Functional Identification of Two Types of Carotene Hydroxylases from the Green Alga Dunaliella bardawil Rich in Lutein.

The salt-tolerant unicellular alga Dunaliella bardawil FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, i.e., ß-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from D. bardawil. Their substrate specificities and enzyme activities were tested through functional complementation assays in Escherichia coli. It was showed that DbBCH could catalyze the hydroxylation of the ß-rings of both ß- and α-carotene, and displayed a low level of ε-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate ß-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the ß-ring and ε-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the ß-ring of α-carotene. The high accumulation of lutein in D. bardawil may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga D. bardawil FACHB-847.

Overexpression of a glycogenin, CmGLG2, enhances floridean starch accumulation in the red alga Cyanidioschyzon merolae.

Microalgae accumulate energy-reserved molecules, such as triacylglycerol and carbohydrates, which are suitable feedstocks for renewable energies such as biodiesel and bioethanol. However, the molecular mechanisms behind the microalgae accumulating these molecules require further elucidation. Recently, we have reported that the target of rapamycin (TOR)-signaling is a major pathway to regulate floridean starch synthesis by changing the phosphorylation status of CmGLG1, a glycogenin generally required for the initiation of starch/glycogen synthesis, in the unicellular red alga Cyanidioschyzon merolae. In the present study, we confirmed that another glycogenin, CmGLG2, is also involved in the floridean starch synthesis in this alga, since the CmGLG2 overexpression resulted in a two-fold higher floridean starch content in the cell. The results indicate that both glycogenin isoforms play an important role in floridean starch synthesis in C. merolae, and would be a potential target for improvement of floridean starch production in microalgae.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

Cell-Type Transcriptomes of the Multicellular Green Alga Volvox carteri Yield Insights into the Evolutionary Origins of Germ and Somatic Differentiation Programs.

Germ-soma differentiation is a hallmark of complex multicellular organisms, yet its origins are not well understood. Volvox carteri is a simple multicellular green alga that has recently evolved a simple germ-soma dichotomy with only two cell-types: large germ cells called gonidia and small terminally differentiated somatic cells. Here, we provide a comprehensive characterization of the gonidial and somatic transcriptomes of V. carteri to uncover fundamental differences between the molecular and metabolic programming of these cell-types. We found extensive transcriptome differentiation between cell-types, with somatic cells expressing a more specialized program overrepresented in younger, lineage-specific genes, and gonidial cells expressing a more generalist program overrepresented in more ancient genes that shared striking overlap with stem cell-specific genes from animals and land plants. Directed analyses of different pathways revealed a strong dichotomy between cell-types with gonidial cells expressing growth-related genes and somatic cells expressing an altruistic metabolic program geared toward the assembly of flagella, which support organismal motility, and the conversion of storage carbon to sugars, which act as donors for production of extracellular matrix (ECM) glycoproteins whose secretion enables massive organismal expansion. V. carteri orthologs of diurnally controlled genes from C. reinhardtii, a single-celled relative, were analyzed for cell-type distribution and found to be strongly partitioned, with expression of dark-phase genes overrepresented in somatic cells and light-phase genes overrepresented in gonidial cells- a result that is consistent with cell-type programs in V. carteri arising by cooption of temporal regulons in a unicellular ancestor. Together, our findings reveal fundamental molecular, metabolic, and evolutionary mechanisms that underlie the origins of germ-soma differentiation in V. carteri and provide a template for understanding the acquisition of germ-soma differentiation in other multicellular lineages.

Alga-PrAS (Algal Protein Annotation Suite): A Database of Comprehensive Annotation in Algal Proteomes.

Algae are smaller organisms than land plants and offer clear advantages in research over terrestrial species in terms of rapid production, short generation time and varied commercial applications. Thus, studies investigating the practical development of effective algal production are important and will improve our understanding of both aquatic and terrestrial plants. In this study we estimated multiple physicochemical and secondary structural properties of protein sequences, the predicted presence of post-translational modification (PTM) sites, and subcellular localization using a total of 510,123 protein sequences from the proteomes of 31 algal and three plant species. Algal species were broadly selected from green and red algae, glaucophytes, oomycetes, diatoms and other microalgal groups. The results were deposited in the Algal Protein Annotation Suite database (Alga-PrAS; http://alga-pras.riken.jp/), which can be freely accessed online.

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms.

Identification and phylogeny of putative PEPC genes in three toxin-producing Karenia (Dinophyta) species.

Dense blooms of toxin-producing Karenia brevis increase local surface ocean pH through CO2 uptake. To identify genes that may contribute to bloom-related environmental pH and pCO2 changes, transcriptomes with RNA from K. brevis Wilson cultures that had been acclimated to low CO2 (250 ppm) or recent CO2 (350 ppm) pCO2 levels were assembled. Among the annotated transcripts were PEPC, PPDK, and PEPCK enzymes found in the model C4 carbon fixation pathway. Previous studies have demonstrated that the enzymatic activity of PEPC, PPDK, and/or PEPCK in some algae species, including marine diatoms, is influenced by variations in dissolved inorganic carbon. We found significantly similar PEPC, PPDK, and PEPCK enzymes in the transcriptomes of K. brevis and two sister species Karenia papilionacea, and Karenia mikimotoi. One or more isoforms of PEPC were also identified in the transcriptomes of thirty additional photosynthetic phytoplankton species from nine phyla. Phylogenetic trees were constructed with neighbor joining and maximum likelihood techniques to characterize the evolutionary relationship among phytoplankton, terrestrial plant C4, and terrestrial plant C3 PEPC sequences. Based on the nucleotide trees constructed during this study, the Karenia PEPC transcripts were more closely related to the terrestrial C4 genes than the terrestrial C3 genes. Furthermore, PEPC phylogeny among phytoplankton closely resembles phylogenetic trees constructed with ribosomal RNA. This study confirmed that the toxin-producing dinoflagellates K. brevis, K. mikimotoi, and K. papilionacea express putative PEPC, PEPCK, and PPDK transcripts.

Bioinformatic Identification and Analysis of Extensins in the Plant Kingdom.

Extensins (EXTs) are a family of plant cell wall hydroxyproline-rich glycoproteins (HRGPs) that are implicated to play important roles in plant growth, development, and defense. Structurally, EXTs are characterized by the repeated occurrence of serine (Ser) followed by three to five prolines (Pro) residues, which are hydroxylated as hydroxyproline (Hyp) and glycosylated. Some EXTs have Tyrosine (Tyr)-X-Tyr (where X can be any amino acid) motifs that are responsible for intramolecular or intermolecular cross-linkings. EXTs can be divided into several classes: classical EXTs, short EXTs, leucine-rich repeat extensins (LRXs), proline-rich extensin-like receptor kinases (PERKs), formin-homolog EXTs (FH EXTs), chimeric EXTs, and long chimeric EXTs. To guide future research on the EXTs and understand evolutionary history of EXTs in the plant kingdom, a bioinformatics study was conducted to identify and classify EXTs from 16 fully sequenced plant genomes, including Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Volvox carteri, Klebsormidium flaccidum, Physcomitrella patens, Selaginella moellendorffii, Pinus taeda, Picea abies, Brachypodium distachyon, Zea mays, Oryza sativa, Glycine max, Medicago truncatula, Brassica rapa, Solanum lycopersicum, and Solanum tuberosum, to supplement data previously obtained from Arabidopsis thaliana and Populus trichocarpa. A total of 758 EXTs were newly identified, including 87 classical EXTs, 97 short EXTs, 61 LRXs, 75 PERKs, 54 FH EXTs, 38 long chimeric EXTs, and 346 other chimeric EXTs. Several notable findings were made: (1) classical EXTs were likely derived after the terrestrialization of plants; (2) LRXs, PERKs, and FHs were derived earlier than classical EXTs; (3) monocots have few classical EXTs; (4) Eudicots have the greatest number of classical EXTs and Tyr-X-Tyr cross-linking motifs are predominantly in classical EXTs; (5) green algae have no classical EXTs but have a number of long chimeric EXTs that are absent in embryophytes. Furthermore, phylogenetic analysis was conducted of LRXs, PERKs and FH EXTs, which shed light on the evolution of three EXT classes.

MARINE SULFUR CYCLE. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle.

Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.

A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry.

The blooms of toxic phototrophic microorganisms, such as microalgae and cyanobacteria, which are typically found in freshwater and marine environments, are becoming more frequent and problematic in aquatic systems. Due to accumulation of toxic algae, harmful algal blooms (HABs) exert negative effects on aquatic systems. Therefore, rapid detection of harmful microalgae is important for monitoring the occurrence of HABs. Mass spectrometry-based methods have become sensitive, specific techniques for the identification and characterization of microorganisms. Matrix-assisted laser desorption/ionization (MALDI) with time-of-flight (TOF) mass spectrometry (MS) allows us to measure a unique molecular fingerprint of highly abundant proteins in a microorganism and has been used for the rapid, accurate identification of bacteria and fungi in clinical microbiology. Here, we tested the specificity of MALDI-TOF MS using microalgal strains (Heterocapsa, Alexandrium, Nannochloropsis, Chaetoceros, Chlorella, and Dunaliella spp.). Our research suggested that this method was comparable in terms of the rapid identification of microalgea to conventional methods based on genetic information and morphology. Thus, this efficient mass spectrometry-based technique may have applications in the rapid identification of harmful microorganisms from aquatic environmental samples.

A novel astaxanthin-binding photooxidative stress-inducible aqueous carotenoprotein from a eukaryotic microalga isolated from asphalt in midsummer.

Water-soluble orange carotenoid proteins (OCPs) that bind 3'-hydroxyechinenone are found in cyanobacteria, and are thought to play a key role in photoprotection. The distribution of OCPs in eukaryotes remains largely unknown. In this study, we identified a novel OCP that predominantly binds astaxanthin from a eukaryotic microalga, strain Ki-4, isolated from a dry surface of heated asphalt in midsummer. A purified astaxanthin-binding OCP, named AstaP, shows high solubility in water with an absorption peak at 484 nm, and possesses a heat-stable activity that quenches singlet oxygen. The deduced amino acid sequence of AstaP comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of unknown function are distributed mainly in organisms from the hydrosphere, such as marine bacteria, cyanobacteria, sea anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI) value among homologs. The gene encoding AstaP, as well as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high light exposure. As a unique aqueous carotenoprotein, AstaP will provide a novel function of OCPs in protection against extreme photooxidative stresses.

Flagellated algae protein evolution suggests the prevalence of lineage-specific rules governing evolutionary rates of eukaryotic proteins.

Understanding the general rules governing the rate of protein evolution is fundamental to evolutionary biology. However, attempts to address this issue in yeasts and mammals have revealed considerable differences in the relative importance of determinants for protein evolutionary rates. This phenomenon was previously explained by the fact that yeasts and mammals are different in many cellular and genomic properties. Flagellated algae species have several cellular and genomic characteristics that are intermediate between yeasts and mammals. Using partial correlation analyses on the evolution of 6,921 orthologous proteins from Chlamydomonas reinhardtii and Volvox carteri, we examined factors influencing evolutionary rates of proteins in flagellated algae. Previous studies have shown that mRNA abundance and gene compactness are strong determinants for protein evolutionary rates in yeasts and mammals, respectively. We show that both factors also influence algae protein evolution with mRNA abundance having a larger impact than gene compactness on the rates of algae protein evolution. More importantly, among all the factors examined, coding sequence (CDS) length has the strongest (positive) correlation with protein evolutionary rates. This correlation between CDS length and the rates of protein evolution is not due to alignment-related issues or domain density. These results suggest no simple and universal rules governing protein evolutionary rates across different eukaryotic lineages. Instead, gene properties influence the rate of protein evolution in a lineage-specific manner.

The purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features.

The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits.

Eukaryotic and eubacterial contributions to the establishment of plastid proteome estimated by large-scale phylogenetic analyses.

Plastids including chloroplasts arose from a cyanobacterial endosymbiont and have retained their own genome, but the size has been reduced to less than one-tenth of the original bacterial genome. Over time, genes essential to plastid function have been transferred from the ancestral plastid genome to the nucleus, and the gene products are now targeted into the plastid from the host cytosol. However, phylogenetic analyses have suggested that the functions of certain original proteins encoded by the endosymbiont genome have been replaced by nucleus-encoded proteins of noncyanobacterial origin and that several proteins have been newly added to maintain and control plastids. In order to evaluate the rate and origin of noncyanobacterial proteins that have contributed to the establishment of the plastid proteome, we performed phylogenetic analyses of plastid-targeted proteins that are shared by the red alga Cyanidioschyzon merolae (455 proteins) and the Viridiplanta Arabidopsis thaliana (744 proteins). Our results show that approximately 40% of the plastid proteome common to red algae and green plants originated from genes of both the ancestral eukaryotic host and various lineages of bacteria (eubacteria) other than cyanobacteria. The replacement or addition of components was frequently observed for most of the plastid functions except for the light reaction of photosynthesis and the translation and degradation of proteins in the plastid. These results suggest that a considerable amount of bacterial metagenomic material, as well as the genomes of the host and the endosymbiont, has contributed to the establishment of the plastid before the split of the red and green algae.

Evolution and diversity of glutaredoxins in photosynthetic organisms.

The genome sequencing of prokaryotic and eukaryotic photosynthetic organisms enables a comparative genomic study of the glutaredoxin (Grx) family. The analysis of 58 genomes, using a specific motif composed of the active site sequence and of amino acids involved in glutathione binding, led to an updated classification of Grxs into six classes. Only two classes (I and II) are common to all photosynthetic organisms. Eukaryotes and cyanobacteria have two specific Grx classes (classes III and IV and classes V and VI, respectively). The classes IV, V and VI have not yet been identified and contain multimodular Grx fusions. In addition, putative Grx partners were identified from the presence of fusion proteins, the conservation of gene order in bacterial operons, and the gene co-occurrence. The genes encoding class II Grxs and BolA/YrbA proteins are frequently adjacent, in the same transcriptional orientation in prokaryote genomes and present in the same organisms.

Photosensory functions of channelrhodopsins in native algal cells.

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light-gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin-2) is biochemical activation of secondary Ca(2+)-channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.

Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salina.

In green algae, the final step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR; EC: 1.17.1.2), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 1959-bp Dunaliella salina HDR (DsHDR) cDNA encoding a deduced polypeptide of 474 amino acid residues. Phylogenetic analysis implied a cyanobacterial origin for plant and algal HDR genes. Steady-state DsHDR transcript levels were higher in D. salina cells submitted to nutritional depletion, high salt and/or high light, suggesting that DsHDR may respond to the same environmental cues as genes involved in carotenoid biosynthesis.

A gender-specific retinoblastoma-related protein in Volvox carteri implies a role for the retinoblastoma protein family in sexual development.

Here, we describe the cloning and characterization of RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development.