Characterization of a maize isochromosome 8S*8S.

An isochromosome was found in the maize HiII Parent B line during somatic karyotyping with a multiprobe fluorescence in situ hybridization (FISH) system. Cytological analyses showed that it pairs with the short arm of chromosome 8 during the pachytene stage of meiosis. The chromosome 8 short arm origin of this isochromosome was also confirmed by FISH at mitotic metaphase. Knob heterochromatin signals were present at the short arms of chromosome 8 when subjected to prolonged exposure and also observed at both ends of the isochromosome. This isochromosome can be a univalent or a trivalent by pairing with the normal chromosome 8 short arms during meiosis. At anaphase and telophase, the isochromosome lagged behind other chromosomes. It had a transmission rate of 17%-20% from both male and female gametes. One plant homozygous for the isochromosome contained 2 isochromosomes that differed in the quantity of their CentC centromere repeat sequence. Both variations of the isochromosome were transmitted to the next generation. Because the 2 isochromosomes should be identical by descent, these observations document a radical change in copy number of the centromere repeat array within 1 generation. Plants with 1 isochromosome were not normal as compared with the original HiII Parent B plants. Those that contained a pair of this isochromosome (6 total copies of 8S) were even more abnormal and had reduced fertility. The results indicate the ability of the somatic karyotyping system to recognize and characterize chromosomal aberrations.     

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.     

Rapid analysis of Saccharomyces cerevisiae genome rearrangements by multiplex ligation-dependent probe amplification

Aneuploidy and gross chromosomal rearrangements (GCRs) can lead to genetic diseases and the development of cancer. We previously demonstrated that introduction of the repetitive retrotransposon Ty912 onto a nonessential chromosome arm of Saccharomyces cerevisiae led to increased genome instability predominantly due to increased rates of formation of monocentric nonreciprocal translocations. In this study, we adapted Multiplex Ligation-dependent Probe Amplification (MLPA) to analyze a large numbers of these GCRs. Using MLPA, we found that the distribution of translocations induced by the presence of Ty912 in a wild-type strain was nonrandom and that the majority of these translocations were mediated by only six translocation targets on four different chromosomes, even though there were 254 potential Ty-related translocation targets in the S. cerevisiae genome. While the majority of Ty912-mediated translocations resulted from RAD52-dependent recombination, we observed a number of nonreciprocal translocations mediated by RAD52-independent recombination between Ty1 elements. The formation of these RAD52-independent translocations did not require the Rad51 or Rad59 homologous pairing proteins or the Rad1-Rad10 endonuclease complex that processes branched DNAs during recombination. Finally, we found that defects in ASF1-RTT109-dependent acetylation of histone H3 lysine residue 56 (H3K56) resulted in increased accumulation of both GCRs and whole-chromosome duplications, and resulted in aneuploidy that tended to occur simultaneously with GCRs. Overall, we found that MLPA is a versatile technique for the rapid analysis of GCRs and can facilitate the genetic analysis of the pathways that prevent and promote GCRs and aneuploidy.     

Ultrafine anaphase bridges, broken DNA and illegitimate recombination induced by a replication fork barrier

Most DNA double-strand breaks (DSBs) in S- and G2-phase cells are repaired accurately by Rad51-dependent sister chromatid recombination. However, a minority give rise to gross chromosome rearrangements (GCRs), which can result in disease/death. What determines whether a DSB is repaired accurately or inaccurately is currently unclear. We provide evidence that suggests that perturbing replication by a non-programmed protein-DNA replication fork barrier results in the persistence of replication intermediates (most likely regions of unreplicated DNA) into mitosis, which results in anaphase bridge formation and ultimately to DNA breakage. However, unlike previously characterised replication-associated DSBs, these breaks are repaired mainly by Rad51-independent processes such as single-strand annealing, and are therefore prone to generate GCRs. These data highlight how a replication-associated DSB can be predisposed to give rise to genome rearrangements in eukaryotes.     

The Rad1-Rad10 complex promotes the production of gross chromosomal rearrangements from spontaneous DNA damage in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) have been observed in many cancers. Previously, we have demonstrated many mechanisms for suppression of GCR formation in yeast. However, pathways that promote the formation of GCRs are not as well understood. Here, we present evidence that the Rad1-Rad10 endonuclease, which plays an important role in nucleotide excision and recombination repairs, has a novel role to produce GCRs. A mutation of either the RAD1 or the RAD10 gene reduced GCR rates in many GCR mutator strains. The inactivation of Rad1 or Rad10 in GCR mutator strains also slightly enhanced methyl methanesulfonate sensitivity. Although the GCRs induced by treatment with DNA-damaging agents were not reduced by rad1 or rad10 mutations, the translocation- and deletion-type GCRs created by a single double-strand break are mostly replaced by de novo telomere-addition-type GCR. Results presented here suggest that Rad1-Rad10 functions at different stages of GCR formation and that there is an alternative pathway for the GCR formation that is independent of Rad1-Rad10.     

Molecular characterization of de novo secondary trisomy 13.

Unbalanced Robertsonian translocations are a significant cause of mental retardation and fetal wastage. The majority of homologous rearrangements of chromosome 21 in Down syndrome have been shown to be isochromosomes. Aside from chromosome 21, very little is known about other acrocentric homologous rearrangements. In this study, four cases of de novo secondary trisomy 13 are presented. FISH using alpha-satellite sequences, rDNA, and a pTRI-6 satellite I sequence specific to the short arm of chromosome 13 showed all four rearrangements to be dicentric and apparently devoid of ribosomal genes. Three of four rearrangements retained the pTRI-6 satellite I sequence. Case 1 was the exception, showing a deletion of this sequence in the rearrangement, although both parental chromosomes 13 had strong positive hybridization signals. Eleven microsatellite markers from chromosome 13 were also used to characterize the rearrangements. Of the four possible outcomes, one maternal Robertsonian translocation, two paternal isochromosomes, and one maternal isochromosome were observed. A double recombination was observed in the maternally derived rob(13q13q). No recombination events were detected in any isochromosome. The parental origins and molecular chromosomal structure of these cases are compared with previous studies of de novo acrocentric rearrangements.     

Formation of supernumerary euchromatic short arm isochromosomes: parent and cell stage of origin in new cases and review of the literature.

In order to get insight in the formation of isochromosomes we analysed different supernumerary euchromatic short arm isochromosomes for the parent and cell stage of origin. After cytogenetic detection and confirmation by fluorescence-in-situ hybridization we performed short tandem repeat typing in a child with i(9p), three with i(12p) and three with i(18p). The extra chromosomes were monocentric in each case, the i(9p) and i(12p) constitutions were found in mosaic with normal cell lines. Our results and those of other groups indicate a strong role of maternal meiosis in isochromosome formation: in one i(8p), 4 out of 5 i(9p), 7 out of 12 i(12p) and 18 out of 23 i(18p) families a maternal meiotic nondisjunction had occurred prior to the centromere misdivision. For chromosome 18, the majority of isochromosomes originated from a maternal meiosis II error (16/18). For the other tetrasomic constitutions the isochromosomes could be delineated from paternal as well as from maternal origin, the short tandem repeat typing patterns being consistent with meiotic or mitotic cell stages of formation. Thus, independently of the chromosomal origin, in the majority of cases with additional euchromatic isochromosomes maternal meiosis nondisjunction is the initial step followed by centromeric misdivision. Postzygotic nondisjunction as suggested previously due to mosaics observed in tetrasomies 9p and 12p seems to be of minor importance. The observed origin of isochromosomes 18 corresponds to that of trisomy 18, where the majority of cases can be delineated from maternal meiosis II errors.     

A genetic and structural study of genome rearrangements mediated by high copy repeat Ty1 elements

Ty elements are high copy number, dispersed repeated sequences in the Saccharomyces cerevisiae genome known to mediate gross chromosomal rearrangements (GCRs). Here we found that introduction of Ty912, a previously identified Ty1 element, onto the non-essential terminal region of the left arm of chromosome V led to a 380-fold increase in the rate of accumulating GCRs in a wild-type strain. A survey of 48 different mutations identified those that either increased or decreased the rate of Ty-mediated GCRs and demonstrated that suppression of Ty-mediated GCRs differs from that of both low copy repeat sequence- and single copy sequence-mediated GCRs. The majority of the Ty912-mediated GCRs observed were monocentric nonreciprocal translocations mediated by RAD52-dependent homologous recombination (HR) between Ty912 and a Ty element on another chromosome arm. The remaining Ty912-mediated GCRs appeared to involve Ty912-mediated formation of unstable dicentric translocation chromosomes that were resolved by one or more Ty-mediated breakage-fusion-bridge cycles. Overall, the results demonstrate that the Ty912-mediated GCR assay is an excellent model for understanding mechanisms and pathways that suppress genome rearrangements mediated by high copy number repeat sequences, as well as the mechanisms by which such rearrangements occur.     

A molecular genetic approach to the identification of isochromosomes of chromosome 21

Summary The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.     

Stabilization of Dicentric Translocations through Secondary Rearrangements Mediated by Multiple Mechanisms in S. cerevisiae

Background

The gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instability

Methodology/Principal Findings

The structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.

Conclusions/Significance

HR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.     

将大赖草种质转移给普通小麦的研究——Ⅶ.一个普通小麦-大赖草等臂染色体异附加系的选育与鉴定

通过花粉母细胞减数分裂中期Ⅰ染色体配对构型分析、Giemsa C-分带,从普通小麦-大赖草第7条染色体二体异附加系自交后代中选育并鉴定出了93G51-9和93G52-8 2个等臂染色体异附加系。该异附加系在花粉母细胞减数分裂中期Ⅰ,其等臂染色体自身两臂配对频率高,染色体易发生断裂,且又携带有较抗赤霉病的基因,是向小麦转移大赖草赤霉病抗性基因的有用中间材料。     

Robertsonian translocations: mechanisms of formation,aneuploidy, and uniparental disomy and diagnostic considerations

Robertsonian translocations (ROBs) are rearrangements of the acrocentric chromosomes 13-15 and 21-22. Cytologically, ROBs between homologous chromosomes cannot be distinguished from isochromosomes that originate through duplication of a single homologue. Both types of rearrangements can be involved in aneuploidy. A conceptus with a trisomy or a monosomy can be rescued, and in a proportion of cases, a uniparental disomy (UPD) would result. If there are regions of genome imprinting on a uniparental chromosome pair, phenotypic consequences can result. Chromosomes 14 and 15 are imprinted, and UPD of these are known to result in abnormalities. Thus, prenatal testing should be considered in all pregnancies when one of the parents is a balanced carrier of a ROB because of the risk for aneuploidy, and UPD testing should be considered in fetuses found to carry a balanced ROB or isochromosome that involves chromosomes 14 or 15. Additionally, infants or children with congenital anomalies who carry a ROB should also be considered for UPD testing.     

Tetrasomy 18p de novo: Identification by FISH with conventional and microdissection probes and analysis of parental origin and formation by short sequence repeat typing

We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared by chromosome microdissection and in vitro amplification. The maternal origin as well as the most probable cell stages of formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool to investigate the nature and the origin of de novo marker chromosomes. Received: 28 August 1995 / Revised: 3 November 1995; 20 December 1995     

Mitotic Instability in Cancer: Is There Method in the Madness?

It has been known for more than a century that neoplastic cells often exhibit disturbances of the mitotic process, but the causes have only recently been thoroughly explored. In many cancers, a combination of cell cycle checkpoint deficiency and abnormal shortening of telomeres predisposes to unbalanced chromosome segregation at cell division and the development of complex genomic rearrangements. Shortening of telomeric repeats beyond normal limits leads to fusion of chromosome ends and the formation of chromatin bridges at anaphase. In turn, these bridges may trigger at least three types of chromosomes mutation: (1) structural rearrangements of chromosomes through extensive chromatin fragmentation beyond the centromeric sequences, typically leading to the formation of isochromosomes and whole-arm translocations, (2) loss of whole chromosomes through mechanical detachment from the mitotic spindle machinery, and (3) failure of cytokinesis, leading to polyploidisation and supernumerary centrosomes, which may in turn orchestrate multipolar spindle configurations at a subsequent mitosis. Anaphase bridging rarely hinders further survival of tumour daughter cells. In contrast, multipolar mitoses may lead to extensive reshuffling of chromosome copies that compromise further clonal expansion. The telomere-dependent instability can be partly counteracted by expression of telomerase during tumour progression, but genomic stabilisation is rarely, if ever, complete.     

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.     

Determining the origin of human X isochromosomes by use of DNA sequence polymorphisms and detection of an apparent i(Xq) with Xp sequences

Summary The parental origin of five X isochromosomes were determined using 11 DnA markers. The isochromosome was derived from a maternal X chromosome in three cases and from a paternal X chromosome in two. Unexpected heterozygosity was detected for the proximal Xp region in one individual in whom the i(Xq) chromosome was paternally derived. This was confirmed by in situ hybridisation. A mode of formation of isochromosomes by breakage and reunion between the sister chromatids of the arms of an X chromosome is proposed to account for this. Sister chromatid breakage and reunion can be considered as a significant mechanism for the origin of i(Xq) chromosomes.     

Two dicentric Y isochromosomes,one without the Yqh heterochromatic segment

Summary Two women with primary amenorrhoea and few other stigmata of Turner's syndrome were found to be chromosome mosaics: 45,X/46,X,idic(Y). In Case 1, the dicentric isochromosome Y was found to have a long-arm breakpoint of formation. This structure was interpreted as containing two Y short arms and centromeres separated by a region derived from the proximal Y long arm. One of the centromeres in the Case 1 —idic(Y) was suppressed in 80% of cells in blood, and in these cells it appeared as a regular Y-shaped chromosome. In Case 2 the idic(Y) was derived by a short-arm breakpoint of formation. In all the dicentrics of this case with one primary constriction (functional monocentrics) there was a single Cd band. In the 10% of dicentrics with two primary constrictions, there were two Cd bands. It is argued that the instability of sex isochromosomes is due to this functional dicentricity in some cells. These cases are compared with 42 other Y isochromosomes with various short- and long-arm breakpoints of formation. It is suggested that some of the nonheterochromatic, nonfluorescent Y chromosomes previously reported may be explained as dicentric i(Y) with proximal long-arm breakpoints of formation and one suppressed centromere.     

Induction of genome instability by DNA damage in Saccharomyces cerevisiae

The accumulation of gross chromosomal rearrangements (GCRs) is a characteristic of many types of cancer cells, although it is unclear what defects cause these rearrangements and how the different types of GCRs observed are formed. In the present study, we have used a Saccharomyces cerevisiae system for measuring GCRs to analyze the ability of a variety of DNA damaging agents to induce GCRs. The two most potent inducers of GCRs observed were methyl methane sulfonate (MMS) and HO-endonuclease-induced double strand breaks (DSBs). Bleomycin, camptothecan and gamma-irradiation induced intermediate levels of GCRs and cisplatin induced very low levels of GCRs whereas N-methyl-NPRIME;-nitro-N-nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS) primarily induced base substitution mutations. MMS treatment primarily induced rearrangements in which the end of a chromosome was deleted and a new telomere was added (telomere additions) and also induced translocations. Consistent with this GCR spectrum, the formation of MMS-induced GCRs was primarily dependent on telomere maintenance functions and were completely eliminated in mutants that were defective for both telomere maintenance functions and non-homologous end joining (NHEJ). In contrast, HO-endonuclease DSBs induced mostly translocations and interstitial deletions whereas few telomere additions were observed. Genetic analysis indicated that HO DSB-induced GCRs were suppressed by a number of pathways including the DNA damage checkpoints, DSB repair pathways and NHEJ.     

47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.     

Meiotic synapsis of the Allium porrum B chromosome: evidence for a derived isochromosome origin.

Ultrastructural analysis of B chromosome synapsis in surface-spread (2B) pollen mother cells of the leek, Allium porrum, has clarified their structural organization and shed new light on their origin. In pachytene cells containing two B chromosomes, these chromosomes either formed a pair of univalents showing foldback hairpin loops or synapsed together to form bivalents of several different types. The synaptic configurations of univalents and bivalents indicate that these B chromosomes have a basically isochromosome organization, but this is modified by a slight centric shift giving an arm ratio of 1.1:1. This analysis adds to the growing number of B chromosomes that have been shown to be isochromosomes or isochromosome derivatives. Key words : Allium porrum, B chromosomes, synapsis, synaptonemal complex, isochromosome.