Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.

Pollen selection: a transgenic reconstruction approach.

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene--present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny.

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.

Polymer Models of Meiotic and Mitotic Chromosomes

Polymers tied together by constraints exhibit an internal pressure; this idea is used to analyze physical properties of the bottle-brush–like chromosomes of meiotic prophase that consist of polymer-like flexible chromatin loops, attached to a central axis. Using a minimal number of experimental parameters, semiquantitative predictions are made for the bending rigidity, radius, and axial tension of such brushes, and the repulsion acting between brushes whose bristles are forced to overlap. The retraction of lampbrush loops when the nascent transcripts are stripped away, the oval shape of diplotene bivalents between chiasmata, and the rigidity of pachytene chromosomes are all manifestations of chromatin pressure. This two-phase (chromatin plus buffer) picture that suffices for meiotic chromosomes has to be supplemented by a third constituent, a chromatin glue to understand mitotic chromosomes, and explain how condensation can drive the resolution of entanglements. This process resembles a thermal annealing in that a parameter (the affinity of the glue for chromatin and/or the affinity of the chromatin for buffer) has to be tuned to achieve optimal results. Mechanical measurements to characterize this protein–chromatin matrix are proposed. Finally, the propensity for even slightly chemically dissimilar polymers to phase separate (cluster like with like) can explain the apparent segregation of the chromatin into A+T- and G+C-rich regions revealed by chromosome banding.

Going mobile: microtubule motors and chromosome segregation.

Proper chromosome segregation in eukaryotes depends upon the mitotic and meiotic spindles, which assemble at the time of cell division and then disassemble upon its completion. These spindles are composed in large part of microtubules, which either generate force by controlled polymerization and depolymerization or transduce force generated by molecular microtubule motors. In this review, we discuss recent insights into chromosome segregation mechanisms gained from the analyses of force generation during meiosis and mitosis. These analyses have demonstrated that members of the kinesin superfamily and the dynein family are essential in all organisms for proper chromosome and spindle behavior. It is also apparent that forces generated by microtubule polymerization and depolymerization are capable of generating forces sufficient for chromosome movement in vitro; whether they do so in vivo is as yet unclear. An important realization that has emerged is that some spindle activities can be accomplished by more than one motor so that functional redundancy is evident. In addition, some meiotic or mitotic movements apparently occur through the cooperative action of independent semiredundant processes. Finally, the molecular characterization of kinesin-related proteins has revealed that variations both in primary sequence and in associations with other proteins can produce motor complexes that may use a variety of mechanisms to transduce force in association with microtubules. Much remains to be learned about the regulation of these activities and the coordination of opposing and cooperative events involved in chromosome segregation; this set of problems represents one of the most important future frontiers of research.

Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

The present study explores a “hydrophobic” energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its “hydrophobicity” and the number of contacts it makes, i.e., E(h⃗, c⃗) = −Σi=1N cihi = −(h⃗.c⃗) is the negative scalar product between two vectors in N-dimensional cartesian space: h⃗ = (h1, … , hN), which represents monomer hydrophobicities and is sequence-dependent; and c⃗ = (c1, … , cN), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c⃗, the best sequence is a vector h⃗ on the direction upon which the projection of c⃗ − c̄⃗ is maximal, where c̄⃗ is the diagonal vector with components equal to c̄, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated kcat of 1.5 min−1. DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPγS.

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5′-ATGACGT-3′. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1–4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.

Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Δ null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction

The yeast genome encodes four proteins (Pms1 and Mlh1–3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to ≈70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.