In domesticated populations of Saccharomyces cerevisiae, whole chromosome aneuploidy is very uncommon.

Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts

Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

Repositioning of aurora B promoted by chiasmata ensures sister chromatid mono-orientation in meiosis I.

During meiosis I, kinetochores of sister chromatids are juxtaposed or fused and mono-orient, while homologous chromosomes that are paired by chiasmata (bivalents) have to biorient. In the absence of chiasmata, biorientation of sister chromatids (univalents), which carries a risk of aneuploidy, has been occasionally detected in several species, including humans. We show in fission yeast that biorientation of fused sister kinetochores predominates during early prometaphase I. Without chiasmata, this undesirable biorientation of univalents persists and eventually evades the spindle assembly checkpoint, provoking abnormal anaphase. When univalents are connected by chiasmata or by an artificial tether, this erroneous attachment is converted to monopolar attachment and stabilized. This stabilization is apparently achieved by a chromosome configuration that brings kinetochores to the outer edge of the bivalent, while bringing Aurora B, a destabilizer of kinetochore-microtubule attachment, inward. Our results elucidate how chiasmata favor biorientation of bivalents over that of univalents at meiosis I.

in polyploid species

We developed the Yeast Gene Order Browser (YGOB; http://wolfe.gen.tcd.ie/ygob) to facilitate visual comparisons and computational analysis of synteny relationships in yeasts. The data presented in YGOB, currently covering seven species, are based on sets of homologous genes that have been intensively manually curated based on both sequence similarity and genomic context (synteny). We reconciled different laboratories' lists of paralogous Saccharomyces cerevisiae gene pairs formed by genome duplication (ohnologs), and present near-exhaustive lists of the ohnolog pairs retained in S. cerevisiae (551, including 22 previously unidentified), Saccharomyces castellii (599), and Candida glabrata (404).

Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts

During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts-Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe-also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10(-8), which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms.

Genome-wide genetic analysis of polyploidy in yeast

Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described ‘ploidy-specific lethality’, where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome-wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher-order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin-dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin-mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle-dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Human aneuploidy: mechanisms and new insights into an age-old problem

Trisomic and monosomic (aneuploid) embryos account for at least 10% of human pregnancies and, for women nearing the end of their reproductive lifespan, the incidence may exceed 50%. The errors that lead to aneuploidy almost always occur in the oocyte but, despite intensive investigation, the underlying molecular basis has remained elusive. Recent studies of humans and model organisms have shed new light on the complexity of meiotic defects, providing evidence that the age-related increase in errors in the human female is not attributable to a single factor but to an interplay between unique features of oogenesis and a host of endogenous and exogenous factors.

Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis.

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.

Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae.

We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

Rare Variants Create Synthetic Genome-Wide Associations

Genome-wide association studies (GWAS) have now identified at least 2,000 common variants that appear associated with common diseases or related traits (http://www.genome.gov/gwastudies), hundreds of which have been convincingly replicated. It is generally thought that the associated markers reflect the effect of a nearby common (minor allele frequency >0.05) causal site, which is associated with the marker, leading to extensive resequencing efforts to find causal sites. We propose as an alternative explanation that variants much less common than the associated one may create "synthetic associations" by occurring, stochastically, more often in association with one of the alleles at the common site versus the other allele. Although synthetic associations are an obvious theoretical possibility, they have never been systematically explored as a possible explanation for GWAS findings. Here, we use simple computer simulations to show the conditions under which such synthetic associations will arise and how they may be recognized. We show that they are not only possible, but inevitable, and that under simple but reasonable genetic models, they are likely to account for or contribute to many of the recently identified signals reported in genome-wide association studies. We also illustrate the behavior of synthetic associations in real datasets by showing that rare causal mutations responsible for both hearing loss and sickle cell anemia create genome-wide significant synthetic associations, in the latter case extending over a 2.5-Mb interval encompassing scores of "blocks" of associated variants. In conclusion, uncommon or rare genetic variants can easily create synthetic associations that are credited to common variants, and this possibility requires careful consideration in the interpretation and follow up of GWAS signals.

Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago

From Darwin's study of the Galapagos and Wallace's study of Indonesia, islands have played an important role in evolutionary investigations, and radiations within archipelagos are readily interpreted as supporting the conventional view of allopatric speciation. Even during the ongoing paradigm shift towards other modes of speciation, island radiations, such as the Lesser Antillean anoles, are thought to exemplify this process. Geological and molecular phylogenetic evidence show that, in this archipelago, Martinique anoles provide several examples of secondary contact of island species. Four precursor island species, with up to 8 mybp divergence, met when their islands coalesced to form the current island of Martinique. Moreover, adjacent anole populations also show marked adaptation to distinct habitat zonation, allowing both allopatric and ecological speciation to be tested in this system. We take advantage of this opportunity of replicated island coalescence and independent ecological adaptation to carry out an extensive population genetic study of hypervariable neutral nuclear markers to show that even after these very substantial periods of spatial isolation these putative allospecies show less reproductive isolation than conspecific populations in adjacent habitats in all three cases of subsequent island coalescence. The degree of genetic interchange shows that while there is always a significant genetic signature of past allopatry, and this may be quite strong if the selection regime allows, there is no case of complete allopatric speciation, in spite of the strong primae facie case for it. Importantly there is greater genetic isolation across the xeric/rainforest ecotone than is associated with any secondary contact. This rejects the development of reproductive isolation in allopatric divergence, but supports the potential for ecological speciation, even though full speciation has not been achieved in this case. It also explains the paucity of anole species in the Lesser Antilles compared to the Greater Antilles.

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

Using the yeast gene deletion collection to customize gene expression.

Substitute Teacher Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF imm...

Genome downsizing in polyploid plants

All else being equal, polyploids are expected to have larger C-values (amount of DNA in the unreplicated gametic nucleus) than their diploid progenitors, increasing in direct proportion with ploidy. This expectation is observed in some polyploid series, especially those newly formed, but there are examples suggesting that C-values in particular polyploids are less than expected. The availability of the Angiosperm DNA C-values database (http:// www.rbgkew.org.uk/cvavhomepage.html) has allowed this question to be addressed across a broad range of angiosperms and has revealed striking results deviating from expectation: (i) mean 1C DNA amount did not increase in direct proportion with ploidy, and (ii) mean DNA amount per basic genome (calculated by dividing the 2C value by ploidy) tended to decrease with increasing ploidy. These results suggest that loss of DNA following polyploid formation, or genome downsizing, may be a widespread phenomenon of considerable biological significance. Recent advances in our understanding of the molecular events that take place following polyploid formation together with new data on how DNA amounts can both increase and decrease provide some insights into how genome downsizing may take place. The nature of the evolutionary forces that may be driving DNA loss are also discussed.

Mapping Meiotic Single-Strand DNA Reveals a New Landscape of DNA Double-Strand Breaks in Saccharomyces cerevisiae

DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.

Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin

Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes.

Roads to polyploidy: the megakaryocyte example.

Polyploidy, recognized by multiple copies of the haploid chromosome number, has been described in plants, insects, and in mammalian cells such as, the platelet precursors, the megakaryocytes. Several of these cell types reach high ploidy via a different cell cycle. Megakaryocytes undergo an endomitotic cell cycle, which consists of an S phase interrupted by a gap, during which the cells enter mitosis but skip anaphase B and cytokinesis. Here, we review the mechanisms that lead to this cell cycle and to polyploidy in megakaryocytes, while also comparing them to those described for other systems in which high ploidy is achieved. Overall, polyploidy is associated with an orchestrated change in expression of several genes, of which, some may be a result of high ploidy and hence a determinant of a new cell physiology, while others are inducers of polyploidization. Future studies will aim to further explore these two groups of genes.

Separation of mother and daughter cells.

Publisher Summary The budding yeast Saccharomyces cerevisiae ( S. cerevisiae ) divides asymmetrically. In vegetative growth, yeast cells reproduce by budding, and the position where the bud forms ultimately determines the plane of cell division. This chapter describes the detailed procedures for the separation and isolation of mothers and daughters. These protocols have been used by investigators studying aging, bud site selection, and other aspects of asymmetric cell division. The chapter describes the procedures for performing life span analysis by micromanipulation and the steps for the large-scale collection of old cells. At the beginning and the end of a life span, it can be difficult to distinguish mothers from daughters. At most points in the life span, daughter cells are smaller than the mothers that produced them. In addition, mother cells will generally bud a second time before their daughter cells form their first bud. One method for effective isolation of virgin daughter cells from mother cells, but not for recovery of old mothers, is called a “baby machine. ” Mother cells are attached to a membrane and allowed to divide. Daughter cells from these attached cells are eluted continuously by washing the membrane.

The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization.

Neither the restoration of the centrosome during fertilization nor its reduction during gametogenesis is fully understood, but both are pivotal events in development. During each somatic cell cycle, the chromosomes, cytoplasm, and centrosomes duplicate in interphase, and all three split in two during each cell division. While it has long been recognized that both the sperm and the egg contribute equal haploid genomes during fertilization and that the vast majority of the cytoplasm is contributed by the egg, the relative contributions of the centrosome by each gamete are still in question. This article explores centrosome inheritance patterns and considers nine integral and secondarily derived activities of the centrosome. Boveri once hypothesized that "The ripe egg possesses all of the elements necessary for development save an active division-center. The sperm, on the other hand, possesses such a center but lacks the protoplasmic substratum in which to operate. In this respect the egg and sperm are complementary structures; their union in syngamy thus restores to each the missing element necessary to further development. " This article reviews the evidence gathered from 11 experimental strategies used to test this theory. While the majority of these approaches supports the hypothesis that the sperm introduces the centrosome at fertilization, the pattern did not reveal itself as universal, since parthenogenesis occurs in nature and can be induced artificially, since centrosome and centriole form de novo in extracts from unfertilized eggs and since the centrosome is derived from maternal sources during fertilization in some systems--notably, in mice. Models of the centrosome are proposed, along with speculative mechanisms which might lead to the cloaking of the reproducing element of the maternal centrosome during oogenesis and the retention of this structure by the paternal centrosome during spermatogenesis. Proteins essential for microtubule nucleation, like gamma-tubulin, are retained in the cytoplasm during oogenesis, but are largely lost during spermatogenesis. It is further postulated that the restoration of the zygotic centrosome at fertilization requires the attraction of maternal centrosomal components (in particular, gamma-tubulin and the 25S "gamma-some" particle) to the paternal reproducing element; this, along with post-translational modifications (including phosphorylation, disulfide reduction, and calcium ion binding), creates a functional zygote centrosome by blending both maternal and paternal constituents.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct visualization of the endomitotic cell cycle in living megakaryocytes: differential patterns in low and high ploidy cells.

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (>or=8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos.

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

Cyclin A Regulates Kinetochore-Microtubules to Promote Faithful Chromosome Segregation

The most conspicuous event in the cell cycle is the alignment of chromosomes in metaphase. Chromosome alignment fosters faithful segregation through the formation of bi-oriented attachments of kinetochores to spindle microtubules. Notably, numerous kinetochore-microtubule (k-MT) attachment errors are present in early mitosis (prometaphase), and the persistence of those errors is the leading cause of chromosome mis-segregation in aneuploid human tumour cells that continually mis-segregate whole chromosomes and display chromosomal instability. How robust error correction is achieved in prometaphase to ensure error-free mitosis remains unknown. Here we show that k-MT attachments in prometaphase cells are considerably less stable than in metaphase cells. The switch to more stable k-MT attachments in metaphase requires the proteasome-dependent destruction of cyclin A in prometaphase. Persistent cyclin A expression prevents k-MT stabilization even in cells with aligned chromosomes. By contrast, k-MTs are prematurely stabilized in cyclin-A-deficient cells. Consequently, cells lacking cyclin A display higher rates of chromosome mis-segregation. Thus, the stability of k-MT attachments increases decisively in a coordinated fashion among all chromosomes as cells transit from prometaphase to metaphase. Cyclin A creates a cellular environment that promotes microtubule detachment from kinetochores in prometaphase to ensure efficient error correction and faithful chromosome segregation.

A 3D Map of the Yeast Kinetochore Reveals the Presence of Core and Accessory Centromere-Specific Histone

The budding yeast kinetochore is ~68 nm in length with a diameter slightly larger than a 25 nm microtubule. The kinetochores from the 16 chromosomes are organized in a stereotypic cluster encircling central spindle microtubules. Quantitative analysis of the inner kinetochore cluster (Cse4, COMA) reveals structural features not apparent in singly attached kinetochores. The cluster of Cse4-containing kinetochores is physically larger perpendicular to the spindle axis relative to the cluster of Ndc80 molecules. If there was a single Cse4 (molecule or nucleosome) at the kinetochore attached to each microtubule plus end, the cluster of Cse4 would appear geometrically identical to Ndc80. Thus, the structure of the inner kinetochore at the surface of the chromosomes remains unsolved. We have used point fluorescence microscopy and statistical probability maps to deduce the two-dimensional mean position of representative components of the yeast kinetochore relative to the mitotic spindle in metaphase. Comparison of the experimental images to three-dimensional architectures from convolution of mathematical models reveals a pool of Cse4 radially displaced from Cse4 at the kinetochore and kinetochore microtubule plus ends. The pool of displaced Cse4 can be experimentally depleted in mRNA processing pat1Δ or xrn1Δ mutants. The peripheral Cse4 molecules do not template outer kinetochore components. This study suggests an inner kinetochore plate at the centromere-microtubule interface in budding yeast and yields information on the number of Ndc80 molecules at the microtubule attachment site.

A Mechanism Linking Extra Centrosomes to Chromosomal Instability

Chromosomal instability (CIN) is a hallmark of many tumours and correlates with the presence of extra centrosomes. However, a direct mechanistic link between extra centrosomes and CIN has not been established. It has been proposed that extra centrosomes generate CIN by promoting multipolar anaphase, a highly abnormal division that produces three or more aneuploid daughter cells. Here we use long-term live-cell imaging to demonstrate that cells with multiple centrosomes rarely undergo multipolar cell divisions, and the progeny of these divisions are typically inviable. Thus, multipolar divisions cannot explain observed rates of CIN. In contrast, we observe that CIN cells with extra centrosomes routinely undergo bipolar cell divisions, but display a significantly increased frequency of lagging chromosomes during anaphase. To define the mechanism underlying this mitotic defect, we generated cells that differ only in their centrosome number. We demonstrate that extra centrosomes alone are sufficient to promote chromosome missegregation during bipolar cell division. These segregation errors are a consequence of cells passing through a transient 'multipolar spindle intermediate' in which merotelic kinetochore-microtubule attachment errors accumulate before centrosome clustering and anaphase. These findings provide a direct mechanistic link between extra centrosomes and CIN, two common characteristics of solid tumours. We propose that this mechanism may be a common underlying cause of CIN in human cancer.

Post-transcriptional regulation of IME1 determines initiation of meiosis in Saccharomyces cerevisiae.

The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis.

The Evolution of Dominance in Sporophytic Self-Incompatibility Systems. II. Mate Availability and Recombination

Sporophytic self-incompatibility (SSI) is a self-pollen recognition system that enforces outcrossing in plants. Recognition in SSI systems is typically controlled by a complex locus (5-locus) with separate genes that determine pollen and stigma specificity. Experimental studies show that S-alleles can be dominant, recessive, or codominant, and that the dominance level of a given S-allele can depend upon whether pollen or stigma specificity is examined. Here and in the companion paper by Llaurens and colleagues, the evolution of dominance in single-locus SSI is explored using numerical models and simulation. Particular attention is directed at factors that can cause S-allele dominance to differ in pollen versus stigma. The effect of recombination between the S-locus and modifier locus is also examined. The models predict that limitation in the number of compatible mates is required for the evolution of S-allele dominance in the stigma but not in the pollen. Tight linkage between the S-locus and modifier promotes the evolution of S-allele dominance hierarchies. Model results are interpreted with respect to published information on the molecular basis of dominance in SSI systems, and reported S-allele dominance relationships in a variety of species. These studies show that dominant S-alleles are more common in the pollen than in the stigma, a pattern that when interpreted in light of model predictions, suggests that mate limitation may be relatively infrequent in natural populations with SSI.

Single-Cell RNA-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells

Expression from both alleles is generally observed in analyses of diploid cell populations, but studies addressing allelic expression patterns genome-wide in single cells are lacking. Here, we present global analyses of allelic expression across individual cells of mouse preimplantation embryos of mixed background (CAST/EiJ × C57BL/6J). We discovered abundant (12 to 24%) monoallelic expression of autosomal genes and that expression of the two alleles occurs independently. The monoallelic expression appeared random and dynamic because there was considerable variation among closely related embryonic cells. Similar patterns of monoallelic expression were observed in mature cells. Our allelic expression analysis also demonstrates the de novo inactivation of the paternal X chromosome. We conclude that independent and stochastic allelic transcription generates abundant random monoallelic expression in the mammalian cell.

Dominance relationships between S-alleles in self-incompatible Brassica campestris L.

Dominance relationships were studied for 249 out of 276 possible pair-wise combinations between 24 S-alleles of Brassica campestris that had been isolated from two natural populations from Turkey and Japan. Each F1 hybrid was test-crossed reciprocally against its respective parental S-homozygotes to determine the dominance relationships between the pair of S-alleles it contained. The 24 S-alleles were classified into two groups on the stigma side and three groups on the pollen side. In the stigma, codominance occurred frequently, and dominance or recessiveness seemed to appear according to the combination of S-alleles. In the pollen, codominance was less frequent, and there seemed to be a certain hierarchy of the dominance relationships as a whole, although dominance appeared with certain specific combinations of S-alleles. Interactions among 24 S-alleles were different in the stigma and in the pollen. Independent weakening of S-alleles was found between 20 pairs in the pollen, but only two in the stigma. This interaction seems to be correlated with recessiveness of S-alleles.

Siblings with opposite chromosome constitutions, dup(2q)/del(7q) and del(2q)/dup(7q).

Chromosome 7q36 microdeletion syndrome is a rare genomic disorder characterized by underdevelopment of the brain, microcephaly, anomalies of the sex organs, and language problems. Developmental delay, intellectual disability, autistic spectrum disorders, BDMR syndrome, and unusual facial morphology are the key features of the chromosome 2q37 microdeletion syndrome. A genetic screening for two brothers with global developmental delay using high-resolution chromosomal analysis and subtelomeric multiplex ligation-dependent probe amplification revealed subtelomeric rearrangements on the same sites of 2q37.2 and 7q35, with reversed deletion and duplication. Both of them showed dysmorphic facial features, severe disability of physical and intellectual development, and abnormal genitalia with differential abnormalities in their phenotypes. The family did not have abnormal genetic phenotypes. According to the genetic analysis of their parents, adjacent-1 segregation from their mother's was suggested as a mechanism of their gene mutation. By comparing the phenotypes of our patients with previous reports on similar patients, we tried to obtain the information of related genes and their chromosomal locations.

Getting started with yeast.

Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

Heterochromatin integrity affects chromosome reorganization after centromere dysfunction.

The centromere is essential for the inheritance of genetic information on eukaryotic chromosomes. Epigenetic regulation of centromere identity has been implicated in genome stability, karyotype evolution, and speciation. However, little is known regarding the manner in which centromere dysfunction affects the chromosomal architectures. Here we show that in the fission yeast Schizosaccharomyces pombe, the conditional deletion of the centromere produces survivors that carry either a neocentromere-acquired chromosome at the subtelomeric region or an acentric chromosome rescued by intertelomere fusion with either of the remaining chromosomes. The ratio of neocentromere formation to telomere fusion is considerably decreased by the inactivation of genes involved in RNA interference-dependent heterochromatin formation. By affecting the modes of chromosomal reorganization, the genomic distribution of heterochromatin may influence the fate of karyotype evolution.

Perspective: repression of competition and the evolution of cooperation.

Repression of competition within groups joins kin selection as the second major force in the history of life shaping the evolution of cooperation. When opportunities for competition against neighbors are limited within groups, individuals can increase their own success only by enhancing the efficiency and productivity of their group. Thus, characters that repress competition within groups promote cooperation and enhance group success. Leigh first expressed this idea in the context of fair meiosis, in which each chromosome has an equal chance of transmission via gametes. Randomized success means that each part of the genome can increase its own success only by enhancing the total number of progeny and thus increasing the success of the group. Alexander used this insight about repression of competition in fair meiosis to develop his theories for the evolution of human sociality. Alexander argued that human social structures spread when they repress competition within groups and promote successful group-against-group competition. Buss introduced a new example with his suggestion that metazoan success depended on repression of competition between cellular lineages. Maynard Smith synthesized different lines of thought on repression of competition. In this paper, I develop simple mathematical models to illustrate the main processes by which repression of competition evolves. With the concepts made clear, I then explain the history of the idea. I finish by summarizing many new developments in this subject and the most promising lines for future study.

Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast

The eukaryotic genome consists of DNA molecules far longer than the cells that contain them. They reach their greatest compaction during chromosome condensation in mitosis. This process is aided by condensin, a structural maintenance of chromosomes (SMC) family member. The spatial organization of mitotic chromosomes and how condensin shapes chromatin architecture are not yet fully understood. Here we use chromosome conformation capture (Hi-C) to study mitotic chromosome condensation in the fission yeast Schizosaccharomyces pombe. This showed that the interphase landscape characterized by small chromatin domains is replaced by fewer but larger domains in mitosis. Condensin achieves this by setting up longer-range, intrachromosomal DNA interactions, which compact and individualize chromosomes. At the same time, local chromatin contacts are constrained by condensin, with profound implications for local chromatin function during mitosis. Our results highlight condensin as a major determinant that changes the chromatin landscape as cells prepare their genomes for cell division.

Telomeres and telomerase: more than the end of the line

Early studies of telomerase suggested that telomeres are maintained by an elegant but relatively simple and highly conserved mechanism of telomerase-mediated replication. As we learn more, it has become clear that the mechanism is elegant but not as simple as first thought. It is also evident that, although many species use similar, sometimes identical, DNA sequences for telomeres, these species express their own individuality in the way they regulate these sequences and, perhaps, in the additional tasks that they have imposed on their telomeric DNA. The striking similarities between telomeres in different species have revealed much about chromosome ends; the differences are proving to be equally informative. In addition to the differences between species that use telomerase, there are also a few exceptional organisms with atypical telomeres for which no telomerase activity has been detected. This review addresses recent studies, the insights they offer, and, perhaps more importantly, the questions they raise.

Exploitative and Hierarchical Antagonism in a Cooperative Bacterium

Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation. Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.

Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells

A long-standing hypothesis on tumorigenesis is that cell division failure, generating genetically unstable tetraploid cells, facilitates the development of aneuploid malignancies. Here we test this idea by transiently blocking cytokinesis in p53-null (p53-/-) mouse mammary epithelial cells (MMECs), enabling the isolation of diploid and tetraploid cultures. The tetraploid cells had an increase in the frequency of whole-chromosome mis-segregation and chromosomal rearrangements. Only the tetraploid cells were transformed in vitro after exposure to a carcinogen. Furthermore, in the absence of carcinogen, only the tetraploid cells gave rise to malignant mammary epithelial cancers when transplanted subcutaneously into nude mice. These tumours all contained numerous non-reciprocal translocations and an 8–30-fold amplification of a chromosomal region containing a cluster of matrix metalloproteinase (MMP) genes. MMP overexpression is linked to mammary tumours in humans and animal models. Thus, tetraploidy enhances the frequency of chromosomal alterations and promotes tumour development in p53-/- MMECs.

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

Isolation of a Fusion Transcript, AMLl IETO, With Similarity to

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Hepatic polyploidy and liver growth control.

The onset of cellular polyploidy is recognized in all differentiated mammalian tissues. Polyploidy has been noted frequently in the normal liver, as well as in pathophysiological states of the liver. As insights into the significance of polyploidy accumulate gradually, it is becoming clear that cells belonging to high ploidy classes exhibit advancement toward terminal differentiation and cellular senescence with greater probabilities of apoptosis. Involvement of specific genetic abnormalities, such as impaired DNA repair, may lead to hepatocellular polyploidy. Working models indicate that extensive polyploidy could lead to organ failure, as well as to oncogenesis with activation of precancerous cell clones.

Common variants in DGKK are strongly associated with risk of hypospadias

Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase κ, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 × 10−11 and OR = 2.3, P = 2.9 × 10−9, respectively) and in the Dutch (OR = 3.9, P = 2.4 × 10−5 and OR = 3.8, P = 3.4 × 10−5) and Swedish (OR = 2.5, P = 2.6 × 10−8 and OR = 2.2, P = 2.7 × 10−6) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.

Temporal regulation of topoisomerase IV activity in E. coli.

We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.

Single-RNA counting reveals alternative modes of gene expression in yeast

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

Identification of an allele of VAM3/SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana.

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis.

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during C. elegans oocyte meiosis

In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.

The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 13

The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.

Global analysis of ATM polymorphism reveals significant functional constraint.

ATM, the gene that is mutated in ataxia-telangiectasia, is associated with cerebellar degeneration, abnormal proliferation of small blood vessels, and cancer. These clinically important manifestations have stimulated interest in defining the sequence variation in the ATM gene. Therefore, we undertook a comprehensive survey of sequence variation in ATM in diverse human populations. The protein-encoding exons of the gene (9,168 bp) and the adjacent intron and untranslated sequences (14,661 bp) were analyzed in 93 individuals from seven major human populations. In addition, the coding sequence was analyzed in one chimpanzee, one gorilla, one orangutan, and one Old World monkey. In human ATM, 88 variant sites were discovered by denaturing high-performance liquid chromatography, which is 96%-100% sensitive for detection of DNA sequence variation. ATM was compared to 14 other autosomal genes for nucleotide diversity. The noncoding regions of ATM had diversity values comparable to other genes, but the coding regions had very low diversity, especially in the last 29% of the protein sequence. A test of the neutral evolution hypothesis, through use of the Hudson/Kreitman/Aguadé statistic, revealed that this region of the human ATM gene was significantly constrained relative to that of the orangutan, the Old World monkey, and the mouse, but not relative to that of the chimpanzee or the gorilla. ATM displayed extensive linkage disequilibrium, consistent with suppression of meiotic recombination at this locus. Seven haplotypes were defined. Two haplotypes accounted for 82% of all chromosomes analyzed in all major populations; two others carrying the same D126E missense polymorphism accounted for 33% of chromosomes in Africa but were never observed outside of Africa. The high frequency of this polymorphism may be due either to a population expansion within Africa or to selective pressure.

Tolerance of whole-genome doubling propagates chromosomal instability and accelerates cancer genome evolution.

UNLABELLED The contribution of whole-genome doubling to chromosomal instability (CIN) and tumor evolution is unclear. We use long-term culture of isogenic tetraploid cells from a stable diploid colon cancer progenitor to investigate how a genome-doubling event affects genome stability over time. Rare cells that survive genome doubling demonstrate increased tolerance to chromosome aberrations. Tetraploid cells do not exhibit increased frequencies of structural or numerical CIN per chromosome. However, the tolerant phenotype in tetraploid cells, coupled with a doubling of chromosome aberrations per cell, allows chromosome abnormalities to evolve specifically in tetraploids, recapitulating chromosomal changes in genomically complex colorectal tumors. Finally, a genome-doubling event is independently predictive of poor relapse-free survival in early-stage disease in two independent cohorts in multivariate analyses [discovery data: hazard ratio (HR), 4.70, 95% confidence interval (CI), 1.04-21.37; validation data: HR, 1.59, 95% CI, 1.05-2.42]. These data highlight an important role for the tolerance of genome doubling in driving cancer genome evolution. SIGNIFICANCE Our work sheds light on the importance of whole-genome–doubling events in colorectal cancer evolution. We show that tetraploid cells undergo rapid genomic changes and recapitulate the genetic alterations seen in chromosomally unstable tumors. Furthermore, we demonstrate that a genome-doubling event is prognostic of poor relapse-free survival in this disease type.

Fission Yeast Scm3: A CENP-A Receptor Required for Integrity of Subkinetochore Chromatin

The mechanisms ensuring specific incorporation of CENP-A at centromeres are poorly understood. Mis16 and Mis18 are required for CENP-A localization at centromeres and form a complex that is conserved from fission yeast to human. Fission yeast sim1 mutants that alleviate kinetochore domain silencing are defective in Scm3(Sp), the ortholog of budding yeast Scm3(Sc). Scm3(Sp) depends on Mis16/18 for its centromere localization and like them is recruited to centromeres in late anaphase. Importantly, Scm3(Sp) coaffinity purifies with CENP-A(Cnp1) and associates with CENP-A(Cnp1) in vitro, yet localizes independently of intact CENP-A(Cnp1) chromatin and is differentially released from chromatin. While Scm3(Sc) has been proposed to form a unique hexameric nucleosome with CENP-A(Cse4) and histone H4 at budding yeast point centromeres, we favor a model in which Scm3(Sp) acts as a CENP-A(Cnp1) receptor/assembly factor, cooperating with Mis16 and Mis18 to receive CENP-A(Cnp1) from the Sim3 escort and mediate assembly of CENP-A(Cnp1) into subkinetochore chromatin.

Evidence that the Ipl1-Sli15 (Aurora Kinase-INCENP) Complex Promotes Chromosome Bi-orientation by Altering Kinetochore-Spindle Pole Connections

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. We investigated IPL1 function in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin.

Cytochemical Analysis of Pollen Development in Wild-Type Arabidopsis and a Male-Sterile Mutant.

Microsporogenesis has been examined in wild-type Arabidopsis thaliana and the nuclear male-sterile mutant BM3 by cytochemical staining. The mutant lacks adenine phosphoribosyltransferase, an enzyme of the purine salvage pathway that converts adenine to AMP. Pollen development in the mutant began to diverge from wild type just after meiosis, as the tetrads of microspores were released from their callose walls. The first indication of abnormal pollen development in the mutant was a darker staining of the microspore wall due to an incomplete synthesis of the intine. Vacuole formation was delayed and irregular in the mutant, and the majority of the mutant microspores failed to undergo mitotic divisions. Enzyme activities of alcohol dehydrogenase and esterases decreased in the mutant soon after meiosis and were undetectable in mature pollen grains of the mutant. RNA accumulation was also diminished. These results are discussed in relation to the possible role(s) of adenine salvage in pollen development.

A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07–1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

SMC complexes differentially compact mitotic chromosomes according to genomic context

Structural maintenance of chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids, while condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate that this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead, it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes.

Deregulated DNA polymerase beta induces chromosome instability and tumorigenesis.

To reach the biological alterations that characterize cancer, the genome of tumor cells must acquire increased mutability resulting from a malfunction of a network of genome stability systems, e.g., cell cycle arrest, DNA repair, and high accuracy of DNA synthesis during DNA replication. Numeric chromosomal imbalance, referred to as aneuploidy, is the most prevalent genetic changes recorded among many types of solid tumors. We report here that ectopic expression in cells of DNA polymerase beta, an error-prone enzyme frequently over-regulated in human tumors, induces aneuploidy, an abnormal localization of the centrosome-associated gamma-tubulin protein during mitosis, a deficient mitotic checkpoint, and promotes tumorigenesis in nude immunodeficient mice. Thus, we find that alteration of polymerase beta expression appears to induce major genetic changes associated with a malignant phenotype.

Localization of a gene for partial epilepsy to chromosome 10q

There is strong evidence for a genetic contribution to epilepsy, but it is commonly assumed that this genetic contribution is limited to ‘generalized’ epilepsies, and that most forms of ‘partial’ epilepsy are nongenetic. In a linkage analysis of a single family containing 11 affected individuals, we obtained strong evidence for localization of a gene for partial epilepsy. This susceptibility gene maps to chromosome 10q, with a maximum two–point lod score for D10S192 of 3.99 at θ=0.0. All affected individuals share a single haplotype for seven tightly linked contiguous markers; the maximum lod score for this haplotype is 4.83 at θ=0.0. Key recombinants place the susceptibility locus within a 10 centimorgan interval.

A forkhead-domain gene is mutated in a severe speech and language disorder

Individuals affected with developmental disorders of speech and language have substantial difficulty acquiring expressive and/or receptive language in the absence of any profound sensory or neurological impairment and despite adequate intelligence and opportunity1. Although studies of twins consistently indicate that a significant genetic component is involved1,2,3, most families segregating speech and language deficits show complex patterns of inheritance, and a gene that predisposes individuals to such disorders has not been identified. We have studied a unique three-generation pedigree, KE, in which a severe speech and language disorder is transmitted as an autosomal-dominant monogenic trait4. Our previous work mapped the locus responsible, SPCH1, to a 5.6-cM interval of region 7q31 on chromosome 7 (ref. 5). We also identified an unrelated individual, CS, in whom speech and language impairment is associated with a chromosomal translocation involving the SPCH1 interval6. Here we show that the gene FOXP2, which encodes a putative transcription factor containing a polyglutamine tract and a forkhead DNA-binding domain, is directly disrupted by the translocation breakpoint in CS. In addition, we identify a point mutation in affected members of the KE family that alters an invariant amino-acid residue in the forkhead domain. Our findings suggest that FOXP2 is involved in the developmental process that culminates in speech and language.

Ria1p (Ynl163c), a protein similar to elongation factors 2, is involved in the biogenesis of the 60S subunit of the ribosome in Saccharomyces cerevisiae

RIA1 (YNL163c) is a quasi-essential gene that encodes a protein with strong similarities to elongation factors 2. Small C-terminal deletions in the protein lead to a severe growth defect. In the case of a 22-residue C-terminal deletion this can be suppressed by intragenic mutations in the RIA1 gene or dominant extragenic mutations in TIF6, which is thought to be involved in the biogenesis of the 60S subunit of the ribosome. The dominant TIF6 alleles can also suppress the phenotype associated with a complete deletion of the RIA1 gene. Depletion of Ria1p has a dramatic effect on the polysome profile: there is a severe reduction in the level of the 80S monosomes, an imbalance in the 40S/60S ratio, and halfmers appear. Dissociation of the monosomes and polysomes in the Ria1p depletion mutant revealed a specific reduction in the amount of 60S subunits. Localization experiments with HA-tagged derivatives of Ria1p did not detect any stable association of Ria1p with ribosome subunits, 80S monosomes or polysomes. Cell fractionation experiments show that Ria1p is found in both the cytoplasmic fraction and the nuclear fraction. Taken together, these data suggest that Ria1p is involved in the biogenesis of the 60S subunit of the ribosome.

The embryonic cell lineage of the nematode Caenorhabditis elegans.

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.

A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis

Although assembly of acentrosomal meiotic spindles has been extensively studied, little is known about the segregation of chromosomes on these spindles. Here, we show in Caenorhabditis elegans oocytes that the kinetochore protein, KNL-1, directs assembly of meiotic kinetochores that orient chromosomes. However, in contrast to mitosis, chromosome separation during meiotic anaphase is kinetochore-independent. Before anaphase, meiotic kinetochores and spindle poles disassemble along with the microtubules on the poleward side of chromosomes. During anaphase, microtubules then form between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between kinetochores during pre-anaphase spindle assembly and anaphase separation. These proteins are localized by the chromosomal passenger complex, which regulates the loss of meiotic chromosome cohesion. Thus, meiotic segregation in C. elegans is a two-stage process, where kinetochores orient chromosomes, but are then dispensable for their separation. We suggest that separation is controlled by a meiosis-specific chromosomal domain to coordinate cohesin removal and chromosome segregation.

A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia

Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p. Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; Pcombined = 4.88 × 10−94). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10−4). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.

Sperm cross-over activity in regions of the human genome showing extreme breakdown of marker association.

Population diversity data have recently provided profound, albeit inferential, insights into meiotic recombination across the human genome, revealing a landscape dominated by thousands of cross-over hotspots. However, very few of these putative hotspots have been directly analyzed for cross-over activity. We now describe a search for very active hotspots, by using extreme breakdown of marker association as a guide for high-resolution sperm cross-over analysis. This strategy has led to the isolation of the most active cross-over hotspots yet described. Their morphology, sequence attributes, and cross-over processes are very similar to those seen at less active hotspots, but their activity in sperm is poorly predicted from population diversity information. Several of these hotspots showed evidence for biased gene conversion accompanying cross-over, in some cases associated with variation between men in cross-over activity and with two hotspots showing complete presence/absence polymorphism in different men. Hotspot polymorphism is very common at less active hotspots but curiously was not seen at any of the most active hotspots. This contrasts with the prediction that extreme hotspots should be the most vulnerable to attenuation by meiotic drive in favor of mutations that suppress recombination and should therefore show rapid rate evolution and thus variation in activity between men. Finally, these very intense hotspots provide a valuable resource for dissecting meiotic recombination processes and pathways in humans.

PGAP2 mutations, affecting the GPI-anchor-synthesis pathway, cause hyperphosphatasia with mental retardation syndrome.

Recently, mutations in genes involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor have been identified in a new subclass of congenital disorders of glycosylation (CDGs) with a distinct spectrum of clinical features. To date, mutations have been identified in six genes (PIGA, PIGL, PIGM, PIGN, PIGO, and PIGV) encoding proteins in the GPI-anchor-synthesis pathway in individuals with severe neurological features, including seizures, muscular hypotonia, and intellectual disability. We developed a diagnostic gene panel for targeting all known genes encoding proteins in the GPI-anchor-synthesis pathway to screen individuals matching these features, and we detected three missense mutations in PGAP2, c.46C>T, c.380T>C, and c.479C>T, in two unrelated individuals with hyperphosphatasia with mental retardation syndrome (HPMRS). The mutations cosegregated in the investigated families. PGAP2 is involved in fatty-acid GPI-anchor remodeling, which occurs in the Golgi apparatus and is required for stable association between GPI-anchored proteins and the cell-surface membrane rafts. Transfection of the altered protein constructs, p. Arg16Trp (NP_001243169.1), p. Leu127Ser, and p. Thr160Ile, into PGAP2-null cells showed only partial restoration of GPI-anchored marker proteins, CD55 and CD59, on the cell surface. In this work, we show that an impairment of GPI-anchor remodeling also causes HPMRS and conclude that targeted sequencing of the genes encoding proteins in the GPI-anchor-synthesis pathway is an effective diagnostic approach for this subclass of CDGs.

All dosage compensation is local: Gene-by-gene regulation of sex-biased expression on the chicken Z chromosome

Recent reports have suggested that birds lack a mechanism of wholesale dosage compensation for the Z sex chromosome. This discovery was rather unexpected, as all other animals investigated with chromosomal mechanisms of sex determination have some method to counteract the effects of gene dosage of the dominant sex chromosome in males and females. Despite the lack of a global mechanism of avian dosage compensation, the pattern of gene expression difference between males and females varies a great deal for individual Z-linked genes. This suggests that some genes may be individually dosage compensated, and that some less-than-global pattern of dosage compensation, such as local or temporal, exists on the avian Z chromosome. We used global gene expression profiling in males and females for both somatic and gonadal tissue at several time points in the life cycle of the chicken to assess the pattern of sex-biased gene expression on the Z chromosome. Average fold-change between males and females varied somewhat among tissue time-point combinations, with embryonic brain samples having the smallest gene dosage effects, and adult gonadal tissue having the largest degree of male bias. Overall, there were no neighborhoods of overall dosage compensation along the Z. Taken together, this suggests that dosage compensation is regulated on the Z chromosome entirely on a gene-by-gene level, and can vary during the life cycle and by tissue type. This regulation may be an indication of how critical a given gene's functionality is, as the expression level for essential genes will be tightly regulated in order to avoid perturbing important pathways and networks with differential expression levels in males and females.

The developmental potential of iPSCs is greatly influenced by reprogramming factor selection.

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

IGF2 is parentally imprinted during human embryogenesis and in the Beckwith–Wiedemann syndrome

The phenomenon of parental imprinting involves the preferential expression of one parental allele of a subset of chromosomal genes and has so far only been documented in the mouse. We show here, by exploiting sequence polymorphisms in exon nine of the human insulin–like growth factor 2 (IGF2) gene, that only the paternally–inherited allele is active in embryonic and extra–embryonic cells from first trimester pregnancies. In addition, only the paternal allele is expressed in tissues from a patient who suffered from Beckwith–Wiedemann syndrome. Thus the parental imprinting of IGF2 appears to be evolutionarily conserved from mouse to man and has implications for the generation of the Beckwith–Wiedemann syndrome.

Functional reprogramming of polyploidization in megakaryocytes.

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells.

AIR-2: An Aurora/Ipl1-related Protein Kinase Associated with Chromosomes and Midbody Microtubules Is  Required for Polar Body Extrusion and Cytokinesis  in Caenorhabditis elegans Embryos

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I–arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms.

BACKGROUND Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. CONCLUSIONS Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae.

In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

Translational control of intron splicing in eukaryotes

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.

Apolipoprotein E genotype and concussion in college athletes.

OBJECTIVE To evaluate the association between apolipoprotein E (APOE) polymorphisms (E2, C/T Arg158Cys; E4, T/C Cys112Arg; and promoter, g-219t) and the history of concussion in college athletes. We hypothesized that carrying 1 or more APOE rare (or minor) allele assessed in this study would be associated with having a history of 1 or more concussions. DESIGN Multicenter cross-sectional study. SETTING University athletic facilities. PARTICIPANTS One hundred ninety-six male football (n = 163) and female soccer (n = 33) college athletes volunteered. INTERVENTIONS Written concussion history questionnaire and saliva samples for genotyping. MAIN OUTCOME MEASURES Self-reported history of a documented concussion and rare APOE genotype (E2, E4, promoter). RESULTS There was a significant association (Wald χ² = 3.82; P = 0.05; odds ratio = 9.8) between carrying all APOE rare alleles and the history of a previous concussion. There was also a significant association (Wald χ² = 3.96, P = 0.04, odds ratio = 8.4) between carrying the APOE promoter minor allele and experiencing 2 or more concussions. CONCLUSIONS Carriers of all 3 APOE rare (or minor) alleles assessed in this study were nearly 10 times more likely to report a previous concussion and may be at a greater risk of concussion versus noncarriers. Promoter minor allele carriers were 8.4 times more likely to report multiple concussions and may be at a greater risk of multiple concussions versus noncarriers. Research involving larger samples of individuals with multiple concussions and carriers of multiple APOE rare alleles is warranted.

Rapid recent growth and divergence of rice nuclear genomes.

By employing the nuclear DNA of the African rice Oryza glaberrima as a reference genome, the timing, natures, mechanisms, and specificities of recent sequence evolution in the indica and japonica subspecies of Oryza sativa were identified. The data indicate that the genome sizes of both indica and japonica have increased substantially, >2% and >6%, respectively, since their divergence from a common ancestor, mainly because of the amplification of LTR-retrotransposons. However, losses of all classes of DNA sequence through unequal homologous recombination and illegitimate recombination have attenuated the growth of the rice genome. Small deletions have been particularly frequent throughout the genome. In >1 Mb of orthologous regions that we analyzed, no cases of complete gene acquisition or loss from either indica or japonica were found, nor was any example of precise transposon excision detected. The sequences between genes were observed to have a very high rate of divergence, indicating a molecular clock for transposable elements that is at least 2-fold more rapid than synonymous base substitutions within genes. We found that regions prone to frequent insertions and deletions also exhibit higher levels of point mutation. These results indicate a highly dynamic rice genome with competing processes for the generation and removal of genetic variation.

Loss of gametophytic self-incompatibility with evolution of inbreeding depression.

Gametophytic self-incompatibility (SI) in plants is a widespread mechanism preventing self-fertilization and the ensuing inbreeding depression, but it often evolves to self-compatibility. We analyze genetic mechanisms for the breakdown of gametophytic SI, incorporating a dynamic model for the evolution of inbreeding depression allowing for partial purging of nearly recessive lethal mutations by selfing, and accounting for pollen limitation and sheltered load linked to the S-locus. We consider two mechanisms for the breakdown of gametophytic SI: a nonfunctional S-allele and an unlinked modifier locus that inactivates the S-locus. We show that, under a wide range of conditions, self-compatible alleles can invade a self-incompatible population. Conditions for invasion are always less stringent for a nonfunctional S-allele than for a modifier locus. The spread of self-compatible genotypes is favored by extremely high or low selfing rates, a small number of S-alleles, and pollen limitation. Observed parameter values suggest that the maintenance of gametophytic SI is caused by a combination of high inbreeding depression in self-incompatible populations coupled with intermediate selfing rates of the self-compatible genotypes and sheltered load linked to the S-locus.

A general model to explore complex dominance patterns in plant sporophytic self-incompatibility systems.

We developed a general model of sporophytic self-incompatibility under negative frequency-dependent selection allowing complex patterns of dominance among alleles. We used this model deterministically to investigate the effects on equilibrium allelic frequencies of the number of dominance classes, the number of alleles per dominance class, the asymmetry in dominance expression between pollen and pistil, and whether selection acts on male fitness only or both on male and on female fitnesses. We show that the so-called "recessive effect" occurs under a wide variety of situations. We found emerging properties of finite population models with several alleles per dominance class such as that higher numbers of alleles are maintained in more dominant classes and that the number of dominance classes can evolve. We also investigated the occurrence of homozygous genotypes and found that substantial proportions of those can occur for the most recessive alleles. We used the model for two species with complex dominance patterns to test whether allelic frequencies in natural populations are in agreement with the distribution predicted by our model. We suggest that the model can be used to test explicitly for additional, allele-specific, selective forces.

Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis.

Using differential interference contrast optics, combined with cinematography, we have studied the morphological changes that the living, syncytial embryo undergoes from stage 10 through 14 of Drosophila embryogenesis, that is just prior to and during formation of the cellular blastoderm. We have supplemented these studies with data collected from fixed, stained, whole embryos. The following information has been obtained. The average duration of nuclear cycles 10, 11, 12 and 13 is about 9, 10, 12 and 21 min, respectively (25 degrees C). In these four cycles, the duration of that portion of the mitotic period that lacks a discrete nuclear envelope is 3, 3, 3 and 5 min, respectively. The length of nuclear cycle 14 varies in a position-specific manner throughout the embryo, the shortest cycles being of 65 min duration. During nuclear cycles 10 through 13, it is commonly observed in living embryos that the syncytial blastoderm nuclei enter (and leave) mitosis in one of two waves that originate nearly simultaneously from the opposite anterior and posterior poles of the embryo, and terminate in its midregion. From our preparations of quick-frozen embryos, we estimate that these mitotic waves take on average about half a minute to travel over the embryonic surface from pole to equator. The yolk nuclei, which remain in the core of the embryo when the rest of the nuclei migrate to the periphery, divide in synchrony with the migrating nuclei at nuclear cycles 8 and 9, and just after the now peripherally located nuclei at nuclear cycle 10. After cycle 10, these yolk nuclei cease dividing and become polyploid. The syncytial embryo has at least three distinct levels of cytoskeletal organization: structured domains of cytoplasm are organized around each blastoderm nucleus; radially directed tracks orient colchicine-sensitive saltatory transport throughout the peripheral cytoplasm; and a long-range organization of the core of the embryo makes possible coherent movements of the large inner yolk mass in concert with each nuclear cycle. This highly organized cytoplasm may be involved in providing positional information for the important process of nuclear determination that is known to occur during these stages.

The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae.

Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.

Age-Associated Sperm DNA Methylation Alterations: Possible Implications in Offspring Disease Susceptibility

Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

The transcriptional program of sporulation in budding yeast

Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

A bird's-eye view of sex chromosome dosage compensation.

Intensive study of a few genetically tractable species with XX/XY sex chromosomes has produced generalizations about the process of sex chromosome dosage compensation that do not fare well when applied to ZZ/ZW sex chromosome systems, such as those in birds. The inherent sexual imbalance in dose of sex chromosome genes has led to the evolution of sex-chromosome-wide mechanisms for balancing gene dosage between the sexes and relative to autosomal genes. Recent advances in our knowledge of avian genomes have led to a reexamination of sex-specific dosage compensation (SSDC) in birds, which is less effective than in known XX/XY systems. Insights about the mechanisms of SSDC in birds also suggest similarities to and differences from those in XX/XY species. Birds are thus offering new opportunities for studying dosage compensation in a ZZ/ZW system, which should shed light on the evolution of SSDC more broadly.

Full-length transcriptome assembly from RNA-Seq data without a reference genome.

Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.

CDE-1 Affects Chromosome Segregation through Uridylation of CSR-1-Bound siRNAs

We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface.

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Caenorhabditis elegans cyclin A- and B-type genes: a cyclin A multigene family, an ancestral cyclin B3 and differential germline expression.

We have cloned cDNAs for Caenorhabditis elegans cyclins A1, B and B3. While cyclins A1 and B are most closely related to either A- or B-type cyclins of other species, cyclin B3 is less related to these cyclins. However, this cyclin is most similar to the recently identified chicken cyclin B3. Our identification of a Caenorhabditis homolog demonstrates that cyclin B3 has been conserved in evolution. Cyclin A1 is a member of an A-type multigene family; however the cyclin A1 cDNA only recognizes a single band on northern blots. A single-sized RNA is also observed for the cyclin B3 cDNA. In contrast, three different transcripts are observed for the cyclin B cDNA. Based on our analyses using RNAs from germline-defective mutants and from populations enriched for males, one cyclin B transcript is specific to the paternal germline. The two other cyclin B transcripts, as well as the cyclin A1 and cyclin B3 transcripts, are most abundant in the maternal germline and are only present at low levels in other tissues. Moreover, the 3' untranslated regions of each Caenorhabditis cyclin cDNA possess several copies of potential translational control elements shown in Xenopus and Drosophila maternal cyclin mRNAs to function during oogenesis and early embryogenesis.

Population genomics of the honey bee reveals strong signatures of positive selection on worker traits.

Most theories used to explain the evolution of eusociality rest upon two key assumptions: mutations affecting the phenotype of sterile workers evolve by positive selection if the resulting traits benefit fertile kin, and that worker traits provide the primary mechanism allowing social insects to adapt to their environment. Despite the common view that positive selection drives phenotypic evolution of workers, we know very little about the prevalence of positive selection acting on the genomes of eusocial insects. We mapped the footprints of positive selection in Apis mellifera through analysis of 40 individual genomes, allowing us to identify thousands of genes and regulatory sequences with signatures of adaptive evolution over multiple timescales. We found Apoidea- and Apis-specific genes to be enriched for signatures of positive selection, indicating that novel genes play a disproportionately large role in adaptive evolution of eusocial insects. Worker-biased proteins have higher signatures of adaptive evolution relative to queen-biased proteins, supporting the view that worker traits are key to adaptation. We also found genes regulating worker division of labor to be enriched for signs of positive selection. Finally, genes associated with worker behavior based on analysis of brain gene expression were highly enriched for adaptive protein and cis-regulatory evolution. Our study highlights the significant contribution of worker phenotypes to adaptive evolution in social insects, and provides a wealth of knowledge on the loci that influence fitness in honey bees.

A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome.

Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

The First Myriapod Genome Sequence Reveals Conservative Arthropod Gene Content and Genome Organisation in the Centipede Strigamia maritima

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.

Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

The Drosophila spaghetti squash ( sqh ) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh -A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh -A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh -E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh -A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.

A DNA test to sex most birds.

Birds are difficult to sex. Nestlings rarely show sex-linked morphology and we estimate that adult females appear identical to males in over 50% of the world's bird species. This problem can hinder both evolutionary studies and human-assisted breeding of birds. DNA-based sex identification provides a solution. We describe a test based on two conserved CHD (chromo-helicase-DNA-binding) genes that are located on the avian sex chromosomes of all birds, with the possible exception of the ratites (ostriches, etc.; Struthioniformes). The CHD-W gene is located on the W chromosome; therefore it is unique to females. The other gene, CHD-Z, is found on the Z chromosome and therefore occurs in both sexes (female, ZW; male, ZZ). The test employs PCR with a single set of primers. It amplifies homologous sections of both genes and incorporates introns whose lengths usually differ. When examined on a gel there is a single CHD-Z band in males but females have a second, distinctive CHD-W band.

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast

A yeast strain lacking Met4p, the primary transcriptional regulator of the sulfur assimilation pathway, cannot synthesize methionine. This apparently simple auxotroph did not grow well in rich media containing excess methionine, forming small colonies on yeast extract/peptone/dextrose plates. Faster-growing large colonies were abundant when overnight cultures were plated, suggesting that spontaneous suppressors of the growth defect arise with high frequency. To identify the suppressor mutations, we used genome-wide single-nucleotide polymorphism and standard genetic analyses. The most common suppressors were loss-of-function mutations in OPI1, encoding a transcriptional repressor of phospholipid metabolism. Using a new system that allows rapid and specific degradation of Met4p, we could study the dynamic expression of all genes following loss of Met4p. Experiments using this system with and without Opi1p showed that Met4 activates and Opi1p represses genes that maintain levels of S-adenosylmethionine (SAM), the substrate for most methyltransferase reactions. Cells lacking Met4p grow normally when either SAM is added to the media or one of the SAM synthetase genes is overexpressed. SAM is used as a methyl donor in three Opi1p-regulated reactions to create the abundant membrane phospholipid, phosphatidylcholine. Our results show that rapidly growing cells require significant methylation, likely for the biosynthesis of phospholipids.

Stepwise Histone Replacement by SWR1 Requires Dual Activation with Histone H2A.Z and Canonical Nucleosome

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.

Epigenetic instability in ES cells and cloned mice.

Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

Stromal p16 expression differentiates endometrial polyp from endometrial hyperplasia

Endometrial polyps are very common benign endometrial lesions, but their pathogenesis is poorly understood, except for a few studies indicating the possibility of benign stromal neoplasm. Although the histopathological diagnosis of endometrial polyp on a surgical specimen is straightforward, it is often difficult to differentiate endometrial polyp from endometrial hyperplasia on a biopsy or curettage specimen. Presently, there is no immunohistochemical marker helpful in this differential diagnosis. In this study, we examined p16 expression in 35 endometrial polyps and 33 cases of endometrial hyperplasia that included 16 simple hyperplasias, 14 complex atypical hyperplasias, and 3 complex hyperplasias without atypia. Stromal p16 expression differed significantly between the two groups; it was seen in 31 (89 %) endometrial polyps, but in only 1 (3 %) endometrial hyperplasia. The percentage of p16-positive stromal cells ranged from 10 to 90 % (mean, 47 %) and the positive cells tended to be distributed around glands. Six cases of endometrial hyperplasia within an endometrial polyp were also examined and all cases showed stromal p16 expression. There was no difference in glandular p16 expression between endometrial polyps 33 (94 %) and hyperplasia 27 (82 %). The p16-immunoreactivity was mostly confined to metaplastic epithelial cells in both groups. Stromal p16 expression might be a peculiar characteristic of endometrial polyp and constitute a useful marker for the diagnosis, especially in fragmented specimens from biopsy or curettage. Stromal p16 expression might be a reflection of p16-induced cellular senescence, which has been documented in several benign mesenchymal neoplasms.

The yeast ribosomal protein L32 and its gene.

The yeast ribosomal protein gene RPL32 of Saccharomyces cerevisiae is of particular interest for two reasons: 1) it is adjacent to another ribosomal protein gene, RP29, whose divergent transcription may be driven from the same control sequences, and 2) it appears that the splicing of its transcript is regulated by the product of the gene, ribosomal protein in L32. RPL32 has been analyzed in detail. It is essential for cell growth. Its sequence predicts L32 to be a protein of 105 amino acids, somewhat basic near the NH2 terminus, rather acidic near the COOH terminus, and homologous to ribosomal protein L30 of mammals. The reading frame has been confirmed by partial NH2-terminal analysis of L32. The nucleotide sequence also predicts an intron of 230 nucleotides, which begins with the unusual sequence GTCAGT and ends 40 nucleotides downstream of the consensus sequence TAC-TAAC. The intron has been confirmed by determination of the sequence of a cDNA clone. Transcription initiates 58 nucleotides upstream of the AUG initiation codon, and the polyadenylation site occurs 100 nucleotides downstream of the termination codon. Regulation of the transcription of ribosomal protein genes has been linked to two related consensus sequences. Analysis of the intergenic region between RP29 and RPL32 reveals three copies of these sequences. A deletion removing all three sequences reduces synthesis of a L32-LacZ fusion protein by more than 90%. Some residual activity, however, remains.

Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.

Deficiency in l-serine deaminase results in abnormal growth and cell division of Escherichia coli K-12.

The loss of the ability to deaminate l-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three l-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l-serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.

Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with a lack of H19 expression.

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.