A genetic analysis of DNA sequence requirements for Dissociation state I activity in tobacco.

Our objective was to test whether the double Ds structure correlated with Dissociation state I activity (i.e., high frequency of chromosome breakage and low frequency of reversion) in maize exhibited similar properties in tobacco. A genetic assay was established to test double Ds and related structures for their ability to cause loss of the linked marker genes streptomycin phosphotransferase and beta-glucuronidase in transgenic tobacco. An engineered double Ds element and a simple Ds element showed behavior consistent with that of state I and state II Ds elements, respectively, as described for maize. DNA structural rearrangements accompanied marker gene loss. Dissection of the double Ds structure showed that a left end and a right end of Ds in direct orientation were sufficient for the instability observed. This result suggested that left and right ends of Ds in direct orientation can participate in aberrant transposition events, consistent with two different models for double Ds-induced chromosome breakage proposed previously. Both models predict that the inversion of a half Ds element accompanies the aberrant transposition event. Such an inversion was detected by polymerase chain reaction experiments in tobacco and maize only when Activator activity was present in the genome.     

Reversed end Ds element: a novel tool for chromosome engineering in Arabidopsis

The maize Ac/Ds transposable element (TE) transposes by a "cut and paste" mechanism. Previous studies in maize showed that when the TE ends are in reversed orientation with respect to each other, alternative transposition reactions can occur resulting in large scale genome rearrangements including deletions and inversions. To test whether similar genome rearrangements can also occur in other plants, we studied the efficacy of such alternative transposition-mediated genome rearrangements in Arabidopsis. Here we present our analysis of 33 independent chromosome rearrangements. Transposition at the reversed ends Ds element can cause deletions over 1 Mbp, and inversions up to 2.4 Mbp in size. We identified additional rearrangements including a reciprocal translocation and a putative ring chromosome. Some of the deletions and inversions are germinally transmitted.     

Molecular evidence that chromosome breakage by Ds elements is caused by aberrant transposition.

The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.     

Shugoshin1 may play important roles in separation of homologous chromosomes and sister chromatids during mouse oocyte meiosis

Background

Homologous chromosomes separate in meiosis I and sister chromatids separate in meiosis II, generating haploid gametes. To address the question why sister chromatids do not separate in meiosis I, we explored the roles of Shogoshin1 (Sgo1) in chromosome separation during oocyte meiosis.

Methodology/Principal Findings

Sgo1 function was evaluated by exogenous overexpression to enhance its roles and RNAi to suppress its roles during two meioses of mouse oocytes. Immunocytochemistry and chromosome spread were used to evaluate phenotypes. The exogenous Sgo1 overexpression kept homologous chromosomes and sister chromatids not to separate in meiosis I and meiosis II, respectively, while the Sgo1 RNAi promoted premature separation of sister chromatids.

Conclusions

Our results reveal that prevention of premature separation of sister chromatids in meiosis I requires the retention of centromeric Sgo1, while normal separation of sister chromatids in meiosis II requires loss of centromeric Sgo1.     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

Inverted meiosis and its place in the evolution of sexual reproduction pathways

Inverted meiosis is observed in plants (Cyperaceae and Juncaceae) and insects (Coccoidea, Aphididae) with holocentric chromosomes, the centromeres of which occupy from 70 to 90% of the metaphase chromosome length. In the first meiotic division (meiosis I), chiasmata are formed and rodlike bivalents orient equationally, and in anaphase I, sister chromatids segregate to the poles; the diploid chromosome number is maintained. Non-sister chromatids of homologous chromosomes remain in contact during interkinesis and prophase II and segregate in anaphase II, forming haploid chromosome sets. The segregation of sister chromatids in meiosis I was demonstrated by example of three plant species that were heterozygous for chromosomal rearrangements. In these species, sister chromatids, marked with rearrangement, segregated in anaphase I. Using fluorescent antibodies, it was demonstrated that meiotic recombination enzymes Spo11 and Rad5l, typical of canonical meiosis, functioned at the meiotic prophase I of pollen mother cells of Luzula elegance and Rhynchospora pubera. Moreover, antibodies to synaptonemal complexes proteins ASY1 and ZYP1 were visualized as filamentous structures, pointing to probable formation of synaptonemal complexes. In L. elegance, chiasmata are formed by means of chromatin threads containing satellite DNA. According to the hypothesis of the author of this review, equational division of sister chromatids at meiosis I in the organisms with inverted meiosis can be explained by the absence of specific meiotic proteins (shugoshins). These proteins are able to protect cohesins of holocentric centromeres from hydrolysis by separases at meiosis I, as occurs in the organisms with monocentric chromosomes and canonical meiosis. The basic type of inverted meiosis was described in Coccoidea and Aphididae males. In their females, the variants of parthenogenesis were also observed. Until now, the methods of molecular cytogenetics were not applied for the analysis of inverted meiosis in Coccoidea and Aphididae. Evolutionary, inverted meiosis is thought to have appeared secondarily as an adaptation of the molecular mechanisms of canonical meiosis to chromosome holocentrism.     

Transposon Ac/Ds-induced chromosomal rearrangements at the rice OsRLG5 locus

Previous studies have shown that pairs of closely-linked Ac/Ds transposable elements can induce various chromosomal rearrangements in plant genomes. To study chromosomal rearrangements in rice, we isolated a line (OsRLG5-161) that contains two inversely-oriented Ds insertions in OsRLG5 (Oryza sativa Receptor like kinase Gene 5). Among approximately 300 plants regenerated from OsRLG5-161 heterozygous seeds, 107 contained rearrangements including deletions, duplications and inversions of various sizes. Most rearrangements were induced by previously identified alternative transposition mechanism. Furthermore, we also detected a new class of rearrangements that contain juxtaposed inversions and deletions on the same chromosome. We propose that these novel alleles were generated by a previously unreported type of alternative transposition reactions involving the 5' and 3' termini of two inversely-oriented Ds elements located on the same chromatid. Finally, 11% of rearrangements contained inversions resulting from homologous recombination between the two inverted Ds elements in OsRLG5-161. The high frequency inheritance and great variety of rearrangements obtained suggests that the rice regeneration system results in a burst of transposition activity and a relaxation of the controls which normally limit the transposition competence of individual Ds termini. Together, these results demonstrate a greatly enlarged potential of the Ac/Ds system for plant chromosome engineering.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Chromosome Breakage by Pairs of Closely Linked Transposable Elements of the Ac-Ds Family in Maize

Chromosome breaks and hence chromosomal rearrangements often occur in maize stocks harboring transposable elements (TEs), yet it is not clear what types of TE structures promote breakage. We have shown previously that chromosomes containing a complex transposon structure consisting of an Ac (Activator) element closely linked in direct orientation to a terminally deleted or fractured Ac (fAc) element have a strong tendency to break during endosperm development. Here we show that pairs of closely linked transposons with intact ends, either two Ac elements--a common product of Ac transposition--or an Ac and a Ds (Dissociation) element, can constitute chromosome-breaking structures, and that the frequency of breakage is inversely related to intertransposon distance. Similar structures may also be implicated in chromosome breaks in other eukaryotic TE systems known to produce chromosomal rearrangements. The present findings are discussed in light of a model of chromosome breakage that is based on the transposition of a partially replicated macrotransposon delimited by the outside ends of the two linked TEs.     

Regulation of activator/dissociation transposition by replication and DNA methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.     

Evolution of meiosis of unicellulate and multicellular eucaryotes. Aromorphosis at the cellular level

An attempt was undertaken to apply the concept elaborated for the evolution of multicellular organisms to that of unicellular eucaryotes. The latter's meiosis was formed on the basis of combination on three intracellular processes: 1) homologous DNA recombination, 2) chromosome disjunction with the assistance of mitotic apparatus, and 3) formation of "linear" chromosome elements consisting of specific proteins. Mechanism of homologous chromosome recombination was inherited from the archibacteria, while both the mitotic apparatus and "linear" chromosome elements emerged de novo. These elements appeared (resulting from appearance of the meiosis-specific proteins) as a complication of cohesion filaments, arising at the boundary between the sister chromatids after DNA replication. Homologous chromosome recombination made it possible for the chromosomes of diploid organisms to join pairwise by means of Holliday structures, while temporary blocking of hydrolysis of the linear elements at centromeres made it possible for the kinetochores to acquire unipolarity and for the sister chromatids to move to the same pole. All these provided for reduction of the chromosome number. Such a type of the reduction of chromosome number was retained by the extant imperfect ascomycetes Schizosaccharomyces pombe and Aspergillus nidulans, and by the infusorian Tetrahyrmena thermophila. It was the derivative of specific proteins, i.e. synaptonemal complexes (SCs). that appeared to be aromorphosis; they came to existence due to the pairwise joining of the chromosome "linear" elements by means of protein "zipper". The SCs join homologous chromosomes temporarily at the prophase of meiotic reduction division, thus optimizing condition for the crossing over and chiasma formation. The latter and the kinetochore unipolarity both provide for the chromosome disjunction. Kinetochore unipolarity is caused by the protein shugoshin which appears at meiotic prophase I and blocks cohesin hydrolysis at centromeres when anaphase I begins. This type of reductional division became the basis of the classical meiosis in the overwhelming majority of unicellular and multicellular organisms over all eucaryote kingdoms.     

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.     

Characteristics of the cis- and trans-positions of chromatids of human acrocentric chromosomes in extreme old age

Cis- and trans-positions of chromatid associations of human acrocentric chromosomes were examined at extreme old age. Lymphocyte cultures were prepared by the usual method, from peripheral blood of 9 subjects aged 80-90 years (analysis of 179 metaphases) and 7 subjects aged 20-48 years (analysis of 124 metaphases). The functional difference between stalks of the sister chromatids was found. In the subjects at the age 80-90 years satellite stalks of chromatids-1 (in all DNA strands thymidine was substituted by 5-BrdU) of the D chromosome in cis-position are included into associations with lower frequency, as compared with the satellite stalks of chromatids-2 (thymidine+ is only substituted by 5-BrdU in a half DNA strands of the chromosome). This apparently reflects variability of regulation of functional activity of satellite stalks of sister chromatids.     

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.     

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.     

Detection of sister chromatid exchanges by 4′-6-diamidino-2-phenylindole fluorescence

This paper describes a 4-6-diamidino-2-phenylindole (DAPI) fluorescent technique for differentiation of sister chromatids and for the study of sister chromatid exchanges (SCE) in mouse chromosomes. The advantages of the DAPI fluorescent technique are also described. Differences in the occurrence of SCE between the centromeric heterochromatin (C-banded) and the chromosomal arm chromatin were studied in mouse cells (RAG) with or without mitomycin C treatment. Single strand exchanges between the DNA double helices in the sister chromatids were not detected. SCE and chromosome breakage appeared to occur more frequently in the centromeric region than in the chromosomal arm. This might play an important role in chromosome evolution in mice.     

Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation

The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases. Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss. In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere. Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules. This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching. Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split. Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p.     

Barley chromosome arms longer than half of the spindle axis interfere with nuclear divisions

We have tested the influence of recombinantly-elongated chromosome arms on nuclear divisions in barley and confirmed a rule according to which half the length of the average spindle axis defines the upper tolerance limit for chromosome arm length. A slightly longer chromosome arm caused incomplete separation of sister chromatids in approximately 70% of mitotic telophase cells and >2.5% of daughter cells showing a micronucleus, due to disruption of non-separated sister chromatids by the newly forming cell wall. In homozygous condition, this elongated chromosome mediated a slower growth and reduced fertility of the carrier plants. Its meiotic transmission was not impaired because of the larger spindle dimensions in meiocytes as compared to those in mitotic cells.     

Fluorometrical detection of thymine base differences in complementary strands of satellite DNA in human metaphase chromosomes.

Using the fluorochrome "Hoechst 33258", intensity of fluorescence was found to differ distinctly between the sister chromatids in the paracentric regions of chromosomes 1, 16, and 19, after one round of replication in medium containing BUdR. Thus the effect of fluorescence asymmetry is not limited to the part of the Y chromosomes that fluoresces intensely with quinacrine; it can also be determined in the weakly Q-fluorescent pericentric regions of chromosomes, which are known to be the sites where highly reiterated sequences of satellite DNA are located. However, an exception is the paracentric region of chromosome 9 which does not show the effect of lateral asymmetry. The difference of fluorescence intensity in the heterochromatic regions of the sister chromatids of human chromosome 1 is measured by densitometric tracement along the long axes of chromosomes; this is obtained from two individuals with an "uncoiler" heterchomatic block (type III) having a relative intensity of 1:1.93 in an average of the total measured blocks. This corresponds to the uneven distribution of thymine base of 22.8 and 43.2 in the two strands of the DNA double hexlix. A chromatid exchange rate of 9 in 100 metaphases per cell cycle was found within the uncoiler region of chromosome 1.