Cytogenetic maps of lampbrush chromosomes of newts of the genus Pleurodeles: an algorithm of lampbrush chromosome identification in Pleurodeles waltl by immunocytochemical marker-loop staining with polyclonal anti-Ro52 antisera

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodeles newts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoprotein (RNP) marker loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain newt lampbrush chromosome preparations. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker loops along the chromosome length were constructed.     

Meiosis in four trisomic and one double trisomic males of the newt Pleurodeles waltlii

In the newt Pleurodeles waltlii, meiosis was studied in four trisomic and one double trisomic males. Study of first prophase shows that trivalent frequencies and trivalent configurations are correlated with chromosome length; moreover, trivalent configurations indicate that long chromosomes have multiple points of initiation of synapsis whereas two points might be adequate to secure synapsis of short chromosomes. From the study of metaphase II it appears that the extra chromosomes segregate in half of the spermatocytes II. Some abnormal spermatocytes, resulting from nondisjunction of chromosomes at mitosis or at first division of meiosis, or from precocious division of chromosomes at first division of meiosis were observed. In the male double trisomic meiosis fails at anaphase of second division; this accounts for the sterility of the individual.     

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.     

Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division

Chromosome segregation requires centromeres on every sister chromatid to correctly form and attach the microtubule spindle during cell division. Even though centromeres are essential for genome stability, the underlying centromeric DNA is highly variable in sequence and evolves quickly. Epigenetic mechanisms are therefore thought to regulate centromeres. Here, we show that the 359-bp repeat satellite III (SAT III), which spans megabases on the X chromosome of Drosophila melanogaster, produces a long noncoding RNA that localizes to centromeric regions of all major chromosomes. Depletion of SAT III RNA causes mitotic defects, not only of the sex chromosome but also in trans of all autosomes. We furthermore find that SAT III RNA binds to the kinetochore component CENP-C, and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. In conclusion, our data reveal that SAT III RNA is an integral part of centromere identity, adding RNA to the complex epigenetic mark at centromeres in flies.     

Chromosome condensation and Hoechst 33258 fluorescence in meiotic chromosomes of the grasshopper Spathosternum prasiniferum (Walker)

Patterns of Hoechst 33258 fluorescence have been studied in grasshopper chromosomes. At metaphase of mitotic as well as meiotic divisions — when chromosomes were maximally compact — all the chromosomes fluoresced brightly but no differentially fluorescing regions were detected. However, when all the chromosomes, except the X, were highly extended at pachytene and diplotene stages a distinct differential fluorescence was observed: only the centromeres of the autosomal bivalents fluoresced brightly whereas the entire X univalent showed bright fluorescence. Restriction of differentially bright fluorescence to the more condensed regions of chromosomes suggests a modulatory role for chromosome condensation in the production of Hoechst fluorescence. This suggestion was further strengthened by the substantial quenching of fluorescence caused by removal of chromosomal proteins following treatment with H₂SO₄. Similarly, post-C-band-treatment staining with Hoechst also led to quenching, though now the centromeres of the chromosomes, including the X, retained their differential fluorescence. It is proposed, therefore, that in grasshopper chromosomes, H-fluorescence is modulated by chromosome condensation brought about by differential ratios of DNA/protein at different chromosome regions and at different division stages.     

Nuclear and territorial topography of chromosome telomeres in human lymphocytes

Nuclear and territorial positioning of p- and q-telomeres and centromeres of chromosomes 3, 8, 9, 13, and 19 were studied by repeated fluorescence in situ hybridization, high-resolution cytometry, and three-dimensional image analysis in human blood lymphocytes before and after stimulation. Telomeres were found on the opposite side of the territories as compared with the centromeres for all chromosome territories investigated. Mutual distances between telomeres of submetacentric chromosomes were very short, usually shorter than centromere-to-telomere distances, which means that the chromosome territory is nonrandomly folded. Telomeres are, on average, much nearer to the center of the cell nucleus than centromeres; q-telomeres were found, on average, more centrally localized as compared with p-telomeres. Consequently, we directly showed that chromosome territories in the cell nucleus are (1) polar and (2) partially oriented in cell nuclei. The distributions of genetic elements relative to chromosome territories (territorial distributions) can be either narrower or broader than their nuclear distributions, which reflects the degree of adhesion of an element to the territory or to the nucleus. We found no tethering of heterologous telomeres of chromosomes 8, 9, and 19. In contrast, both pairs of homologous telomeres of chromosome 19 (but not in other chromosomes) are tethered (associated) very frequently.     

Immunocytochemical study of lampbrush chromosomes of the urodele Pleurodeles waltl: Axial granules are recognized by the mitosis-specific monoclonal antibody MPM-2

The lampbrush chromosomes of the urodele Pleurodeles waltl have been studied using the mitosis-specific monoclonal antibody MPM-2. Immunofluorescence studies revealed that MPM-2 stains structures associated with axial granules, numerous other chromomeres, telomeres and certain chiasmata. These structures showed a negative reaction with the anti-DNA monoclonal antibody AC-30-10. In course of meiotic condensation of the chromosomes, in growing and maturating oocytes, the number of such structures associated with the chromosome axis was found to diminish progressively. These granular structures have been found to be formed by fine fibrils about 5 nm in diameter. Immunogold labeling confirmed the results of immunofluorescence studies. MPM-2 was also found to stain two other types of structures observed in association with the lampbrush chromosome axis in P waltl, viz the sphere organelle (only in later stages of oogenesis) and the structure known as ‘M’ which is singular to this material.     

Differential kinetochore protein requirements for establishment versus propagation of centromere activity in Saccharomyces cerevisiae

Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface. Using a conditionally functional dicentric chromosome in vivo, we demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage. Mutations in chl4/mcm17/ctf17 segregate dicentric chromosomes through successive cell divisions without breakage, indicating that only one of the two centromeres is functional. Centromere DNA introduced into the cell is unable to promote kinetochore assembly in the absence of CHL4. In contrast, established centromeres retain their segregation capacity for greater than 25 generations after depletion of Chl4p. The persistent mitotic stability of established centromeres reveals the presence of an epigenetic component in kinetochore segregation. Furthermore, this study identifies Chl4p in the initiation and specification of a heritable chromatin state.     

Stretching the rules: monocentric chromosomes with multiple centromere domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.     

Flow cytometry measurements of human chromosome kinetochore labeling

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.     

Meiotic Disjunction of Homologs in Saccharomyces Cerevisiae Is Directed by Pairing and Recombination of the Chromosome Arms but Not by Pairing of the Centromeres

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis. The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III. In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses. In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome. Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third. In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome. Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed. Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.     

Localisation et signification des zones Q observées sur les chromosomes mitotiques de l'amphibien Pleurodeles waltlii Michah. après coloration par la moutarde de quinacrine

Metaphase plates from tailbuds of Pleurodeles waltlii embryos (stage 34) with or without preceding cold treatment were obtained by squashing followed by quinacrine mustard staining. In both cases, caryograms were established and sites of Q bands located. — Most of the secondary constrictions exhibit very high fluorescence. In general, it is the same for the centromeric parts, but their fluorescence is quenched very strongly by cold treatment. — The proximal part of the long arm of chromosome VII and satellites of chromosomes III and XI exhibit dull fluorescence. All these sites are compared with heterochromatin localization. The relation between banding and base composition or non-histone proteins interactions is discussed.

---

---

Remerciements. Ce travail a été effectué dans le cadre d'un contrat D.G.B.S.T. n 73-1-1145.     

Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations

This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.by H.F. Willard     

Autoradiographic study of the DNA synthesis process along metaphasic chromosomes of Pleurodeles waltlii Michah (urodele amphibian)]

Chromosomes from embryos (stage 34) of the amphibian urodele Pleurodeles waltlii were labeled by 3H-thymidine at different times of the DNA synthesis cycle and studied at metaphase. Terminal segments of the chromosomes, induced secondary constrictions, and satellites were found to be late-labeling. Therefore, they may be considered as formed at least partially by heterochromatin. However, after other treatments or stainings, they do not present other features considered characteristic of heterochromatin. The question of the heterogeneity of their structures is discussed and the ambiguity of the term heterochromatin is emphasized. The proximal part of the long arm of chromosome VII shows characteristics similar to satellites, which may indicate the presence of a previously unrecognized satellite.     

Genetic events associated with an insertion mutation in yeast

The his4-912 mutation shares similar genetic properties with mutations promoted by procaryotic insertion elements. This mutation lacks all three his4 functions. Many different classes of His+ revertants have been obtained from his4-912. The most frequent class of His+ revertants results from a site mutation which confers a cold-sensitive His- phenotype. Other classes of revertants contain translocations (one between chromosomes I and III and the other between chromosomes III and XII), a transposition of the his4 region to chromosome VIII, and an inversion of most of the left arm of chromosome III. Another class contains deletions which extend from his4-912 into the his4 region. In each of these classes of revertants, the his4 region is closely linked to the chromosomal aberration. Many of these revertants contain additional changes in chromosome structure (duplication, deletion and aneuploidy) that are unrelated to the reversion of his4-912 to His4+.     

Fragmentation par les rayons X de l'organisateur d'une différenciation de chromosome en écouvillon (lampbrush), chez Pleurodeles waltlii

X-rays have been used to induce heritable changes in the specific morphology of the lampbrush chromosomes in the newt Pleurodeles waltlii. The karyotype organization of female progeny of irradiated males was studied. Nine out of ten females were found to have chromosomal aberrations. In one of the nine, one of the breaks occurred at the sphere organizer, the sphere being part of the striking morphological features of chromosome IV. On irradiation the normal sphere organizer had been broken into two fragments each of which, when recombined with other chromosome breaks, still forms a sphere. The relationship of these observations to genome redundancy is discussed.     

The maps of the lampbrush chromosomes of Triturus (Amphibia urodela)

The lampbrush chromosomes of Triturus vulgaris meridionalis were isolated from the germinal vesicle of medium and large-sized oocytes and studied with phase-contrast microscope. The maps were constructed on the basis of the lengths and major morphological features of the chromosomes. The length of each map is equal to the mean of the relative lengths of the corresponding chromosome from different oocytes (the relative length of each chromosome is represented by the ratio between its absolute length and that of chromosome XII from the same complement, conventionally considered as 100 units long). The maps arranged in decreasing length order, were oriented according to the most frequent position of chiasmata, as centromeres were not always evident. — Chromosomes VI and XI bear a sphere in subterminal position. Landmarks typical for T. vulgaris meridionalis are the loops inserted on chromosomes VIII (47 units), X (23 units), XI (34 units) and XII (34 units) frequently presenting themselves under the form of double loop bridges of considerable extension. On chromosomes I (4 units), VI (13 units), X (4 units) and XI (36 units) giant bodies were found that are sometimes comparable to dense-matrix loops. Chromosome XI includes a nucleolus-organizing region, sometimes identifiable by the presence of an inserted nucleolus. Normal and granular loops (much extended at times), axial granules, globules, and loopless bars supplement the morphology of the lampbrush chromosomes of this species.     

Cytogenetic characterization of the invasive mussel species Xenostrobus securis Lmk. (Bivalvia: Mytilidae)

The chromosomes of the invasive black-pigmy mussel (Xenostrobus securis (Lmk. 1819)) were analyzed by means of 4',6-diamidino-2-phenylindole (DAPI) / propidium iodide (PI) and chromomycin A3 (CMA) / DAPI fluorescence staining and fluorescent in situ hybridization using major rDNA, 5S rDNA, core histone genes, linker histone genes, and telomeric sequences as probes. The diploid chromosome number in this species is 2n = 30. The karyotype is composed of seven metacentric, one meta/submetacentric, and seven submetacentric chromosome pairs. Telomeric sequences appear at both ends of every single chromosome. Major rDNA clusters appear near the centromeres on chromosome pairs 1 and 3 and are associated with bright CMA fluorescence and dull DAPI fluorescence. This species shows five 5S rDNA clusters close to the centromeres on four chromosome pairs (2, 5, 6, and 8). Three of the four core histone gene clusters map to centromeric positions on chromosome pairs 7, 10, and 13. The fourth core histone gene cluster occupies a terminal position on chromosome pair 8, also bearing a 5S rDNA cluster. The two linker histone gene clusters are close to the centromeres on chromosome pairs 12 and 14. Therefore, the use of these probes allows the unequivocal identification of 11 of the 15 chromosome pairs that compose the karyotype of X. securis.     

The compact Brachypodium genome conserves centromeric regions of a common ancestor with wheat and rice

The evolution of five chromosomes of Brachypodium distachyon from a 12-chromosome ancestor of all grasses by dysploidy raises an interesting question about the fate of redundant centromeres. Three independent but complementary approaches were pursued to study centromeric region homologies among the chromosomes of Brachypodium, wheat, and rice. The genes present in pericentromeres of the basic set of seven chromosomes of wheat and the Triticeae, and the 80 rice centromeric genes spanning the CENH3 binding domain of centromeres 3, 4, 5, 7, and 8 were used as “anchor” markers to identify centromere locations in the B. distachyon chromosomes. A total of 53 B. distachyon bacterial artificial chromosome (BAC) clones anchored by wheat pericentromeric expressed sequence tags (ESTs) were used as probes for BAC-fluorescence in situ hybridization (FISH) analysis of B. distachyon mitotic chromosomes. Integrated sequence alignment and BAC-FISH data were used to determine the approximate positions of active and inactive centromeres in the five B. distachyon chromosomes. The following syntenic relationships of the centromeres for Brachypodium (Bd), rice (R), and wheat (W) were evident: Bd1-R6, Bd2-R5-W1, Bd3-R10, Bd4-R11-W4, and Bd5-R4. Six rice centromeres syntenic to five wheat centromeres were inactive in Brachypodium chromosomes. The conservation of centromere gene synteny among several sets of homologous centromeres of three species indicates that active genes can persist in ancient centromeres with more than 40 million years of shared evolutionary history. Annotation of a BAC contig spanning an inactive centromere in chromosome Bd3 which is syntenic to rice Cen8 and W7 pericentromeres, along with BAC FISH data from inactive centromeres revealed that the centromere inactivation was accompanied by the loss of centromeric retrotransposons and turnover of centromere-specific satellites during Bd chromosome evolution.