Yeast 2.0-connecting the dots in the construction of the world's first functional synthetic eukaryotic genome.

Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the biosafety, bioethics and regulatory aspects of their pioneering work. This article presents a shared vision of constructing a synthetic eukaryotic genome in a safe model organism by using novel concepts and advanced technologies. This multidisciplinary and collaborative project is conducted under a sound governance structure that does not only respect the scientific achievements and lessons from the past, but that is also focussed on leading the present and helping to secure a brighter future for all.

Distributed synergistic plasticity and cerebellar learning.

Studies on synaptic plasticity in the context of learning have been dominated by the view that a single, particular type of plasticity forms the underlying mechanism for a particular type of learning. However, emerging evidence shows that many forms of synaptic and intrinsic plasticity at different sites are induced conjunctively during procedural memory formation in the cerebellum. Here, we review the main forms of long-term plasticity in the cerebellar cortex that underlie motor learning. We propose that the different forms of plasticity in the granular layer and the molecular layer operate synergistically in a temporally and spatially distributed manner, so as to ultimately create optimal output for behaviour.

Visuo-vestibular information processing by unipolar brush cells in the rabbit flocculus.

The unipolar brush cell (UBC) is a glutamatergic granular layer interneuron that is predominantly located in the vestibulocerebellum and parts of the vermis. In rat and rabbit, we previously found using juxtacellular labeling combined with spontaneous activity recording that cells with highly regular spontaneous activity belong to the UBC category. Making use of this signature, we recorded from floccular UBCs in both anesthetized and awake rabbits while delivering visuo-vestibular stimulation by using sigmoidal rotation of the whole animal. In the anesthetized rabbit, the activity of the presumed UBC units displayed a wide variety of modulation profiles that could be related to aspects of head velocity or acceleration. These modulation profiles could also be found in the awake rabbit where, in addition, they could also carry an eye position signal. Furthermore, units in the awake rabbit could demonstrate rather long response latencies of up to 0.5 s. We suggest that the UBCs recorded in this study mostly belong to the type I UBC category (calretinin-positive) and that they can play diverse roles in floccular visuo-vestibular information processing, such as transformation of velocity-related signals to acceleration-related signals.

Enhanced AMPA receptor function promotes cerebellar long-term depression rather than potentiation.

Ampakines are allosteric modulators of AMPA receptors that facilitate hippocampal long-term potentiation (LTP) and learning, and have been considered for the treatment of cognition and memory deficits. Here, we show that the ampakine CX546 raises the amplitude and slows the decay time of excitatory postsynaptic currents (EPSCs) at cerebellar parallel fiber (PF) to Purkinje cell synapses, thus resembling CX546 effects described at hippocampal synapses. Using the fluorescent calcium indicator dye Oregon Green BAPTA-2 and an ultra-high-speed CCD camera, we also monitored calcium transients in Purkinje cell dendrites. In the presence of CX546 in the bath, PF-evoked calcium transients were enhanced and prolonged, suggesting that CX546 not only enhances synaptic transmission, but also boosts dendritic calcium signaling at cerebellar synapses. In contrast to previous observations in the hippocampus, however, CX546 applied during cerebellar recordings facilitates long-term depression (LTD) rather than LTP at PF synapses. These findings show that ampakines selectively modify the LTP-LTD balance depending on the brain area and type of synapse, and may provide tools for the targeted regulation of synaptic memories.

Retrotransposition mechanisms.

Recent developments in the area of the transposition mechanisms used by retrotransposons and related retroviral pathways are discussed. In particular, advances in the areas of retrotransposon gene expression, virus-like particle assembly, reverse transcription, and integration are reviewed.

A general method for the chromosomal amplification of genes in yeast.

The yeast retrotransposon Ty can be used to insert multiple copies of a gene at new sites in the genome. The gene of interest is inserted into a GALI-Ty fusion construct; the entire "amplification cassette" is then introduced into yeast on a high copy number plasmid vector. Transposition of the Ty element carrying the gene occurs at multiple sites in the genome. Two genes, a bacterial neomycin phosphotransferase gene and the yeast TRPl gene, were amplified in this way. Although the amplified genes were about 1 kilobase in length, they were amplified to about the same extent as a 40-base pair segment. The benefit of this "shotgun" approach is that amplification can be achieved in one set of manipulations.

Avian pollination studies: a simple scanning electron microscopic technique.

Identifying ornithophilous plant species utilized by several different flower-visiting birds is simplified by the scanning electron microscope. The technique involves comparing pollen samples taken from the birds' head feathers with pollen reference standards collected in the same area, which simplifies analysis of pollination patterns in a complex community.

A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae.

We describe a microarray-based screen performed by imposing different genetic selections on thousands of yeast mutants in parallel, representing most genes in the yeast genome. The presence or absence of mutants was detected by oligonucleotide arrays that hybridize to 20-nucleotide "barcodes." We used this method to screen for components of the nonhomologous end-joining (NHEJ) pathway. Known components of the pathway were identified, as well as a gene not previously known to be involved in NHEJ, NEJ1. Nej1 protein interacts with the amino terminus of LIF1/XRCC4, a recently recognized "guardian of the genome" against cancer.

Reverse transcriptase encoded by a human transposable element.

L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

A critical role for the C-terminus of Nej1 protein in Lif1p association, DNA binding and non-homologous end-joining.

A predominant pathway implicated in repair of DNA double-strand breaks (DSBs) is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1p, for which a biochemical function has yet to be determined. In this work we demonstrate that Nej1p exhibits a DNA binding activity (KD approximately 1.8 microM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short approximately 20 bp substrates can suffice. This DNA binding activity is the first biochemical evidence supporting the idea that Nej1p plays a direct role in the repair of double-strand breaks. The C-terminus of Nej1p is required for interaction with Lif1p and is sufficient for DNA binding. Structural characterization reveals that Nej1p exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Nej1p (aa 1-244) is shown to be defective in end-joining in vivo. Preliminary functional and structural studies on the Nej1p-Lif1p complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1p and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1p, XLF/Cernunnos.

Climbing fiber-evoked endocannabinoid signaling heterosynaptically suppresses presynaptic cerebellar long-term potentiation.

Endocannabinoid signaling has been demonstrated to mediate depolarization-induced suppression of excitation at climbing fiber (CF) and parallel fiber (PF) synapses onto cerebellar Purkinje cells. Here, we show that CF-evoked release of cannabinoids (CBs) additionally suppresses a presynaptic form of long-term potentiation (LTP) at PF synapses. PF-LTP can be induced by 8 Hz PF tetanization but is blocked when the PF tetanization is paired with 4 or 1 Hz CF coactivation. CF activity can be substituted for by bath application of the CB receptor agonist WIN55,212-2 [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone]. In the presence of the CB1 receptor antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], CF activity no longer suppresses PF-LTP. Presynaptic potentiation can also be obtained by the adenylyl cyclase activator forskolin. WIN55,212-2 blocked this forskolin-mediated enhancement, showing that CB1 receptor activation interferes with the adenylyl cyclase-protein kinase A cascade, which participates in LTP induction. CF activity has been described to promote the induction of postsynaptic PF-long-term depression (LTD) and to impair postsynaptic PF-LTP. Our observation that CF activity blocks the induction of presynaptic LTP suggests that the CF input controls all forms of presynaptic and postsynaptic PF plasticity and that CF activity provides a "safety lock" to prevent an enhancement of transmitter release while postsynaptic AMPA receptor function is downregulated during LTD.

Yeast retrotransposons and tRNAs.

The role of tRNAs in protein synthesis seems routine when compared with the novel ways in which the Ty retrotransposons of Saccharomyces cerevisiae use these interpreters of the genetic code. tRNAs and tRNA genes control essential steps in the retrotransposon life cycle by regulating protein expression, priming DNA synthesis and specifying integration target sites.

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.

In vitro integration of retrotransposon Ty1: a direct physical assay.

Retrotransposon Ty1 of Saccharomyces cerevisiae inserts a double-stranded Ty1 cDNA into the yeast genome by a reaction analogous to the integration mechanism used by retroviruses. A quantitative in vitro integration assay that directly detects integrative recombination products was developed for Ty1. Blunt-ended artificial radioactive substrates bearing Ty1 termini integrate into circular or linear target DNAs. The reaction is specific for native integrase isolated in the form of virus-like particles; virus-like particles prepared from integrase mutants were completely inactive in this assay. The products are radioactive, allowing direct detection after gel electrophoresis by autoradiography. Using this simple and amenable system, we characterized the biochemical requirements of the system and the structures of the major integration products. Two classes of products were detected: those that were the result of bona fide complete integration events (concerted reactions) and single-end joinings of substrate to target (half-reactions). Additionally, we used a genetic selection scheme to identify and characterize target sites of complete integration events into a circular target plasmid; a 5-bp target site duplication flanking the inserted DNA resembling the duplication characteristic of in vivo integration was observed.

Inhibition of Ty1 transposition by mating pheromones in Saccharomyces cerevisiae.

The Ty1 elements in the yeast Saccharomyces cerevisiae are a family of retrotransposons which transpose via a process similar to that of retroviral replication. We report here that the Ty1 transposition process can be blocked posttranscriptionally by treatment of cells with mating pheromones. When haploid yeast cells are treated with appropriate mating pheromones, the transposition frequency of a marked Ty1 element driven by the GAL1 promoter is greatly diminished. Ty1 viruslike particles (VLPs), the putative intermediates for transposition, can be isolated from mating pheromone-treated cells. These VLPs accumulate to normal levels but are aberrant in that they produce very few reverse transcripts of Ty1 RNA both in vivo and in vitro and contain subnormal amounts of p90-TYB and related proteins. In addition, a TYA phosphoprotein product accumulates in treated cells, and some species of TYB proteins have decreased stability. We also show that decreased transposition in mating pheromone-treated cells is not a consequence of simply blocking cell division, since Ty1 transposes at a nearly normal rate in yeast cells arrested in G2 by the drug nocodazole.

Ty1 in vitro integration: effects of mutations in cis and in trans.

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.

Two related families of retrotransposons from Schizosaccharomyces pombe.

Two related families of transposons were isolated from schizosaccharomyces pombe, an organism which has been the object of extensive genetic studies which had previously produced no evidence for the existence of such elements. These two classes of repeated DNAs, dubbed Tf1 (transposon of fission yeast 1) and Tf2 have many properties of retrotransposons. Tf1 and Tf2 both possess long terminal repeats and predicted protein sequences that resemble the protease, reverse transcriptase, and integrase domains of retroviruses. The chromosomal locations and total numbers of Tf1 and Tf2 differ greatly in various isolates of S. pombe. The Tf elements are expressed in the form of 4.5-kb mRNAs. The complete sequence of Tf1 was determined and suggests that a novel mechanism for regulating its gene expression may be used.

Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements.

Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element. The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101. Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F. Winston, K. J. Durbin, and G. R. Fink, Cell 39:675-682, 1984). Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent. Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid. In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.