Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components.

Evolutionary experiments with microbes are a powerful tool to study mutations and natural selection. These experiments, however, are often limited to the well-mixed environments of a test tube or a chemostat. Since spatial organization can significantly affect evolutionary dynamics, the need is growing for evolutionary experiments in spatially structured environments. The surface of a Petri dish provides such an environment, but a more detailed understanding of microbial growth on Petri dishes is necessary to interpret such experiments. We formulate a simple deterministic reaction-diffusion model, which successfully predicts the spatial patterns created by two competing species during colony expansion. We also derive the shape of these patterns analytically without relying on microscopic details of the model. In particular, we find that the relative fitness of two microbial strains can be estimated from the logarithmic spirals created by selective sweeps. The theory is tested with strains of the budding yeast Saccharomyces cerevisiae for spatial competitions with different initial conditions and for a range of relative fitnesses. The reaction-diffusion model also connects the microscopic parameters like growth rates and diffusion constants with macroscopic spatial patterns and predicts the relationship between fitness in liquid cultures and on Petri dishes, which we confirmed experimentally. Spatial sector patterns therefore provide an alternative fitness assay to the commonly used liquid culture fitness assays.

BACKGROUND: We investigated the determinants of sexual identity in the budding yeast Saccharomyces cerevisiae. The higher fungi are divided into the ascomycetes and the basidiomycetes. Most ascomycetes have two mating types: one (called α in yeasts and MAT1-1 in filamentous fungi) produces a small, unmodified, peptide pheromone, and the other (a in yeasts and MAT1-2 in filamentous fungi) produces a peptide pheromone conjugated to a C-terminal farnesyl group that makes it very hydrophobic. In the basidiomycetes, all pheromones are lipid-modified, and this difference is a distinguishing feature between the phyla. We asked whether the asymmetry in pheromone modification is required for successful mating in ascomycetes. RESULTS: We cloned receptor and pheromone genes from a filamentous ascomycete and a basidiomycete and expressed these in the budding yeast, Saccharomyces cerevisiae, to generate novel, alternative mating pairs. We find that two yeast cells can mate even when both cells secrete a-like or α-like peptides. Importantly, this is true regardless of whether the cells express the a- or α-mating-type loci, which control the expression of other, sex-specific genes, in addition to the pheromones and pheromone receptors. CONCLUSIONS: We demonstrate that the asymmetric pheromone modification is not required for successful mating of ascomycete fungi and confirm that, in budding yeast, the primary determinants of mating are the specificity of the receptors and their corresponding pheromones.

The spindle checkpoint monitors chromosome alignment on the mitotic and meiotic spindle. When the checkpoint detects errors, it arrests progress of the cell cycle while it attempts to correct the mistakes. This perspective will present a brief history summarizing what we know about the checkpoint, and a list of questions we must answer before we understand it.

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation.

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.

We use the budding yeast, Saccharomyces cerevisiae, to investigate one model for the initial emergence of multicellularity: the formation of multicellular aggregates as a result of incomplete cell separation. We combine simulations with experiments to show how the use of secreted public goods favors the formation of multicellular aggregates. Yeast cells can cooperate by secreting invertase, an enzyme that digests sucrose into monosaccharides, and many wild isolates are multicellular because cell walls remain attached to each other after the cells divide. We manipulate invertase secretion and cell attachment, and show that multicellular clumps have two advantages over single cells: they grow under conditions where single cells cannot and they compete better against cheaters, cells that do not make invertase. We propose that the prior use of public goods led to selection for the incomplete cell separation that first produced multicellularity.

Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother-daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother-daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother-daughter mating might not be as significant as was considered.

BACKGROUND: Subtelomeres, regions proximal to telomeres, exhibit characteristics unique to eukaryotic genomes. Genes residing in these loci are subject to epigenetic regulation and elevated rates of both meiotic and mitotic recombination. However, most genome sequences do not contain assembled subtelomeric sequences, and, as a result, subtelomeres are often overlooked in comparative genomics. RESULTS: We studied the evolution and functional divergence of subtelomeric gene families in the yeast lineage. Our computational results show that subtelomeric families are evolving and expanding much faster than families that do not contain subtelomeric genes. Focusing on three related subtelomeric MAL gene families involved in disaccharide metabolism that show typical patterns of rapid expansion and evolution, we show experimentally how frequent duplication events followed by functional divergence yield novel alleles that allow the metabolism of different carbohydrates. CONCLUSIONS: Taken together, our computational and experimental analyses show that the extraordinary instability of eukaryotic subtelomeres supports rapid adaptation to novel niches by promoting gene recombination and duplication followed by functional divergence of the alleles.

BACKGROUND: Evolution depends on mutations: rare errors in the transmission of genetic information. Experimentally, mutations have been found by detecting altered phenotypes or sequencing complete genomes, but most mutations do not have overt phenotypes, and sequencing is expensive and has limited time resolution. The major source of mutations is DNA replication errors. Nearly all mistakes in DNA replication are detected and repaired by the mismatch repair machinery. RESULTS: We use a functional, fluorescently labeled derivative of one of the key mismatch repair proteins (MutL) to see and count the small fraction of errors in Escherichia coli that does not get repaired and is converted into stable mutations by the next round of DNA replication. Over a 300-fold range, there is a linear relationship between the frequency of fluorescent foci and the genetically measured mutation frequency, and the mean frequency of fluorescent foci agrees well with estimates of the global mutation rate. CONCLUSION: We describe a method for detecting the majority of genomic mutations emerging in living cells, independently of their potential phenotype. The distribution of emerging mutations per cell is roughly Poisson distributed, suggesting that all the cells in the population have roughly the same mutation rate.

Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate how, we engineered a synthetic library of 154 genes that varied randomly at synonymous sites, but all encoded the same green fluorescent protein (GFP). When expressed in Escherichia coli, GFP protein levels varied 250-fold across the library. GFP messenger RNA (mRNA) levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained more than half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.

Cell cycle transitions are controlled, in part, by ubiquitin-dependent proteolysis. In mitosis, the metaphase to anaphase transition is governed by an E3 ubiquitin ligase called the cyclosome or Anaphase-Promoting Complex (APC), and a WD40-repeat protein co-factor called Cdc20. In vitro Cdc20-dependent APC (APC(Cdc20)) assays have been useful in the identification and validation of target substrates, and in the study of APC enzymology and regulation. Many aspects of the regulation of cell cycle progression have been discovered in the budding yeast Saccharomyces cerevisiae, and proteins purified from this model organism have been employed in a wide variety of in vitro assays. Here we outline a quantitative in vitro mitotic APC(Cdc20) assay that makes use of a highly active form of the APC that is purified from budding yeast cells arrested in mitosis.

Accurate chromosome segregation depends on the kinetochore, which is the complex of proteins that link microtubules to centromeric DNA. The kinetochore of the budding yeast Saccharomyces cerevisiae consists of more than 80 proteins assembled on a 125-bp region of DNA. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1-DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, bi-orient sister kinetochores at metaphase during the mitotic cycle, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers assembly of the complex structure that directs mitotic chromosome segregation.

Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes differ significantly (3.80x10(-10) at URA3 and 6.44x10(-10) at CAN1) and we propose a definition for the effective target size of genes (the probability that a mutation inactivates the gene) that acknowledges that the mutation rate is nonuniform across the genome.

BACKGROUND: Chromosomes must biorient on the mitotic spindle, with the two sisters attached to opposite spindle poles. The spindle checkpoint detects unattached chromosomes and monitors biorientation by detecting the lack of tension between two sisters attached to the same pole. After the spindle has been depolymerized and allowed to reform, budding yeast sgo1 mutants fail to biorient their sister chromatids and die as cells divide. RESULTS: In sgo1 mutants, chromosomes attach to microtubules normally but cannot reorient if both sisters attach to the same pole. The mutants' fate depends on the position of the spindle poles when the chromosomes attach to microtubules. If the poles have separated, sister chromatids biorient, but if the poles are still close, sister chromatids often attach to the same pole, missegregate, and cause cell death. CONCLUSIONS: These observations argue that budding yeast mitotic chromosomes have an intrinsic, geometric bias to biorient on the spindle. When the poles have already separated, attaching one kinetochore to one pole predisposes its sister to attach to the opposite pole, allowing the cells to segregate the chromosomes correctly. When the poles have not separated, the second kinetochore eventually attaches to either of the two poles randomly, causing orientation errors that are corrected in the wild-type but not in sgo1 mutants. In the absence of spindle damage, sgo1 cells divide successfully, suggesting that kinetochores only make stable attachments to microtubules after the cells have entered mitosis and separated their spindle poles.

BACKGROUND: Bistability in genetic networks allows cells to remember past events and to make discrete decisions in response to graded signals. Bistable behavior can result from positive feedback, but feedback loops can have other roles in signal transduction as well. RESULTS: We introduced positive feedback into the budding-yeast pheromone response to convert it into a bistable system. In the presence of feedback, transient induction with high pheromone levels caused persistent pathway activation, whereas at lower levels a fraction of cells became persistently active but the rest inactivated completely. We also generated mutations that quantitatively tuned the basal and induced expression levels of the feedback promoter and showed that they qualitatively changed the behavior of the system. Finally, we developed a simple stochastic model of our positive-feedback system and showed the agreement between our simulations and experimental results. CONCLUSIONS: The positive-feedback loop can display several different behaviors, including bistability, and can switch between them as a result of simple mutations.

BACKGROUND: The rate at which beneficial mutations accumulate determines how fast asexual populations evolve, but this is only partially understood. Some recent clonal-interference models suggest that evolution in large asexual populations is limited because smaller beneficial mutations are outcompeted by larger beneficial mutations that occur in different lineages within the same population. This analysis assumes that the important mutations fix one at a time; it ignores multiple beneficial mutations that occur in the lineage of an earlier beneficial mutation, before the first mutation in the series can fix. We focus on the effects of such multiple mutations. RESULTS: Our analysis predicts that the variation in fitness maintained by a continuously evolving population increases as the logarithm of the population size and logarithm of the mutation rate and thus yields a similar logarithmic increase in the speed of evolution. To test these predictions, we evolved asexual budding yeast in glucose-limited media at a range of population sizes and mutation rates. CONCLUSIONS: We find that their evolution is dominated by the accumulation of multiple mutations of moderate effect. Our results agree with our theoretical predictions and are inconsistent with the one-by-one fixation of mutants assumed by recent clonal-interference analysis.

Improper meiotic chromosome segregation causes conditions such as Down's syndrome. Recombination promotes proper chromosome segregation in meiosis I; chromosomes without crossovers near the centromere are more likely to segregate to the same spindle pole (nondisjoin). Here we have used budding yeast to determine whether the spindle checkpoint promotes segregation of such chromosomes. In checkpoint-defective mad2Delta cells, properly segregating chromosomes have more crossovers near the centromere than their wild-type counterparts, and an artificial tether that holds chromosomes together suppresses nondisjunction as long as the tether is near the centromere. The tether partially rescues the segregation of chromosomes that lack crossovers.

The spindle checkpoint delays cell cycle progression until microtubules attach each pair of sister chromosomes to opposite poles of the mitotic spindle. Following sister chromatid separation, however, the checkpoint ignores chromosomes whose kinetochores are attached to only one spindle pole, a state that activates the checkpoint prior to metaphase. We demonstrate that, in budding yeast, mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the checkpoint, leads to sustained inactivation of the spindle checkpoint. Mps1 protein abundance decreases in anaphase, and Mps1 is a target of the APC. Furthermore, expression of Mps1 in anaphase, or repression of the APC in anaphase, reactivates the spindle checkpoint. This APC-Mps1 feedback circuit allows cells to irreversibly inactivate the checkpoint during anaphase.

Assortative mating, when individuals of similar phenotypes mate, likely plays a key role in preventing gene flow during speciation. Reinforcement occurs when two previously geographically separated (allopatric) groups meet after having evolved partial postzygotic isolation; they are selected to evolve or enhance assortative mating to prevent costly intergroup matings that produce only maladaptive or sterile hybrids. Studies in Drosophila have shown that the genetic architectures of mating discrimination could differ significantly with or without reinforcement, suggesting that the evolution of assortative mating may be more complicated than expected. To study the evolution of assortative mating, we evolved mating discrimination in populations of the budding yeast, Saccharomyces cerevisiae. After 36 cycles of selection, these cells are five times more likely to mate with each other than to their ancestors, despite detectable one-way gene flow between the selected and reference populations. Several individual cultures evolved mating discrimination by changing their mating kinetics, with some mating more rapidly and others more slowly than the ancestral population. Genetic analysis indicates that multiple mutations have accumulated to produce the altered mating preference. Our results show that subtle details of mating behavior can play an important role in the evolution of reproductive isolation.