Evidence for a new chromosome in Saccharomyces cerevisiae.

The current yeast map has 16 chromosomes, each originally defined by a centromere-linked gene unlinked to previously defined centromere markers. We examined four genes, cly2, KRB1, AMY2, and tsm0115, each centromere linked, but previously thought to be not on chromosomes I to XVI. We found that AMY2 is linked to cly2, and both are on chromosome II. tsm0115 is on the left arm of chromosome XVI. We confirm the earlier evidence that KRB1 is not on chromosomes I through XVI. This gene thus defines a new chromosome XVII. We also report meiotic linkage of met4 and pet8 (on chromosome XIV), confirming the connection between the petx-kex2 fragment of XIV and the centromere of XIV.     

Chromosomal Assignment of Mutations by Specific Chromosome Loss in the Yeast Saccharomyces Cerevisiae

Yeast 2-microns plasmids were integrated near the centromere of a different chromosome in each of 16 cir0 mapping strains of Saccharomyces cerevisiae. The specific chromosomes containing the integrated 2-microns plasmid DNA were lost at a high frequency after crossing the cir0 strains to cir+ strains. A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-microns plasmid and the unmapped mutation on homologous chromosomes.     

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.     

Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast

By cloning centromere-linked genes followed by partial overlapping hybridization, we constructed a 210-kb map encompassing the centromere in chromosome II and a 60-kp map near the centromere of chromosome I in the fission yeast Schizosaccharomyces pombe which has three chromosomes. Integration of the cloned sequences into the chromosome and subsequent analyses of tetrads and dyads revealed an approximately 50 kb long domain located in the middle of the 210-kb map, tightly linked to the centromere and greatly reduced in meiotic recombination. This domain contained at least two classes of repetitive sequences. One, designated yn1, was specifically present in a particular chromosome and repeated three times in the 210-kb map of chromosome II. The other, designated dg, was located in all the centromere regions of three chromosomes. One (dgI) and two (dgIIa, dgIIb) copies of the dg were found in the maps of chromosomes I and II, respectively. The dgIIa and dgIIb were arranged with a 20-kb interval within the repetitive domain. In the centric region of chromosome II, 3-4 copies of the dg appeared to exist. By determining the nucleotide sequences of dgI and dgIIa, the dg was identified to be 3.8 kb long. The sequence homology was 99% between dgI and dgIIa. These extraordinarily homologous sequences seemed not to be transcribed into RNA nor to be encoding any protein. The larger part of the dg sequence was internally non-repetitious, a 600-bp region existed which consisted of stretches of several short repeating units. The structures in or surrounding the centromeres of S. pombe appear to be much more complex than those of the budding yeast Saccharomyces cerevisiae.     

Regional Bivalent-Univalent Pairing versus Trivalent Pairing of a Trisomic Chromosome in Saccharomyces Cerevisiae

In meiosis I, homologous chromosomes pair, recombine and segregate to opposite poles. These events and subsequent meiosis II ensure that each of the four meiotic products has one complete set of chromosomes. In this study, the meiotic pairing and segregation of a trisomic chromosome in a diploid (2n + 1) yeast strain was examined. We find that trivalent pairing and segregation is the favored arrangement. However, insertions near the centromere in one of the trisomic chromosomes leads to preferential pairing and segregation of the ``like' centromeres of the remaining two chromosomes, suggesting that bivalent-univalent pairing and segregation is favored for this region.     

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.     

Rapid evolution of yeast centromeres in the absence of drive

To find the most rapidly evolving regions in the yeast genome we compared most of chromosome III from three closely related lineages of the wild yeast Saccharomyces paradoxus. Unexpectedly, the centromere appears to be the fastest-evolving part of the chromosome, evolving even faster than DNA sequences unlikely to be under selective constraint (i.e., synonymous sites after correcting for codon usage bias and remnant transposable elements). Centromeres on other chromosomes also show an elevated rate of nucleotide substitution. Rapid centromere evolution has also been reported for some plants and animals and has been attributed to selection for inclusion in the egg or the ovule at female meiosis. But Saccharomyces yeasts have symmetrical meioses with all four products surviving, thus providing no opportunity for meiotic drive. In addition, yeast centromeres show the high levels of polymorphism expected under a neutral model of molecular evolution. We suggest that yeast centromeres suffer an elevated rate of mutation relative to other chromosomal regions and they change through a process of "centromere drift," not drive.     

Chromatin conformation of yeast centromeres

The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and XI. A 289-base pair centromere (CEN3) sequence can stabilize yeast chromosome III through mitosis and meiosis. The orientation of the centromeric fragments within chromosome III has no effect on the normal mitotic or meiotic behavior of the chromosome. The structural integrity of the centromere region in these genomic substitution strains was examined by mapping nucleolytic cleavage sites within the chromatin DNA. A nuclease-protected centromere core of 220-250 base pairs was evident in all of the genomic substitution strains. The position of the protected region is determined strictly by the centromere DNA sequence. These results indicate that the functional centromere core is contained within 220- 250 base pairs of the chromatin DNA that is structurally distinct from the flanking nucleosomal chromatin.     

The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin.

In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided. The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin. Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function. We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ). In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4. A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus. In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes. We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)). Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.     

Distributive Disjunction of Authentic Chromosomes in Saccharomyces Cerevisiae

Distributive disjunction is defined as the first division meiotic segregation of either nonhomologous chromosomes that lack homologs or homologous chromosomes that have not recombined. To determine if chromosomes from the yeast Saccharomyces cerevisiae were capable of distributive disjunction, we constructed a strain that was monosomic for both chromosome I and chromosome III and analyzed the meiotic segregation of the two monosomic chromosomes. In addition, we bisected chromosome I into two functional chromosome fragments, constructed strains that were monosomic for both chromosome fragments and examined meiotic segregation of the chromosome fragments in the monosomic strains. The two nonhomologous chromosomes or chromosome fragments appeared to segregate from each other in approximately 90% of the asci analyzed, indicating that yeast chromosomes were capable of distributive disjunction. We also examined the ability of a small nonhomologous centromere containing plasmid to participate in distributive disjunction with the two nonhomologous monosomic chromosomes. The plasmid appeared to efficiently participate with the two full length chromosomes suggesting that distributive disjunction in yeast is not dependent on chromosome size. Thus, distributive disjunction in S. cerevisiae appears to be different from Drosophila melanogaster where a different sized chromosome is excluded from distributive disjunction when two similar size nonhomologous chromosomes are present.     

A comparative linkage and physical map of bovine chromosome 24 with human chromosome 18

Polymorphic microsatellites have been developed in the vicinity of nine genes on bovine chromosome (BTA) 24, all orthologous to genes on human chromosome (HSA) 18. The microsatellites have been isolated from bacterial and yeast artificial chromosome clones containing the genes. A linkage map was developed including these polymorphic markers and four anonymous, published microsatellites. Yeast artificial chromosomes containing six of these genes have also been mapped using fluorescent in situ hybridization (FISH), thereby tying the linkage map together with the physical map of BTA24. Comparing gene location on HSA18 and BTA24 identifies four regions of conserved gene order, each containing at least two genes. These genes identify six regions of conserved order between human and mouse, two more than in the human-bovine comparison. The breakpoints between regions of conserved order for human-bovine are also breakpoints in the human-mouse comparison. The centromere identifies a fifth conserved region if the BTA24 centromere is orthologous with the HSA18 centromere. Received: 17 September 1998 / Accepted: 4 December 1998     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Meiosis in Saccharomyces Cerevisiae Mutants Lacking the Centromere-Binding Protein Cp1

CP1 (encoded by the CEP1 gene) is a centromere binding protein of Saccharomyces cerevisiae that binds to the conserved DNA element I (CDEI) of yeast centromeres. To investigate the function of CP1 in yeast meiosis, we analyzed the meiotic segregation of CEN plasmids, nonessential chromosome fragments (CFs) and chromosomes in cep1 null mutants. Plasmids and CFs missegregated in 10-20% of meioses with the most frequent type of aberrant event being precocious sister segregation at the first meiotic division; paired and unpaired CFs behaved similarly. An unpaired chromosome I homolog (2N + 1) also missegregated at high frequency in the cep1 mutant (7.6%); however, missegregation of other chromosomes was not detected by tetrad analysis. Spore viability of cep1 tetrads was significantly reduced, and the pattern of spore death was nonrandom. The inviability could not be explained solely by chromosome missegregation and is probably a pleiotropic effect of cep1. Mitotic chromosome loss in cep1 strains was also analyzed. Both simple loss (1:0 segregation) and nondisjunction (2:0 segregation) were increased, but the majority of loss events resulted from nondisjunction. We interpret the results to suggest that CP1 generally promotes chromatid-kinetochore adhesion.     

Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast.

Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry. The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations. This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase. We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays. This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31). Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance. The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity. Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays. We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.     

Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.     

Direct FISH of 5S rDNA on tomato pachytene chromosomes places the gene at the heterochromatic knob immediately adjacent to the centromere of chromosome 1.

We describe a molecular cytogenetic procedure for high resolution physical mapping of DNA markers, an essential step toward construction of an integrated molecular-classical-cytological map. Tomato was selected as material because its pachytene chromosomes are amenable for study and because detailed molecular, classical, and cytological maps are available. Karyotyping of acetocarmine-stained pachytene chromosomes showing detailed cytogenetic landmarks was combined with direct FISH of the 5S rDNA gene. This enabled us to pinpoint the 5S rDNA gene to the first heterochromatic knob immediately adjacent to the centromere in the short arm of chromosome 1. Thus the position of the 5S rDNA gene on the molecular map was related to the position of the 5S rDNA on the cytogenetic map. The results also provide conclusive evidence of the location of a functional gene in the pericentric heterochromatic region, a rare event to date in plants. We conclude that karyotyping of pachytene chromosomes can be combined with FISH to map a DNA sequence to a cytogenetically defined region and to determine the chromatin origin of an expressed gene. Key words : direct fluorescence in situ hybridization, 5S rDNA, pachytene chromosomes, heterochromatic gene.     

Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat

We used gametocidal (Gc) chromosomes 2C and 3C(SAT) to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.