A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.     

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.     

Mutational Analysis of Meiotic and Mitotic Centromere Function in Saccharomyces cerevisiae

A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.     

mus(3)312D1, a mutagen sensitive mutant with profound effects on female meiosis in Drosophila melanogaster

The third chromosome, mutagen sensitive mutant mus(3)312^D1 impairs the meiotic process in females by increasing the frequency of first division nondisjunction and decreasing the frequency of meiotic crossing over. These genetic properties connote 312 to be defective in DNA replication and/or repair intimately associated with the crossing over exchange process. The mutant maps to the left arm of chromosome III between ru and h, and represents a new genetic site for a meiotic mutant. It is a pleasure and honor to dedicate this paper to my longtime younger friend and collaborator Wolfgang Beermann, cytologist par excellence, on the occasion of his 60th birthday     

Chromosome stability in saccharomycete yeasts

Mutants with high instability of chromosome III designated Chl+ (chromosome loss) were obtained after irradiation with UV the Z4221-3c1 haploid disomic for chromosome III. The Chl+ mutants can be divided into two classes: 1) CL2, CL3, CL7, CL9, CL11, CL12, CL13 with elevated level of spontaneous inter- and intragenic recombination; 2) CL4, CL8 which unstable maintenance of chromosome III not accompanied with elevation of mitotic recombination frequency. The CL4 and CL8 mutants also reveal, in contrast to other mutants, unstable maintenance of artificial mini-chromosomes with chromosomal replicator ARS1 and centromeric loci CEN3, CEN4, CEN5, CEN6, CEN11. Substitution of ARS1 for other yeast replicators (ARS2, ARS of 2 micron plasmid) leads to no stabilization of mini-chromosomes in mutants. The noncentromeric plasmids containing homologous replicator (or replicators) from Candida maltosa are maintained with the same frequency both in wild type and in mutants. So, the stability of mini-chromosomes in CL4 and CL8 is not connected with uneffective replication of these chromosomes. Instability of chromosome III and mini-chromosomes in CL4 and CL8 is controlled by two nonallelic genes designated chl14 and chl18. We suppose that these genes control the process of centromere interaction with mitotic spindle microtubules.     

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.     

Analysis of a Recombination Hotspot for Gene Conversion Occurring at the His2 Gene of Saccharomyces Cerevisiae

The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites. The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot. The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2. Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed. Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure. Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion. This contrasts with other reports about the role of heterologies at the MAT locus.     

The Effect of Recombination-Defective Meiotic Mutants on Fourth-Chromosome Crossing over in DROSOPHILA MELANOGASTER

Crossing over was measured on the normally achiasmate fourth chromosome in females homozygous for one of our different recombination-defective meiotic mutants. Under the influence of those meiotic mutants that affect the major chromosomes by altering the spatial distribution of exchanges, meiotic fourth-chromosome recombinants were recovered irrespective of whether or not the meiotic mutant decreases crossing over on the other chromosomes. No crossing over, on the other hand, was detected on chromosome 4 in either wild type or in the presence of a meiotic mutant that decreases the frequency, but does not affect the spatial distribution, of exchange on the major chromosomes. It is concluded from these observations that (a) in wild type there are regional constraints on exchange that can be attenuated or eliminated by the defects caused by recombination-defective meiotic mutants; [b] these very constraints account for the absence of recombination on chromosome 4 in wild type; and [c] despite being normally achiasmate, chromosome 4 responds to recombination-defective meiotic mutants in the same way as do the other chromosomes.     

Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae.

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.     

Genetic Analysis of a Meiotic Recombination Hotspot on Chromosome III of Saccharomyces Cerevisiae

In a previous study, we analyzed meiotic recombination events that occurred in the 22-kb region (LEU2 to CEN3) of chromosome III of Saccharomyces cerevisiae. We found one region with an enhanced level of crossovers (a hotspot) and one region with a depressed level of crossovers. In this study, we show that about one-third of the crossovers that occur between LEU2 and CEN3 are initiated in a 1.3-kb region located approximately 6 kb from the centromere. Both crossovers and gene conversion events are initiated at this site. Events initiated at this position can be resolved as crossovers in regions located either centromere-distally or centromere-proximally from the initiation site.     

Properties of natural double-strand-break sites at a recombination hotspot in Saccharomyces cerevisiae

This study addresses three questions about the properties of recombination hotspots in Saccharomyces cerevisiae: How much DNA is required for double-strand-break (DSB) site recognition? Do naturally occurring DSB sites compete with each other in meiotic recombination? What role does the sequence located at the sites of DSBs play? In S. cerevisiae, the HIS2 meiotic recombination hotspot displays a high level of gene conversion, a 3''-to-5'' conversion gradient, and two DSB sites located approximately 550 bp apart. Previous studies of hotspots, including HIS2, suggest that global chromosome structure plays a significant role in recombination activity, raising the question of how much DNA is sufficient for hotspot activity. We find that 11.5 kbp of the HIS2 region is sufficient to partially restore gene conversion and both DSBs when moved to another yeast chromosome. Using a variety of different constructs, studies of hotspots have indicated that DSB sites compete with one another for DSB formation. The two naturally occurring DSBs at HIS2 afforded us the opportunity to examine whether or not competition occurs between these native DSB sites. Small deletions of DNA at each DSB site affect only that site; analyses of these deletions show no competition occurring in cis or in trans, indicating that DSB formation at each site at HIS2 is independent. These small deletions significantly affect the frequency of DSB formation at the sites, indicating that the DNA sequence located at a DSB site can play an important role in recombination initiation.     

Chromosome size-dependent control of meiotic reciprocal recombination in Saccharomyces cerevisiae: the role of crossover interference.

In the yeast Saccharomyces cerevisiae, small chromosomes undergo meiotic reciprocal recombination (crossing over) at rates (centimorgans per kilobases) greater than those of large chromosomes, and recombination rates respond directly to changes in the total size of a chromosomal DNA molecule. This phenomenon, termed chromosome size-dependent control of meiotic reciprocal recombination, has been suggested to be important for ensuring that homologous chromosomes cross over during meiosis. The mechanism of this regulation was investigated by analyzing recombination in identical genetic intervals present on different size chromosomes. The results indicate that chromosome size-dependent control is due to different amounts of crossover interference. Large chromosomes have high levels of interference while small chromosomes have much lower levels of interference. A model for how crossover interference directly responds to chromosome size is presented. In addition, chromosome size-dependent control was shown to lower the frequency of homologous chromosomes that failed to undergo crossovers, suggesting that this control is an integral part of the mechanism for ensuring meiotic crossing over between homologous chromosomes.     

Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Puzzling chromosome behavior in triploid females of Drosophila melanogaster

Based on a particular formation of the chromocenter and trivalents in triploid Drosophila females, as well as on asynapsis in pericentromeric regions (which is a result of trivalent competition), an explanation for the increased frequency of crossing over and nonrandom segregation of the X chromosomes and autosomes in the first meiotic division is suggested. It is proposed that a delay in pairing of the pericentromeric heterochromatic chromosome regions combined into a single chromocenter leads to the following: (1) formation of the heteroduplex structures (X structures) takes more time and, consequently, their number and the frequency of crossing over in the paired chromosome regions increases; (2) in nonhomologous chromosomes, the chromocentral connections, which normally degrade in prometaphase, are retained to fulfill a function of coorientation during the first meiotic division.     

Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae

Homologous chromosome synapsis (``homosynapsis') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to ~55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level.     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

Meiotic Disjunction of Homologs in Saccharomyces Cerevisiae Is Directed by Pairing and Recombination of the Chromosome Arms but Not by Pairing of the Centromeres

We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on the pattern of disjunction and recombination of chromosome III during meiosis. The segregation of deletion chromosomes lacking part or all (telocentric) of one arm was analyzed in the presence of one or two copies of a normal chromosome III. In strains containing one normal and any one deletion chromosome, the two chromosomes disjoined in most meioses. In strains with one normal chromosome and both a left and right arm telocentric chromosome, the two telocentrics preferentially disjoined from the normal chromosome. Homology on one arm was sufficient to direct chromosome disjunction, and two chromosomes could be directed to disjoin from a third. In strains containing one deletion chromosome and two normal chromosomes, the two normal chromosomes preferentially disjoined, but in 4-7% of the tetrads the normal chromosomes cosegregated, disjoining from the deletion chromosome. Recombination between the two normal chromosomes or between the deletion chromosome and a normal chromosome increased the probability that these chromosomes would disjoin, although cosegregation of recombinants was observed. Finally, we observed that a derivative of chromosome III in which the centromeric region was deleted and CEN5 was integrated at another site on the chromosome disjoined from a normal chromosome III with fidelity. These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs, that directs meiotic chromosome segregation.     

Meiotic Behavior of Compound Autosomes in Females of DROSOPHILA MELANOGASTER: Interchromosomal Effects and the Source of Spontaneous Nonsegregation

In females of Drosophila melanogaster, compound autosomes enter the repulsion phase of meiosis uncommitted to a particular segregation pattern because their centromeres are not restricted to a bivalent pairing complex as a consequence of crossing over. Their distribution at anaphase, therefore, is determined by some meiotic property other than exchange pairing, a property that for many years has been associated with the concept of nonhomologous pairing. In the absence of heterologous rearrangements or a free Y chromosome, C(3L) and C(3R) are usually recovered in separate gametes, that is as products of meiotic segregation. Nevertheless, there is a regular, albeit infrequent, recovery of reciprocal meiotic products (the nonsegregational products) that are disomic and nullosomic for compound thirds. The frequency of these exceptions, which is normally between 0.5 and 5.0%, differs for the various strains examined, but remains constant for any given strain. Since previous studies have not uncovered a cause for this base level of nonsegregation, it has been referred to as the spontaneous frequency. In this study, crosses between males and females whose X chromosomes, as well as compound autosomes, are differentially marked reveal a highly significant positive correlation between the frequency of compound-autosome nonsegregation and the frequency of X-chromosome nondisjunction. However, an inverse correlation is found when the frequency of nondisjunction is related to the frequency of crossing over in the proximal region of the X chromosome. These findings have been examined with reference to the distributive pairing and the chromocentral models and interpreted as demonstrating (1) that nonsegregational meiotic events arise primarily as a result of nonhomologous interactions, (2) that forces responsible for the segregation of nonhomologous chromosomes are properties of the chromocentral region, and (3) that these forces come into expression after the exchange processes are complete.     

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.