Human chromosome polymorphism and disordered reproductive function. II. C-variant chromosomes]

C-stained polymorphic variants of chromosomes 1, 9, 13--16, 21, 22 and Y were studied in married couples with reproductive failure (200 individuals) and in control couples having normal children and no spontaneous abortions and stillbirths. Location of heterochromatic segments, their size and heteromorphism of homologues were estimated. The individuals with reproductive failure were carriers of variants of chromosomes 9 and acrocentrics with higher content of heterochromatic material as well as with heterochromatic chromosome 9 significantly more frequently as compared with control individuals.     

Intra-individual polymorphism of human Y chromosome as a result of deletion in the heterochromatin region

Two equal cell populations with Y-heterochromatin of different lengths were found in a sterile male with azoospermia. There was no evidence for translocation of the heterochromatic material to other chromosomes. Both cell lines have the same Q-, C- and Ag-NOR patterns of chromosomal differential staining. The Y-chromosomes of both the father and brother were as long as the longest of the two populations in the proband. This intraindividual heteromorphism of Y-chromosome is, probably, a result of Y-heterochromatin deletion during the first mitotic division of the zygote, with the loss of a fragment as long as the difference between the long and the short Y populations in the proband. Intraindividual chromosomal heteromorphism is a convenient model to study reasons for variability in the heterochromatin regions of chromosomes.     

Centric-fusion translocation and whole-arm heterochromatin in the karyotype of the blue fox (Alopex lagopus L.): synaptonemal complex analysis.

Conventional observations of mitotic chromosomes from two male blue foxes, revealing a centric-fusion translocation and whole-arm heterochromatin, were verified by synaptonemal complex analysis. This analysis revealed that the centric fusion had been preceded by a conspicuous loss of chromosome material in the two one-armed chromosomes involved, but the chromosomal origin of the centric-fusion kinetochore could not be established. The nontranslocated chromosomes of the trivalent, which in all cells but one were in cis configuration, had reached by early pachytene a stage in which almost complete homologous pairing and nonhomologous association or pairing of the free ends of the chromosomes could be observed. In later stages, complete pairing of the nontranslocated chromosomes with the corresponding arms of the centric-fusion translocation was seen occasionally. One to six autosomal bivalents demonstrated unpaired heterochromatic arms in early pachytene, and the heterochromatic chromosome arms were sometimes unpaired even in late pachytene. Some of them showed a distinct size heteromorphism in late zygotene and early pachytene. In most late-pachytene cells, however, the heteromorphic chromosomes were completely length-adjusted. Only a small fraction of the cells showed pairing interference between nonhomologous chromosomes.     

A radiation analysis of a comstockiella chromosome system: destruction of heterochromatic chromosomes during spermatogenesis in Parlatoria oleae (Coccoidea: Diaspididae)

In the males of the olive scale insect, Parlatoria oleae (2n=8), the paternal set of chromosomes becomes heterochromatic during late cleavage or early blastula and remains so until spermatogenesis. Immediately before the onset of meiosis in the males one or more heterochromatic chromosomes disappear from each primary spermatocyte. At prophase four euchromatic and from one to three heterochromatic chromosomes are present in each cell. The disappearance of the heterochromatic chromosomes before meiosis could be due either to the dehetero-chromatization of the heterochromatic chromosomes and their subsequent pairing with their euchromatic homologues, or to the destruction of the heterochromatic chromosomes. — The alternative interpretations of spermatogenesis in P. oleae were tested by using chromosome aberrations, which had been induced in the heterochromatic set by paternal X-irradiation, as genetic markers in breeding tests of about 400 X₁ males. Meiosis was examined in X₁ males which showed conspicuous chromosomal rearrangements in their somatic cells. The absence of either heteromorphic chromosome pairs or multivalents at spermatogenesis and the failure of the X₁ males to transmit any form of chromosome aberration induced by paternal irradiation is strong evidence that the heterochromatic chromosomes are destroyed in P. oleae. — The evolutionary relationships of the chromosome systems in the coccids are considered. Models are outlined for the derivation of a Comstockiella system involving chromosome destruction either from a lecanoid sequence or from a hypothetical Comstockiella sequence involving chromosome pairing. Problems concerning the control of chromosome destruction are discussed.From a dissertation submitted in partial fulfillment of the requirements of Doctor of Philosophy in Genetics.This work was supported by grant GB 8196 from the National Science Foundation to Dr. Spencer W. Brown, and by a National Institutes of Health Fellowship 1 F02 CA 44173-01 to the author from the National Cancer Institute.Dedicated to Dr. Sally Hughes-Schrader on the occasion of her seventy-fifth birthday.     

DNA methylation and chromosome instability in lymphoblastoid cell lines

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.     

Intranuclear destruction of heterochromatin in two species of armored scale insects

Spermatogenesis is described in two species of armored scale insects,Parlatoria proteus andParlatoria ziziphus. In the males of both species, a haploid set of four chromosomes becomes heterochromatic during early embryogeny. The heterochromatic chromosomes are lost later by two different mechanisms during spermatogenesis. Just before meiosis begins one or more heterochromatic chromosomes disappear from each primary spermatocyte as a consequence of a rapid intranuclear chromosome destruction. Meiosis consists of a single achiasmatic division. At prophase four euchromatic and from one to three heterochromatic chromosomes are present in each cell. Although both the euchromatic and remaining heterochromatic chromosomes divide, the heterochromatic chromosomes are later eliminated by posttelophase ejection; the eliminated chromosomes then disintegrate slowly in the cytoplasm. Each of the two species displays a species specific level of heterochromatin retention and both differ in this regard from the previously describedParlatoria oleae. The evolution of a chromosome system involving intranuclear chromosome destruction is discussed.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Karyotype, C-banding, and Ag-NOR analysis in Diplodus bellottii (Sparidae, Perciforms). Intra-individual polymorphism involving heterochromatic regions.

A karyotype analysis was carried out in nine specimens of the Sparid species Diplodus bellottii using conventional staining, as well as C-banding and Ag-NOR banding techniques, showing, respectively, 2n = 46 and fundamental number (FN) = 54, and scarce heterochromatic areas irregularly distributed and up to four NOR active regions that were C positive. When compared with the karyotypes of other related species, one centric fusion giving rise to a large metacentric pair and several pericentric inversions seem to have been involved in the karyotype evolution. An intra-individual polymorphism was detected in one specimen, resulting in two karyotypic forms in roughly identical proportion, owing to a larger C-band by the NOR regions, appearing either in a terminal position of the short arms of pair 2 or in telomeric position of pair 3. These findings suggest that the extra heterochromatic segment responsible for the heteromorphism apparently only involves associated heterochromatin and not the NORs themselves. This C-positive block seems to have eventually been transferred between heterologous NOR chromosomes by a somatic event, facilitated by the physical proximity of NOR pairs in the nucleolus.     

Characterization of a rare short arm heteromorphism of chromosome 22 in a girl with down-syndrome like facies

Chromosomal heteromorphisms are described as interindividual variation of chromosomes without phenotypic consequence. Chromosomal polymorphisms detected include most regions of heterochromatin of chromosomes 1, 9, 16 and Y and the short arms of all acrocentric chromosomes. Here, we report a girl with Down-syndrome such as facies and tremendously enlarged short arm of a chromosome 22. Fluorescence in situ hybridization (FISH) with a probe specific for all acrocentric short arms revealed that the enlargement p arms of the chromosome 22 in question contained exclusively heterochromatic material derived from an acrocentric short arm. Parental studies identified a maternal origin of this heteromorphism. Cryptic trisomy 21 of the Down-syndrome critical region was excluded by a corresponding FISH-probe. Here, we report, to the best of our knowledge, largest ever seen chromosome 22 short arm, being ~×1.5 larger than the normal long arm.     

A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura,Dicroglossidae)

Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.     

Mitomycin C induced structural changes in heterochromatic regions of human chromosomes 1, 9, 16 and Y

Aberrations and variations in the heterochromatic blocks of chromosomes 1, 9, 16 and Y were found under the influence of mitomycin C in cultured lymphocytes of peripheral human blood. Lymphocytes were cultured during 96 hours, mitomycin C in final concentration of 0.3 mkg/ml was present in the culture during the latest 24 hours of culturing. Different changes in the heterochromatic regions of chromosomes were found in approximately 30% of cells: in 6.3% of cells mitotic chiasmata were indicated. In 9.5% of cells isolocus breaks were observed in heterochromatic region of chromosome 1 in segment 1q11. In the latter case this may be a fragile site detected under the influence of mitomycin C on the lymphocytes.     

Eu-heterochromatic rearrangements induce replication of heterochromatic sequences normally underreplicated in polytene chromosomes of Drosophila melanogaster

In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.     

A karyological study of Asphodelus L. (Asphodelaceae) from the Western Mediterranean

The following aspects of Asphodelus karyology are analysed: base number, polyploidy, chromosome size, chromosome morphology, satellited chromosomes, structural heteromorphism, karyotype asymmetry and karyotype evolution. The base number 0 ×= 14 is common to all species except for A. refractus , which has the derived ×= 13. Three ploidy levels occur, often in the same species; diploid, tetraploid and hexaploid, with 2n = 28, 56 and 84. Chromosomes are generally small to medium-small, with the occasional presence of medium-large chromosomes. The most frequent chromosome types are metacentric of type m and submetacentric. Metacentric chromosomes of type M occur only in sections. Verineopsis, Verinea and Plagiasphodelus ; subtelocentric chromosomes occur only in sections Asphodelus and Plagiasphodelus. There is a wide variability in relation to the number of satellited chromosomes, relative to ploidy level. There are usually two to four in diploids, four to eight in tetraploids and usually six, exceptionally up to 12, in the hexaploid. Satellites are present on the shortest arm, exceptionally on the longest arm. There is a high degree of structural heteromorphism in practically all the species which affects satellited and non satellited chromosomes. Karyotype asymmetry is generally of type 2B. Inter-and intra-chromosomal differences are estimated by the A1 and A2 indexes. Both indices vary in the karyotype evolution of the genus, with a decrease of A1 and an increase of A2. The role of polyploidy, hybridization, asymmetry and decrease of chromosome size in the evolution of Asphodelus is discussed.     

Sex determination in the European eel (Anguilla anguilla, L.). A hypothesis based on cytogenetic results, correlated with the findings of skewed sex ratios in eel culture ponds

Metaphase chromosomes from cultured blood cells of female, male, and hermaphroditic European eels were analyzed. In addition, both gonads from each of the specimens were examined microscopically to ensure correct sexing. The karyological investigation revealed that in some of the specimens a heteromorphic chromosome pair was present. This heteromorphism appeared in both sexes and in the hermaphrodite. C-banding and silver nitrate staining demonstrated that the heteromorphism was due to quantitative differences in constitutive heterochromatin and nucleolar organizing regions in the short arm of chromosome 8. In G-banded preparations it was demonstrated that, except for the heteromorphism mentioned, the karyotypes from both sexes and the hermaphrodite were identical. With the G-band technique it was also easily demonstrated that both the largest metacentric (No. 1) and the smallest metacentric (No. 11) had homologs. Therefore, in contrast to some earlier reports which claimed that these two chromosomes were a heteromorphic pair of sex chromosomes, it is concluded that Anguilla anguilla has no heteromorphic sex chromosomes. The implication of these findings are discussed in relation to the many reports of strongly skewed sex ratios found in commercial eel farms. It is tentatively hypothesized that sex determination in A. anguilla may be metagamic and that sex inversion may occur in this species.     

Sex chromosome evolution in frogs—helpful insights from chromosome painting in the genus Engystomops

The differentiation of sex chromosomes is thought to be interrupted by relatively frequent sex chromosome turnover and/or occasional recombination between sex chromosomes (fountain-of-youth model) in some vertebrate groups as fishes, amphibians, and lizards. As a result, we observe the prevalence of homomorphic sex chromosomes in these groups. Here, we provide evidence for the loss of sex chromosome heteromorphism in the Amazonian frogs of the genus Engystomops, which harbors an intriguing history of sex chromosome evolution. In this species complex composed of two named species, two confirmed unnamed species, and up to three unconfirmed species, highly divergent karyotypes are present, and heteromorphic X and Y chromosomes were previously found in two species. We describe the karyotype of a lineage estimated to be the sister of all remaining Amazonian Engystomops (named Engystomops sp.) and perform chromosome painting techniques using one probe for the Y chromosome and one probe for the non-centromeric heterochromatic bands of the X chromosome of E. freibergi to compare three Engystomops karyotypes. The Y probe detected the Y chromosomes of E. freibergi and E. petersi and one homolog of chromosome pair 11 of Engystomops sp., suggesting their common evolutionary origin. The X probe showed no interspecific hybridization, revealing that X chromosome heterochromatin is strongly divergent among the studied species. In the light of the phylogenetic relationships, our data suggest that sex chromosome heteromorphism may have occurred early in the evolution of the Amazonian Engystomops and have been lost in two unnamed but confirmed candidate species.Subject terms: Cytogenetics, Evolutionary genetics     

Satellited Y chromosomes: Structure,origin, and clinical significance

Summary Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunction and/or to translocations of other chromosomes is discussed.     

Occurrence of two cytotypes in <Emphasis Type="Italic">Bryconamericus</Emphasis> aff. <Emphasis Type="Italic">iheringii</Emphasis> (Characidae): karyotype analysis by C- and G-banding and replication bands

Cytogenetic analyses of Bryconamericus aff. iheringii specimens from the upper Paraná River basin (State of Paraná, Brazil) are provided. They had 2n = 52 chromosomes and two cytotypes with variations in their karyotypic formulae: cytotype I with 12 metacentric, 18 submetacentric, 8 subtelocentric and 14 acrocentric chromosomes with a fundamental number (FN) of 90; cytotype II with 8 metacentric, 28 submetacentric, 6 subtelocentric and 10 acrocentric chromosomes with a fundamental number (FN) of 94. Differences in C- and G-band patterns between the cytotypes, distinguishing marker chromosomes for each karyotype, were reported. The R-band pattern by 5-bromodeoxyuridine incorporation was obtained in chromosomes of the cytotype II sample. In some metaphases, the second pair of submetacentric chromosomes is distinctive: its short arm is heterochromatic (positive C-band), corresponding to a late replication region. In the same cytotype, a G- and R-band size heteromorphism w as recorded in the long arm of pair 9 (submetacentric). These methodologies revealed an actual karyotypic differentiation in the B. aff. iheringii population analyzed. Morphometrical comparative analyses and a discussion of evolutionary aspects of chromosome diversification in species of this genus are provided as well.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.     

Somatic synapsis of eu- and heterochromatic regions of Drosophila melanogaster chromosomes

Quantitative cytogenetical analysis has been used to study the synapsis of D. melanogaster neuroblast mitotic chromosomes from normal females, flies with heterozygous deletions, duplications or inversions in the heterochromatic regions of chromosome 2 and in triploid females. In all these genotypes chromocentric fusion of heterochromatic regions of heterologous chromosomes is observed. Eu- and heterochromatic regions of homologous chromosomes are intimately paired at the same time during the cell cycle. The structural rearrangements lead to reduced frequencies of chromocentric association as well as of homologous synapsis compared with the frequencies in the wild-type. The results obtained are discussed with respect to the general problem of the homologous interaction of chromosomes and the significance of heterochromatin for these processes.     

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.