Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery.

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.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription.

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.

Periodic cyclin-Cdk activity entrains an autonomous Cdc14 release oscillator.

One oscillation of Cyclin-dependent kinase (Cdk) activity, largely driven by periodic synthesis and destruction of cyclins, is tightly coupled to a single complete eukaryotic cell division cycle. Tight linkage of different steps in diverse cell-cycle processes to Cdk activity has been proposed to explain this coupling. Here, we demonstrate an intrinsically oscillatory module controlling nucleolar release and resequestration of the Cdc14 phosphatase, which is essential for mitotic exit in budding yeast. We find that this Cdc14 release oscillator functions at constant and physiological cyclin-Cdk levels, and is therefore independent of the Cdk oscillator. However, the frequency of the release oscillator is regulated by cyclin-Cdk activity. This observation together with its mechanism suggests that the intrinsically autonomous Cdc14 release cycles are locked at once-per-cell-cycle through entrainment by the Cdk oscillator in wild-type cells. This concept may have broad implications for the structure and evolution of eukaryotic cell-cycle control.

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.

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.

Expanding and improving nanobody repertoires using a yeast display method: Targeting SARS-CoV-2.

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.

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii.

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.

Cleavage-furrow formation without F-actin in Chlamydomonas.

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.

Comprehensive Discovery of Cell-Cycle-Essential Pathways in Chlamydomonas reinhardtii.

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 ∼20% for the Chlamydomonas genome overall. Findings about these mutants will likely have direct relevance to essential cell biology in land plants.

Interregulation of CDKA/CDK1 and the Plant-Specific Cyclin-Dependent Kinase CDKB in Control of the Chlamydomonas Cell Cycle.

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.

F-actin homeostasis through transcriptional regulation and proteasome-mediated proteolysis.

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.

Oscillatory stimuli differentiate adapting circuit topologies.

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.

Multiple sequence-specific factors generate the nucleosome-depleted region on CLN2 promoter.

Nucleosome-depleted regions (NDRs) are ubiquitous on eukaryotic promoters. The formation of many NDRs cannot be readily explained by previously proposed mechanisms. Here, we carry out a focused study on a physiologically important NDR in the yeast CLN2 promoter (CLN2pr). We show that this NDR does not result from intrinsically unfavorable histone-DNA interaction. Instead, we identified eight conserved factor binding sites, including that of Reb1, Mcm1, and Rsc3, that cause the local nucleosome depletion. These nucleosome-depleting factors (NDFs) work redundantly, and simultaneously mutating all their binding sites eliminates CLN2pr NDR. The loss of the NDR induces unreliable "on/off" expression in individual cell cycles, but in the presence of the NDR, NDFs have little direct effect on transcription. We present bioinformatic evidence that the formation of many NDRs across the genome involves multiple NDFs. Our findings also provide significant insight into the composition and spatial organization of functional promoters.

Cyclin-Dependent Kinase Regulation of Diurnal Transcription in Chlamydomonas.

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 ∼2300 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.

A microbial avenue to cell cycle control in the plant superkingdom.

Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.

Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes.

In eukaryotes, cell cycle progression is controlled by cyclin/cyclin-dependent kinase (CDK) pairs. To better understand the details of this process, it is necessary to dissect the CDK's substrate pool in a cyclin- and cell cycle stage-specific way. Here, we report a mass spectrometry-based method that couples rapid isolation of native kinase-substrate complexes to on-bead phosphorylation with heavy-labeled ATP (ATP-γ-(18)O4). This combined in vivo/in vitro method was developed for identifying cyclin/CDK substrates together with their sites of phosphorylation. We used the method to identify Clb5 (S-cyclin)/Cdc28 and Cln2 (G1/S-cyclin)/Cdc28 substrates during S phase in Saccharomyces cerevisiae (Cdc28 is the master CDK in budding yeast). During the work, we discovered that Clb5/Cdc28 specifically phosphorylates S429 in the disordered tail of Cdc14, an essential phosphatase antagonist of Cdc28. This phosphorylation severely decreases the activity of Cdc14, providing a means for modulating the balance of CDK and phosphatase activity.

The Chlamydomonas cell cycle.

The position of Chlamydomonas within the eukaryotic phylogeny makes it a unique model in at least two important ways: as a representative of the critically important, early-diverging lineage leading to plants; and as a microbe retaining important features of the last eukaryotic common ancestor (LECA) that has been lost in the highly studied yeast lineages. Its cell biology has been studied for many decades and it has well-developed experimental genetic tools, both classical (Mendelian) and molecular. Unlike land plants, it is a haploid with very few gene duplicates, making it ideal for loss-of-function genetic studies. The Chlamydomonas cell cycle has a striking temporal and functional separation between cell growth and rapid cell division, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth and the cell division cycle; it also exhibits a highly choreographed interaction between the cell cycle and its centriole-basal body-flagellar cycle. Here, we review the current status of studies of the Chlamydomonas cell cycle. We begin with an overview of cell-cycle control in the well-studied yeast and animal systems, which has yielded a canonical, well-supported model. We discuss briefly what is known about similarities and differences in plant cell-cycle control, compared with this model. We next review the cytology and cell biology of the multiple-fission cell cycle of Chlamydomonas. Lastly, we review recent genetic approaches and insights into Chlamydomonas cell-cycle regulation that have been enabled by a new generation of genomics-based tools.

Stochastic expression and epigenetic memory at the yeast HO promoter.

Eukaryotic gene regulation usually involves sequence-specific transcription factors and sequence-nonspecific cofactors. A large effort has been made to understand how these factors affect the average gene expression level among a population. However, little is known about how they regulate gene expression in individual cells. In this work, we address this question by mutating multiple factors in the regulatory pathway of the yeast HO promoter (HOpr) and probing the corresponding promoter activity in single cells using time-lapse fluorescence microscopy. We show that the HOpr fires in an "on/off" fashion in WT cells as well as in different genetic backgrounds. Many chromatin-related cofactors that affect the average level of HO expression do not actually affect the firing amplitude of the HOpr; instead, they affect the firing frequency among individual cell cycles. With certain mutations, the bimodal expression exhibits short-term epigenetic memory across the mitotic boundary. This memory is propagated in "cis" and reflects enhanced activator binding after a previous "on" cycle. We present evidence that the memory results from slow turnover of the histone acetylation marks.

Positive feedback of G1 cyclins ensures coherent cell cycle entry.

In budding yeast, Saccharomyces cerevisiae, the Start checkpoint integrates multiple internal and external signals into an all-or-none decision to enter the cell cycle. Here we show that Start behaves like a switch due to systems-level feedback in the regulatory network. In contrast to current models proposing a linear cascade of Start activation, transcriptional positive feedback of the G1 cyclins Cln1 and Cln2 induces the near-simultaneous expression of the approximately 200-gene G1/S regulon. Nuclear Cln2 drives coherent regulon expression, whereas cytoplasmic Cln2 drives efficient budding. Cells with the CLN1 and CLN2 genes deleted frequently arrest as unbudded cells, incurring a large fluctuation-induced fitness penalty due to both the lack of cytoplasmic Cln2 and insufficient G1/S regulon expression. Thus, positive-feedback-amplified expression of Cln1 and Cln2 simultaneously drives robust budding and rapid, coherent regulon expression. A similar G1/S regulatory network in mammalian cells, comprised of non-orthologous genes, suggests either conservation of regulatory architecture or convergent evolution.

Origin of irreversibility of cell cycle start in budding yeast.

Budding yeast cells irreversibly commit to a new division cycle at a regulatory transition called Start. This essential decision-making step involves the activation of the SBF/MBF transcription factors. SBF/MBF promote expression of the G1 cyclins encoded by CLN1 and CLN2. Cln1,2 can activate their own expression by inactivating the Whi5 repressor of SBF/MBF. The resulting transcriptional positive feedback provides an appealing, but as yet unproven, candidate for generating irreversibility of Start. Here, we investigate the logic of the Start regulatory module by quantitative single-cell time-lapse microscopy, using strains in which expression of key regulators is efficiently controlled by changes of inducers in a microfluidic chamber. We show that Start activation is ultrasensitive to G1 cyclin. In the absence of CLN1,2-dependent positive feedback, we observe that Start transit is reversible, due to reactivation of the Whi5 transcriptional repressor. Introduction of the positive feedback loop makes Whi5 inactivation and Start activation irreversible, which therefore guarantees unidirectional entry into S phase. A simple mathematical model to describe G1 cyclin turn on at Start, entirely constrained by empirically measured parameters, shows that the experimentally measured ultrasensitivity and transcriptional positive feedback are necessary and sufficient dynamical characteristics to make the Start transition a bistable and irreversible switch. Our study thus demonstrates that Start irreversibility is a property that arises from the architecture of the system (Whi5/SBF/Cln2 loop), rather than the consequence of the regulation of a single component (e.g., irreversible protein degradation).

The effects of molecular noise and size control on variability in the budding yeast cell cycle.

Molecular noise in gene expression can generate substantial variability in protein concentration. However, its effect on the precision of a natural eukaryotic circuit such as the control of cell cycle remains unclear. We use single-cell imaging of fluorescently labelled budding yeast to measure times from division to budding (G1) and from budding to the next division. The variability in G1 decreases with the square root of the ploidy through a 1N/2N/4N ploidy series, consistent with simple stochastic models for molecular noise. Also, increasing the gene dosage of G1 cyclins decreases the variability in G1. A new single-cell reporter for cell protein content allows us to determine the contribution to temporal G1 variability of deterministic size control (that is, smaller cells extending G1). Cell size control contributes significantly to G1 variability in daughter cells but not in mother cells. However, even in daughters, size-independent noise is the largest quantitative contributor to G1 variability. Exit of the transcriptional repressor Whi5 from the nucleus partitions G1 into two temporally uncorrelated and functionally distinct steps. The first step, which depends on the G1 cyclin gene CLN3, corresponds to noisy size control that extends G1 in small daughters, but is of negligible duration in mothers. The second step, whose variability decreases with increasing CLN2 gene dosage, is similar in mothers and daughters. This analysis decomposes the regulatory dynamics of the Start transition into two independent modules, a size sensing module and a timing module, each of which is predominantly controlled by a different G1 cyclin.