Effect of mitomycin C on the structure of human chromosomes observed in metaphase after labelling by moderate thermal denaturation]

Mitomycin C induced chromosome rearrangements were analysed in cultured human leukocytes by reverse banding technique. Breaks and chromosomal exchanges involved preferentialy the entromeric region of some chromosomes (1, 5, 9, 16, and 20). Associations between acrocentric chromosomes was not found to be increased. But acrocentric associations with centromeric regions were frequently present. The differences between the mechanism of exchanges and breaks are discussed. The part of heterochromatin in post replication DNA repair is considered.     

Molecular mechanisms of production of symmetrical and asymmetrical chromosome exchanges

The formation of a heteroduplex is probably the first step leading to chromosome exchanges. Heteroduplexes occur by complementary association of two single DNA strands from different chromosomes. Therefore, repetitive DNA is the most common region involved in heteroduplex formation. DNA repeats are defined as polarized when they run the same, and antipolarized when they run opposite from centromere to telomere in two different chromosomes or chromosome arms. Paracentric inversions may easily account for the origin of antipolarized repeats. Palindromes are a special type of reversed repeats which always run the same from centromere to telomere independently of the existence or not of chromosomal rearrangements. Heteroduplexes leading to symmetrical exchanges can only occur by association of DNA strands with polarized repeats. On the other hand, antipolarized repeats are essential for the occurrence of asymmetrical rearrangements. Accordingly, the frequency of induced symmetrical and asymmetrical exchanges in a cell population may partially depend on the frequency of polarized and antipolarized repeats in the genome.     

Restriction endonuclease and molecular analyses of three rat genomes with special reference to chromosome rearrangement and speciation problems

When differences are found between related species of organisms, it is often assumed that the differences themselves are causal factors either in speciation itself or in processes related to speciation. Two recent proposals on the functions of satellite DNA (Hatch et al., 1976 and Fry and Salser, 1977) are that (a) large amounts of satellite DNA are important in facilitating chromosome rearrangements and hence cytogenetic evolution, and (b) satellite DNA differences between homologous chromosomes lead to pairing difficulties and are important in generating infertility barriers and hence speciation. If these proposals were to have some generality, one could expect organisms with very low amounts of highly repeated DNA to exhibit few chromosome rearrangements and to be evolutionarily conservative in a cyto-genetic sense. — We have chosen two very closely related species of rat which are phenotypically almost indistinguishable and which have undergone massive genome reorganization. They differ by 11 major centric rearrangements (2n=32, 2n=50). We have characterised their genomes by restriction endonuclease digestions, thermal denaturations, analytical ultracentrifugations and reassociation techniques, and have found that they have virtually no highly repeated DNA. Thus the 11 major chromosomal rearrangements have been fixed in present day genomes with hardly any highly repeated DNA, centric or otherwise. — It appears therefore that a large amount of highly repeated DNA is not obligatory for the formation and fixation of chromosome rearrangements. In addition, the existing literature reveals that one can find almost any situation at all, from species groups with high amounts of satellite DNA and no gross chromosomal rearrangements, to ones such as those described here, with tiny amounts of highly repeated DNA and massive chromosomal reorganisation. Since direct experimental data indicates that satellite DNA differences per se between homologous chromosomes do not cause infertility, speculations concerning modes of speciation based on satellite DNA differences between otherwise homologous chromosomes would appear to be ill founded.     

A high degree of macronuclear chromosome polymorphism is generated by variable DNA rearrangements in Paramecium primaurelia during macronuclear differentiation.

DNA rearrangements in Paramecium lead to the formation of macronuclear chromosomes, the sizes of which range from 50 and 800 kb (1 kb is 10(3) base-pairs). This process does not appear to be a simple size reduction of the micronuclear chromosomes by specific and reproducible DNA sequence elimination and chromosomal breakage followed by chromosomal amplification. On the contrary, this process generates a variety of different, but sequence-related, macronuclear chromosomes from a unique set of micronuclear chromosomes. This paper describes an attempt to understand the nature of the diversity of the macronuclear chromosomes and the mechanisms of their production. The structure of three macronuclear chromosomes, 480, 250 and 230 kb in size, have been determined utilizing chromosome-jumping and YAC-cloning techniques. The two smallest chromosomes correspond roughly to the two halves of the longest chromosome. The main contribution to the diversity arises from the chromosomal ends and is due to variable positions of the telomere addition sites and/or to variable rearrangements of DNA sequences. The 480 kb chromosome contains a region of variable length, which is likely to be due to a variable deletion, located at the position of telomerization seen in the two small chromosomes. A model of chromosomal breakage is proposed to rationalize this result where micronuclear DNA is first amplified, broken and degraded to various extent from the newly formed ends, which subsequently are either telomerized or religated. Potential implications of these processes for gene expression is discussed. Known phenotypes that have a macronuclear determinism could be explained by this type of process.     

Studies on the Mitotic Prophase Chromosomal Fibers in Vicia faba

The observations have been made on the structures of mitotic prophase nuclei and chromosomes in Vicia faba root meristematic cells. Two methods, i.e., the cell squashing and the nucleus isolation methods, were applied in present study to prepare the specimen of chromosomes and nuclei. Chromosomal fibers 0.3—0.5 μm in diameter were observed in the squashed preparations stained with Giemsa, and in the isolated nucleus preparations treated with 0.05% EDTA followed by Giemsa staining. Using Feulgen reaction, it has been demonstrated that these fibers are nuclear origin containing DNA. The results suggest that this order of chromosomal fiber may be one structural level in the chromosomes in Vicia faba. This conclusion is in support of the view which holds that there exists an intermediate level of structure between the 250–300Å chromatin fiber and the chromosome.     

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.     

Frequency and sites of sister chromatid exchanges in rat kangaroo chromosomes

The frequency of sister chromatid exchanges in the chromosomes of a cell line from the Tasmanian rat kangaroo was determined to be 0.79 exchanges per chromosome for two cell cycles. Twenty-five percent of these exchanges occurred at the kinetochore. The mean frequency of exchanges per chromosomal arm was roughly proportional to the length of the chromosome, with the exception of a mean frequency of 0.20 exchanges per chromosome found at the kinetochore of all chromosomes, regardless of length. Thus, the kinetochore is a highly preferential site for sister chromatid exchanges. Compared to the main portion of the chromosomal arms the exchange frequency was somewhat lower adjacent to the kinetochore and at chromosome ends. The number of exchanges per unit length also tended to be lower for the short arm of chromosome 1. No correlation was found between the frequency of exchanges and late-replicating DNA.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

I-R hybrid dysgenesis in Drosophila melanogaster. Use of in situ hybridization to show the association of I factor DNA with induced sex-linked recessive lethals.

The purpose of this paper is the genetic visualization by in situ hybridization of 130 sex-linked recessive lethals plus a non-lethal induced by I-R dysgenesis. This collection of lethals involves inducer strains which differ in the position of the I elements on the X chromosomes. The I-R interaction was strong. Our previous results have shown that about 30% of the induced recessive lethals are associated with cytologically visible chromosomal rearrangements. (1) The rearrangements induced by I-R-type hybrid dysgenesis often exhibit homology with the I factor at the level of one or both junction points, depending on the types of chromosome rearrangements. These results suggest that the chromosome rearrangements arise directly from the transposition of I elements. However, the breakpoints of some types of cytologically non-visible deficiencies and of 2 small cytologically visible deficiencies do not present detectable homology with the I factor. (2) The majority of rearrangements do not involve the I elements already present on the paternal X chromosome. (3) The hybridization signal distributions on the X chromosome are not uniform. They present peaks of various heights which may correspond to specific anchoring areas of copies of I in the course of integration. (4) The data presented here agree with the literature with respect to the mean number of copies of I per X chromosome and to the excess of copies of I at locus 1A. Two rearrangement formation mechanisms are envisaged: crossing-over and 'target' exchanges.     

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.     

The role of chromosomal rearrangements in the evolution of <Emphasis Type="Italic">Silene latifolia</Emphasis> sex chromosomes

Silene latifolia is a model plant for studies of the early steps of sex chromosome evolution. In comparison to mammalian sex chromosomes that evolved 300 mya, sex chromosomes of S. latifolia appeared approximately 20 mya. Here, we combine results from physical mapping of sex-linked genes using polymerase chain reaction on microdissected arms of the S. latifolia X chromosome, and fluorescence in situ hybridization analysis of a new cytogenetic marker, Silene tandem repeat accumulated on the Y chromosome. The data are interpreted in the light of current genetic linkage maps of the X chromosome and a physical map of the Y chromosome. Our results identify the position of the centromere relative to the mapped genes on the X chromosome. We suggest that the evolution of the S. latifolia Y chromosome has been accompanied by at least one paracentric and one pericentric inversion. These results indicate that large chromosomal rearrangements have played an important role in Y chromosome evolution in S. latifolia and that chromosomal rearrangements are an integral part of sex chromosome evolution.     

The presence of interstitial telomeric sequences in constitutional chromosome abnormalities.

We describe a novel chromosome structure in which telomeric sequences are present interstitially, at the apparent breakpoint junctions of structurally abnormal chromosomes. In the linear chromosomes with interstitial telomeric sequences, there were three sites of hybridization of the telomere consensus sequence within each derived chromosome: one at each terminus and one at the breakpoint junction. Telomeric sequences also were observed within a ring chromosome. The rearrangements examined were constitutional chromosome abnormalities with a breakpoint assigned to a terminal band. In each case (with the exception of the ring chromosome), an acentric segment of one chromosome was joined to the terminus of an apparently intact recipient chromosome. One case exhibited apparent instability of the chromosome rearrangement, resulting in somatic mosaicism. The rearrangements described here differ from the telomeric associations observed in certain tumors, which appear to represent end-to-end fusion of two or more intact chromosomes. The observed interstitial telomeric sequences appear to represent nonfunctional chromosomal elements, analogous to the inactivated centromeres observed in dicentric chromosomes.     

Common fragile sites as targets for chromosome rearrangements

Common fragile sites are large chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed. Fragile site instability in cultured cells is well documented and includes gaps and breaks on metaphase chromosomes, translocation and deletions breakpoints, and sister chromosome exchanges. In recent years, much has been learned about the genomic structure at fragile sites and the cellular mechanisms that monitor their stability. The study of fragile sites has merged with that of cell cycle checkpoints and DNA repair, with multiple proteins from these pathways implicated in fragile site stability, including ATR, BRCA1, CHK1, and RAD51. Since their discovery, fragile sites have been implicated in constitutional and cancer chromosome rearrangements in vivo and recent studies suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early tumorigenesis. Here we review the relationship of fragile sites to chromosome rearrangements, particularly in tumor cells, and discuss the mechanisms that may be involved.     

The three-dimensional fine structure of chromosomes in a prophaseDrosophila nucleus

It has been possible to identify the chromosomes in electron micrographs of a prophase neuroblast nucleus inDrosophila melanogaster by using stereophotographs of stacked transparencies made from serial sections. Depth was determined by increasing or decreasing the stereo angle. Identification was facilitated by the use of a stock carrying chromosomal rearrangements. In this stock only chromosome 2 is metacentric. Compound chromosomes [symbolized C(3L)RM and C(3R)RM] were formed from the left and from the right arms of chromosome 3, and the X chromosome was intercalated in an inverted position between the two arms of the Y (Y^SX·Y^L).—Three-dimensional aspects of the ultrastructure of these chromosomes were observed as follows. The compacted centric regions are composed of fibers coiled in irregular gyres. In the extended regions, the fibers subdivide and appear uncoiled except for regions of compaction—“chromomeres”—which are seen in both homologues. Each homologue appears to be composed of four fibers of 130 Å diameter, a total of eight for the prophase chromosome. Since the larger neuroblast cells are 8C in the G2 phase, it would seem that the 130 Å fiber is equivalent to the cytological chromatid.     

应用基因组原位杂交鉴定蓝粒小麦及其诱变后代

应用基因组原位杂交技术（GISH）对普通小麦（Triticum aestivumL.)和长穗偃麦草[Agropyron elongatum(Host)Beauv,2n=10x=70]杂交后选育出的蓝粒小麦蓝-58及其诱变后代的染色体组成进行了鉴定。结果表明,GISH可方便地检测到小麦遗传背景中的长穗偃麦草染色体或易位的片段。如前人报道,蓝-58（2n=42)是一个具有2条长穗偃麦草4E染色体的异代换系（4E/4D）。LW004可能是一个具有两对相互易位染色体的纯合系,其田间表现磷高效特性,LW43-3-4为41条染色体的蓝单体（40W 1’4E）,种子颜色为浅蓝色,通过此法还检测出一些染色体结构发生很大变异的材料如4E的单端体（40W 1‘4E),种子颜色为浅蓝色,通过此法还检测出一些染色结构发生很大变异的材料如4E的单端体（40W 1‘t4E)以及组型为39W 1‘4E 1‘t4E的个体,此项研究结果更为直观地表明控制蓝粒体状的基因的确在来自长穗偃麦草的染色体上。同时说明有效的突变方法与灵活方便的检测手段的有机结合在染色体工程材料的创制和染色体工程育种中起着至关重要的作用。     

Reorganization of the X chromosome in voles of the genus Microtus

Comparative chromosomal analysis is a powerful tool in the investigation of the mechanisms of chromosomal evolution. The accuracy of the analysis depends on the availability of region-specific markers to follow the fate of the particular chromosomal region through the evolution of species. We have assigned 12 unique sequences to the euchromatic part of the vole X chromosome, which serve as reliable markers of chromosomal segments. Together with region-specific libraries and GTG banding, these markers allow us to delineate the homologous regions of the X chromosomes in five species of the genus Microtus. We found that X chromosomes of these species differ by numerous rearrangements and all rearrangements are clustered at specific breakpoints. Moreover, these breakpoints were found to colocalise with repetitive and/or duplicated DNA sequences. We suggest that clusters of repeated and/or duplicated DNA sequences have played a crucial role in the formation of rearrangement hot spots during evolution of the X chromosome in the subgenus Microtus.     

Further observations on the nature of radiation-induced chromosomal interchanges recovered from Drosophila sperm

The induction and analysis of numerous translocations (identified genetically and characterized cytologically) between chromosomes 2 and 3 of Drosophila melanogaster have allowed us to reexamine three issues concerning the nature of radiation-induced interchanges in spermatozoa. First, our results support the idea that, relative to their mitotic metaphase length, all major chromosomal regions are similar in their breakability, whether euchromatic (proximal or distal) or heterochromatic. Second, analysis of all our reciprocal exchanges between the two chromosomes shows a statistically significant dependence of the position of the chromosome 2 breakpoint on that of the chromosome 3 breakpoint. Thirdly, our combined cytological and genetic approach strengthens the results of previous analyses, which suggested a strong tendency for chromosomal interchanges to be of the reciprocal type in multiple-break rearrangements. This indicates that if radiation induces chromosome breaks, then the resulting broken ends tend to rejoin in pairs rather than independently.     

Telomere functions: lessons from yeast

Telomeres are specialized DNA protein structures that form the ends of eukaryotic chromosomes. In yeast, loss of even a single telomere causes a prolonged, but transitory, cell-cycle arrest. During this arrest, many broken chromosomes acquire a new telomere by one of three pathways, although at the cost of a partial loss of heterozygosity. In addition, a substantial fraction of the chromosomes lacking a telomere is lost, which generates an aneuploid cell. In these cases, the broken chromosome is usually replicated and segregated for ten or more cell divisions in unstable form. Extrapolation from yeast suggests that the gradual loss of telomeric DNA that accompanies ageing in humans may initiate the kinds of chromosomal rearrangements and genetic changes that are associated with tumorigenesis.     

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.     

The fate of DNA statellites I,II, III and ribosomal DNA in a familial dicentric chromosome 13:14

Summary In a family with a stable dicentric 13:14 translocation chromosome, the distribution of DNA sequences complementary to satellite DNAs I, II and III and ribosomal RNA were studied. The translocation chromosome showed a loss of sequences complementary to all three satellite DNAs, located in the short arms of all the acrocentric chromosomes, but slightly more of the sequences complementary to satellite I were retained than of the other two satellite DNAs. The fact that material was lost from all three satellites indicates that they are not present as single discrete blocks in these chromosomes, when we would expect to find the distal sequences lost and the proximal ones retained, but consist of interspersed blocks with each sequence represented by more than one, and probably several blocks. There was a total loss of ribosomal DNA from the nucleolar organiser regions of the chromosomes involved in the 13:14 translocation, but an interesting finding was the presence of extra ribosomal DNA and satellite DNAs I, II and III in one chromosome 22 which was found in seven out of nine individuals of the family with the 13:14 translocation, and in only one of five individuals without the translocation. There may be a compensatory mechanism present when certain sequences are eliminated during chromosomal rearrangements. The relationship of such mechanisms to reproductive fitness is discussed.