High FST Research Articles

The evolution and population genetics of the ALDH2 locus: random genetic drift, selection, and low levels of recombination.

The catalytic deficiency of human aldehyde dehydrogenase 2 (ALDH2) is caused by a nucleotide substitution (G1510A; Glu487Lys) in exon 12 of the ALDH2 locus. This SNP, and four non-coding SNPs, including one in the promoter, span 40 kb of ALDH2; these and one downstream STRP have been tested in 37 worldwide populations. Only four major SNP-defined haplotypes account for almost all chromosomes in all populations. A fifth haplotype harbours the functional variant and is only found in East Asians. Though the SNPs showed virtually no historic recombination, LD values are quite variable because of varying haplotype frequencies, demonstrating that LD is a statistical abstraction and not a fundamental aspect of the genome, and is not a function solely of recombination. Among populations, different sets of tagging SNPs, sometimes not overlapping, can be required to identify the common haplotypes. Thus, solely because haplotype frequencies vary, there is no common minimum set of tagging SNPs globally applicable. The Fst values of the promoter region SNP and the functional SNP were about two S.D. above the mean for a reference distribution of 117 autosomal biallelic markers. These high Fst values may indicate selection has operated at these or very tightly linked sites.

Allele frequency-based analyses robustly map sequence sites under balancing selection in a malaria vaccine candidate antigen.

The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading candidate for a malaria vaccine. Here, within-population analyses of alleles from 50 Thai P. falciparum isolates yield significant evidence for balancing selection on polymorphisms within the disulfide-bonded domains I and III of the surface accessible ectodomain of AMA1, a result very similar to that seen previously in a Nigerian population. Studying the frequency of nucleotide polymorphisms in both populations shows that the between-population component of variance (F(ST)) is significantly lower in domains I and III compared to the intervening domain II and compared to 11 unlinked microsatellite loci. A nucleotide site-by-site analysis shows that sites with exceptionally high or low F(ST) values cluster significantly into serial runs, with four runs of low values in domain I and one in domain III. These runs may map the sequences that are consistently under the strongest balancing selection from naturally acquired immune responses.

Local differentiation in Plasmodium falciparum drug resistance genes in Sudan.

Studies of population genetic structure of parasites can be used to infer which parasite genes are under selection. Here, the population structure of 4 genes associated with drug resistance of Plasmodium falciparum (the chloroquine resistance transporter, pfcrt, dihydrofolate reductase, dhfr, dihydropteroate synthase, dhps, and multi-drug resistance, pfmdr-1) were examined in parasite populations in 3 villages in eastern Sudan and in an urban area of Khartoum, the capital. In order to differentiate the effects of drug selection from neutral influences on population structure, parasites were also genotyped for 3 putatively neutral microsatellite loci (polyalpha, TA81 and pfg377), and for 2 antigenic loci that are either under balancing selection or neutral, merozoite surface protein 1 and 2, (MSP-1 and MSP-2). Cross-sectional surveys were carried out during the peak transmission (wet) season and in the ensuing dry season. No significant variation in frequencies of MSP-1 and MSP-2 alleles was seen among villages in the eastern region and between the villages and Khartoum, nor between the wet and dry season. However, the drug resistance genes, pfmdr-1, pfcrt and dhfr and to a lesser extent the microsatellite loci showed high FST values when comparing villages with Khartoum, indicating strong geographical differentiation at these loci. Moreover, variation in frequencies of the drug resistance genes, pfmdr-1, pfcrt and dhfr, was observed between the wet and dry season. These differences most probably reflect the variation in drug pressure between each region, and in drug usage between the wet and dry season in a given region.

Genetic structure of Aedes aegypti populations in Chiang Mai (Thailand) and relation with dengue transmission.

We analysed the population genetic structure and differentiation regarding vector competence for a dengue virus of 15 Aedes aegypti samples collected from Chiang Mai in northern Thailand. Based on polymorphism of 10 isoenzyme loci, genetic differentiation was confirmed among samples collected in different subdistricts (high FST values and P < 0.05). Based on infection rate for a dengue 2 virus, susceptibilities were similar in mosquitoes collected in San Nuea subdistrict and in Choeng Doi subdistrict, and were heterogeneous in populations sampled in other subdistricts. These findings are discussed and related to insecticide treatments.

A Global Perspective on Genetic Variation at the ADH Genes Reveals Unusual Patterns of Linkage Disequilibrium and Diversity

Variants of different Class I alcohol dehydrogenase (ADH) genes have been shown to be associated with an effect that is protective against alcoholism. Previous work from our laboratory has shown that the two sites showing the association are in linkage disequilibrium and has identified the ADH1B Arg47His site as causative, with the ADH1C Ile349Val site showing association only because of the disequilibrium. Here, we describe an initial study of the nature of linkage disequilibrium and genetic variation, in population samples from different regions of the world, in a larger segment of the ADH cluster (including the three Class I ADH genes and ADH7). Linkage disequilibrium across approximately 40 kb of the Class I ADH cluster is moderate to strong in all population samples that we studied. We observed nominally significant pairwise linkage disequilibrium, in some populations, between the ADH7 site and some Class I ADH sites, at moderate values and at a molecular distance as great as 100 kb. Our data indicate (1) that most ADH-alcoholism association studies have failed to consider many sites in the ADH cluster that may harbor etiologically significant alleles and (2) that the relevance of the various ADH sites will be population dependent. Some individual sites in the Class I ADH cluster show Fst values that are among the highest seen among several dozen unlinked sites that were studied in the same subset of populations. The high Fst values can be attributed to the discrepant frequencies of specific alleles in eastern Asia relative to those in other regions of the world. These alleles are part of a single haplotype that exists at high (>65%) frequency only in the eastern-Asian samples. It seems unlikely that this haplotype, which is rare or unobserved in other populations, reached such high frequency because of random genetic drift alone.

Polymorphic microsatellites in the African freshwater snail, Bulinus forskalii (Gastropoda, Pulmonata)

AbstractEleven microsatellites were isolated in the freshwater snail Bulinus forskalii, intermediate host for the medically important trematode Schistosoma intercalatum. Characterization in 60 snails from three populations of B. forskalii from Cameroon revealed 4 to 18 alleles per locus. Low observed heterozygosity but higher expected heterozygosity, high FIS estimates, significant departures from Hardy–Weinberg equilibrium and genotypic linkage disequilibria all indicate that B. forskalii is a preferential selfer. High FST estimates suggest that effective dispersal is limited and genetic drift is an important determinant of genetic structure. The potential utility of the microsatellite primers in other closely related Bulinus species was explored.

Isozymes of the Isoetes riparia Complex, I. Genetic Variation and Relatedness of Diploid Species

Abstract Isozymes were examined in the six aquatic (including amphibious) diploid species of Isoetes of eastern North America in order to quantify their levels and patterns of genetic variation, to determine their genetic distinctness, and to evaluate the utility of allozymes in addressing ancestry in the I. riparia complex. Twenty-six populations representing diploid species, I. echinospora, I. engelmannii, I. flaccida, I. mattaponi, I. prototypus, and I. valida were compared for eleven enzymes coded by 16 interpretable loci. Levels of genetic variation in populations tended to be low ranging from none (means: P = 0.0, A = 1.0, H = 0.0) in I. echinospora to moderate (means: P = 22.9, A = 1.4, H = 0.093) in I. flaccida. Genetic variation in polymorphic species tended to be distributed more among than within populations, as indicated by high Fst values (Fst > 0.607 in all polymorphic species except I.mattaponi, for which Fst = 0.272). The genetic identities of conspecific populations were high, with mean v...

Investigations into the Genetic Variation, Population Structure, and Breeding Systems of the Fern Asplenium trichomanes subsp. quadrivalens.

The genetic structure of five populations of the tetraploid fern Asplenium trichomanes subsp. quadrivalens in northern Switzerland was analyzed. Asplenium trichomanes subsp. quadrivalens is one of the most common and most widespread ferns in Europe. In this study we have combined genetic investigations, spatial autocorrelation, and breeding experiments to investigate in detail five populations from natural rock faces. Enzyme electrophoresis revealed very low genetic variability within and among the populations. The small amount of variation was partitioned mainly among the localities, indicated by high Fst values up to 0.764. Overall means of the proportion of polymorphic loci (P=0.076), the mean number of alleles per locus (A=1.086), and the mean expected heterozygosity (H=0.018) were low compared with other ferns (e.g., Kirkpatrick et al. 1990). Very few heterozygous individuals were found. Values of the fixation index (F) were high, ranging between 0.732 and 1.000 and indicating substantial inbreeding. Spatial autocorrelation showed different patterns of substructure in populations of A. trichomanes subsp. quadrivalens with a tendency for patches in short distances (up to 1.5 m). The breeding experiments with isolated prothalli and prothalli pairs showed that a mean of 56.4% of the isolated prothalli were successful in sporophyte formation. The highest rate in one population was 83.3%. We conclude that genetic load must be low in A. trichomanes subsp. quadrivalens. Sporophyte formation was statistically more successful in the experiments with gametophyte pairs than in isolates, indicating that additional cross-fertilization occurred. The latter agreed with the occurrence of few heterozygote samples and the small number of multilocus phenotypes found in natural habitats. Asplenium trichomanes subsp. quadrivalens is shown to be a highly inbreeding taxon with the capability of single spore colonization and subsequent founding of new populations. Such features can be hypothesized to have contributed to the postglacial colonization and the widespread distribution of this taxon in Europe.

Bay‐scale population structure in coastal Atlantic cod in Labrador and Newfoundland, Canada

Polymorphisms at five microsatellite DNA loci provide evidence that Atlantic cod Gadus morhua inhabiting Gilbert Bay, Labrador are genetically distinguishable from offshore cod on the north‐east Newfoundland shelf and from inshore cod in Trinity Bay, Newfoundland. Antifreeze activity in the blood suggests that Gilbert Bay cod overwinter within the Bay. Gilbert Bay cod are also smaller (weight and length) for their age and consequently less fecund for their age, than cod elsewhere within the northern cod complex. The productivity and recruitment potential of coastal cod off Labrador may thus be much lower than that of offshore northern cod or of inshore cod farther south, implying that a more conservative management strategy may be required for cod from coastal Labrador than traditionally practised for northern cod inhabiting less harsh environments. Relatively high FST and RST measures of population structure suggest that important barriers to gene flow exist among five components that include two inshore (Gilbert and Trinity Bay) and three offshore cod aggregations on the north‐east Newfoundland Shelf and the Grand Bank. DA and DSW estimates of genetic distance that involve Gilbert Bay cod are approximately three‐ and 10–fold larger, respectively, than estimates not involving Gilbert Bay cod. The differences between inshore cod from Gilbert Bay and Trinity Bay raise the possibility that other genetically distinguishable coastal populations may exist, or may have existed prior to the northern cod fishery collapse. Harvesting strategies for northern cod should recognize the existence of genetic diversity between inshore and offshore components as well as among coastal components.

Aedes aegypti in Tahiti and Moorea (French Polynesia): isoenzyme differentiation in the mosquito population according to human population density.

Genetic differences at five polymorphic isoenzyme loci were analyzed by starch gel electrophoresis for 28 Aedes aegypti samples. Considerable (i.e., high Fst values) and significant (i.e., P values >10(-4)) geographic differences were found. Differences in Ae. aegypti genetic structure were related to human population densities and to particularities in mosquito ecotopes in both Tahiti and Moorea islands. In highly urbanized areas (i.e., the Papeete agglomeration), mosquitoes were highly structured. Recurrent extinction events consecutive to insecticidal treatments during dengue outbreaks tend to differentiate mosquito populations. In less populated zones (i.e., the east coast of Moorea and Tahiti), differences in ecotope characteristics could explain the lack of differentiation among mosquitoes from rural environments such as the east coast of Tahiti where natural breeding sites predominate. When the lowest populated zones such as Tahiti Iti and the west coast of Moorea are compared, mosquito are less differentiated in Moorea. These results will be discussed in relation to the recent findings of variation in mosquito infection rates for dengue-2 virus.

INSIGHT INTO SPECIATION FROM HISTORICAL DEMOGRAPHY IN THE PHYTOPHAGOUS BEETLE GENUS OPHRAELLA.

Speciation in phytophagous insects is commonly associated with shifts in host use. Using a phylogenetic framework to identify recently diverged taxa that have undergone a radical host shift, this study focuses on how reconstruction of the historical demography of a species, in conjunction with branching patterns between species, provides insight into mode of speciation. Analyses of mitochondrial cytochrome oxidase I sequences indicate that the leaf beetle Ophraella communa exhibits significant population structure, as shown by patterns of genealogical relationships among mitochondrial haplotypes and high FST -values. However, the absence of regional localization of old clades of haplotypes, negative Tajima's D, and unimodal rather than bimodal frequency distribution of the number of pairwise differences between sequences suggests an absence of long-term barriers to gene flow. Furthermore, we found no evidence of isolation by distance. This pattern of genetic variation is consistent with episodes of gene flow on a large geographic scale, perhaps owing to Pleistocene changes in climate. Ophraella communa and its sister species O. bilineata diverged during the early Pleistocene. The evidence of dynamic population structure in O. communa, potentially including episodic but massive gene flow, suggests that reproductive isolation evolved quite rapidly on a localized geographic scale, because speciation would probably have been reversed by gene flow if the evolution of reproductive isolation had been prolonged. That is, gene flow occasioned by range shifts during the Pleistocene would likely have interrupted speciation unless it occurred very rapidly. Sequence diversity implies a large effective population size (> 106 ) in both O. communa and O. bilineata. However, a model based on a drastic bottleneck did not have a lower likelihood than a model with no bottleneck, simply because the time since speciation has been great enough for coalescence to a single ancestor that existed after the speciation event. Sequence diversity in itself, without reference to the time since speciation, cannot provide evidence on the demography of speciation.

Reply from J.L. Bossart and D. Pashley Prowell

We thank Bohonak et al. for the opportunity to expand upon our perspective of gene flow estimates[1xBossart, J.L. and Prowell, D.P. Trends Ecol. Evol. 1998; 13: 202–206Abstract | Full Text PDF | PubMed | Scopus (375)See all References][1]. We are comfortable with our portrayal of the state of this field based on data from Evolution. Evolution was selected because it has a long history as a top journal focusing on empirically based population genetics, has a vast readership and high citation rates. Molecular Ecology, however, is a new, more specialized and less available journal.We agree that most indirect studies of gene flow report data for multiple loci and many use resampling techniques or regression to conduct sensitivity analyses. The issue is whether these are informative comparisons. For example, have estimates averaged over loci advanced our understanding of gene frequency evolution when estimates among loci vary, not uncommonly, by orders of magnitude[2xTempleton, A.R. Mol. Ecol. 1998; 7: 381–398CrossRef | PubMed | Scopus (898)See all References][2]?Our perspective acknowledged problems associated with direct measures of dispersal. Like others[3xSlatkin, M. Annu. Rev. Ecol. Syst. 1985; 16: 393–430CrossRefSee all References][3], however, we emphasized the essential information these estimates provide. Correlational approaches between Fst and life history traits to assess accuracy of genetic methods, as advocated by Bohonak et al., are statistically weak and confounded by evolutionary history—for example, higher Fst values in sessile versus vagile organisms may reflect vicariant events.Application of conventional indirect methods to address ecological questions is inappropriate for two reasons. First, there is no temporal reference point. Do data reveal processes operating over ten years, 100 years, 1000 years or 10 000 years? Second, Nm changes only trivially between Nm=2 and Nm=infinity, so Nm=2 is virtually indistinguishable from Nm=2000 (Ref. [2xTempleton, A.R. Mol. Ecol. 1998; 7: 381–398CrossRef | PubMed | Scopus (898)See all References][2]). Thus, conventional approaches have insufficient power to address relative rates of gene flow among populations impinged upon by human activities, metapopulation dynamics, trophic interactions or relative magnitude of gene flow versus selection.Our overall conclusion that indirect methods `provide a useful starting point'[1xBossart, J.L. and Prowell, D.P. Trends Ecol. Evol. 1998; 13: 202–206Abstract | Full Text PDF | PubMed | Scopus (375)See all References][1]was mirrored by Bohonak et al. in their statement that indirect methods `provide a valuable first approximation'. We part company, however, on the widespread use of new analytical models, as our review validated. As this changes, the field will advance.

Genetic evidence of closed life-cycles for some coral reef fishes within Taiaro Lagoon (Tuamotu Archipelago, French Polynesia)

Taiaro Lagoon has no permanent or regular connection with the ocean (ingress is restricted to episodes of high sea-level and/or strong wave action) raising the question of how fish populations with normally dispersive larvae are maintained inside this lagoon. We compared the genetic population structures of two coral reef fishes, Acanthurus triostegus and Chaetodon ulietensis, present on both sides of the atoll rim to determine whether there is evidence of reproductive isolation. Genetic surveys showed that the lagoonal and oceanic populations were statistically different at five loci (AAT * -3, GDA *, HPD *, MDH * and SDH *) in A. triostegus and three loci (PGI-2*, IDH * and PGD *) in C. ulietensis, producing high F st values of 0.055 and 0.021, respectively. Our genetic and demographic data on these species suggest that both may be completing their life-cycles inside the lagoon, which leads us to question the common assumption that coral reef fishes require oceanic conditions for larval development.

Differential Structuring of Human Populations for Homologous X and Y Microsatellite Loci

The global pattern of variation at the homologous microsatellite loci DYS413 (Yq11) and DXS8174 and DXS8175 (Xp22) was analyzed by examination of 30 world populations from four continents, accounting for more than 1,100 chromosomes per locus. The data showed discordant patterns of among- and within-population gene diversity for the Y-linked and the X-linked microsatellites. For the Y-linked polymorphism, all groups of populations displayed high FST values (the correlation between random haplotypes within subpopulations, relative to haplotypes of the total population) and showed a general trend for the haplotypes to cluster in a population-specific way. This was especially true for sub-Saharan African populations. The data also indicated that a large fraction of the variation among populations was due to the accumulation of new variants associated with the radiation process. Europeans exhibited the highest level of within-population haplotype diversity, whereas sub-Saharan Africans showed the lowest. In contrast, data for the two X-linked polymorphisms were concordant in showing lower FST values, as compared with those for DYS413, but higher within-population variances, for African versus non-African populations. Whereas the results for the X-linked loci agreed with a model of greater antiquity for the African populations, those for DYS413 showed a confounding pattern that is apparently at odds with such a model. Possible factors involved in this differential structuring for homologous X and Y microsatellite polymorphisms are discussed.

MITOCHONDRIAL-GENE TREES VERSUS NUCLEAR-GENE TREES, A REPLY TO HOELZER.

MITOCHONDRIAL-GENE TREES VERSUS NUCLEAR-GENE TREES, A REPLY TO HOELZER.

Variation in genetic traits of the lugworm Arenicola marina:temperature related expression of mitochondrial allozymes?

Genetic traits of the lugworm Arenicola marina were determined for four Atlantic populations from France to Norway and compared with a population from the sub-arctic White Sea in Russia. Seven loci were analysed using horizontal starch gel electrophoresis. A low heterozygosity (0.09-0.17) and a non-significant heterozygote deficiency were found in all populations. The genetic identity between lugworms of European Atlantic populations was high, whereas similarity of the Atlantic populations with the population from the White Sea was low. The gene flow between the Atlantic and the White Sea populations must be considered negligible, as deduced from the average high and significant gene differentiation FST. In particular, differences in allele frequencies of glucose phosphate isomerase (Gpi) and phosphoglucomutase (Pgm) showed that the White Sea population differed significantly from the others. A very strong correlation existed between the frequency of the alleles of isocitrate dehydrogenases 2-A and -B (Idh2-A and Idh2-B) and the average water temperature. It is concluded that temperature had a selective influence on isocitrate dehydrogenase 2, which, in contrast to isocitrate dehydrogenase 1, was identified as a mitochondrial enzyme. These findings support the hypothesis that mitochondria play a key role in temperature adaptation and the adjustment of critical temperatures.

Allozyme differentiation of populations of the dogwhelk Nucella lapillus, (L.): the relative effects of geographic distance and variation in chromosome number

Geographic patterns of genic differentiation were compared with differentiation between karyotypes in the intertidal snail Nucella lapillus. Samples from 24 sites covering the species range in Europe and North America were analysed for allozyme variation at 16 soluble enzyme loci. Two homokaryotypes have been identified with diploid numbers 2n = 26 and 2n = 36 (variation is Robertsonian and hybrids have intermediate chromosome numbers) and samples were classified (on the basis of published data) according to karyotype. Group 1 consisted of samples from three English Channel populations of higher chromosome number (on average 2n > 32) and Group 2 consisted of the remaining 21 samples (presumed to be 2n = 26). Karyotype variation accounts for roughly the same amount of the absolute allozyme variance as geographic variation (46.3% and 53.7% respectively). Yet the patterns of differentiation seen between karyotypes and with geographic separation are very different. In samples classified as 2n = 26 (Group 2), while there is a significant amount of heterogeneity (FST per locus averaged 0.128 for 10 polymorphic loci), allozyme variation occurs independently at different loci so mean genetic identity (Nei) is high: 0.972. There is only a slight decline in genetic identity with distance (genetic identity averaged 0.965 for amphi-atlantic comparisons) indicating that passive transport of juveniles or adults may contribute significantly to gene flow. Conversely, allozyme variation between karyotypes was concordant. High chromosome number populations possessed a suite of alleles at four allozyme loci (Est-3, Lap-2, Mdh-1 and Pep-2) which were absent or rare in Group 2 samples resulting in high FST values for these loci (from 0.294 to 0.472) when karyotypic classes were combined. Consequently the mean genetic identity between these Robertsonian rares is low, 0.856, and falls within the range more usually associated with congeneric comparisons than with con-specific comparisons. The mechanisms maintaining this genic difference are unclear. However the distribution of the karyotypes and physiological and morphological differences (in shell shape) between them strongly suggest that karyotypic variation in Nucella is adaptive.

Latitudinal allozymic variability in natural populations ofZaprionus indianus from India

Twelve natural populations of Z. indianus from India, collected along lines of latitude, were analyzed electrophoretically and the geographical pattern of variation was compared at 15 allozyme loci. Except for Mdh-2, all the loci revealed extensive polymorphism. The allele frequency variation revealed a statistically significant correlation as well as regression coefficients with the latitude. The populations revealed extensive clinical variation at all the polymorphic loci, which depicted moderate to higher FST values. Thus, the observed genetic divergence in colonizing populations of Z. indianus shows the role of natural selection that varies spatially along the north-south axis of the Indian subcontinent.

VOCAL DIALECTS AND THE STRUCTURE OF GEOGRAPHIC VARIATION IN MORPHOLOGICAL AND ALLOZYMIC CHARACTERS IN THE RUFOUS-COLLARED SPARROW, ZONOTRICHIA CAPENSIS.

The geographical patterns of variation shown at 20 allozyme and non-enzymatic protein-coding loci, in 8 external, and in 12 skeletal morphological characters in the rufous-collared sparrow, Zonotrichia capensis, were analyzed in order to test the local (genetic) adaptation hypothesis regarding the origin and maintenance of vocal dialects in birds. Approximately 20 males were collected from each of four sites within each of six different dialect zones. There was significant variability in both external and skeletal morphology among all 24 sites and among dialect groups. Average Wright's corrected fixation coefficient (FST ) was 0.118, indicating significant genetic differentiation among all sites, regardless of dialect. Hierarchical F statistics indicated that only 50% of among site variability was due to a dialect effect. Puna dialect sites were highly differentiated from all other sites with respect to both morphology (external and skeletal measures) and allozyme frequencies. Heterogeneity at the PGM-1 locus among puna scrub sites was the major cause of the high average FST across all sites, and within the puna scrub dialect. Average genetic differentiation among non-puna sites (FST = 0.018) was similar to differentiation among sites within each of the five non-puna dialect groups (mean FST = 0.0132 ± 0.0069). Hierarchical F statistics indicated that none of the among-site differentiation in this subset of samples was due to a dialect effect. These observations are not consistent with the local adaptation hypothesis. All significant genetic heterogeneity occurred among sites in mountainous habitats, and we suggest that topography and patchiness of habitat may have been major factors involved in population differentiation, rather than vocal dialects.

Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT-4 and HL-60 cells are revealed by altered DNA sensitivity to denaturation

The antitumor drug fostriecin (phosphotrienin, FST) has been reported to exert its cytostatic and cytotoxic effects via inhibition of DNA topoisomerase II. The sensitivity of human lymphocytic leukemic MOLT-4 and promyelocytic HL-60 leukemic cells to a wide range of FST concentrations was studied by analyzing the cell cycle-specific effects and changes in nuclear chromatin induced by this inhibitor. The latter was evaluated by assaying the sensitivity of DNA in situ to acid-induced denaturation cytofluorimetrically, with the use of the metachromatic fluorochrome acridine orange (AO), which differentially stains double-stranded and denatured DNA. The cytostatic effects were observed soon after addition of FST (at concentrations of 1–30 μ M for MOLT-4 cultures and 1–5 μ M for HL-60 cultures) as a perturbation of cell progression through S and G 2 phases of the cell cycle. Cell progression through the cycle was halted at >30 μ M FST in MOLT-4 cultures and at >5 μ M in HL-60 cultures; the effect was instantaneous and affected all phases of the cycle, so that no changes in the cell cycle distribution were apparent with increasing length of exposure to the drug. Instead, at these high FST concentrations, immediate cytotoxic effects became evident, manifesting either as cell apoptosis or necrosis. Apoptosis was observed only in the case of HL-60 cells, at FST concentrations of 5–100 μ M, and was characterized by markedly increased sensitivity of DNA to denaturation combined with a decrease in overall DNA stainability, either with the DNA-specific dye DAPI or with AO, indicative of the activation of endogenous nucleases. Necrotic cell death was observed at FST concentrations of 1 m M and at >30 μ M for HL-60 and MOLT-4 cells, respectively: in both cases the overall DNA stainability, with either DAPI or AO, was unchanged compared to the control, but their DNA was very sensitive to denaturation. Interestingly, DNA in G 2 and late S phase MOLT-4 cells, which were undergoing necrotic death, was much more sensitive to denaturation than was DNA in G 1 cells of this lineage. The data indicate that chromatin changes induced by DNA topoisomerase II inhibitors in cells that undergo apoptotic or necrotic death can be conveniently monitored by the assay of DNA in situ sensitivity to denaturation.