RESUMEN
Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (false discovery rate [FDR] < 0.11) 70 poorly characterized genes required for photosynthesis. We then enable the functional characterization of these genes by providing a resource of proteomes of mutant strains, each lacking one of these genes. The data allow assignment of 34 genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Further analysis uncovers biogenesis/regulatory roles for at least seven proteins, including five photosystem I mRNA maturation factors, the chloroplast translation factor MTF1, and the master regulator PMR1, which regulates chloroplast genes via nuclear-expressed factors. Our work provides a rich resource identifying regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.
Asunto(s)
Chlamydomonas reinhardtii , Fotosíntesis , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Fotosíntesis/genética , Regulación de la Expresión Génica , Proteínas/genética , Proteínas/metabolismo , Mutación , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genéticaRESUMEN
COVID-19, caused by the coronavirus SARS-CoV-2, represents a serious worldwide health issue, with continually emerging new variants challenging current therapeutics. One promising alternate therapeutic avenue is represented by nanobodies, small single-chain antibodies derived from camelids with numerous advantageous properties and the potential to neutralize the virus. For identification and characterization of a broad spectrum of anti-SARS-CoV-2 Spike nanobodies, we further optimized a yeast display method, leveraging a previously published mass spectrometry-based method, using B-cell complementary DNA from the same immunized animals as a source of VHH sequences. Yeast display captured many of the sequences identified by the previous approach, as well as many additional sequences that proved to encode a large new repertoire of nanobodies with high affinities and neutralization activities against different SARS-CoV-2 variants. We evaluated DNA shuffling applied to the three complementarity-determining regions of antiviral nanobodies. The results suggested a surprising degree of modularity to complementarity-determining region function. Importantly, the yeast display approach applied to nanobody libraries from immunized animals allows parallel interrogation of a vast number of nanobodies. For example, we employed a modified yeast display to carry out massively parallel epitope binning. The current yeast display approach proved comparable in efficiency and specificity to the mass spectrometry-based approach, while requiring none of the infrastructure and expertise required for that approach, making these highly complementary approaches that together appear to comprehensively explore the paratope space. The larger repertoires produced maximize the likelihood of discovering broadly specific reagents and those that powerfully synergize in mixtures.
Asunto(s)
Anticuerpos Neutralizantes , SARS-CoV-2 , Anticuerpos de Dominio Único , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Regiones Determinantes de Complementariedad , Saccharomyces cerevisiae/genética , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.
Asunto(s)
Chlamydomonas , Ciclina B , Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/genética , Mitosis/genética , Huso Acromático/metabolismoRESUMEN
DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions ('multiple fission'). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Componente 4 del Complejo de Mantenimiento de Minicromosoma/genética , Mitosis/genética , Animales , División Celular/genética , Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , Quinasas Ciclina-Dependientes/genética , Componente 6 del Complejo de Mantenimiento de Minicromosoma/genética , Mutación/genética , Complejo de Reconocimiento del Origen/genética , Fosforilación/genética , Plásmidos/genética , Proteolisis , Ribonucleótido Reductasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.
Asunto(s)
Actinas/metabolismo , Chlamydomonas/citología , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , División Celular , Chlamydomonas/química , Citocinesis , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Unión ProteicaRESUMEN
The green alga Chlamydomonas proliferates by "multiple fission": a long G1 with >10-fold cell growth followed by multiple rapid divisions. Cells above a critical size threshold are "committed" and will divide independent of light and further cell growth. The number of divisions carried out depends on the initial size of the committed mother cell. Here, I show that CDKA1, the ortholog of the yeast and animal mitotic inducer CDK1, regulates the critical size for commitment. The Rb/E2F/Dp1 pathway regulates division number as well as commitment size. Epistasis analysis indicated that CDKA1 and Rb/E2F/Dp1 regulate multiple fission by distinct mechanisms. Rb-E2F/Dp1 regulates G1/S gene expression in animals and land plants. Transcriptome analysis showed that mat3 or dp1 disruption altered regulation of a large group of cell-division-associated genes with respect to cell size, but not with respect to synchronization timing. In contrast, cdka1 inactivation disturbed both temporal and cell-size regulation of expression. These defects were enhanced by double inactivation of cdka1 and dp1, suggesting interaction between CDKA1 and the Rb-E2F/Dp1 pathways in regulating cell-cycle-specific gene expression and cell-cycle initiation. In the context of a theoretical model for regulation of Chlamydomonas multiple fission, these results suggest that CDKA1 may promote a switch into a division-competent state, and E2F/Dp1 may promote maintenance of this state.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclo Celular/fisiología , Chlamydomonas reinhardtii/metabolismo , Factores de Transcripción E2F/metabolismo , Proteína Quinasa CDC2/genética , Factores de Transcripción E2F/genética , Regulación de la Expresión Génica , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción DP1/genética , Factor de Transcripción DP1/metabolismo , Transcripción GenéticaRESUMEN
Most eukaryotic cells execute binary division after each mass doubling in order to maintain size homeostasis by coordinating cell growth and division. By contrast, the photosynthetic green alga Chlamydomonas can grow more than 8-fold during daytime and then, at night, undergo rapid cycles of DNA replication, mitosis, and cell division, producing up to 16 daughter cells. Here, we propose a mechanistic model for multiple-fission cycles and cell-size control in Chlamydomonas. The model comprises a light-sensitive and size-dependent biochemical toggle switch that acts as a sizer, guarding transitions into and exit from a phase of cell-division cycle oscillations. This simple "sizer-oscillator" arrangement reproduces the experimentally observed features of multiple-fission cycles and the response of Chlamydomonas cells to different light-dark regimes. Our model also makes specific predictions about the size dependence of the time of onset of cell division after cells are transferred from light to dark conditions, and we confirm these predictions by single-cell experiments. Collectively, our results provide a new perspective on the concept of a "commitment point" during the growth of Chlamydomonas cells and hint at intriguing similarities of cell-size control in different eukaryotic lineages.
Asunto(s)
Ciclo Celular/efectos de la radiación , Chlamydomonas reinhardtii/fisiología , Luz , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/efectos de la radiaciónRESUMEN
Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.
Asunto(s)
Actinas/biosíntesis , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transcripción Genética , Actinas/genética , Chlamydomonas reinhardtii/genética , Proteínas de Plantas/genética , Complejo de la Endopetidasa Proteasomal/genéticaRESUMEN
We generated a large collection of temperature-sensitive lethal mutants in the unicellular green alga Chlamydomonas reinhardtii, focusing on mutations specifically affecting cell cycle regulation. We used UV mutagenesis and robotically assisted phenotypic screening to isolate candidates. To overcome the bottleneck at the critical step of molecular identification of the causative mutation ("driver"), we developed MAPS-SEQ (meiosis-assisted purifying selection sequencing), a multiplexed genetic/bioinformatics strategy. MAPS-SEQ allowed us to perform multiplexed simultaneous determination of the driver mutations from hundreds of neutral "passenger" mutations in each member of a large pool of mutants. This method should work broadly, including in multicellular diploid genetic systems, for any scorable trait. Using MAPS-SEQ, we identified essential genes spanning a wide range of molecular functions. Phenotypic clustering based on DNA content analysis and cell morphology indicated that the mutated genes function in the cell cycle at multiple points and by diverse mechanisms. The collection is sufficiently complete to allow specific conditional inactivation of almost all cell-cycle-regulatory pathways. Approximately seventy-five percent of the essential genes identified in this project had clear orthologs in land plant genomes, a huge enrichment compared with the value of â¼20% for the Chlamydomonas genome overall. Findings about these mutants will likely have direct relevance to essential cell biology in land plants.
Asunto(s)
Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/genética , Mutación/genética , FenotipoRESUMEN
The cyclin-dependent kinase CDK1 is essential for mitosis in fungi and animals. Plant genomes contain the CDK1 ortholog CDKA and a plant kingdom-specific relative, CDKB. The green alga Chlamydomonas reinhardtii has a long G1 growth period followed by rapid cycles of DNA replication and cell division. We show that null alleles of CDKA extend the growth period prior to the first division cycle and modestly extend the subsequent division cycles, but do not prevent cell division, indicating at most a minor role for the CDK1 ortholog in mitosis in Chlamydomonas. A null allele of cyclin A has a similar though less extreme phenotype. In contrast, both CDKB and cyclin B are essential for mitosis. CDK kinase activity measurements imply that the predominant in vivo complexes are probably cyclin A-CDKA and cyclin B-CDKB. We propose a negative feedback loop: CDKA activates cyclin B-CDKB. Cyclin B-CDKB in turn promotes mitotic entry and inactivates cyclin A-CDKA. Cyclin A-CDKA and cyclin B-CDKB may redundantly promote DNA replication. We show that the anaphase-promoting complex is required for inactivation of both CDKA and CDKB and is essential for anaphase. These results are consistent with findings in Arabidopsis thaliana and may delineate the core of plant kingdom cell cycle control that, compared with the well-studied yeast and animal systems, exhibits deep conservation in some respects and striking divergence in others.
Asunto(s)
Proteínas Algáceas/metabolismo , Ciclo Celular/genética , Chlamydomonas reinhardtii/genética , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , ADN de Algas/genética , Mitosis/genética , Proteínas Algáceas/genética , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/fisiología , Ciclina B/genética , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Biology emerges from interactions between molecules, which are challenging to elucidate with current techniques. An orthogonal approach is to probe for 'response signatures' that identify specific circuit motifs. For example, bistability, hysteresis, or irreversibility are used to detect positive feedback loops. For adapting systems, such signatures are not known. Only two circuit motifs generate adaptation: negative feedback loops (NFLs) and incoherent feed-forward loops (IFFLs). On the basis of computational testing and mathematical proofs, we propose differential signatures: in response to oscillatory stimulation, NFLs but not IFFLs show refractory-period stabilization (robustness to changes in stimulus duration) or period skipping. Applying this approach to yeast, we identified the circuit dominating cell cycle timing. In Caenorhabditis elegans AWA neurons, which are crucial for chemotaxis, we uncovered a Ca2+ NFL leading to adaptation that would be difficult to find by other means. These response signatures allow direct access to the outlines of the wiring diagrams of adapting systems.
Asunto(s)
Adaptación Fisiológica/fisiología , Retroalimentación Fisiológica/fisiología , Modelos Biológicos , Animales , Caenorhabditis elegans , Ciclo Celular/fisiología , Regulación de la Expresión Génica/fisiología , Neuronas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in Chlamydomonas and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with Arabidopsis; severity of the mutation relative to Chlamydomonas wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of "best reciprocal BLAST" (BRB) relationships among Chlamydomonas and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to Arabidopsis.
Asunto(s)
Arabidopsis/genética , Chlamydomonas/genética , Genoma de Planta , Meiosis , Modelos Genéticos , Mutación , Teorema de BayesRESUMEN
B-type cyclins promote mitotic entry and inhibit mitotic exit. In Saccharomyces cerevisiae, four B-type cyclins, Clb1-4, carry out essential mitotic roles, with substantial but incomplete overlap of function among them. Previous work in many organisms has indicated that B-type cyclin-dependent inhibition of mitotic exit imposes a requirement for mitotic destruction of B-type cyclins. For instance, precise genomic removal of the Clb2 destruction box (D box) prevents mitotic proteolysis of Clb2, and blocks mitotic exit. Here, we show that, despite significant functional overlap between Clb2 and Clb3, D-box-dependent Clb3 proteolysis is completely dispensable for mitotic exit. Removal of the Clb3 D box results in abundant Clb3 protein and associated kinase throughout the cell cycle, but mitotic exit occurs with close to normal timing. Clb3 degradation is required for pre-Start G1 control in the succeeding cell cycle. Deleting the CLB3 D box essentially eliminates all time delay before cell cycle Start following division, even in very small newborn cells. CLB3∆db cells show no cell cycle arrest response to mating pheromone, and CLB3∆db completely bypasses the requirement for CLN G1 cyclins, even in the absence of the early expressed B-type cyclins CLB5,6 Thus, regulated mitotic proteolysis of Clb3 is specifically required to make passage of Start in the succeeding cell cycle "memoryless"-dependent on conditions within that cycle, and independent of events such as B-type cyclin accumulation that occurred in the preceding cycle.
Asunto(s)
Ciclina B/metabolismo , Mitosis/genética , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ciclina B/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Técnicas Biosensibles/métodos , Proteínas Cdh1/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas , Proteínas Cdh1/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Carioferinas/metabolismo , Proteínas Luminiscentes , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. However, the current reference genome contains â¼1000 blocks of unknown sequence ('N-islands'), which are frequently placed in introns of annotated gene models. We developed a strategy to search for previously unknown exons hidden within such blocks, and determine the sequence, and exon/intron boundaries, of such exons. These methods are based on assembly and alignment of short cDNA and genomic DNA reads, completely independent of prior reference assembly or annotation. Our evidence indicates that a substantial proportion of the annotated intronic N-islands contain hidden exons. For most of these, our algorithm recovers full exonic sequence with associated splice junctions and exon-adjacent intronic sequence. These new exons represent de novo sequence generally present nowhere in the assembled genome, and the added sequence improves evolutionary conservation of the predicted encoded peptides.
Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas/genética , ADN Complementario/genética , Genoma de Planta , Análisis de Secuencia de ADN/métodos , Transcriptoma , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Chlamydomonas/metabolismo , Biología Computacional/métodos , ADN Complementario/metabolismo , Exones , Intrones , Anotación de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Throughout cell-cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell-cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low.
Asunto(s)
Relojes Biológicos , Ciclo Celular , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ciclinas/metabolismo , Dineínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Análisis de la Célula IndividualRESUMEN
Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.
Asunto(s)
Chlamydomonas reinhardtii/genética , Genes Letales , Ensayos Analíticos de Alto Rendimiento/métodos , Mutación , Robótica/métodos , Temperatura , Alelos , Genes Esenciales , FenotipoRESUMEN
Actin is one of the most conserved eukaryotic proteins. It is thought to have multiple essential cellular roles and to function primarily or exclusively as filaments ("F-actin"). Chlamydomonas has been an enigma, because a null mutation (ida5-1) in its single gene for conventional actin does not affect growth. A highly divergent actin gene, NAP1, is upregulated in ida5-1 cells, but it has been unclear whether NAP1 can form filaments or provide actin function. Here, we used the actin-depolymerizing drug latrunculin B (LatB), the F-actin-specific probe Lifeact-Venus, and genetic and molecular methods to resolve these issues. LatB-treated wild-type cells continue to proliferate; they initially lose Lifeact-stained structures but recover them concomitant with upregulation of NAP1. Thirty-nine LatB-sensitive mutants fell into four genes (NAP1 and LAT1-LAT3) in which we identified the causative mutations using a novel combinatorial pool-sequencing strategy. LAT1-LAT3 are required for NAP1 upregulation upon LatB treatment, and ectopic expression of NAP1 largely rescues the LatB sensitivity of the lat1-lat3 mutants, suggesting that the LAT gene products comprise a regulatory hierarchy with NAP1 expression as the major functional output. Selection of LatB-resistant revertants of a nap1 mutant yielded dominant IDA5 mutations that presumably render F-IDA5 resistant to LatB, and nap1 and lat mutations are synthetically lethal with ida5-1 in the absence of LatB. We conclude that both IDA5 and the divergent NAP1 can form filaments and redundantly provide essential F-actin functions and that a novel surveillance system, probably responding to a loss of F-actin, triggers NAP1 expression and perhaps other compensatory responses.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/genética , Proteínas Algáceas/genética , Alelos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Chlamydomonas reinhardtii/metabolismo , Mutaciones Letales Sintéticas , Tiazolidinas/química , Regulación hacia ArribaRESUMEN
The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. The annotated genome is very rich in open reading frames upstream of the annotated coding sequence ('uORFs'): almost three quarters of the assigned transcripts have at least one uORF, and frequently more than one. This is problematic with respect to the standard 'scanning' model for eukaryotic translation initiation. These uORFs can be grouped into three classes: class 1, initiating in-frame with the coding sequence (CDS) (thus providing a potential in-frame N-terminal extension); class 2, initiating in the 5' untranslated sequences (5UT) and terminating out-of-frame in the CDS; and class 3, initiating and terminating within the 5UT. Multiple bioinformatics criteria (including analysis of Kozak consensus sequence agreement and BLASTP comparisons to the closely related Volvox genome, and statistical comparison to cds and to random sequence controls) indicate that of â¼4000 class 1 uORFs, approximately half are likely in vivo translation initiation sites. The proposed resulting N-terminal extensions in many cases will sharply alter the predicted biochemical properties of the encoded proteins. These results suggest significant modifications in â¼2000 of the â¼20,000 transcript models with respect to translation initiation and encoded peptides. In contrast, class 2 uORFs may be subject to purifying selection, and the existent ones (surviving selection) are likely inefficiently translated. Class 3 uORFs are found in more than half of transcripts, frequently multiple times per transcript; however, they are remarkably similar to random sequence expectations with respect to size, number, and composition, and therefore may in most cases be selectively neutral.
Asunto(s)
Regiones no Traducidas 5' , Chlamydomonas/genética , Genoma de Planta , Genómica , Sistemas de Lectura Abierta , Evolución Biológica , Codón Iniciador , Biología Computacional/métodos , Secuencia de Consenso , Genómica/métodos , Anotación de Secuencia Molecular , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Biosíntesis de ProteínasRESUMEN
We analyzed global transcriptome changes during synchronized cell division in the green alga Chlamydomonas reinhardtii. The Chlamydomonas cell cycle consists of a long G1 phase, followed by an S/M phase with multiple rapid, alternating rounds of DNA replication and segregation. We found that the S/M period is associated with strong induction of â¼2300 genes, many with conserved roles in DNA replication or cell division. Other genes, including many involved in photosynthesis, are reciprocally downregulated in S/M, suggesting a gene expression split correlating with the temporal separation between G1 and S/M. The Chlamydomonas cell cycle is synchronized by light-dark cycles, so in principle, these transcriptional changes could be directly responsive to light or to metabolic cues. Alternatively, cell-cycle-periodic transcription may be directly regulated by cyclin-dependent kinases. To distinguish between these possibilities, we analyzed transcriptional profiles of mutants in the kinases CDKA and CDKB, as well as other mutants with distinct cell cycle blocks. Initial cell-cycle-periodic expression changes are largely CDK independent, but later regulation (induction and repression) is under differential control by CDKA and CDKB. Deviation from the wild-type transcriptional program in diverse cell cycle mutants will be an informative phenotype for further characterization of the Chlamydomonas cell cycle.