Further studies on effects of X-irradiation on prespermatid stages of the Northern vole Microtus oeconomus: Low induction of sex-chromosomal nondisjunction and very high induction of diploid spermatids

Microtus males have been irradiated with X-ray doses of 25, 50, 100 and 200 rad and early spermatids were then analyzed for evidence of induction of sex-chromosomal nondisjunction and diploid spermatids at 1, 4, 6, 7, 9 and 12 days after treatment. In contrast to earlier findings, there was no induction of nondisjunction above control levels. A possible explanation for the differences in results of old and new experiments might be that genetic changes have taken place in the Microtus colony that was initiated with animals trapped in the wild, but which has now become highly inbred.In the present experiment, diploid spermatids were frequently induced. The dose—effect relationships at the different time intervals were linear, but the slopes were different, indicating stage-specific differences in sensitivity. The average doubling dose is of the order of 12 rad with a range of 5–30 rad for the individual time intervals.When diploid spermatozoa in man are also inducible by such low doses of X-rays, the consequence would be an increase of triploid abortions which would constitute and undesirable form of personal or family harddhip.     

The response of spermatogonia and spermatocytes of the Northern vole Microtus oeconomus to the induction of sex-chromosome nondisjunction, diploidy and chromosome breakage by X-rays and fast fission neutrons

Microtus males were exposed to different doses of 250 kV X-rays or fast fission neutrons of 1 MeV mean energy. Early (= round) spermatids were analyzed for the presence of extra sex chromosomes, diploidy and micronuclei at different time intervals corresponding with treated differentiating spermatogonia and spermatocytes. Induction of nondisjunction of sex chromosomes could not be detected. In contrast, induction of diploids by both types of radiation was statistically significant at all sampling times. Dose-effect relationships for most of the sampling times were linear and sometimes linear-quadratic concave upward or downward. There were pronounced stage-specific differences in sensitivity as reflected by differences in doubling doses that ranged from 4 to 22 cGy for X-rays and from 0.4 to 4 cGy for neutrons. Spermatocytes at pachytene were the most sensitive cells and proliferating spermatogonia the least sensitive ones. The relative biological effectiveness (RBE) of neutrons depended on the cell stage treated and fluctuated between 1.4 and 9.2. Evidence for radiation-induced chromosomal breakage events was obtained via detection of micronuclei. Induction of micronuclei by X-rays or neutrons was statistically significant at all spermatocyte stages tested. There was no effect in spermatogonia. With a few exceptions dose-effect relationships were linear. Differences in stage sensitivity were clearly present as evidenced by doubling dose which ranged from 5 to 29 cGy for X-rays and from 1 to 3 cGy for neutrons. RBE values varied from 5.2 to 12.7. Maximum sensitivity was detected in spermatocytes at diakinesis, MI and MII. Resting primary spermatocytes (G1 and S phase) were somewhat less sensitive and actively proliferating spermatogonia were the least sensitive cells. The pattern of stage sensitivity for induction of diploids was distinctly different from that for induction of chromosomal breakage.     

Induced nondisjunction in drosophila oocytes.

Nondisjunction of the sex chromosomes and compound-second chromosomes was induced in Drosophila melanogaster by irradiation of immature oocytes and cold treatment of mature oocytes. These two treatments cause nondisjunction by different mechanisms. This results in a differential for stage sensitivity and the production of relatively different frequencies of the various exceptional progeny classes.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes. Received: 19 January 1998; in revised form: 28 May 1998 / Accepted: 30 June 1998     

Isolation and cytogenetic characterization of male meiotic mutants of Drosophila melanogaster

Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (<10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing >10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometa-phase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.     

The behaviour and possible cytoevolutionary significance of B chromosomes in Dampiera linearis (Angiospermae: Goodeniaceae)

Nineteen percent of diploid plants at the ecotonal junction between laterite inhabiting diploid and coastal sandplain inhabiting tetraploid forms of Dampiera linearis carry from 1 to 6 B chromosomes. The B chromosomes usually form temporary pseudovalent associations at diplotene and often A chromosome/B chromosome connections can be seen. Some plants with high numbers of B chromosomes exhibit A bivalent nondisjunction. Although undivided B chromosomes are distributed to TI poles at random, the nondisjoined bivalents show a polarised movement to poles with high numbers of Bs. It is suggested that the pre-metaphase-1 A/B associations provide a basis for non-random assortment in genomically hybrid individuals and that nonreduction, associated with wholesale nondisjunction induced by very high numbers of B chromosomes, resulted in the generation of polyploids which were reproductively isolated from their diploid progenitors, heterotic and adapted to the coastal plain environment.     

Increased rate of nondisjunction in sex cells derived from low-quality semen

The relationship between chromosomal nondisjunction and semen quality was studied in two groups of males who differ highly in their semen quality: 12 individuals with low-quality semen caused by varicocele, and 8 subjects with high-quality semen, selected from sperm donors for in vitro fertilization. Chromosomal nondisjunction was inferred from the rate of disomy found in mature sperm cells. To determine the rate of disomy, we applied fluorescence in situ hybridization using satellite-specific probes for chromosomes 1, 15, 18, X and Y. In sperm cells of males with low-quality semen, the mean rate of disomy for each of the autosomes and of hetero-disomy for the sex chromosomes (XY) was significantly higher than that observed in the high-quality semen samples: more than 15-fold higher for chromosomes 1 and 15, and 7-fold higher for chromosomes 18 and XY. Yet, the homo-disomy rate for each of the sex chromosomes (XX and YY) was almost the same in both types of semen. The large discrepancy between the low- and high-quality semen in the rate of sex chromosome hetero-disomy versus the similar rate of homo-disomy strongly suggests that the abnormal chromosomal segregation in meiocytes of males with low-quality semen resulted from chromosomal nondisjunction at the first meiotic division. The results indicate that men showing poor semen quality are at an increased risk for meiotic nondisjunction, similar to women at the end of their reproductive years. Received: 30 June 1997 / Accepted: 17 September 1997     

Fifty years of cytogenetics: a parallel view of the evolution of cytogenetics and genotoxicology

A parallelism exists between human cytogenetics and cytogenetic toxicology. The breakthroughs, mostly coming from and used in clinical genetics, are widely used in genetic toxicology. The birth of human cytogenetics occurred in 1956 when it was published that the diploid number of chromosomes in humans is 46. The first stage in chromosome-induced mutagenesis began in 1938 when Sax published the effects of X-rays on the chromosomes of Drosophila. In 1959, the cytogenetic anomalies for Down, Klinefelter, and Turner syndromes were described, and parallelly in 1960, the first publication on chromosomal aberrations in man caused by ionizing radiation appeared. The cytogenetic analysis of chromosomal aberrations in cell cultures is considered one of the primary methods to evaluate induced mutagenesis. At the end of the 1960s, banding techniques allowed chromosomes to be individually identified, in parallel, the sister chromatid exchange analysis technology was described. Another milestone in the history of induced mutagenesis was the discovery that mutagenic agents were able to alter chromosomal division and segregation in gonads inducing meiotic nondisjunction. Here we review new approaches and applications such as biological dosimetry, translocation scoring using FISH, and micronucleus test. Chromosomal aberrations and micronucleus test are now effective cytogenetic biomarkers of early effect used as cancer predictors. Human cytogenetics has proven to be effective over its 50-year lifespan and, although each new technique that has appeared seemed to announce its end, the fact is that the current state of cytogenetics is in reality a collection of techniques that, while common, are cheap, fast, and wide-ranging. Therefore, in genotoxicology, they continue to be useful to identify mutagenic agents as well as to evaluate and analyze exposed populations.     

Nondisjunction and chromosome breakage in mouse oocytes after various X-ray doses

Summary The effect of varying X-ray doses (0.05–0.80 Gy) on preovulatory mouse oocytes was studied by measuring nondisjunction during the first meiotic division, as well as structural chromosome anomalies in ovulated oocytes at metaphase stage II. The incidence of nondisjunction (0.1% hyperploid oocytes) found in oocytes from nonirradiated NMRI-Han female mice was in accordance with the results previously obtained with the same strain. Significantly (P<0.05) more hyperploid oocytes (0.9%) were ovulated following irradiation with 0.8 Gy. There was no statistically significant increase of nondisjunction after low doses. Structural chromosome anomalies occurred, however, even after an irradiation dose as low as 0.05 Gy. The dose response for structural chromosome anomalies is altogether different from that of radiation-induced hyperpoidy. We consider that irradiation of mature oocytes might well be less hazardous with regard to its potency for increasing nondisjunction during the first meiotic division when compared with the effect of chemical mutagens.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Induction and transmission of wheat-Haynaldia villosa chromosomal translocations

In order to develop more wheat-Haynaldia villosa translocations involving different chromosomes and chromosome segments of H. villosa, T. durum-H, villosa amphiploid was irradiated with ^60Co γ-rays at doses of 800, 1,200, and 1,600 rad. Pollen collected from the spikes 1, 2, and 3 days after irradiation were transferred to emasculated spikes of the common wheat cv. ‘Chinese Spring＇. Genomic in situ hybridization was used to identify wheat-H, villosa chromosome translocations in the M1 generation. Transmission of the identified translocation chromosomes was analyzed in the BC1, BC2, and BC3 generations. The results indicated that all three irradiation doses were highly efficient for inducing wheat-alien translocations without affecting the viability of the M1 seeds. Within the range of 800-1,600 rad, both the efficiency of translocation induction and the frequency of interstitial chromosome breakage-fusion increased as the irradiation dosage increased. A higher translocation induction frequency was observed using pollen collected from the spikes 1 day after irradiation over that of 2 or 3 days after irradiation. More than 70% of the translocations detected in the M1 generation were transmitted to the BC1 through the female gametes. All translocations recovered in the BC1 generation were recovered in the following BC2, and BC3 generations. The transmission ability of different translocation types in different genetic backgrounds showed an order of ‘whole-arm translocation 〉 small alien segment translocation 〉 large alien segment translocation＇, through either male or female gametes, In general, the transmission ability through the female gametes was higher than that through the male gametes. By this approach, 14 translocation lines that involved different H. villosa chromosomes have been identified in the BC3 using EST-STS markers, and eight of them were homozygous.     

Nondisjunction Rates and Abnormal Embryonic Development in a Mouse Cross between Heterozygotes Carrying a (7, 18) Robertsonian Translocation Chromosome

Mice bearing Robertsonian translocation chromosomes frequently produce aneuploid gametes. They are therefore excellent tools for studying nondisjunction in mammals. Genotypic analysis of embryos from a mouse cross between two different strains of mice carrying a (7, 18) Robertsonian chromosome enabled us to measure the rate of nondisjunction for chromosomes 7 and 18. Embryos (429) were harvested from 76 litters of mice and the parental origin of each chromosome 7 and 18 determined. Genotyping these embryos has allowed us to conclude the following: (1) there were 96 embryos in which at least one nondisjunction event had taken place; (2) the rate of maternal nondisjunction was greater than paternal nondisjunction for the chromosomes sampled in these mice; (3) a bias against chromosome 7 and 18 nullisomic gametes was observed, reflected in a smaller than expected number of uniparental disomic embryos; (4) nondisjunction events did not seem to occur at random throughout the 76 mouse litters, but were clustered into fewer than would be expected by chance; and (5) a deficiency of paternal chromosome 18 uniparental disomic embryos was observed along with a higher than normal rate of developmental retardation at 8.5 days post coitum, raising the possibility that this chromosome has at least one imprinted gene.     

Meiosis in Male DROSOPHILA MELANOGASTER I. Isolation and Characterization of Meiotic Mutants Affecting Second Chromosome Disjunction

Two second chromosome, EMS-induced, meiotic mutants which cause an increase in second chromosome nondisjunction are described. The first mutant is recessive and causes an increase in second chromosome nondisjunction in both males and females. It causes no increase in nondisjunction of the sex chromosomes in either sex, nor of the third chromosome in females. No haplo-4-progeny were recovered from either sex. Thus, it appears that this mutant, which is localized to the second chromosome, affects only second chromosome disjunction and acts in both sexes.-The other mutant affects chromosome disjunction in males and has no effect in females. Nondisjunction occurs at the first meiotic division. Sex chromosome disjunction in the presence of this mutant is similar to that of sc(4)sc(8), with an excess of X and nullo-XY sperm relative to Y and XY sperm. In some lines, there is an excess of nullo-2 sperm relative to diplo-2 sperm, which appears to be regulated, in part, by the Y chromosome. A normal Y chromosome causes an increase in nullo-2 sperm, where B(s)Y does not. There is also a high correlation between second and sex chromosome nondisjunction. Nearly half of the second chromosome exceptions are also nondisjunctional for the sex chromosomes. Among the double exceptions, there is an excess of XY nullo-2 and nullo-XY diplo-2 gametes. Meiotic drive, chromosome loss and nonhomologous pairing are considered as possible explanations for the double exceptions.     

Aneuploidy in human sperm: the use of multicolor FISH to test various theories of nondisjunction.

While it is known that all chromosomes are susceptible to meiotic nondisjunction, it is not clear whether all chromosomes display the same frequency of nondisjunction. By use of multicolor FISH and chromosome-specific probes, the frequency of disomy in human sperm was determined for chromosomes 1, 2, 4, 9, 12, 15, 16, 18, 20, and 21, and the sex chromosomes. A minimum of 10,000 sperm nuclei were scored from each of five healthy, chromosomally normal donors for every chromosome studied, giving a total of 418,931 sperm nuclei. The mean frequencies of disomy obtained were 0.09% for chromosome 1; 0.08% for chromosome 2; 0.11% for chromosome 4; 0.14% for chromosome 9; 0.16% for chromosome 12; 0.11% for chromosomes 15, 16, and 18; 0.12% for chromosome 20; 0.29% for chromosome 21; and 0.43% for the sex chromosomes. Data for chromosomes 1, 12, 15, and 18, and the sex chromosomes have been published elsewhere. When the mean frequencies of disomy were compared, the sex chromosomes and chromosome 21 had significantly higher frequencies of disomy than that of any other autosome studied. These results corroborate the pooled data obtained from human sperm karyotypes and suggest that the sex chromosome bivalent and the chromosome 21 bivalent are more susceptible to nondisjunction during spermatogenesis. From these findings, theories proposed to explain the variable incidence of nondisjunction can be supported or discarded as improbable.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Coincident maternal meiotic nondisjunction of chromosomes X and 21 without evidence of autosomal aysnapsis

Summary A family in which the proband showed phenotypic signs of both the Turner and Down syndromes was studied cytogenetically and with restriction fragment length polymorphisms. The proband's karyotype was 46,X,+21, showing double aneuploidy without any signs of mosaicism. The single X and one chromosome 21 were of paternal origin while two chromosomes 21 were of maternal origin. The nondisjunction of chromosome 21 took place in maternal meiosis II. If it is assumed that the absence of mosaicism renders postzygotic mitotic loss of the X chromosome unlikely, then the X chromosome would have been lost in maternal meiosis I or II. Recombination had occurred between the nondisjoined chromosomes 21. We conclude that double nondisjunction took place in one parent and that asynapsis was not a prerequisite for the autosomal nondisjunction.     

HALDANE'S RULE IS EXTENDED TO PLANTS WITH SEX CHROMOSOMES

Haldane's rule is an empirical phenomenon that has been observed in animals with sex chromosomes. The rule states that the heterogametic sex (XY or ZW) will be “absent, rare, or sterile” following hybridization between two species. Despite the near ubiquity of Haldane's rule in animal hybridizations, it has not been documented in organisms other than animals. Here, we show evidence for both rarity and sterility in hybrid male but not female offspring in crosses between three dioecious plant species from the genus Silene with heteromorphic (XY) sex chromosomes. Our results are consistent with Haldane's rule, extending its applicability to plants with sex chromosomes.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.     

Comparative genetic mapping points to different sex chromosomes in sibling species of wild strawberry (Fragaria)

Separate sexes have evolved repeatedly from hermaphroditic ancestors in flowering plants, and thus select taxa can provide unparalleled insight into the evolutionary dynamics of sex chromosomes that are thought to be shared by plants and animals alike. Here we ask whether two octoploid sibling species of wild strawberry--one almost exclusively dioecious (males and females), Fragaria chiloensis, and one subdioecious (males, females, and hermaphrodites), F. virginiana--share the same sex-determining chromosome. We created a genetic map of the sex chromosome and its homeologs in F. chiloensis and assessed macrosynteny between it and published maps of the proto-sex chromosome of F. virginiana and the homeologous autosome of hermaphroditic diploid species. Segregation of male and female function in our F. chiloensis mapping population confirmed that linkage and dominance relations are similar to those in F. virginiana. However, identification of the molecular markers most tightly linked to the sex-determining locus in the two octoploid species shows that, in both, this region maps to homeologues of chromosome 6 in diploid congeners, but is located at opposite ends of their respective chromosomes.     

The mechanism of secondary nondisjunction in Drosophila melanogaster females

Bridges (1916) observed that X chromosome nondisjunction was much more frequent in XXY females than it was in genetically normal XX females. In addition, virtually all cases of X nondisjunction in XXY females were due to XX <--> Y segregational events in oocytes in which the two X chromosomes had failed to undergo crossing over. He referred to these XX <--> Y segregation events as "secondary nondisjunction." Cooper (1948) proposed that secondary nondisjunction results from the formation of an X-Y-X trivalent, such that the Y chromosome directs the segregation of two achiasmate X chromosomes to opposite poles on the first meiotic spindle. Using in situ hybridization to X and YL chromosomal satellite sequences, we demonstrate that XX <--> Y segregations are indeed presaged by physical associations of the X and Y chromosomal heterochromatin. The physical colocalization of the three sex chromosomes is observed in virtually all oocytes in early prophase and maintained at high frequency until midprophase in all genotypes examined. Although these XXY associations are usually dissolved by late prophase in oocytes that undergo X chromosomal crossing over, they are maintained throughout prophase in oocytes with nonexchange X chromosomes. The persistence of such XXY associations in the absence of exchange presumably facilitates the segregation of the two X chromosomes and the Y chromosome to opposite poles on the developing meiotic spindle. Moreover, the observation that XXY pairings are dissolved at the end of pachytene in oocytes that do undergo X chromosomal crossing over demonstrates that exchanges can alter heterochromatic (and thus presumably centromeric) associations during meiotic prophase.