Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance

Results of karyological analysis of cells CHL V-79 RJK selected for resistance to ethidium bromide (EB) causing multidrug resistance (MDR) (line Vebr-5) were compared with the data of microfluorimetric determination of DNA content in individual chromosomes of the karyotype. The analysis was performed at the 11th and 88th passages. Karyotyping of Vebr-5 has shown the presence of an additional genetic material (ADM) in the form of homogenously or differentially stained regions (HSRs and DSRs, respectively) in two chromosomes (Z1 and Z6, loci 1 p29-31 and 1q26, respectively). HSRs in Z6, in the region of localization of the wild type of gene mdr, had unstable length and structure characteristic of morphological markers of amplification of genes of the family mdr. During long cultivation of Vebr-5 in the presence of EB (88 passages), the instability of HSRs in Z6 increased. Results of microfluorimetric analysis of Vebr-5 at the 11th passage have shown an increase in the DNA content not only in chromosomes Z1 and Z6 marked by HSRs, but also in three chromosomes (Z5, Z12 and Z13) that have no visual morphological changes. The corresponding analysis at the 88th passage has also revealed non-random changes in the DNA content in four more chromosomes: an increase in Z14, while a decrease in chromosomes 8, Z7, and Z9. A decrease of the DNA content in chromosomes is considered to be a result of a partial loss of genetic material, while its increase is a result of its translocation and (or) amplification. Coefficient of variation of the DNA content changes for large chromosomes amounted to about 9%. while for small chromosomes it is about 26%, which indicates that small chromosomes have greater potential for instability than the large ones. The data obtained not only confirm, but also enlarge the concept of directions and character of destabilization of the cell genetic apparatus in the process of neoplastic transformation due to the MDR acquisition by cells.     

Chromosomal polymorphism of mammalian cells resistant to drugs in multiple passages. I. Karyotype analysis of Chinese hamster cells resistant to ethidium bromide in the early passages of the first steps of selection

G-banded metaphase chromosomes of Chinese hamster V-79 RJK cells resistant to ethidium bromide (2.5 and 10 mcg/ml) have been analysed. The cells of the first selection step (clone IVerb-2, the 9th passage) revealed definite karyotypical instabilities. Amplifications or, in rare cases, deletions were found in chromosomes Z1 and Z6 which appear to have derived from chromosome 1. The amplified region in chromosome Z6 varied considerably in morphology. The chromosome instability, detected in Z1 and Z6, was reproducible in cells throughout the eight independent clones isolated from clone IVebr-2 under non-selective conditions. The data obtained allow to suggest a genetically conditioned mechanism of the above chromosome instability. In the population of resistant cells on the second step of selection (clone I Vebr-5, the 9th passage) the frequency of the cells with amplification increased up to 100%. The length of amplifications increased in the majority of cells. In the cells of the third step of selection (clone IVerb-10, the 12th passage) with near-tetraploid chromosome composition, besides amplifications some specific rearrangements of chromosomes 2 and 7 (markers Z16, Z17) were revealed. The above rearrangements are indicative of the karyotypical destabilization in the drug resistant cells, and may be evaluated as secondary phenomena casually connected with amplifications found at the earlier steps of selection.     

DNA image-fluorimetry of individual human chromosomes

A microfluorimetric method has been developed for determination of DNA content in individual human chromosomes. The method is based on a preliminary identification of chromosomes with Hoechst 33258 followed by staining of the chromosomes with Feulgen reaction by using Schiff’s reagent type ethidium bromide-SO₂ and then by measuring the fluorescence intensity of the chromosomes by using an image analyzer. The method allows determining the DNA content of individual chromosomes with an accuracy up to 4.5 fg. The DNA content of individual human chromosomes and their p-and q-arms, as well as homologous chromosomes, were measured by using the developed method. It has been shown that the DNA content in chromosomes of the normal human karyotype is unstable and can fluctuate in some chromosomes within 35–40 fg.     

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.     

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010)     

人鼻咽癌克隆株体外传代过程的群体变化

将人低分化鼻咽癌克隆株CNE-2 Z-5-2-B_7在体外连续传代培养,从群体的角度观察了不同代数的细胞形态定量、DNA含量和体外增殖能力的变化。结果:(1)各细胞形态参数、DNA含量出现异质性,为多种瘤细胞亚群所构成,而且随传代过程变化、消长。(2)41代以细胞面积大、核大、核浆比小、DNA含量高、异质性明显的大细胞群体占优势。其形态特征与鼻咽低分化鳞癌的大核型相似。(3)81代主要为胞、核面积小、核浆比大、DNA含量高的小细胞群体,其体外增殖能力较第1代明显为高,形态及生长特征与鼻咽未分化癌相似。提示随着肿瘤的演进,如不给予影响(治疗),瘤细胞有恶性发展的内在倾向。     

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Increased DNA content of HSR-marker chromosomes of human neuroblastoma cells

Scanning cytophotometry demonstrates that the DNA content of human neuroblastoma chromosomes containing a specific abnormal segment—the Homogeneously Staining Region (HSR)—is significantly greater than that of homologous normal chromosomes without this segment. The potential significance of this finding and its relationship to another chromosome abnormality, the double minutes, is discussed.     

Stable alterations in HeLa cell mitochondria following ethidium bromide treatment

This study describes the isolation of three HeLa cell clones after exposure of HeLa cells to ethidium bromide (EB) in culture medium for either 14 days, or 14 days plusreexposure for 30 days. All three EB-induced clones differed from the parental HeLa cell in various physical properties of their mitochondria. The ratio of mitochondrial DNA component I to component II was altered in clone HeLa-2A. In addition, the cytochrome content of the respiratory chain a + a₃, b and C₁ decreased, while the cytochroms c content remained unchanged. The amount of cytochromes b and c₁; which were not reduced by KCN treatment, but were reduced by dithionite, increased in clone HeLa-2A. The ultrastructure of HeLa-2A cells revealed several alterations characteristic of EB treatment. Some mitochondria had enlarged profiles, a reduced number of cristae and a more lucent electron density of the matrix. Other mitochondria were tightly packed with cristae, which occasionally showed a whorled configuration. These changes were observed 4 months (20–25 passages) after the omission of EB from the medium.     

Effect of zearalenone and estradiol benzoate on serum concentrations of LH, FSH and prolactin in ovariectomized gilts

Six ovariectomized gilts were given zearalenone (Z), estradiol benzoate (EB) or vehicle in a replicated 3 x 3 Latin square design. Zearalenone was added to 2.3 kg of a corn-soybean ration at a dose of 1 mg Z/kg body weight; EB was given intramuscularly at 0.1 mg EB/kg body weight. Control gilts received vehicle solvent for both Z and EB. Blood samples were collected from indwelling jugular cannulas at 6-h intervals for 48 h before Z, EB or vehicle was given. After treatment, blood samples were drawn at 6-h intervals for an additional 84 h. Serum concentrations of luteinizing hormone (LH) decreased (P<0.001) from 4.67 ng/ml to 0.29 ng/ml within 6 h of EB. From 54 to 84 h after EB, serum concentrations of LH rose to 15.60 ng/ml (P<0.001). Serum concentrations of LH were reduced (P<0.001) in a similar pattern after Z (3.70 ng/ml to 0.49 ng/ml), but a rise in serum LH was not observed 54 to 84 h after Z (1.30 ng/ml). Serum concentrations of LH remained unchanged (P=0.55) in gilts given vehicle. Serum concentrations of follicle stimulating hormone (FSH) were suppressed (P<0.03) at 6 h in EB (19.10 vs 11.35 ng/ml) and Z gilts (16.16 vs 11.41 ng/ml) but remained unchanged in vehicle gilts. Serum concentrations of FSH did not change in EB or Z gilts during the next 36 h. These data indicate that the suppressive action of Z on serum concentrations of LH and FSH was similar to that of EB, while the biphasic stimulatory effect of EB for LH was not manifested by Z.     

DNS-Replikationsmuster der Speicheldrüsen-Chromosomen von Chironomiden

The pattern of DNA-synthesis of the salivary gland chromosomes of Chironomus thummi thummi, Ch. th. piger, Ch. annularius, Ch. plumosus and Ch. melanotus was studied using H³-thymidine-autoradiography. Contrary to the previous conception the bands of the salivary gland chromosomes of Chironomus do not begin replication simultaneously. H³-thymidine incorporation in bands of high DNA content begins later than in bands with a lesser amount of DNA. This difference in time is very small in bands outside the kinetochore regions and not comparable to the asynchrony in replication of typical heterochromatin in the salivary gland chromosomes of Chironomus melanotus. Differences in the amount of DNA in homologous bands do not affect the onset of replication. — Bands of high DNA content are replicating during a longer time than those having less DNA. However, certain chromosome regions behave differently. In these regions bands of very low DNA content are synthesizing DNA during the whole replication cycle. Since no excessive increase of DNA could be observed in these regions it is supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which disappears immediately from the chromosome. — At the end of the replication cycle in the salivary gland nuclei of the hybrid Chironomus th. thummi X Ch. th. piger a labeling pattern is found in the chromosomes of Ch. th. thummi which differs from that in the parental subspecies Ch. th. thummi.     

FISH studies on the telomeric regions of the T-cell acute lymphoblastic leukemia cell line CCRF-CEM

So far, the problem of an influence of translocations on the telomeres of the involved chromosomes has not been addressed yet in human cells. Therefore, the telomeres of a karyotypically rather well characterized T-cell acute lymphoblastic leukemia (T-ALL) cell line (CCRF-CEM) with several marker chromosomes were examined using peptide nucleic acid (PNA) telomere FISH probes to compare the telomere length of these markers with that of the chromosome arms of their origin. In addition, chromosome libraries, centromeric probes, and subtelomeric DNA probes were used to further define the marker chromosomes. Two markers could be newly defined and a concise karyotype of the cell line could be obtained by these detailed examinations: 42-47,X,-X,del(5) (q35?),t(5;15)(q14;q13.2),t(8;9)(p11;p24),del(9)(:p13-->qter)/inv(9)(pter-->p12::q21-->p12::q21-->qter),+13,+20,+der(22)(p+ [HSR?])[cp]. The relative telomere length of all chromosomes showed considerable interchromosomal, intercellular, and inter-passage variation. However, it could be shown, that in four different passages of the examined cell line the observed differences between relative telomere lengths of the markers and the chromosomes of their origin, with two exceptions (short arms of del/inv9 and der22), were not significant. On the other hand, because of its mentioned variability, telomere length alone is not sufficient to reliably define the derivation of markers.     

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     

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis,Lacertidae) and elucidation of karyotype evolution in lacertid lizards

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n?=?38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.     

Rare Circulating Cells in Familial Waldenstr?m Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization

The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10⁻⁷). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.     

Molecular characterisation of the species of the genus Zygosaccharomyces

The restriction fragments polymorphisms of the mitochondrial DNA and the PCR fragment that comprised the internal transcribes spacers and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 40 strains from the 10 species of the genus Zygosaccharomyces, including the new species Z. lentus were examined. The RFLP's of the ITS-5.8S region showed a specific restriction pattern for each species, including the new species Z. lentus. The only exception were the species Z. cidri and Z. fermentati that produced identical restriction profiles. The electrophoretic chromosome patterns confirmed the differences between the species of this genus, including the phylogenetic closest species Z. cidri and Z. fermentati. They present few chromosomes ranging from 3 bands (4 or 5 chromosomes) for Z. florentinus to 7 bands (8 to 10 chromosomes) for Z. cidri and Z. fermentati. The strain level resolution power of RFLP's of mtDNA of this genus enabled the characterisation of strains from the same species, even where they are isolated from the same substrate. However, in the cases of Z. bailii and Z. lentus, electrophoretic karyotyping there was considerable variation.     

Cytogenetic changes in an explanted mouse rhabdomyosarcoma during prolonged cultivation

Quantitative and qualitative chromosome rearrangements, dynamics of distribution of double-minute chromosomes (DMs), and morphological characteristics of tumor rhabdomyoblasts MH-82 during explantation and following in vitro cultivation are analysed. Cells of the 13th and 27th passages of cultivation were characterized by the epithelial type of growth, although their form and size varied. Chromosome analysis of tumor rhabdomyoblasts was carried out on passages 4, 14, 20, 25 and 30 of in vitro cultivation. The modal class with 53-55 chromosomes was established within 20 passages. Heterogeneity of cell population in concern to the chromosome number and content of hypotetraploid cells (72-78) diminished during cultivation. Chromosome rearrangements (marker chromosomes) in hyperdiploid and hypotetraploid cell subpopulations differed. The number of cells with DMs and the number of DMs per cell decreased till the full disappearance by the 30th passage. It is concluded that the establishment of the MH-82 cell line was completed up to the 30th passage of cultivation.     

Pleiotropic changes in karyotype structure of Chinese hamster fibroblast cell lint CHL V-79 RJK in relation to their acquisition of resistance to etoposide, an inducer of apoptosis]

Chinese hamster fibroblasts CHL V-79 RJK were subjected to multistep selection in the presence of etoposide, known as an inhibitor of topoisomerase II and inductor of apoptosis. The karyotype of cells stably resistant to etoposide was analysed at progressive stages of selection using G-type staining of metaphase chromosomes. Multiple changes in the karyotype of resistant cells were observed at an early stage of selection (0.2 mg/ml of etoposide) and included: random chromosome breaks leading to formation of new chromosome markers, high frequency of aneuploid and polyploid cells, morphological instability and extracopy of q-shoulder of chromosome 1. On advanced stages of selection we observed an increased frequency of specific minute chromosome and the appearance of chromosomes with homogeneously or differentially stained regions (HSR and DSR). These data suggest that different mechanisms may be involved in developing cellular resistance to etoposide at progressive stages of selection.     

The volumes and morphology of human chromosomes in mitotic reconstructions

Summary Ten unpretreated normal human male fibroblast cells in mitosis were completely reconstructed from micrographs of between 82 and 119 consecutive serial sections. All 46 chromosomes and their centromeres could be reconstructed in every cell. Measurements of chromosome volumes and centromere indices are presented. The data enabled allocation of all chromosomes to their groups (A to G), and chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and Y were individually identified. Comparisons with published karyotypes showed that volume measurements correlated well with measurements of DNA content and chromosome length. Centromere indices also showed good correlation, but the acrocentric chromosomes were more unequally armed than found by length measurement. Secondary constrictions at the nucleolar organising region were visible on about a third of the acrocentric chromosomes. One chromosome of the C group, number 9, had a diffuse subcentromeric region (DSR) on the long arm, at the position of the constitutive heterochromatin and (in meiotic cells) the paramere.