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Previously, Tuller et al. found that the first 30 to 100 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons, and are presumably translated somewhat slowly. They argued, based on informatics, that this initial slow translation “ramp” was adaptive; and that it increased the efficiency of translation by provided a queuing mechanism to prevent ribosome collisions and traffic jams. Today, the translational speeds of the various codons are known, and there is a strong correlation between rare codon usage and slow translation speed. We have re-examined the slow translation ramp. We confirm the finding of Tuller et al. that a region of slow translation exists. However, we also find that the 5’ ends of yeast genes are poorly conserved, suggesting that they are unstable in evolution and turn over relatively rapidly. When a new 5’ end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5’ ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated 5’ ends are translated poorly compared to genes with rapidly translated 5’ ends. Thus we conclude that slow 5’ translation may be a “spandrel”; it is a non-adaptive consequence of something else, in this case the turnover of 5’ ends in evolution.

Background and Issues: Acute strokes are medical emergencies that require rapid assessment and treatment. Following treatments, focused and frequent monitoring is essential to quickly identify and mitigate any physiological deterioration. However, hospital capacity and decreased availability of monitored beds often causes delay in admission and prolonged stays in the emergency department and the potential increase in morbidity and mortality. Purpose: The purpose of this quality improvement initiative is to reduce the length of time from a patient presenting to the emergency department with acute stroke symptoms and treated with alteplase, to the time that the patient is being monitored utilizing “comprehensive specialized stroke care” level monitoring. This level of monitoring allows for the patient to be assessed closely and frequently to ensure the absence of physiological or neurological deterioration as well as for any adverse thrombolytic reaction. Methods: To improve the process of patient progression within the hospital, the care team utilized the ADKAR® Change Management Model. This model focuses on sponsoring awareness, promoting desire, providing knowledge, ensuring ability, and reinforcing changes. Each of these five components is critical to implement the proposed changes and ensure the longevity of the process changes. Results: Since the implementation of the process change, the Primary Stroke Center has experienced twenty-four months of mean “door-to-monitored bed” times below the 180 minute benchmark. In addition, the mean “door to monitored bed” time has decreased from 210 minutes in the three months preceding the process change (n=20), to 113 minutes during the twenty-four months following the change (n=150). Conclusions: During this process change, the Stroke Center successfully reduced the time between patient arrival and being in a monitored bed. The use of the ADKAR® Change Management Model is particularly advantageous in implementing a process change that is expected to be sustained into the future.

Yeast sporulation is a highly regulated developmental program by which diploid cells generate haploid gametes, termed spores. To better define the genetic pathways regulating sporulation, a systematic screen of the set of ~3300 nonessential Schizosaccharomyces pombe gene deletion mutants was performed to identify genes required for spore formation. A high-throughput genetic method was used to introduce each mutant into an h90 background, and iodine staining was used to identify sporulation-defective mutants. The screen identified 34 genes whose deletion reduces sporulation, including 15 that are defective in forespore membrane morphogenesis. In S. pombe, the total number of sporulation-defective mutants is a significantly smaller fraction of coding genes than in S. cerevisiae, which reflects the different evolutionary histories and biology of the two yeasts.

Expression of crs1 pre-mRNA, encoding a meiotic cyclin, is blocked in actively growing fission yeast cells by a multifaceted mechanism. The most striking feature is that crs1 transcripts are continuously synthesized in vegetative cells, but are targeted for degradation rather than splicing and polyadenylation. Turnover of crs1 RNA requires the exosome, similar to previously described nuclear surveillance and silencing mechanisms, but does not involve a non-canonical poly(A) polymerase. Instead, crs1 transcripts are targeted for destruction by a factor previously implicated in turnover of meiotic RNAs in growing cells. Like exosome mutants, mmi1 mutants splice and polyadenylate vegetative crs1 transcripts. Two regulatory elements are located at the 3′ end of the crs1 gene, consistent with the increased accumulation of spliced RNA in polyadenylation factor mutants. This highly integrated regulatory strategy may ensure a rapid response to adverse conditions, thereby guaranteeing survival.

The RNA exosome is a conserved complex for RNA degradation with two ribonucleolytic subunits, Dis3 and Rrp6. Rrp6 is a 3′–5′ exonuclease, but it also has a structural role in helping target RNAs to the Dis3 activity. The relative importance of the exonuclease activity and the targeting activity probably differs between different RNA substrates, but this is poorly understood. To understand the relative contributions of the exonuclease and the targeting activities to the degradation of individual RNA substrates in Schizosaccharomyces pombe, we compared RNA levels in an rrp6 null mutant to those in an rrp6 point mutant specifically defective in exonuclease activity. A wide range of effects was found, with some RNAs dependent mainly on the structural role of Rrp6 (“protein-dependent” targets), other RNAs dependent mainly on the catalytic role (“activity-dependent” targets), and some RNAs dependent on both. Some protein-dependent RNAs contained motifs targeted via the RNA-binding protein Mmi1, while others contained a motif possibly involved in response to iron. In these and other cases Rrp6 may act as a structural adapter to target specific RNAs to the exosome by interacting with sequence-specific RNA-binding proteins.

Simmonds et al. (1) have criticized our paper (2). Two disagreements stand out. First, the authors suggest that our viruses are attenuated by increased “dinucleotide frequencies” (1, 3), but “codon pair deoptimization” they dismiss as “artefact” (3). We believe this is just semantic word juggling. When a protein is recoded, UpA/CpG dinucleotides available for change are largely at codon–codon junctions, where they define codon pairs. In mammals, many disfavored codon pairs have UpA or CpG at their junctions (4). Therefore, altering UpA/CpG dinucleotides simultaneously alters codon pairs, and vice versa. Accordingly, all of the viral constructs in question (1, 2) coordinately alter dinucleotides and codon pairs (see below) and so do not distinguish the phenomena. No mechanism is known for either phenomenon. Nothing about our results, conclusions, or the future uses of these attenuated viruses would change if we called them “dinucleotide deoptimized.”

Mitotic genes are one of the most strongly oscillating groups of genes in the eukaryotic cell cycle. Understanding the regulation of mitotic gene expression is a key issue in cell cycle control but is poorly understood in most organisms. Here, we find a new mitotic transcription factor, Sak1, in the fission yeast Schizosaccharomyces pombe. Sak1 belongs to the RFX family of transcription factors, which have not previously been connected to cell cycle control. Sak1 binds upstream of mitotic genes in close proximity to Fkh2, a forkhead transcription factor previously implicated in regulation of mitotic genes. We show that Sak1 is the major activator of mitotic gene expression and also confirm the role of Fkh2 as the opposing repressor. Sep1, another forkhead transcription factor, is an activator for a small subset of mitotic genes involved in septation. From yeasts to humans, forkhead transcription factors are involved in mitotic gene expression and it will be interesting to see whether RFX transcription factors may also be involved in other organisms.

Despite a high frequency of introns in the fission yeast Schizosaccharomyces pombe, regulated splicing is virtually unknown. We present evidence that splicing constitutes a major mechanism for controlling gene expression during meiosis, as 12 of 96 transcripts tested, which encode known components as well as previously uncharacterized ORFs, retain introns until specific times during differentiation. The meiotically spliced pre-mRNAs include two cyclins, rem1 (discovered by Ayte and Nurse) and crs1. Consistent with the use of regulated splicing to block protein production, expression of crs1 in vegetative cells is toxic. Analyses of gene chimeras indicate that splicing is prevented in mitotically growing cells via inhibition, in contrast to the positive control of meiotic splicing in budding yeast. Most strikingly, splicing of crs1 and rem1 is regulated by sequences located outside the coding regions, far from the target introns, a phenomenon previously observed only in metazoans.

Background: Eukaryotic DNA replication starts at many origins. Some origins are used early in S phase, while others are programmed to fire later. In general, late replication is correlated with transcriptional inactivity and with location near the nuclear periphery. However, the mechanisms that determine replication timing are unclear, and the cause-and-effect relationship between late replication, transcriptional inactivity, and location at the nuclear periphery is unknown. Results: Using budding yeast, we show that a transcriptional silencer, HMR-E , can reset the time of initiation of ARS305 from early to late. This resetting requires Sir proteins, which are silencers of transcription. Resetting can also be achieved by targeting Sir4 to ARS305 . HMR-E sequences and targeted Sir4, both of which cause late replication of ARS305 , also cause transcriptional silencing of the nearby APA1 gene. Conclusions: Sir proteins are sufficient to reprogram an origin from early to late; that is, Sir proteins are a cause of late replication. Presumably, the tight chromatin structure promoted by Sir proteins favors both transcriptional inactivity and late replication.

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell- cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. © 2014 by the Genetics Society of America., Financial support to H.V.M. from the Comisión Interministerial de Ciencia Y Tecnología (Spain)/European Union Fondo Europeo de Desarrollo Regional program (grant BFU2010-15085) made this work possible. M.A.C. and N.d.L were supported by Formación de Personal Universitario fellowships from the Ministry of Science, and M.H. was supported by a Junta de Ampliación de Estudios predoctoral fellowship from the Consejo Superior de Investigaciones Científicas, Spain.

The Schizosaccharomyces pombe gene pch1(+) (pombe cyclin C homology) was isolated in a two-hybrid screen for proteins that interact with Cdc2. The cyclin box region of Pch1 protein shares greatest sequence identity with mammalian and Drosophila C-type cyclins ( approximately 33% identity). Pch1 is significantly less similar to Mcs2 (19% identity), a second member of the C-type cyclin family in S. pombe. Cdc2 co-precipitates with Pch1 in S. pombe cell lysates, although Cdc2 may not be the major catalytic partner of a Pch1 kinase in vivo. Purified Pch1-associated kinase phosphorylated myelin basic protein, histone H1, and a peptide corresponding to the carboxyl-terminal domain repeat of RNA polymerase II. The amount of pch1 mRNA does not oscillate during the cell cycle, as is the case for mRNA transcripts of other C-type cyclin genes. Deltapch1 cells are inviable, therefore S. pombe has two essential genes that encode members of the C-type cyclin family, pch1(+) and mcs2(+). The Deltapch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not result in a cdc cell cycle-arrest phenotype.

In fission yeast, Cdc2 kinase has both positive and negative roles in regulating DNA replication, being first necessary for the transition from G1 to S phase and later required to prevent the re-initiation of DNA replication during G2. We report here that Cdc2 interacts with Orp2, a protein similar to the Orc2 replication factor subunit of Saccharomyces cerevisiae origin recognition complex (ORC). ORC binds chromosomal origins and is essential for chromosomal replication initiation. Fission yeast Orp2 is required for DNA replication and interacts with the rate-limiting replication activator Cdc18. Cells lacking Orp2 undergo aberrant mitosis, indicating that Orp2 is involved in generating a checkpoint signal. These findings suggest that ORC functions are conserved among eukaryotes and provide evidence that Cdc2 controls DNA replication initiation by acting directly at chromosomal origins.

Considerable evidence connects heterochromatin or silenced chromatin with the Origin Recognition Complex (ORC) which is needed for initiation of DNA replication.1-7 In this review we consider biological forces that might be served by this connection. The prevailing view in the literature is that ORC recruits heterochromatin. This seems paradoxical because a replication initiator, ORC, would be recruiting factors which seem to oppose replication by forming inaccessible chromatin structures. Here we suggest a different view, that heterochromatin recruits ORC to facilitate replication of hard-to-replicate heterochromatic regions. We consider how existing data can be reconciled with this viewpoint, and we consider the biological predictions that arise from this perspective.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5′ rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.

We have used micrococcal nuclease (MNase) digestion followed by deep sequencing in order to obtain a higher resolution map than previously available of nucleosome positions in the fission yeast, Schizosaccharomyces pombe. Our data confirm an unusually short average nucleosome repeat length, ∼152 bp, in fission yeast and that transcriptional start sites (TSSs) are associated with nucleosome-depleted regions (NDRs), ordered nucleosome arrays downstream and less regularly spaced upstream nucleosomes. In addition, we found enrichments for associated function in four of eight groups of genes clustered according to chromatin configurations near TSSs. At replication origins, our data revealed asymmetric localization of pre-replication complex (pre-RC) proteins within large NDRs—a feature that is conserved in fission and budding yeast and is therefore likely to be conserved in other eukaryotic organisms.

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

A screen of Saccharomyces cerevisiae histone alanine substitution mutants revealed that mutations in any of three adjacent residues, L97, Y98, or G99, near the C terminus of H4 led to a unique phenotype. The mutants grew slowly, became polyploid or aneuploid rapidly, and also lost chromosomes at a high rate, most likely because their kinetochores were not assembled properly. There was lower histone occupancy, not only in the centromeric region, but also throughout the genome for the H4 mutants. The mutants displayed genetic interactions with the genes encoding two different histone chaperones, Rtt106 and CAF-I. Affinity purification of Rtt106 and CAF-I from yeast showed that much more H4 and H3 were bound to these histone chaperones in the case of the H4 mutants than in the wild type. However, in vitro binding experiments showed that the H4 mutant proteins bound somewhat more weakly to Rtt106 than did wild-type H4. These data suggest that the H4 mutant proteins, along with H3, accumulate on Rtt106 and CAF-I in vivo because they cannot be deposited efficiently on DNA or passed on to the next step in the histone deposition pathway, and this contributes to the observed genome instability and growth defects.

The fission yeast clade-comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus-occupies the basal branch of Ascomycete fungi and is an important model of eukaryote biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.

Summary Transcriptional control is exerted by the antagonistic activities of activator and repressor proteins. In Saccharomyces cerevisiae, transcription factor complexes containing the MADS box protein Mcm1p are key regulators of cell cycle-dependent transcription at both the G2/M and M/G1 transitions. The homeodomain repressor protein Yox1p acts in a complex with Mcm1p to control the timing of gene expression. Here, we show that Yox1p interacts with Mcm1p through a motif located N terminally to its homeodomain. Yox1p functions as a transcriptional repressor by competing with the forkhead transcription activator protein Fkh2p for binding to Mcm1p through protein-protein interactions at promoters of a subset of Mcm1p-regulated genes. Importantly, this competition is not through binding the same DNA site that is commonly observed. Thus, this study describes a different mechanism for determining the timing of cell cycle-dependent gene expression that involves competition between short peptide motifs in repressor and activator proteins for interaction with a common binding partner., Highlights ► The repressor protein Yox1p uses a short hydrophobic motif to bind to Mcm1p ► Yox1p and the forkhead transcriptional activator, Fkh2p, competitively bind to Mcm1p ► Competition is driven through mutually exclusive protein-protein interactions ► Temporal binding of Yox1p and Fkh2p determines the timing of cell-cycle transcription

Cyclin-dependent kinases (CDKs) are subunits of transcription factor (TF) IIH and positive transcription elongation factor b (P-TEFb). To define their functions, we mutated the TFIIH-associated kinase Mcs6 and P-TEFb homologs Cdk9 and Lsk1 of fission yeast, making them sensitive to inhibition by bulky purine analogs. Selective inhibition of Mcs6 or Cdk9 blocks cell division, alters RNA polymerase (Pol) II carboxyl-terminal domain (CTD) phosphorylation, and represses specific, overlapping subsets of transcripts. At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5-a CDK substrate and regulator of elongation-accumulates disproportionately to Pol II when either kinase is inhibited. In contrast, Mcs6 activity is sufficient-and necessary-to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs.

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G(1)/S transcriptional program by directly regulating MBF, the G(1)/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G(1)/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G(1)/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

Background Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe. Results We found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) ~2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50–100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome. Conclusion The origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.

Background PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN. Results Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis. Conclusion We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs.

Considerable evidence connects heterochromatin or silenced chromatin with the Origin Recognition Complex (ORC) which is needed for initiation of DNA replication. In this review we consider biological forces that might be served by this connection. The prevailing view in the literature is that ORC recruits heterochromatin. This seems paradoxical because a replication initiator, ORC, would be recruiting factors which seem to oppose replication by forming inaccessible chromatin structures. Here we suggest a different view, that heterochromatin recruits ORC to facilitate replication of hard-to-replicate heterochromatic regions. We consider how existing data can be reconciled with this viewpoint, and we consider the biological predictions that arise from this perspective

Messenger RNAs don't usually correspond exactly to DNA — portions of the primary transcript, known as introns, are removed by splicing. A study reveals new ways in which splicing can be regulated. Genome-wide analyses in yeast have shown that the switch from mitosis to gamete-forming meiosis is associated with a dramatic change in gene expression profiles. One protein expressed only during meiosis is the cyclin Rem1, which enhances pre-meiotic intragenic recombination and ensures progression through meiosis. Moldon et al. now show that Rem1 expression in the fission yeast Saccharomyces pombe is controlled not only at the level of transcription, but also by splicing. In mitotic cells, binding of the Fkh2 transcription factor to the Rem1 promoter yields a transcript that retains its introns so that only a short protein is produced; this protein affects recombination levels. In meiotic cells, binding of a meiosis-specific transcription factor, Mei4, to the Rem1 promoter results in splicing of Rem1, and the active cyclin. Thus two transcription factors can differentially modify splicing of the same gene.

In a recent issue of Nature, Skotheim et al. (2008) show that a transcriptional positive feedback loop plays a key role in the commitment to enter the yeast cell cycle.

Transcription of a typical eukaryotic gene by RNA polymerase II is activated by proteins bound to sites found near the beginning of the gene as well as to sites, called enhancers, located a great distance from the gene. According to one view, the primary difference between an activator that can work at a large distance and one that cannot is that the former bears a particularly strong activating region; the stronger the activating region, the more readily the activator interacts with its target bound near the transcriptional start site, with the intervening DNA looping out to accommodate the reaction. One alternative view is that the effect of proteins bound to enhancers might require some special aspect of cellular or chromosome structure. Consistent with the first view, an activator bearing an unusually potent activating region can stimulate transcription of a mammalian gene in a HeLa nuclear extract when bound as far as 1.3 kilobase pairs upstream or 320 base pairs downstream of the transcriptional start site.

Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45. Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes. One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a 'clamp-loader' required to lock the MCMs around DNA. The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins. Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. Conclusion The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks.

Genome-wide measures of DNA strand composition have been used to find archaeal DNA replication origins. Archaea seem to replicate using a single origin (as do eubacteria) even though archaeal replication factors are more like those of eukaryotes.

The mammalian type II DNA topoisomerase has been proposed to be the intracellular target of a variety of antitumor agents, including m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide]. Because the bacteriophage T4-encoded topoisomerase resembles the mammalian enzyme, we are using T4 as a simple model system to investigate the mechanism of action of m-AMSA. A mutation that renders T4 growth m-AMSA-resistant is closely linked to an amber mutation in T4 gene 39, which encodes one of the topoisomerase subunits. In addition, the gene 39 subunit from the m-AMSA-resistant mutant phage has an altered net charge, strongly indicating that the drug-resistance mutation is within gene 39. Topoisomerase purified from mutant phage-infected Escherichia coli exhibits drug-insensitive DNA relaxation and DNA cleavage activities. Because a single mutation results in both drug-resistant phage growth and a drug-insensitive viral topoisomerase, we conclude that the T4-encoded type II DNA topoisomerase is the physiological target of m-AMSA in phage-infected E. coli.

GAL4 is a yeast transcriptional activator protein that binds to specific 2-fold rotationally symmetric sites on DNA and stimulates transcription of the genes required for galactose catabolism. The DNA binding region of the protein is located within the first 74 amino acids and contains a "zinc finger" sequence motif. We show that a polypeptide comprising the first 147 amino acids of GAL4, designated GAL4 (1-147), binds DNA as a dimer in vitro. Although a protein containing only the first 74 amino acids, designated GAL4 (1-74), binds DNA specifically, its affinity is reduced relative to GAL4 (1-147). Addition of the strong dimerization domain of lambda repressor to GAL4 (1-74) generates a protein that binds as tightly as GAL4 (1-147). GAL4 (1-147) makes rotationally symmetric contacts with its recognition site when assayed by DNase I, exonuclease III and hydroxyl radical footprinting and by phosphate ethylation interference. Binding of GAL4 (1-147) in vitro requires either zinc or cadmium.

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know., A comprehensive examination of gene expression throughout the cell cycle of fission yeast is compared with recent related studies to highlight robust transcriptional patterns.