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.     

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.     

Meiotic and Mitotic Behavior of Dicentric Chromosomes in SACCHAROMYCES CEREVISIAE

Meiotic recombination between a circular and a linear chromosome in Saccharomyces cerevisiae has been investigated. The circle was a haploid-viable derivative of chromosome III constructed by joining regions near the two chromosome ends via a recombinant DNA construction: (HMR/MAT-URA3-pBR322-MAT/HML) and was also deleted for MAL2 (which therefore uniquely marks a linear chromosome III). Recombination along chromosome III was measured for eight intervals spanning the entire length of the circular derivative. Only 25% of all tetrads from a ring/rod diploid contained four viable spores. These proved to be cases in which there was either no recombination along chromosome III or in which there were two-strand double crossovers or higher order crossovers that would not produce a dicentric chromosome.--At least half of the tetrads with three viable spores included one Ura+ Mal+ spore that was genetically highly unstable. The Ura+ Mal+ spore colonies gave rise to as many as seven genetically distinct, stable ("healed") derivatives, some of which had lost either URA3 or MAL2. Analysis of markers on chromosome III suggests that dicentric chromosomes frequently do not break during meiosis but are inherited intact into a haploid spore. In mitosis, however, the dicentric chromosome is frequently broken, giving rise to a variety of genetically distinct derivatives. We have also shown that dicentric ring chromosomes exhibit similar behavior: at least half the time they are not broken during meiosis but are broken and healed during mitosis.--The ring/rod diploid can also be used to determine the frequency of sister chromatid exchange (SCE) along an entire yeast ring chromosome. We estimate that an unequal number of SCE events occurs in approximately 15% of all cells undergoing meiosis. In contrast, the mitotic instability (and presumably SCE events) of a ring chromosome is low, occurring at a rate of about 1.2 X 10(-3) per cell division.     

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.     

The Role of Replication Bypass Pathways in Dicentric Chromosome Formation in Budding Yeast

Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20–200 bp of homology, separated by ∼1–5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.ALL organisms are prone to large-scale changes (gross chromosomal rearrangements, GCRs) to their genomes that include deletions, inversions, and translocations. These large-scale changes are thought to drive evolutionary events, such as speciation, and contribute to human pathology such as Pelziaeus-Merzbacher syndrome and other genetic disorders (Lee et al. 2007; Stankiewicz and Lupski 2010). Thus, a firm understanding of how cells normally prevent such rearrangements, and how they accumulate, is critical to our understanding of both evolution and pathology.GCRs arise by many different mechanisms, and there is growing evidence that errors during DNA replication are a major source (Myung et al. 2001; Admire et al. 2006; Mizuno et al. 2009). Errors are thought to arise when replication forks encounter “lesions” on the template strand. Lesions can consist of protein complexes bound to DNA or lesions in the DNA itself. Replication forks bypass lesions by several different mechanisms that are still poorly understood (Atkinson and McGlynn 2009; Weinert et al. 2009). We believe that understanding lesion bypass mechanisms is central to understanding both how GCRs are prevented and how they form when lesion bypass mechanisms fail.All lesion bypass pathways utilize sequence homology to restart replication (Atkinson and McGlynn 2009; Weinert et al. 2009). Use of sequence homology during restart may limit the frequency of GCRs, as it lowers the probability of annealing to nonallelic sequences. Repetitive sequences present a problem because lesion bypass at sites near repetitive sequences may lead to annealing of nonallelic sequences and thus to GCR formation (Lemoine et al. 2005; Narayanan et al. 2006; Argueso et al. 2008). Indeed in yeast and in other organisms, GCRs occur frequently in repeat sequences (Dunham et al. 2002; Argueso et al. 2008; Di Rienzi et al. 2009). Some rearrangements do occur between so-called “single-copy sequences” with either no homology or limited homology (microhomologies of 5–9 bp; Myung et al. 2001; Kolodner et al. 2002; Putnam et al. 2005) though evidence suggests these rearrangements occur less frequently than rearrangements between repetitive sequences (Putnam et al. 2009). Interestingly, it has been shown that some genes are required to prevent the fusion of repetitive elements yet have no effect on rearrangements between single-copy sequences (Putnam et al. 2009). Currently it is not clear how these pathways act to suppress repeat-mediated events and why they are not required to prevent rearrangements between single-copy sequences.Our current understanding of the mechanisms underlying GCR formation is mostly derived from assays designed to detect specific changes to yeast chromosomes (Chen and Kolodner 1999; Myung et al. 2001; Huang and Koshland 2003; Lambert et al. 2005; Rattray et al. 2005; Admire et al. 2006; Narayanan et al. 2006; Schmidt et al. 2006; Smith et al. 2007; Pannunzio et al. 2008; Payen et al. 2008; Paek et al. 2009; Mizuno et al. 2009). Previously we reported on GCR formation in the budding yeast Saccharomyces cerevisiae using an assay we developed. We found that a major source of genome instability involves the fusion of nearby inverted repeats (with ∼20–200 bp of sequence homology, separated by 1–5 kb) to form either dicentric or acentric chromosomes (Figure 1D; Paek et al. 2009). We also found that fusion of inverted repeats is general: fusion occurred between inverted repeats at all five different locations tested on four different yeast chromosomes, as well as between synthetic inverted repeats (Paek et al. 2009). Genetic data suggest that these events most likely occur during replication of DNA (Admire et al. 2006). Further genetic analysis suggested that the mechanism of inverted repeat fusion differed from that of direct repeat recombination, in that inverted repeat fusion did not require genes involved in homologous recombination (HR) or single-strand annealing (SSA) pathways (Paek et al. 2009). In addition, fusion events are unlikely to involve double-strand breaks (DSBs), as genes in the nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) are not required for fusion events (Paek et al. 2009). Indeed gene knockouts in the HR (RAD52, RAD51, and RAD59), SSA (RAD52 and RAD1) and postreplication repair (PRR) (RAD18) pathways actually increased the frequency of fusion of an inverted repeat on chromosome (Chr) VII (Paek et al. 2009); these pathways normally suppress inverted repeat fusion.Open in a separate windowFigure 1.—Experimental setup for the detection of inverted repeat fusion and chromosome instability. Objects are not drawn to scale. (A) The starting strain has two copies of Chr VII. One copy contains the CAN1 gene, ADE6, ade3, while the other copy is ade6, ADE3. Cells are plated to canavanine, and three types of colonies are formed: (B) Allelic recombinants are round in appearance and are Ade⁺; (C) colonies that form by loss of Chr VII are round in appearance and Ade⁻; and (D) cells that contain unstable dicentric chromosomes form by the fusion of inverted repeats. One specific case of this fusion (the S2/S3 dicentric) is shown within braces. Cells with dicentrics form mixed colonies, which contain allelic recombinants, chromosome loss events, as well as a translocation between D7 and D11. The bar in the S2/S3 repeat represents a fusion junction. (E) The specific dicentric is detected by dicentric primers DP1 and DP2 and (F) a monocentric translocation that is detected with translocation primers TP1 and TP2.To further our previous studies, we analyzed three groups of genes implicated in the maintenance of genome stability. We tested how these genes affect the overall stability of Chr VII, focusing on the fusion of nearby inverted repeats to form a specific dicentric Chr VII and the resolution of the dicentric into a monocentric translocation (which we term the 403–535 translocation; Figure 1, D–F). First, we analyzed several genes in the PRR pathway and found that error-free bypass, but not translesion synthesis, is required for the prevention of inverted repeat fusion. Surprisingly, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Second, we analyzed several mutations in genes that are associated with replication forks. We found that mutants in POL3 (polymerase delta) and MGS1 (encoding a single-strand annealing protein, which binds polymerase delta) significantly reduced the frequency of dicentric formation and allelic recombinants that arise in the checkpoint mutant rad9 (Giot et al. 1997; Hishida et al. 2001; Paek et al. 2009). Finally we studied genes associated with rearrangements involving repeats or single-copy sequences, as well as a subset of mutants involved in recombination. Generally, we find that the mechanisms of nearby inverted repeat fusion are distinct from mechanisms fusing longer repeats or single-copy sequences.     

Meiotic Recombination on Artificial Chromosomes in Yeast

We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.     

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein^1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.     

A Chlamydomonas Genomic Library in Yeast Artificial Chromosomes

We have constructed and characterized a Chlamydomonas reinhardtii total genomic library in yeast artificial chromosomes (YACs). The library contains 7500 clones with inserts ranging in size from 100-200 kb. The representation of the library was assessed by screening one-third of it with a probe derived from the dispersed repeat, Gulliver, which occurs ~13 times in the genome. At least 10 of these Gulliver loci were isolated within 15 independent YACs. Two of these YACs encompass the Gulliver element designated G, which was reported to map to the uni linkage group (ULG). The end clones of these two YACs have been genetically mapped by RFLP analysis in an interspecific cross and thereby shown to be closely linked to the APM locus on the ULG. A third uni-specific YAC has also been isolated and its ends have been mapped by RFLP analysis. Genetic and RFLP analysis of these and other YACs indicates that the frequency of chimeric YACs in the library is very low. The library was constructed in a second generation vector that enables plasmid rescue of YAC end clones as well as copy number amplification of artificial chromosomes. We provide evidence that amplification of intact YACs requires a rad1:rad52 yeast strain.     

Homologous Recombination between Episomal Plasmids and Chromosomes in Yeast

We have observed genetic recombination between ura3( -) mutations (among them extensive deletions) carried on "episomal" (i.e., 2micro DNA-containing) plasmids and other ura3( -) alleles present at the normal chromosomal URA3 locus. The recombination frequency found was comparable to the level observed for classical mitotic recombination but was relatively insensitive to sunlamp radiation, which strongly stimulates mitotic recombination. Three equally frequent classes could be distinguished among the recombinants. Two of these are the apparent result of gene conversions (or double crossovers) which leave the URA3(+) allele on the chromosome (class I) or on the plasmid (class II). The third class is apparently due to a single crossover that results in the integration of the plasmid into a chromosome. Plasmid-chromosome recombination can be useful in fine structure genetic mapping, since recombination between a chromosomal point mutation and a plasmid-borne deletion mutation only 25 base pairs distant was easily detected.     

Mechanisms Controlling the Integrity of Replicating Chromosomes in Budding Yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

Distribution of Ribosomal Ribonucleic Acid Cistrons Among Yeast Chromosomes

High-molecular-weight deoxyribonucleic acid (DNA) of Saccharomyces carls bergensis has been fractionated by sucrose density gradient centrifugation. The main DNA fraction has an average molecular weight of about 500 x 10(6). A major fraction of the DNA molecules containing sequences homologous to ribosomal ribonucleic acid (RNA) sediments as material of this molecular weight. The remainder sediments as material of a molecular weight of about 250 x 10(6). The latter fraction contains relatively more ribosomal RNA cistrons than the former. Studies on the buoyant density of high-molecular-weight DNA homologous to ribosomal RNA have led to the conclusion that the ribosomal RNA cistrons occur in groups attached to a relatively large amount of nonribosomal RNA and suggest that ribosomal RNA cistrons are distributed over a number of yeast chromosomes.     

Evidence of Preferential Pairing of Chromosomes at Meiosis in Aneuploid Yeast

Meiotic pairing in homothallic S. cerevisiae was studied by tetrad analysis, using strains that were trisomic or tetrasomic for chromosome I. The disomic segregants of these strains produce tetrasomic spore colonies that can be distinguished by their phenotype. Results indicated the existence of preferential pairing and nonrandom assortment of chromosomes at meiosis I. The frequency of crossing over is apparently normal in at least some regions when non-preferred pairing occurs.     

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.     

Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2μm plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.It is well known that the nuclear genomes of most eukaryotes contain integrated fragments of organellar DNA called NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) (26, 29, 44, 45, 57). These fragments are usually pseudogenes, although some NUMTs and NUPTs have become incorporated into functional nuclear genes (38). The NUMTs present in the nuclear genomes of Saccharomycotina yeast species were recently analyzed by Sacerdot et al. (48).In addition to their mitochondrial genomes, yeast species contain a variety of other extranuclear DNA and RNA elements, including viruses and plasmids. These extrachromosomal elements are usually considered to be autonomous entities that do not interact with nuclear DNA. When our laboratory sequenced the genome of the yeast Vanderwaltozyma polyspora (synonym: Kluyveromyces polysporus) (49), we were therefore surprised to find the genomic region we describe here, which contains integrated fragments of several plasmid- and virus-like sequences. We propose that this region was formed by the capture of plasmid and viral sequences by the same mechanism that captures mitochondrial DNA to form NUMTs (43, 65). In a literature search, we could find only one previous report of a similar finding: Utatsu et al. (59) reported the sequences of two regions of nuclear DNA from Zygosaccharomyces rouxii that were highly similar to parts of the 2μm-like plasmid pSR1 from that species, but rearranged.Before describing the V. polyspora region, and similar regions found in other species, we will first briefly introduce the extrachromosomal RNA and DNA entities that are known to exist in yeasts. Extrachromosomal nucleic acids are relatively uncommon in yeasts: a broad survey of 1,800 strains from 600 species by Fukuhara (14) found that 196 strains (11%) contained some sort of extrachromosomal entity. Among these, 105 strains had a double-stranded RNA (dsRNA), 28 had a linear dsDNA plasmid, and 53 had a circular DNA plasmid of the 2μm family. These elements typically also have a patchy distribution within a species, being found in some individuals or strains but not in others. For instance, Nakayashiki et al. (37) surveyed 70 “wild” strains of Saccharomyces (mostly S. cerevisiae) for the presence of five extrachromosomal elements (2μm DNA plasmid, L-A and L-BC helper RNA viruses, and W and T RNA entities) and found each element to be present in between 1 and 38 of the strains, with 1 strain even containing all five elements simultaneously.     

Suppression of Chromosome Healing and Anticheckpoint Pathways in Yeast Postsenescence Survivors

Telomere repeat-like sequences at DNA double-strand breaks (DSBs) inhibit DNA damage signaling and serve as seeds to convert DSBs to new telomeres in mutagenic chromosome healing pathways. We find here that the response to seed-containing DSBs differs fundamentally between budding yeast (Saccharomyces cerevisiae) cells that maintain their telomeres via telomerase and so-called postsenescence survivors that use recombination-based alternative lengthening of telomere (ALT) mechanisms. Whereas telomere seeds are efficiently elongated by telomerase, they remain remarkably stable without de novo telomerization or extensive end resection in telomerase-deficient (est2Δ, tlc1Δ) postsenescence survivors. This telomere seed hyper-stability in ALT cells is associated with, but not caused by, prolonged DNA damage checkpoint activity (RAD9, RAD53) compared to telomerase-positive cells or presenescent telomerase-negative cells. The results indicate that both chromosome healing and anticheckpoint activity of telomere seeds are suppressed in yeast models of ALT pathways.     

Construction and Characterization of aPlasmodium vivaxGenomic Library in Yeast Artificial Chromosomes

Here we describe the construction of a representative YAC library for the human malarial parasitePlasmodium vivax.AsP. vivaxcannot be maintained continuously under laboratory conditions, theP. vivaxDNA necessary for the library construction was isolated from a single human patient presenting himself with vivax malaria to a local hospital in the Brazilian Amazon. Thus, this YAC library is the first of its kind to be generated from patient-derived material. The YAC library consists of 560 clones with an average insert size of 180 kb. Of 9 publishedP. vivaxgenes, 8 were found to be present in the library. In addition, 12P. vivaxtelomeric YAC clones were identified.     

Dicentric Robertsonian translocations in man

Summary The authors studies 17 cases of Robertsonian translocations. In all cases but one C banding showed that a dicentric translocation was involved. Silver staining demonstrated the presence of an NOR between the two centromeres in only one case.     

Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

Spo11-mediated DNA double-strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To elucidate this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thus, we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.