Centromere ultrastructure in germ-line chromosomes of Parascaris

Ultrastructural analysis of the centromere in germ-line mitotic chromosomes of Parascaris univalens and Parascaris equorum revealed that these chromosomes are holocentric. In thin longitudinal sections of both species the kinetochore appeared as a continuous plate (up to 3.8 m long) and displayed a layered structure. This structure consisted of electron-dense inner and outer layers (average width 10 nm) separated by a less dense middle layer (25 nm wide), which had transverse electron-dense bars (10 nm wide) regularly spaced every 25–30 nm. Thus the ladderlike kinetochore profile observed in Parascaris gonial mitotic chromosomes represents a different type of organization from that of the classical trilaminar kinetochore found in both holocentric and monocentric chromosomes.     

Cytological analysis of chromosomes in the two species Parascaris univalens and P. equorum

Chromosome organization and the phenomenon of chromatin diminution in the two species Parascaris univalens (2n=2) and P. equorum (2n=4) were cytologically analysed by a variety of staining techniques (Quinacrine, Hoechst 33258, Chromomycin A₃ and C-banding). The results show that: (1) the chromosomes of the two species differ markedly in both the location and the type of heterochromatin they contain; (2) in both species there is a strong chromosome polymorphism which, however, ranges within a basic species-specific phenotype; (3) the heterochromatin can be eliminated in presomatic cells during early embryogenesis at two different stages and in both cases the consequence of this process is the generation of somatic cells with a 2n=60 karyotype. Moreover, evidence suggesting the sterility of hybrids between the two species is provided.     

Centromere organization in chromosomes of the mouse

The ultrastructure of the centromere region of chromosomes from mouse L929 cells treated with agents that affect centromere condensation have been examined using light, transmission electron, and scanning electron microscopic techniques. Micrographs of expanded centromeres from treated chromosomes illustrate that both the biarmed chromosomes that were generated by Robertsonian fusion during the past history of the strain and the functional centromere of the multicentromeric marker chromosomes display a prominent gap. This gap probably represents the original site of association of the acrocentric chromosomes and is also the site of the kinetochore. Despite the multicentromeric nature of the marker chromosome a single pair of kinetochores were found only at the central heterochromatic region. The functional implications of these structural findings are discussed.     

Kinetochores and chromatin diminution in early embryos of Parascaris univalens

In Parascaris the mitotic chromosomes of gonial germline cells are holocentric and possess a continuous kinetochore along their entire length. By contrast, in meiotic cells, the centromeric activity is restricted to the heterochromatic tips where direct insertion of spindle microtubules into chromatin without any kinetochore plate is seen. In the presomatic cells of early embryos, which undergo heterochromatin elimination, only euchromatin shows kinetic activity. After developing a technique to separate the very resistant egg shell from the embryos, we studied the cell divisions during early embryogenesis by immunochemical and EM approaches. The results reported here show that in presomatic cells microtubules bind only the euchromatin where a continuous kinetochore plate is present. We also report observations suggesting that the binding of the long kinetochores to the mitotic spindle initiates to a limited number of sites and extends along the entire length, during chromosome condensation. The existence of different centromere stages in different cell types, rends Parascaris chromosomes a very good model to study centromere organization.     

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda)

Summary In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An activation model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

The highly variable pentameric repeats of the AT-rich germline limited DNA in Parascaris univalens are the telomeric repeats of somatic chromosomes.

We have characterized the organization of the germline limited DNA of P. univalens by means of sequence analysis. The repeat unit of this satellite DNA is the pentanucleotide 5'TTGCA, although there is a high degree of sequence variation. Repeat variants are not arranged in tandem but in a disperse, nonrandom manner. In the somatic genome which arises from the germline genome through extensive genomic rearrangement early in development, copies of these pentamers represent the telomeric repeats, indicated by their sensitivity to Bal 31 and their presence in a somatic endlibrary. Unlike telomeric sequences from other species the P. univalens telomeres do not display consecutive guanines and no strand bias for that base, recently suggested as universal features of eukaryotic telomeres. Investigation of fragments that carry pentameric repeats along with sequences of different type identifies a 5 bp consensus sequence at the junction point. We suggest a model in which pentameric repeats originate via amplification by a terminal transferase (telomerase) in both the germline and the somatic genome.     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.     

The meiotic chromosomes of man

Summary Information was obtained on the chromosome number, and the behavior of autosomes as well as of the sex chromosomes in meiosis in human male germ cells derived from 25 Japanese patients, 4 to 79 years in age, who were hospitalized mostly due to epididymitis, prostate cancer, undescended testes or infertility.In 16 out of the 25 specimens, the chromosome numbers, 46 in 2n and 23 in n, were consistently established together with an XY sex-determining mechanism based on spermatogonial and spermatocyte divisions. No reliable counts were obtained from the remaining 9 cases, because of that they provided no cells for precise investigation.The X and Y chromosomes during the leptotene stage were observed as two separate heteropycnotic bodies lying along the inner wall of the nucleus, while at pachytene they formed a sex-vesicle after homologous pairing. At the diplotene, diakinesis and first metaphase the X and the Y appeared as an isopycnotic bivalent showing an end-to-end association, though there were some cells in which they remained as two separate entities free from contact. Evidence was presented that the X and the Y seemed to associate with each other at the distal end of the short arm of each element.One or sometimes two smallest autosomal bivalents tended to show rather precociously a chiasma-terminalization at the first metaphase.The metaphase chromosomes of the second spermatocytes were evident by the haploid number as well as by their widely diverged chromatids with a characteristic spiral configuration.The testicular materials under study contained in most cases polyploid cells with a considerable frequency in spermatogonia as well as in first and second spermatocytes. Giant sperm heads were observed not infrequently, mostly being abnormal in shape. No significant correlation was obtained between the frequency of polyploid cells and the age of patients so far studied.Contribution No. 679 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. — It is our pleasure to dedicate this paper to Professor Dr. Hans Bauer, Max-Planck-Institut für Meeresbiologie, Tübingen, in honor of his sixtieth birthday.     

Spatial organization and dynamics of the association of Rec102 and Rec104 with meiotic chromosomes

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.     

The analysis of exchanges in tritium-labelled meiotic chromosomes

The autoradiographic analysis of exchanges in tritium-labelled meiotic chromosomes is potentially a useful approach to the study of meiotic exchange events since this method differentially labels meiotic chromatids along their entire length. The main problem encountered in earlier autoradiographic studies is that of distinguishing label exchanges generated at chiasmata from label exchanges generated by sister chromatid exchange. This problem was overcome in the present study by the choice of a meiotic system (male meiosis of Stethophyma grossum) where chiasmata are limited to just one proximally localised chiasma in each bivalent. This system allows the positive identification of chiasma-generated label exchanges and demonstrates convincingly the origin of chiasmata through breakage and rejoining of homologous non-sister chromatids. Sister chromatid exchanges are also readily detected in labelled meiotic chromosomes of this species, where they occur with a mean frequency of 0.35 per chromosome. This frequency is similar to that found in mitotic spermatogonial cells and the exchanges are randomly distributed both within and between chromosomes. These features of meiotic sister chromatid exchanges suggest that they are unrelated to non-sister chiasmatic exchanges and they probably have no special meiotic significance.     

Folded chromosomes in meiotic yeast cells: analysis of early meiotic events.

Analysis of folded chromosomes in cells under standard sporulation conditions shows that the g₀ form of the folded genome is used as the entry into meiosis. Premeiotic DNA replication is initiated from the g₀ structure. In contrast, mitotic DNA replication is preceded by a characteristic pre-replicative form, g₁. Nonetheless, the mitotic and meiotic replication structures are indistinguishable by sedimentation. Preliminary evidence also suggests that the meiotic equivalent of the mitotic post-replicative structure, g₂, is absent. In strains homozygous for the mating type locus, aa and αα, meiotic replicating structures are not detected, and the folded chromosomes remain in a non-cycling form. However, this non-cycling form is distinguishable from the g₀ form of aα. cells.     

Discontinuities and unsynapsed regions in meiotic chromosomes have a trans effect on meiotic recombination of some chromosomes in human males

During meiosis, homologous chromosome pairing and synapsis are essential for subsequent meiotic recombination (crossing-over). Discontinuous regions (gaps) and unsynapsed regions (splits) were most frequently observed in the heterochromatic regions of bivalent synaptonemal complex (SC) 9, and we have previously demonstrated that gaps and splits significantly altered the distribution of MLH1 recombination foci on SC 9. Here, immunofluorescence techniques (using antibodies against SC proteins and the crossover-associated MLH1 protein) were combined with a centromere-specific fluorescence in situ hybridization technique that allows identification of every individual chromosome. The effect of gaps/splits on meiotic recombination patterns in autosomes other than chromosome 9 during the pachytene stage of meiotic prophase was then examined in 6,026 bivalents from 262 pachytene cells from three human males. In 64 analyzed cells with a gapped SC 9, the frequency of MLH1 foci in SCs 5 and 10 and in SC arms 10q, 11p and 16q was decreased compared to 168 analyzed cells with a normally-synapsed SC 9 (controls). In 24 analyzed cells with splits in SC 9, there was a significant reduction in MLH1 focus frequency for SC 5q and the whole SC5 bivalent. The positioning of MLH1 foci on other SCs in cells with gapped/split SC 9 was not altered. These studies suggest that gaps and splits not only have a cis effect, but may also have a trans effect on meiotic recombination in humans.