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.     

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.     

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.     

TAR载体克隆染色体目的片段的研究进展

分离染色体DNA上的目的片段,对其进行结构及功能的分析,对于发现新基因,研究基因功能都具有很大的意义。利用TAR克隆技术可以直接从基因组中分离所需的目的片段,这比传统的通过筛库获得目的片段的方法简便、快速、准确并且特异性强 。     

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.     

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.     

Recombination Suppression by Heterozygous Robertsonian Chromosomes in the Mouse

Robertsonian chromosomes are metacentric chromosomes formed by the joining of two telocentric chromosomes at their centromere ends. Many Robertsonian chromosomes of the mouse suppress genetic recombination near the centromere when heterozygous. We have analyzed genetic recombination and meiotic pairing in mice heterozygous for Robertsonian chromosomes and genetic markers to determine (1) the reason for this recombination suppression and (2) whether there are any consistent rules to predict which Robertsonian chromosomes will suppress recombination. Meiotic pairing was analyzed using synaptonemal complex preparations. Our data provide evidence that the underlying mechanism of recombination suppression is mechanical interference in meiotic pairing between Robertsonian chromosomes and their telocentric partners. The fact that recombination suppression is specific to individual Robertsonian chromosomes suggests that the pairing delay is caused by minor structural differences between the Robertsonian chromosomes and their telocentric homologs and that these differences arise during Robertsonian formation. Further understanding of this pairing delay is important for mouse mapping studies. In 10 mouse chromosomes (3, 4, 5, 6, 8, 9, 10, 11, 15 and 19) the distances from the centromeres to first markers may still be underestimated because they have been determined using only Robertsonian chromosomes. Our control linkage studies using C-band (heterochromatin) markers for the centromeric region provide improved estimates for the centromere-to-first-locus distance in mouse chromosomes 1, 2 and 16.     

Plasmid-Mediated Induction of Recombination in Yeast

Diploid yeast cells heteroallelic at the HIS3 locus were transformed with a minichromosome (centromeric plasmid) carrying homology to the HIS3 region and containing the same two mutations as were present in the chromosomes. When a double-strand break (DSB) was introduced in the region of homology, an increase in the recombination frequency between heteroalleles (leading to His(+) cells) was observed, although the plasmid was unable to donate wild-type information. This induction of recombination was dependent on the presence of homology between the plasmid sequences and the chromosomes. We show evidence for the physical involvement of the plasmid in tripartite recombination events, and we propose models that can explain the interactions between the plasmid-borne and chromosomal-borne alleles. Our results suggest that the mitotic induction of recombination by DNA damage is due to localized initiation of recombination events, and not to a general induction of recombination enzymes in the cell.     

Stimulation of Meiotic Recombination in Yeast by an Ars Element

In a previous study, meiotic recombination events were monitored in the 22-kb LEU2 to CEN3 region of chromosome III of Saccharomyces cerevisiae. One region (the hotspot) was shown to have an enhanced level of both gene conversion events and reciprocal crossovers, whereas a second region (the coldspot) was shown to have a depressed level of both types of recombination events. In this study we have analyzed the effects of a replication origin, ARS307, located about 2 kb centromere proximal to the hotspot region, on the distribution of meiotic recombination events. We find that a deletion of this origin results in a reduction of both gene conversions and reciprocal crossovers in the hotspot region, and that a 200-bp fragment of this ARS element can stimulate both types of recombination events when relocated to the coldspot region. Although the magnitude of stimulation of these events is similar in both orientations, whether the ARS is functional or not, the distribution of events is dependent upon the orientation of the element.     

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.     

Induction of Intrachromosomal Recombination in Yeast by Inhibition of Thymidylate Biosynthesis

The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.     

Analysis of Meiosis-Defective Mutations in Yeast by Physical Monitoring of Recombination

We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis-defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions.     

Karyotype Variability in Yeast Caused by Nonallelic Recombination in Haploid Meiosis

Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes. About 7% of haploid meioses produced chromosomes that differed by >/=10 kb from their wild-type counterparts. Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences. By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the ~100 kb MAT-HMR segment during repeated rounds of haploid meioses. Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged. Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes. In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent. The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.     

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.     

Ends-in Vs. Ends-Out Recombination in Yeast

Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for ``omega' insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks.     

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.