Microsurgically-extracted metaphase chromosomes of the Indian muntjac examined with phase contrast and scanning electron microscopy.

Metaphase chromosomes are extracted from Indian muntjac cultured fibroblasts either through the use of microneedles or by the application of a droplet of silicone oil onto the cell surface. Interconnecting fibers among the chromosomes allow the entire diploid complement to be extracted from the cell. The seven muntjac chromosomes are brought to the surface of a glass coverslip for analysis. Each chromosome can be identified on the basis of morphology, and particular chromosomes or chromosome parts can be isolated. Many of the fibers which interconnect the chromosomes may be attributed to adhesions formed between the sticky chromosome surfaces during extraction. However, when interchromosomal contacts are avoided during extraction, the chromosomes are found to be arranged radially with the centromeres near the center and interconnected by fibers. This arrangement is similar to that seen inside muntjac cells at metaphase. Scanning electron microscopy reveals the chromosome surfaces to consist of looping fibers, except for regions near the centromeres and the secondary constrictions. Chromosome fibers at these sites are organized into parallel bundles. Chromosome interconnections are strands composed of multiple fibers which seem to be continuous with chromosome fibers.     

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.     

Problem of chromosome formation in T-even phages]

Three basic versions for the formation of circularly permuted and terminally redundant chromosomes with rings, concatemers, or fragments as replicative intermediates were considered. Experimental results show that the chromosome of T-even phage can turn into 4-6 large fragments soon after it penetrates inside the Escherichia coli cell. The fragments are capable for autonomous replication and contribute their material to progeny phage chromosomes. These results confirm the suggestion that circularly permuted and terminally redundant chromosomes of T-even phages are made of fragments. A theoretical analysis of different modes of parental chromosome fragments formation, autonomous replication and ordered association was carried out. In particular, it was emphasized that at a low multiplicity of infection the reassociation of fragments by means of recombination can be accomplished only if breaks in complementary strands of the parental chromosome were made with a shift for about 3000 nucleotides. Complexity is a feature of linear chromosomes that ensures their reproduction without defects at the ends.     

Different mechanisms of telomere length regulation

Eukaryotic chromosomes are linear and have their, ends formed by DNA-protein structures, telomeres. At present more and more facts demonstrate the diversity of telomere functions. Telomeres protect the chromosome ends from degradation, fusion, recombination, and from the repair system that recognizes nicks in DNA strands. As shown recently, shortening of the telomeres is a cause of cell aging. In most organisms, telomeres are elongated by means of a special ribonucleoprotein complex; however, in some insects this takes place by either gene conversion or transposition of mobile elements. Evolutionary relations between different types of telomeres are discussed.     

Telomeres and DNA damage checkpoints

In all eukaryotic organisms, interruptions in duplex DNA molecules elicit a DNA damage response, which includes activation of DNA repair machineries and surveillance mechanisms, known as DNA damage checkpoints. Telomeres and double-strand breaks (DSBs) share the common feature of being physical ends of chromosomes. However, unlike DSBs, telomeres do not activate the DNA damage checkpoints and are usually protected from end-to-end fusions and other processing events that normally promote repair of DNA breaks. This indicates that they are shielded from being recognized and processed as DSBs. On the other hand, chromosome ends resemble damaged DNA, as several factors required for DNA repair and checkpoint networks play important roles in telomere length maintenance. Due to the critical role of both DNA damage checkpoints and telomere homeostasis in maintaining genetic stability and in counteracting cancer development, the knowledge of their interconnections is essential for our understanding of these key cellular controls.     

Telomeres--what's new at the end?

Telomeres are specialized chromatin domains located at the ends of chromosomes. They are involved in chromosome replication, stability and localization in the nucleus. In addition to these functions, recent work suggests that telomeres are involved in such superficially diverse cellular phenomena as ageing, cancer, nuclear architecture and nuclear/cellular division.     

Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion

Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving "protofilaments," strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore "plate" and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.     

Nuclear structures in the telotrophic ovarioles of nocturnal ground beetles (Tenebrionidae, Polyphaga). III. The oocyte nucleus. Electron microscopic data]

The fine structural organization of nuclei was studied in the growing oocytes of Blaps lethifera, B. mortisaga and Gnaptor spinimanus. In the beginning of diplotene the nuclei contain primary fibrillar nucleoli and numerous electron dense globules dispersed all over the nucleus; the loose chromosome material (lampbrush chromosomes) is distributed all over the nucleus. With the oocyte growth the chromosomes are spiralized and join into the karyosphere. A capsule of fibrous material forms around the karyosphere. The karyosphere nucleoli appear on the chromosomes and, then, move to the capsule region and outside its limits, to the nuclear envelope. They are fibrillar and non-active with respect to RNA synthesis. The fibrous material of the capsule is represented by strands which consist of bundles of cross-striated filaments. These latter contact directly with the chromosomes in the karyosphere and with the surface of the karyosphere nucleoli. The fibrillar-granular bodies are distributed along the strands in the capsule; they contain both RNA and DNA. The nature of extrachromosomal DNA in the karyosphere capsule and its participation in the formation of the capsule material are discussed. A suggestion is put forward on the similarity of the capsule strands with the modified central elements of synaptinemal complex.     

Telomeres: more than chromosomal non-sticking ends

Telomeres are specialized natural ends of eukaryotic chromosomes that, contrary to the ends of broken chromosomes, are stable and do not fuse with the ends of other chromosomes. In addition, telomeres protect chromosomal ends from degradation, facilitate completion of chromosomal DNA replication, and contribute to chromosome positioning within nuclei. Telomeric DNA consists of repetitive sequences and specific associated proteins, including the telomere repeat-binding factors TRF1 and TRF2. A lack of TRF2 enables end-to-end chromosome fusion. A structural disruption of telomeres not only causes chromosomal mechanical instability but also activates a programmed cell death cascade.     

Direct imaging of DNA in living cells reveals the dynamics of chromosome formation

Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.     

Absence of true interchromosomal connectives in microsurgically isolated chromosomes

Mitotic chromosomes of the Indian muntjac were isolated from cultured fibroblast-like cells by microsurgery. The entire complement of seven chromosomes could be obtained with the radial array of chromosomes on the mitotic spindle intact. The center of the radial array was occupied by a fibrous network which stained with tubulin antiserum. This network was absent when cells were treated with colchicine or vinblastine sulfate prior to chromosome isolation, and probably represents a remnant of the mitotic spindle. Most isolated chromosomes were connected to the spindle by fibers attached to the centromeres. Such fibers did not stain for DNA and were resistant to DNases but sensitive to proteases. No interconnections were found to run from chromosome to chromosome except for occasional artifactual adhesions resulting from collisions between chromosomes which occurred during micromanipulation. We therefore found no evidence that chromosomes of the Indian muntjac are interconnected at mitosis.     

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis. J. Cell. Biochem. 65:114–130. © 1997 Wiley-Liss, Inc.     

Cytological and functional aspects of telomere maintenance

The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity. Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions. However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions. Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis. Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer. Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.     

端粒和端粒酶的发现及其生物学意义

2009年的诺贝尔生理学或医学奖授予了美国加州大学旧金山分校的Elizabeth H．Blackburn、约翰霍普金斯大学的Carol W．Greider以及哈佛医学院的Jack W．Szostak三位科学家,肯定他们在发现端粒以及端粒酶保护染色体末端方面所做出的贡献。端粒以及端粒酶的发现历经近半个世纪,追溯起端粒和端粒酶的整个发现过程,却是耐人寻味,给人启发。端粒是真核生物中位于染色体末端的DNA和蛋白质的复合物,它对于维持基因组的完整性以及染色体的稳定性都有着至关重要的作用。端粒DNA可以被一种特化的称为“端粒酶”的逆转录酶延伸。端粒长度的维持以及端粒结构的稳定在细胞衰老、癌症发生以及干细胞全能性自我更新能力维持等生命过程中都起重要作用。     

Nonrandom Sister Chromatid Segregation and Nuclear Migration in Hyphae of Aspergillus nidulans

Radioactive conidiospores of Aspergillus nidulans were prepared by growing a purine-requiring mutant with tritiated adenine. When these spores germinated in a nonradioactive medium, the dispersion of the original chromosome set could be followed by treating the hyphae with ribonuclease and preparing radioautograms. Germinating spores with four or eight nuclei contained two highly labeled nuclei and two or six nuclei with much less or no radioactivity. Successive mitotic divisions thus distributed the deoxyribonucleic acid (DNA) of the eight spore chromosomes among only two of the progeny nuclei. The two nuclei containing the original chromosome set were not dispersed at random along the linear hypha but were usually located near the growing tip. These results are compatible with the view that chromatids containing DNA strands of identical age segregate as a unit during mitosis. They further indicate that the mechanism which disperses newly formed nuclei in the growing hypha can distinguish between nuclei containing DNA strands of different ages.     

Chromosome rearrangements resulting from telomere dysfunction and their role in cancer

Telomeres play a vital role in protecting the ends of chromosomes and preventing chromosome fusion. The failure of cancer cells to properly maintain telomeres can be an important source of the chromosome instability involved in cancer cell progression. Telomere loss results in sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles, leading to extensive DNA amplification and large deletions. These B/F/B cycles end primarily when the unstable chromosome acquires a new telomere by translocation of the ends of other chromosomes. Many of these translocations are nonreciprocal, resulting in the loss of the telomere from the donor chromosome, providing a mechanism for transfer of instability from one chromosome to another until a chromosome acquires a telomere by a mechanism other than nonreciprocal translocation. B/F/B cycles can also result in other forms of chromosome rearrangements, including double-minute chromosomes and large duplications. Thus, the loss of a single telomere can result in instability in multiple chromosomes, and generate many of the types of rearrangements commonly associated with human cancer.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

Frequent transpositions of Drosophila melanogaster HeT-A transposable elements to receding chromosome ends.

HeT-A elements are a new family of transposable elements in Drosophila that are found exclusively in telomeric regions and in the pericentric heterochromatin. Transposition of these elements onto broken chromosome ends has been implicated in chromosome healing. To monitor the fate of HeT-A elements that had attached to broken ends of the X chromosome, we examined individual X chromosomes from a defined population over a period of 17 generations. The ends of the X chromosomes with new HeT-A additions receded at the same rate as the broken ends before the HeT-A elements attached. In addition, some chromosomes, approximately 1% per generation, had acquired new HeT-A sequences of an average of 6 kb at their ends with oligo(A) tails at the junctions. Thus, the rate of addition of new material per generation matches the observed rate of terminal loss (70-75 bp) caused by incomplete replication at the end of the DNA molecule. One such recently transposed HeT-A element which is at least 12 kb in length has been examined in detail. It contains a single open reading frame of 2.8 kb which codes for a gag-like protein.