Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin. Received: 1 July 1998 / Accepted: 7 September 1998     

Polymorphism of heterochromatin C-blocks of rye genome chromosomes in rye-wheat amphidiploids and their chromosome substitution lines

Polymorphism of heterochromatin C-blocks in chromosomes of rye genome has been studied in the F1BC1 hybrids and the D/A substitution lines of rye-wheat amphidiploids (Verasen x L374, Novosibirskaya x x L246)--secalotriticum for revealing cytogenetic markers of rye chromosomes. An increase in polymorphism for the presence and value of heterochromatin blocks in chromosomes of rye genome was shown in the F1BC1 hybrids ((Verasen x L374) x L145 x L145) and unstable genotypes of the F6BC1 chromosome substitution lines (line 118 (Novosibirskaya x L246) x Reso x Reso), line 104 ((Verasen x x L374) x Garmoniya x Garmoniya) that was related to activation of mobile genetic elements present in cereals. Heterochromatin markers of all seven chromosomes in rye genome were revealed. The polymorphism system of heterochromatin blocks may serve as a marker for specificity of the linear chromosome structure during reconstruction of synthetic cereal genomes, and as a test for cytological and morphogenetic stability of hybrid polygenomes in a series of generations.     

Relative DNA content of somatic nuclei and chromosomal studies in three genera,Tilapia, Sarotherodon,andOreochromis of the tribe Tilapiini (Pisces,Cichlidae)

Seven Tilapiine species from three generaTilapia, Sarotherodon, andOreochromis were cytogenetically studied for chromosome number, chromosome morphology, and DNA content. The chromosome number 2n=44 was the same in all seven species. Arm number (NF) differences indicate the possible role of pericentric invasions in the karyotypic evolution of these species. C-banding of metaphase chromosomes shows that heterochromatin is localised around the centromere in all species ofOreochromis and Sarotherodon butT. zillii has more heterochromatin with six chromosomes having completely C-positive short arms. DNA values vary between 0.84 pq forO. macrochir and 1.21 pq forO. aureus. No heteromorphic sex chromosome pair could be found in any species. These findings suggest that karyotypic evolution has occurred but does not appear to be associated with speciation in this group.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. n. albomicana

Heterochromatin distribution and differentiation in metaphase chromosomes of two morphologically identical Drosophila races, D. nasuta nasuta and D. n. albomicana, have been studied by C- and N-banding methods. — The total heterochromatin values differ only slightly between these races. However, homologous chromosomes of the two Drosophila forms show striking differences in the size of heterochromatin regions and there is an alternating pattern in D. n. nasuta and D. n. albomicana of chromosomes which contain more, or respectively less heterochromatin than their counterparts in the other race. — Three different N-banding patterns could be obtained depending on the conditions of the method employed: One banding pattern occurs which corresponds to the C-banding pattern. Another pattern is the reverse of the C-band pattern; the euchromatic chromosome regions and the centromeres are stained whereas the pericentric heterochromatin regions remain unstained. In the Y chromosomes of both races and in chromosome 4 of D. n. albomicana, however, the heterochromatin is further differentiated. In the third N-banding pattern only the centromeres are deeply stained. Furthermore, between the races, subtle staining differences in the pericentric heterochromatin regions can be observed as verified in F₁ hybrids. On the basis of C- and N-banding results specific aspects of chromosomal differences between D. n. nasuta and D. n. albomicana are discussed.Dedicated to Prof. W. Beermann on the occasion of his 60th birthday     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Rye chromosome translocations in hexaploid wheat: a re-evaluation of the loss of heterochromatin from rye chromosomes

Summary Using in situ hybridization techniques, we have been able to identify the translocated chromosomes resulting from whole arm interchanges between homoeologous chromosomes of wheat and rye. This was possible because radioactive probes are available which recognize specific sites of highly repeated sequence DNA in either rye or wheat chromosomes. The translocated chromosomes analysed in detail were found in plants from a breeding programme designed to substitute chromosome 2R of rye into commercial wheat cultivars. The distribution of rye highly repeated DNA sequences showed modified chromosomes in which (a) most of the telomeric heterochromatin of the short arm and (b) all of the telomeric heterochromatin of the long arm, had disappeared. Subsequent analyses of these chromosomes assaying for wheat highly repeated DNA sequences showed that in type (a), the entire short arm of 2R had been replaced by the short arm of wheat chromosome 2B and in (b), the long arm of 2R had been replaced by the long arm of 2B. The use of these probes has also allowed us to show that rye heterochromatin has little effect on the pairing of the translocated wheat arm to its wheat homologue during meiosis. We have also characterized the chromosomes resulting from a 1B-1R translocation event.From these results, we suggest that the observed loss of telomeric heterochromatin from rye chromosomes in wheat is commonly due to wheat-rye chromosome translocations.     

Qualitative analysis of constitutive heterochromatin and primate evolution

The results of qualitative heterochromatin analysis in 16 species of primates: Homo sapiens , Pan troglodytes and Gorilla gorilla (F. Hominidae), Hylobates syndactilus (F. Hylobatidae), Macaca fascicularis , M. tibetana , Mandrillus sphinx , M. leucophaeus , Cercopithecus aethiops , C. sabaeus and C. albogularis (F. Cercopithecidae), Cebus apella , Ateles belzebuth hybridus , Aotus azarae , Saimiri sciureus and Lagothrix lagothricha (F. Cebidae) are presented in this work. We characterized heterochromatin using: (a) in situ digestion with restriction enzymes AluI, HaeIII, RsaI and Sau3A, and (b) chromosome staining with DA/DAPI on unbanded chromosomes, on C-banded chromosomes and on sequentially G-C-banded chromosomes. The aim of this work was to relate the qualitative characteristics of constitutive heterochromatin observed with the cytogenetic evolutive processes in the primate group. Results obtained show that (1) in the family Cercopithecidae, Papionini species do not present chromosomal rearrangements when their karyotypes are compared and the heterochromatin characteristics are uniform, while Cercopithecini species show a high number of chromosomal reorganizations, but they have the same heterochromatic characteristics; (2) the Platyrrhini species analysed show variability in their karyological and heterochromatic characteristics; (3) the Hominoidea present two different situations: Pan , Gorilla and Homo with few chromosomal reorganizations among their karyotypes but with a high variability in their heterochromatin characteristics, and Hylobates with low heterochromatin variability and a highly derived karyotype. Speciation processes related to chromosome changes and heterochromatin variations in different groups of primates are discussed.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 107–124.     

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.     

Sister chromatid exchanges in Drosophila melanogaster cell lines in vitro

The frequency of sister chromatid exchanges (SCEs) in two cell lines of Drosophila melanogaster with different karyotypes (XX and XY) was determined, considering (1) the distribution of SCEs within each chromosome, with reference to eu- and heterochromatin and (2) the distribution of SCEs in different chromosomes. A comparison was made between chromosome pairs within each karyotype and between the two different karyotypes. The following results were obtained. The SCEs are not randomly distributed along chromosomes, since exchanges were never observed in heterochromatin. SCEs are more frequent in XY than in XX cells; moreover, in both cell types there exists a significantly higher frequency of SCEs in the X chromosome than in the autosomes. These findings are discussed in relation to chromosome aberrations and mitotic recombination.     

Molecular and cytological characterization of repetitive DNA sequences from the centromeric heterochromatin of <Emphasis Type="Italic">Sciara coprophila</Emphasis>

Sciara coprophila (Diptera, Nematocera) constitutes a classic model to analyze unusual chromosome behavior such as the somatic elimination of paternal X chromosomes, the elimination of the whole paternal, plus non-disjunction of the maternal X chromosome at male meiosis. The molecular organization of the heterochromatin in S. coprophila is mostly unknown except for the ribosomal DNA located in the X chromosome pericentromeric heterochromatin. The characterization of the centromeric regions, thus, is an essential and required step for the establishment of S. coprophila as a model system to study fundamental mechanisms of chromosome segregation. To accomplish such a study, heterochromatic sections of the X chromosome centromeric region from salivary glands polytene chromosomes were microdissected and microcloned. Here, we report the identification and characterization of two tandem repeated DNA sequences from the pericentromeric region of the X chromosome, a pericentromeric RTE element and an AT-rich centromeric satellite. These sequences will be important tools for the cloning of S. coprophila centromeric heterochromatin using libraries of large genomic clones.     

Assessment of intergenomic recombination through GISH analysis of F1, BC1 and BC2 progenies of Tulipa gesneriana and T. fosteriana

Using 23 F1 hybrids, 14 BC1 and 32 BC2 progenies, the genome composition of Darwin hybrid tulips was analysed through genomic in situ hybridisation (GISH) of somatic chromosomes. All plants were diploids (2n = 2x = 24) with the exception of one tetraploid BC1 (2n = 4x = 48) and one aneuploid BC2 (2n = 2x + 1 = 25) hybrid. Morphometric analysis in F1 hybrids revealed a difference in the total length of chromosomes representing genomes of T. gesneriana and T. fosteriana, where the percentage of each genome equaled 55.18 ± 0.8 and 44.92 ± 0.6% respectively. GISH distinguished chromosomes from both parent genomes although there was a lack of consistent chromosome labelling in some cases. In both T. gesneriana and T. fosteriana chromosomes some segments of heterochromatin in the telomeric and intercalary regions exhibited a higher intensity of fluorescence. In situ hybridisation with 5S rDNA and 45S rDNA probes to metaphase chromosomes of F1 hybrids showed that these regions are rich in rDNA. A notable feature was that, despite genome differences, there was a considerable amount of intergenomic recombination between the parental chromosomes of the two species as estimated in both BC1 and BC2 offspring. The number of recombinant chromosomes ranged from 3 to 8 in BC1 and from 1 to 7 in BC2 progenies. All recombinant chromosomes possessed mostly a single recombinant segment derived from either a single crossover event or in a few cases double crossover events. This explains the fact that, unlike the situation in most F1 hybrids of other plant species, certain genotypes of Darwin hybrid tulips behave like normal diploid plants producing haploid gametes and give rise to mostly diploid sporophytes.     

Chromosome plasticity in Ctenomys (Rodentia Octodontidae): chromosome 1 evolution and heterochromatin variation

A chromosome 1 (Cr1) pericentric inversion is described in six of seven species in the genus Ctenomys (tuco-tucos) from Uruguay. The inversion was inferred from G-band analyses of subtelocentric Cr1 hypothesised to be derived from the ancestral metacentric condition. Cr1 varies across species in heterochromatin amount and localisation including a metacentric chromosome without positive C-bands in C. torquatus, a subtelocentric chromosome with heterochromatic short arms in C. rionegrensis, and a subtelocentric chromosome negative after C-banding in five of the species analysed here. Pachytene chromosomes from C. rionegrensis, a species with the highest heterochromatin content, and C. torquatus, one of the species with the lowest heterochromatin content, were analysed in order to assess possible mechanisms of heterochromatin evolution. This analysis revealed the presence of three heterochromatic chromocenters in C. rionegrensis where bivalents converge, while in C. torquatus only one chromocenter was observed. In both species, highly repetitive DNA was observed, localised in chromocenters after “in situ” hybridisation. Heterochromatin associated protein M31 was localised in chromocenters of both species after immuno-detection. The spread of heterochromatin in Ctenomys chromosomes could be produced by chromatin exchanges at the chromocenter level. We propose the exchange of this DNA associated proteins between non-homologous chromosomes in pachytene to be the responsible for the spread of heterochromatin through the karyotypes of species like C. rionegrensis     

Pattern of repetitive DNA of the chromosomes of Microtus agrestis

The distribution of repetitive DNA in the chromosomes of Microtus agrestis was studied with the method for demonstrating constitutive heterochromatin given by Yunis et al. (1971) and the reassociation technique described by Schnedl (1971). All autosomes can be individually recognized by means of the position of their bands. The euchromatic segment of the X1 chromosome shows the same banding pattern as the corresponding segment of X2 which consists of facultative heterochromatin. The short arms of the Y chromosome are not deeply stained with either method and therefore do not contain noticeable amounts of repetitive DNA. The relative distances between the bands remain constant during chromosome contraction in mitosis.     

Heterochromatin (C-bands) in somatic and male germ cells in three species ofMicrotinae

The C-banding patterns in the chromosomes ofMicrotus oeconomus, M. arvalis andM. ochrogaster demonstrate differences in the amount and distribution of heterochromatin. Autosomal centromeric heterochromatin appears as conspicuous blocks or as small dots, and in several chromosomes no heterochromatin was detected; interstitial heterochromatin was observed in one autosome pair ofM. ochrogaster. The sex chromosomes also demonstrate differences in the C-banding pattern. InM. oeconomus, the X chromosome exhibits a block of centromeric heterochromatin which is larger than that of the autosomes; this characteristic helps to recognize the X chromosomes in the karyotype. InM. arvalis no heterochromatin was appreciated in the sex chromosomes. The Y chromosomes ofM. ochrogaster andM. oeconomus are entirely heterochromatic. During male meiosis heterochromatin shows condensation, association and chiasma prevention; the sex chromosomes pair end to end in the three species. At pairing, the Y chromosome ofM. arvalis is despiralized, but it appears condensed again shortly before separation of the bivalent.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

Intercalary heterochromatin in Drosophila

Sites of intercalary heterochromatin (IH) in the complete set of Drosophila melanogaster polytene chromosomes were localized and studied according to the following criteria: tendency to break (weak points), ectopic pairing and late replication, the existence of repeats (in X and 2R) including those enriched with A-T bases. Correlation between these features investigated, the highest correlation coefficients found between weak point behavior, late replication, and ectopic pairing. The frequency of breaks in weak points in some IH bands was shown to be different in different tissues, strains and closely related Drosophila species. Sexual differences in morphology and manifestation of IH features were found in bands of the X chromosome: weak point behavior and participation in ectopic pairing of IH bands are an order of magnitude less frequent in male X chromosomes than in female X chromosomes. In autosomes such differences have not been observed. IH bands in male X chromosomes look more massive than the homologous ones in female X chromosomes: the DNA content of the 11A6-9 region is four times less in females than in males. The hypothesis is proposed that the specific features of intercalary heterochromatin bands are determined by tandem repetitiveness and late replication. The latter, if it occurs in a cluster of repetitions, could cause incomplete polytenization of the region and, as a consequence, breaks (or weak points) and the appearance of adhesive ends which may take part either in realization of ectopic contacts or in fixation of those occurring previously. Breaks caused by chromosome aberrations in regions with repeats may not result in a sharp decline of viability, so that break points of chromosome rearrangements in intercalary heterochromatin may be more frequent than in other regions.     

C-band length variability and reproductive wastage

Summary The possible influence of total Y chromosome length and the C-band size variability of chromosomes 1, 9, 16, and Y, on reproductive wastage was investigated. One hundred couples with recurrent reproductive wastage and 106 control couples with at least two healthy children and no miscarriages were cytogenetically studied. Total Y chromosome length was evaluated as the Y/F index and the C-band size was analyzed quantitatively according to the linear measurement method of Baliek et al. (1977). The different degrees of mitotic contraction were corrected on the basis of the linear correlation found between heterochromatin and euchromatin length. Statistical comparison between results of Y chromosome from both samples demonstrated, in the test group, an increase in the mean value of the Y/F index, but the increase of Y C-band length did not reach significance. In addition mean values of C-band length on chromosomes 1, 9, and 16 in couples from the test group and especially those who had had two or more abortions, were lower than those in the controls. Among the latter the frequency of chromosomes included in the category of very large heterochromatin size is higher. However these length differences have been demonstrated only in specific subgroups, and in each one for a different chromosome. Our results indicated that Y chromosome length as well as C-band size variabilities are not directly related to reproductive wastage.     

Homologous and non-homologous chromosome associations by interchromosomal chromatin connectives in Ornithogalum virens

Based on interchromosomal chromatin connectives, a statistical analysis of homologous and non-homologous chromosome associations was made on mitotic metaphase chromosomes of Ornithogalum virens. The great majority of connectives involve constitutive heterochromatin, and connections between homologous chromosomes are twice as common as would be expected by chance. It is suggested that constitutive heterochromatin with similar DNA is involved in both homologous and non-homologous chromosome associations.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.