小麦染色体轴结构的观察

对普通小麦 ( Triticum aestivum L.)中期染色体进行常规制片银染的结果显示 ,染色体中存在着染色深的轴结构 ,每个染色单体一条 ,轴在有些部位似乎是螺旋的。研究结果对染色体轴结构的真实性提供了证据     

The bending rigidity of mitotic chromosomes

The bending rigidities of mitotic chromosomes isolated from cultured N. viridescens (newt) and Xenopus epithelial cells were measured by observing their spontaneous thermal bending fluctuations. When combined with simultaneous measurement of stretching elasticity, these measurements constrain models for higher order mitotic chromosome structure. We measured bending rigidities of B approximately 10(-22) N. m(2) for newt and approximately 10(-23) N. m(2) for Xenopus chromosomes extracted from cells. A similar bending rigidity was measured for newt chromosomes in vivo by observing bending fluctuations in metaphase-arrested cells. Following each bending rigidity measurement, a stretching (Young's) modulus of the same chromosome was measured in the range of 10(2) to 10(3) Pa for newt and Xenopus chromosomes. For each chromosome, these values of B and Y are consistent with those expected for a simple elastic rod, B approximately YR(4), where R is the chromosome cross-section radius. Our measurements rule out the possibility that chromosome stretching and bending elasticity are principally due to a stiff central core region and are instead indicative of an internal structure, which is essentially homogeneous in its connectivity across the chromosome cross-section.     

Silver staining of histone-depleted metaphase chromosomes

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.     

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.     

Structure of a frequently rearranged rRNA-encoding chromosome in Giardia lamblia.

It has been shown previously that the rRNA encoding chromosomes in Giardia lamblia undergo frequent rearrangements with an estimated rate of approximately 1% per cell per division (Le Blancq et al., 1992, Nucleic Acids Res., 17, 4539-4545). Following these observations, we searched for highly recombinogenic regions in one of the frequently rearranged rRNA encoding chromosomes, that is chromosome 1, a small, 1.1 Mb chromosome. Chromosome 1 undergoes frequent rearrangements that result in size variation of 5-20%. We analyzed the structure of chromosome 1 in clonal lineages from the WB strain. The two ends of chromosome 1 comprise telomere repeat [TAGGG] arrays joined to a truncated rRNA gene and a sequence referred to as '4e', respectively. Comparison of the structure of four polymorphic versions of chromosome 1, resulting from independent rearrangement events in four cloned lines, located a single polymorphic region to the variable rDNA-telomere domain. Chromosome 1 is organized into two domains: a core region spanning approximately 850 kb that does not exhibit size heterogeneity among different chromosome 1 and a variable region that spans 185-450 kb and includes the telomeric rRNA genes, referred to as the variable rDNA-telomere domain. The core region contains a conserved region, spanning approximately 550 kb adjacent to the telomeric 4e sequence, which is only present in the 4e containing chromosomes and a 300 kb region of repetitive sequences that are also components of other chromosomes as well. Changes in the number of rDNA repeats accounted for some, but not all, of the size variation. Since there are four chromosomes that share the core region of chromosome 1, we suggest that the genome is tetraploid for this chromosome.     

红翅皱膝蝗减数分裂染色体的螺旋与轴结构

用低渗处理和苯酚品红染色,在经过卡诺液(甲醇3∶冰醋酸1)固定和未经固定的红翅皱膝蝗减数分裂染色体上都看到了螺旋结构。观察和测量结果表明,每条染色单体都是由430nm左右的染色线螺旋形成的。由染色线到染色体的压缩率为4∶1。低渗处理后固定的材料经过银染,则显示了染色体轴结构。同样,未经低渗处理直接固定的材料银染时也出现了轴结构。银染的轴结构位于每个染色单体的中央,并贯穿整个染色单体。在光镜下,这个轴并不是直径均一的棒状结构,而似乎是由许多大小相近的颗粒相连而成。本文对染色体结构的有关模型、骨架和轴结构的真实性以及轴和螺旋的关系等问题进行了讨论。     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

Immunofluorescent staining of human metaphase chromosomes with monoclonal antibody to histone H2B

A mouse monoclonal IgM antibody against the core histone H2B has been shown, by indirect immunofluorescence, to stain metaphase chromosomes from a variety of cultured cell types. Experiments carried out with human HeLa cells showed that the intensity of staining varied along the length of chromosome arms giving in some cases a rudimentary banded staining pattern. Considerable variation in staining intensity was noted between individual chromosomes and between different metaphase spreads. It was noted that chromosomes having a more swollen appearance stained more intensely than those with a more compact structure, which were often unstained. Preincubation of unfixed metaphase chromosomes in buffered salt solutions virtually eliminated the cell to cell and chromosome to chromosome variation in staining, even when no visible effect on chromosome morphology was caused by such treatment. It is concluded that the determinant recognised by antibody HBC-7 is ubiquitous but is inaccessible in some chromosomes or chromosome regions. Digestion of purified chromatin (primarily interphase) with DNAase 1 or micrococcal nuclease resulted in a several-fold increase in the binding of antibody HBC-7 measured by solid-phase radioimmunoassay. This increase was abolished by subsequent treatment with trypsin, which suggests that the antigenic determinant recognised by antibody HBC-7 lies in the trypsin-sensitive N-terminal region of nucleosomal H2B. As the cationic N-terminal regions of the core histones are involved in DNA binding, it is likely that the accessibility of the determinant recognised by antibody HBC-7 is influenced by the relationship between the core histones and their associated DNA.     

Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici

The genomes of many filamentous fungi consist of a ‘core’ part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage‐specific (LS) chromosomes. In the plant‐pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the ‘effector’ LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1‐Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole‐genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.     

Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores

In a previous report [2] we have described a non-histone protein core which could be isolated from Chinese hamster metaphase chromosomes. This core structure maintained the overall morphology of the metaphase chromosome even after removal of all of the histones, together with many of the non-histone proteins and the bulk of the DNA. As part of our work on the characterization of these core structures, we have developed a novel procedure for the isolation of metaphase chromosomes which avoids the use of high pH buffers and hexylene glycol, as well as eliminating the numerous centrifugation and resuspension steps previously employed. Chromosome cores prepared by 2 M NaCl extraction and DNase I digestion from metaphase chromosomes isolated under these more gentle, quasi-physiological conditions, are shown to contain a relatively simple subset of non-histone proteins. One-dimensional SDS-polyacrylamide gel electrophoresis shows two major groups of polypeptides having molecular weights 48 000-52 000 and 65 000-72 000 D respectively, with similarities in mobilities to the nuclear pore complex-lamina polypeptides and tubulins. However, more detailed analysis by two-dimensional gel electrophoresis and peptide mapping has failed to detect these proteins. A 52 000 D polypeptide component of the core is tentatively identified as the intermediate filament protein vimentin. The in vivo significance of chromosome cores is discussed.     

The Relation between the Axial Complex of Meiotic Prophase Chromosomes and Chromosome Pairing in a Salamander (Plethodon cinereus)

An investigation of the structure of meiotic chromosomes from primary spermatocytes of two salamanders, Plethodon cinereus and Desmognathus fusca, has been made using correlated light and electron microscopy. Feulgen squashes were compared with stained sections and these related to adjacent thin sections in the electron microscope. A transition from the familiar cytological preparation to the electron image was thus effected. A linear complex consisting of three parallel strands has been observed with the electron microscope, passing along the central axis of primary spermatocyte chromosomes. The complex is similar to that found in comparable chromosomes from at least a dozen animal species. The structure in Plethodon is described in detail. Synapsis has been positively identified as the stage of meiotic prophase at which the complex occurs. Thus the complex is a part of bivalent chromosomes. It has not been seen in other stages or other divisions and is thus thought to be exclusively of synaptic occurrence. The term synaptinemal complex is suggested for the entire structure. By virtue of the material condensed around it, the complex is also seen in the light microscope where it appears as a fine, densely Feulgen-positive central core along the chromosome. The complex is thus closely associated with DNA, if not at least in part, composed of it. In the stages studied, homologous chromosomes are not always completely paired. The lateral elements of the complex separate and follow the single chromosome axes at these points. The central element disappears and thus may be a phenomenon of pairing. It is concluded that the lateral elements of the synaptinemal complex may more correctly be a "core" of the single meiotic prophase chromosome, possibly being concerned with its linear organization.     

Chromosome Markers in MUS MUSCULUS: Strain Differences in C-Banding

The mitotic chromosomes of several inbred strains of mice and a series of F(1) hybrids have been analyzed by quinacrine staining and further characterized by the centromeric heterochromatin banding (C-banding). Inbred strains had the same amount of C-banding material on homologous chromosomes but showed variation in the amount on different chromosomes. F(1) hybrids showed characteristics of each parent and it appears that the amount of C-banding on each chromosome is a simple inherited polymorphism. In this study 12 different chromosomes could be distinguished by their C-banding, and these can be used as normal chromosome markers.     

Breakage-fusion-bridge Cycles and Large Insertions Contribute to the Rapid Evolution of Accessory Chromosomes in a Fungal Pathogen

Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens.     

Insights into the kinetochore

The Ndc80 complex is a core component of the kinetochore, which links chromosomes to microtubules. Recently, Ciferri et al. (2008) published an atomic-level structure of the complex with implications for kinetochore architecture and for the generation and control of chromosome movements during mitosis.     

The G-banded prophase chromosomes of man.

Using a simple G-banding technique developed in our laboratory, analysis of late prophases enables the visualization of approximately 1000 bands in the haploid set of human chromosomes. These bands have been classified according to the recommendations of the Paris Conference. The increased resolution offered by this technique is likely to be useful in the study of the structure and molecular organization of chromosomes and in identifying minute chromosome defects in birth defects and neoplasia.     

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.     

The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo

The effect of chromatin structure on the binding of a chemical carcinogen to the genomic DNA was studied. The binding in vivo of the ultimate carcinogen, benzo-pyrene 7,8,-diol,-9,10-epoxide, to various regions of the SV40 chromosome was revealed by an immunological method. Particular attention was given to restriction fragments which include the origin of replication which is "non-nucleosomal" in a significant fraction of the chromosomes. The distribution of (+/-) trans-7,8-dihydrobenzo[alpha]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts was studied in 1) SV40 DNA modified in vitro to a level of 20 adducts/molecule, 2) DNA from SV40 chromosomes modified in vivo to a level of less than 1 adduct, and 3) DNA from only those chromosomes with an open origin of replication. In other experiments, the binding of BPDE to the origin region was compared to the binding to nucleosome core particle DNA from the viral chromosome. The origin region bound 1.7-fold more BPDE than core DNA, while linker DNA is 3-fold more modified than core DNA. However, the origin region was only about 20% more modified than any other region of the chromosome. We conclude that while the conformation of the DNA in chromatin has a slight effect on its accessibility to the carcinogen, the SV40 chromosome does not contain a particular "hot spot" which is preferentially modified by BPDE.     

Human Chromosomes: I. Preparation,Analysis and Diagnostic Implications of Abnormalities

This presentation is designed to help persons unfamiliar with cytogenetic terms and techniques to understand how chromosomes are prepared and analyzed to determine if abnormalities exist. A diagrammatic flow-sheet has been used to show how peripheral blood is treated to stimulate growth of cells, and how chromosomes are dispersed and made readily visible. Technical details have been replaced by simple statements explaining the various steps. By careful attention to differences (over-all length, position of centromere, length of arms, presence or absence of satellites), chromosomes may be arranged in order of length and into definite groups to form a typical karyotype of a person''s cells. Familiarity with chromosomes makes it possible to determine (a) if an extra chromosome is present, (b) if a chromosome is missing, or (c) if an abnormal chromosome exists.     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

A search for protein cores in chromosomes: Is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.