Background In most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe. Results Intron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors. Conclusions Rec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

Background In order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle. Results Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occuring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-γ-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-γ-S. Conclusions Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

Background SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes. Results We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity. Conclusion Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.

Background The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. Results We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to ∼ 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. Conclusions Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.

Background Transcriptional regulation in eukaryotes generally operates at the level of individual genes. Regulation of sets of adjacent genes by mechanisms operating at the level of chromosomal domains has been demonstrated in a number of cases, but the fraction of genes in the genome subject to regulation at this level is unknown. Results Drosophila gene-expression profiles that were determined from over 80 experimental conditions using high-density oligonucleotide microarrays were searched for groups of adjacent genes that show similar expression profiles. We found about 200 groups of adjacent and similarly expressed genes, each having between 10 and 30 members; together these groups account for over 20% of assayed genes. Each group covers between 20 and 200 kilobase pairs of genomic sequence, with a mean group size of about 100 kilobase pairs. Groups do not appear to show any correlation with polytene banding patterns or other known chromosomal structures, nor were genes within groups functionally related to one another. Conclusions Groups of adjacent and co-regulated genes that are not otherwise functionally related in any obvious way can be identified by expression profiling in Drosophila. The mechanism underlying this phenomenon is not yet known.

Background Insertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria. Their activities cause mutations, promoting genetic diversity and sometimes adaptation. Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates. Here, we map most of the IS elements in E. coli B and compare their locations with the published genomes of K-12 and O157:H7. Results The genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7. IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7. IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA. Some sequences specific to B are identified, as reported previously for O157:H7. The extent of nucleotide sequence divergence between B and K-12 is <2% for most sequences adjacent to IS elements. By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci. Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12. Conclusions These data reveal a high level of IS activity since E. coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions. These findings indicate the important role of IS elements in genome plasticity and divergence.

Background: Whole-genome comparisons can provide great insight into many aspects of biology. Until recently, however, comparisons were mainly possible only between distantly related species. Complete genome sequences are now becoming available from multiple sets of closely related strains or species. Results: By comparing the recently completed genome sequences of Vibrio cholerae, Streptococcus pneumoniae and Mycobacterium tuberculosis to those of closely related species - Escherichia coli, Streptococcus pyogenes and Mycobacterium leprae, respectively - we have identified an unusual and previously unobserved feature of bacterial genome structure. Scatterplots of the conserved sequences (both DNA and protein) between each pair of species produce a distinct X-shaped pattern, which we call an X-alignment. The key feature of these alignments is that they have symmetry around the replication origin and terminus; that is, the distance of a particular conserved feature (DNA or protein) from the replication origin (or terminus) is conserved between closely related pairs of species. Statistically significant X-alignments are also found within some genomes, indicating that there is symmetry about the replication origin for paralogous features as well. Conclusions: The most likely mechanism of generation of X-alignments involves large chromosomal inversions that reverse the genomic sequence symmetrically around the origin of replication. The finding of these X-alignments between many pairs of species suggests that chromosomal inversions around the origin are a common feature of bacterial genome evolution.

Background The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. Results While those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a. Conclusions The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

Background Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies. Results This report presents a database () of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains). Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested. Conclusions Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb) density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for polymorphism was sufficient to quickly develop a set of more than fifteen informative markers, some of which show a very high degree of polymorphism. In one instance, the polymorphism information content index reaches 0.82 with allele length covering a wide size range (600-1950 bp), and nine alleles resolved in the small number of independent Bacillus anthracis strains typed here.

Background Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast two-hybrid mating system, will yield valuable information for functional biology on a proteomic scale. Results We have developed an optimized interaction mating protocol for the yeast two-hybrid system, which gives increased mating efficiencies. This significantly reduces the effort and cost of cDNA library screening and allows multiple parallel approaches. Improved preincubation conditions before mating, and optimal cell densities and cell ratios enable almost quantitative mating of the yeast cells carrying the cDNA library. We have proved the applicability of this technology using 20 bait proteins to screen an Arabidopsis thaliana cDNA library, in spite of bait-dependent variations in mating efficiency. Conclusions The improved yeast two-hybrid interaction-mating protocol presented here allows the multiple parallel screening of cDNA libraries. It can be carried out without specialized equipment and has the potential to be standardized and automated.