorescence technique. After its use a banding pattern results which is similar to, but less distinct than, that found after the renaturation procedure.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes.

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

Optimising human chromosome separation for the production of chromosome-specific DNA libraries by flow sorting

Summary A number of cell lines, some containing chromosomes with distinctive heteromorphisms, have been flow karyotyped using a single laser flow sorter in an attempt to select those suitable for sorting all human chromosomes individually. Using the non-base-specific DNA stain ethidium bromide, chromosomes 3,4,5, and 6 form individual peaks in practically all normal subjects, while the right combination of heteromorphisms enables chromosomes 1, 2, 8, 9, 13, 16, 17, 18, 19, 20, 21, 22, and Y to be sorted separately. Two male cell lines, one containing a duplication and one a deletion of the X, produce flow karyotypes suitable for sorting chromosomes 7 and 8. The use of numerical chromosome abnormalities to enrich the sex chromosomes and the autosomes 18 and 21 is also illustrated. The DNA stain Hoechst 33258 binds preferentially to AT base pairs. Flow karyotypes produced with this fluorochrome separate some chromosomes not well separated with ethidium bromide. Chromosomes 5, 6, 8, 13, 14, 15, 17, and 20, and Y can be sorted individually with Hoechst 33258 with the right combination of heteromorphisms. Using these techniques, all human chromosomes apart from 10, 11, and 12 have been found as individual flow karyotype peaks, suitable for sorting with a high degree of purity.     

Anatomy of human genome by restriction endonucleases Alul, Ddel, Haelll, Hinfl, Mobol and Rsal, and their application in clinical cytogenetics

The application of restriction endonucleases Alul, Ddel, Haelll, Hinfl, Mbol and Rsal in clinical cytogenetics is described. The extensive inherent heterogeneity of heterochromatin in the C-band regions revealed by these enzymes provides a valuable tool for describing the origin of trisomies and abnormal chromosomes in humans. The heteromorphic markers identified by these enzymes have tremendous potential in clinical cytogenetics. Unlike the CBG technique, slides can be stained immediately after preparation providing a rapid diagnosis. Characteristic bands induced by each enzyme in the human complement are discussed in detail. Comparative analysis of the present data, with those described earlier, revealed certain discrepancies including chromosomes 19 and 20 by Alul, chromosomes 4, 5, 8 and 22 by Mbol and chromosomes 12 and 20 by Rasl. These controversies are examined in the light of heteromorphisms, technical variables and chromosome identification.     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

An unstable giant satellite associated with chromosomes 21 and 22 in the same individual.

The short arms of the acrocentric chromosomes are among the most common sites in which to find human chromosomal heteromorphisms. Heteromorphic chromosomes are noted for their variability between individuals and populations; however, they generally are consistent within an individual. Contrary to this general rule, a normal female was found to have a giant satellite on the short arm of a chromosome 22 in most lymphocytes and fibroblasts, but in other cells, it was attached to a chromosome 21. Furthermore, in some cells, it was found on multiple chromosomes, that is, on both 22's or on a 21 and a 22. The familial nature of this heteromorphism was established when it was found in the woman's mother, where it was confined exclusively to chromosome 22. These results suggest an unstable giant satellite associated with both G-group chromosomes of a normal individual. Results are discussed in the light of the patient's occupational exposure to insecticides at a mushroom farm.     

Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization

A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique sequence of signals, a result termed the “chromosome bar code”. The reproducibility of the hybridization pattern in various labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques. Received: 18 December 1996 / Accepted: 10 February 1997     

Binding of quinacrine to the human Y chromosome

Human chromosome spreads were stained with 3H-quinacrine and their fluorescence observed. The exact location of specific spreads on each slide was noted and photographs taken. Autoradiographs were then prepared so that the quinacrine fluorescence of any specific chromosome could be compared directly with the distribution of grains over the same chromosome on the autoradiograph. The Y chromosome fluoresced much more intensely than any of the other chromosomes, but there were no more grains over the Y chromosome than over the other chromosomes. Therefore the enhanced fluorescence of the human Y chromosome is not due to an increased binding of quinacrine.     

Heterochromatin recognition and analysis of chromosome variation in Scilla sibirica

The heteroohromatin of Scilla sibirica, consists of two distinct types: 1) showing enhanced Quinacrine fluorescence and located near the centromere of all the chromosomes of the complement, and 2) with reduced Quinacrine fluorescence and located in various positions along the chromosomes. After a denaturation-reannealing treatment both heterochromatin types are stained by Giemsa, and by acetic-orcein. Acetic-orcein, however, tends to stain preferentially the reduced fluorescence segments. An analysis of chromosome variation in a population of twenty plants, reveals that all the plants are unique in their heterochromatic segment endowment. All the chromosomes are polymorphic but there is a certain constancy for band patterns in individual chromosome types, and for the number of bands per chromosome complement.