Healing of Broken Linear Dicentric Chromosomes in Yeast

In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura⁺ Mal⁺ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura⁺ Mal ⁺, others Ura⁺ Mal^- and others Ura ^- Mal⁺. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.     

Time course study of the chromosome-type breakage-fusion-bridge cycle in maize.

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. "Healing" of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.     

The transmission of fragmented chromosomes in Drosophila melanogaster.

We investigated the fate of dicentric chromosomes in the mitotic divisions of Drosophila melanogaster. We constructed chromosomes that were not required for viability and that carried P elements with inverted repeats of the target sites (FRTs) for the FLP site-specific recombinase. FLP-mediated unequal sister-chromatid exchange between inverted FRTs produced dicentric chromosomes at a high rate. The fate of the dicentric chromosome was evaluated in the mitotic cells of the male germline. We found that dicentric chromosomes break in mitosis, and the broken fragments can be transmitted. Some of these chromosome fragments exhibit dominant semilethality. Nonlethal fragments were broken at many sites along the chromosome, but the semilethal fragments were all broken near the original site of sister-chromatid fusion, and retained P element sequences near their termini. We discuss the implications of the recovery and behavior of broken chromosomes for checkpoints that detect double-strand break damage and the functions of telomeres in Drosophila.     

Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.     

INCIPIENT POLYPLOID SPECIATION IN THE MAIDENHAIR FERN (ADIANTUM PEDATUM; ADIANTACEAE)?

Despite the widespread occurrence of polyploids in plant taxa and the many advantages attributed to polyploidy, very little is known about the specific processes that lead to the establishment of polyploids in nature. Classical models suggested that polyploids arise following somatic chromosome doubling in hybrids. However, the production of polyploids from unreduced meiotic products has been receiving greater attention. During an enzyme electrophoretic study of a local population of Adiantum pedatum, a mutant producing viable unreduced spores along with abortive spores was discovered. Studies of sporogenesis showed that a synaptic mutation caused the paired chromosomes to disassociate, with mostly univalents remaining at metaphase I. In such aberrant spore mother cells, one of two pathways was followed in the remaining stages of meiosis. Cells attempting both meiotic divisions formed abortive spores. However, in spore mother cells that bypassed meiosis I and formed a restitution nucleus, meiosis II and subsequent stages of sporogenesis proceeded normally. Unreduced diploid spores resulted from this second pathway. When sown on either agar or sterile soil, these diplospores germinated and produced diploid gametophytes. Tetraploid sporophytes were produced by the gametophytes growing on sterile soil. The discovery of diploid sporophytes producing unreduced spores provided the opportunity to characterize the first step of one possible route to polyploid formation. Continued observations of the natural population may provide insights into the earliest stages of natural polyploid formation.     

Resolution of Dicentric Chromosomes by Ty-Mediated Recombination in Yeast

We have integrated a plasmid containing a yeast centromere, CEN5, into the HIS4 region of chromosome III by transformation. Of the three transformant colonies examined, none contained a dicentric chromosome, but all contained a rearranged chromosome III. In one transformant, rearrangement occurred by homologous recombination between two Ty elements; one on the left arm and the other on the right arm of chromosome III. This event produced a ring chromosome (ring chromosome III) of about 60 kb consisting of CEN3 and all other sequences between the two Ty elements. In addition, a linear chromosome (chromosome IIIA) consisting of sequences distal to the two Ty elements including CEN5, but lacking 60 kb of sequences from the centromeric region, was produced. Two other transformants also contain a similarly altered linear chromosome III as well as an apparently normal copy of chromosome III. These results suggest that dicentric chromosomes cannot be maintained in yeast and that dicentric structures must be resolved for the cell to survive.--The meiotic segregation properties of ring chromosome III and linear chromosome IIIA were examined in diploid cells which also contained a normal chromosome III. Chromosome IIIA and normal chromosome III disjoined normally, indicating that homology or parallel location of the centromeric regions of these chromosomes are not essential for proper meiotic segregation. In contrast, the 60-kb ring chromosome III, which is homologous to the centromeric region of the normal chromosome III, did not appear to pair with fidelity with chromosome III.     

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy.     

Mutations in Cen3 Cause Aberrant Chromosome Segregation during Meiosis in Saccharomyces Cerevisiae

We investigated the structural requirements of the centromere from chromosome III (CEN3) of Saccharomyces cerevisiae by analyzing the ability of chromosomes with CEN3 mutations to segregate properly during meiosis. We analyzed diploid cells in which one or both copies of chromosome III carry a mutant centromere in place of the wild-type centromere and found that some alterations in the length, base composition and primary sequence characteristics of the central A+T-rich region (CDE II) of the centromere had a significant effect on the ability of the chromosome to segregate properly through meiosis. Chromosomes containing mutations which delete a portion of CDE II showed a high rate of premature disjunction at meiosis I. Chromosomes containing point mutations in CDE I or lacking CDE I appeared to segregate properly through meiosis; however, plasmids carrying centromeres with CDE I completely deleted showed an increased frequency of segregation to nonsister spores.     

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.     

Nonrecombinant Meiosis I Nondisjunction in Saccharomyces Cerevisiae Induced by Trna Ochre Suppressors

The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.     

Chromosome length controls mitotic chromosome segregation in yeast

We have examined the effect of physical length on the mitotic segregation of artificial chromosomes and fragments of natural yeast chromosomes. Increasing the length of artificial chromosomes decreases the rate at which they are lost during mitosis. We have made fragments of chromosome III by integrating new telomeres at different positions along the length of the chromosome. Chromosome fragments of 42 and 72 kb behave like artificial chromosomes: they are lost in mitosis much more frequently than natural chromosomes. In contrast, a chromosome fragment of 150 kb is as mitotically stable as the full-length chromosome from which it is derived. The structural instability of a short dicentric artificial chromosome demonstrates that, although short artificial chromosomes segregate poorly in mitosis, they do attach to the mitotic spindle. We discuss these results in the context of a model in which chromosome segregation is directed by the intercatenation of the segregating DNA molecules.     

Meiotic gene conversion and crossing over: their relationship to each other and to chromosome synapsis and segregation

The yeast mer1 mutant produces inviable spores and is defective in both meiotic recombination and chromosome pairing. A gene called MER2 partially suppresses the mer1 phenotype when present in high copy number. Both gene conversion and chromosome pairing are completely restored in mer1 strains overexpressing MER2; however, reciprocal crossing over and spore viability are not restored. The data presented are consistent with a model in which chromosome pairing is a direct consequence of a homology search mediated through gene conversion. Analysis of random viable spores indicates that the crossovers that occur in mer1 strains overexpressing MER2 are more effective in ensuring meiosis I disjunction than those that occur in mer1 strains. One interpretation of this result is that only those crossovers that occur in the context of the synaptonemal complex lead to the establishment of functional chiasmata. The MER2 gene product is essential for meiosis.     

Induction of meiotic chromosomal malsegregation in yeast

A system to detect chromosome number abnormalities occurring during meiosis in Saccharomyces cerevisiae is described. It is based on selection of spores carrying 2 multi-marked chromosomes V. Each step of the technical procedure is critically analyzed and the origin of some biases discussed. Selection and subsequent genetic analysis allow the estimation of the frequency of spontaneous and induced diploid and aneuploid n + 1 (diplo-V) spores. Data are reported concerning the effect of 53 chemical compounds. The great majority of active chemicals induce diploid clones while a minority cause non-disjunction of chromosome V.     

Haploid Meiosis in Arabidopsis: Double-Strand Breaks Are Formed and Repaired but Without Synapsis and Crossovers

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.     

Diploid and triploid offspring of triploid agamosporous fernDryopteris pacifica

A cytological and reproductive study of the diploid and triploid agamosporousDryopteris pacifica was made to elucidate the origin of its infraspecific cytotypes. Some triploids produced 16 spore mother cells (SMCs) sometimes with n=41II+41I chromosomes, in addition to eight SMCs with n=123II, in each sporangium. In the former case the 16 SMCs usually underwent abnormal meiosis to give rise to some 50 spores, some of which were regular-shaped; in the latter the eight SMCs multiplied into 32 spores by normal meiosis. We found that spores from one of the triploid plants developed into either diploid or triploid gametophytes, which further apogamously produced diploid or triploid sporophytes, respectively. This novel mechanism of ploidy reduction is discussed in relation to the origin of diploid agamosporous ferns, the taxonomic complexity of the species, and the correlation of agamospory with polyploidy. The mechanism is also compared to that operating in agamospermous angiosperms.     

Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.     

Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae.

The introduction of a conditional centromere into chromosome III of Saccharomyces cerevisiae provided an opportunity to evaluate phenotypic and karyotypic consequences in cells harboring dicentric chromosomes upon entry into mitosis. A mitotic pause ensued, and monocentric derivatives of chromosome III were generated at a high frequency.     

Quinacrine fluorescence of variant and abnormal human Y chromosomes

Quinacrine fluorescence has been used to examine variant and abnormal human Y chromosomes, at interphase and mitosis. The length difference in variant Y chromosomes has been shown to involve the highly fluorescent segment only. Eight abnormalities of the Y chromosome have been positively identified, two isochromosomes of the long arms of the Y, five pericentric inversions, and a dicentric Y chromosome with two long arms. Contributory evidence for two further abnormalities, a ring Y and a dicentric with two short arms, is also given.     

Chromatid and Chromosome Type Breakage-Fusion-Bridge Cycles in Wheat (Triticum Aestivum L.)

During the development of disomic additions of rye (Secale cereale L.) chromosomes to wheat (Triticum aestivum L.), two reverse tandem duplications on wheat chromosomes 3D and 4A were isolated. By virtue of their meiotic pairing, the reverse tandem duplications initiated the chromatid type of the breakage-fusion-bridge (BFB) cycle. This BFB cycle continued through pollen mitoses and in the early endosperm divisions, but no clear evidence of its presence in embryo mitoses was found. The chromosome type of BFB cycle was initiated by fusion of two broken chromosome ends resulting in a dicentric or a ring chromosome. Chromosome type BFB cycles were detected in embryo mitoses and in root tips, but they did not persist until the next meiosis and were not transmitted to the progeny. Active BFB cycles induced breakage of other wheat chromosomes that resulted in additional reverse tandem duplications and dicentric and ring chromosomes. Four loci, on chromosome arms 2BS, 3DS, 4AL, and most likely on 7DL, were particularly susceptible to breakage. The BFB cycles produced high frequency of variegation for pigmentation of the aleurone layer of kernels and somatic chimeras for a morphological marker. With the exception of low mutation rate, the observed phenomena are consistent with the activity of a Ds-like element. However, it is not clear whether such an element, if indeed present, was of wheat or rye origin.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.