The Saccharomyces cerevisiae Mlh1-Mlh3 Heterodimer Is an Endonuclease That Preferentially Binds to Holliday Junctions

MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX₂EX₄E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.     

Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport

A significant fraction of a plant''s nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.     

Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in Chlamydomonas reinhardtii

Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y₃₁₃F₃₁₄L₃₁₅, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y₃₁₃ or L₃₁₅ fail to rescue. Our immunoprecipitation results indicate that the Y₃₁₃, L₃₁₅, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y₃₁₃, L₃₁₅, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V₃₁₀T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.     

Tetrahymena Pot2 Is a Developmentally Regulated Paralog of Pot1 That Localizes to Chromosome Breakage Sites but Not to Telomeres

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.     

RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.Cytomegalovirus (CMV) is a highly prevalent betaherpesvirus that can cause severe multiorgan disease in immunocompromised individuals (45). The ability of this virus to infect an exceptionally wide variety of different cell types substantially contributes to pathogenesis (5, 68). CMV tropism is largely determined by a finely tuned interplay between cellular and viral factors, many of which act at the earliest stages of infection (30, 68). We recently showed that the cellular protein vimentin is required for efficient onset of infection in primary human foreskin fibroblasts (HF). Interestingly, the degree of reliance on the presence and integrity of vimentin intermediate filaments is dependent on the virus strain, with the broadly tropic strain TB40/E being more negatively affected than the HF-adapted, attenuated strain AD169 (44).Serial passage of clinical isolates in HF or in endothelial cells (EC) has produced strains with different tropisms. The attenuated strains AD169 and Towne were developed as vaccine candidates by propagation in HF for more than 50 (AD169) and 125 (Towne) serial passages (19, 53, 61). During this process, both strains, compared to clinical isolates, accumulated multiple mutations and genomic deletions, resulting in the loss of more than 19 open reading frames (ORFs) (8). The number of serial passages in HF of another commonly used strain, Toledo, has been more moderate (19, 54, 58). This, however, did not prevent the emergence of numerous genomic mutations, including the inversion of an ∼15-kb fragment (8, 16, 56). As a consequence of these changes, productive infections by AD169, Towne, and Toledo are largely restricted to HF. In contrast, propagation of clinical isolates in EC has yielded a series of strains with more-intact genomes and broader tropisms, such as TB40/E, VHL/E, and FIX (VR1814) (25, 60, 71). These strains retain the ability to grow in a wider variety of cell types, including EC, epithelial cells, and dendritic cells (DC), in addition to HF (23, 28, 59, 60, 68).The UL128, UL130, and UL131A gene products were recently identified as essential mediators of CMV infection of EC and epithelial cells (26, 72, 73) and of virus transfer from infected EC to monocyte-derived DC (23). Each of these proteins is independently required for the broader tropisms of EC-propagated CMV isolates (63, 64), and the presence of mutations affecting their functionality has been directly linked to the inability of AD169, Towne, and Toledo to initiate productive infections in EC and epithelial cells (26, 72, 73).We have shown that mature Langerhans-type DC differentiated in vitro from CD34⁺ hematopoietic progenitor cells are highly permissive to direct infection with TB40/E or VHL/E, with 48 to 72% of cells in culture expressing the viral immediate-early genes IE1 and IE2 at 48 h postinfection (hpi) (28). In contrast, only 2 to 5% and 0% of mature Langerhans cells were IE1/IE2 positive after exposure to Towne and Toledo, respectively. However, productive infection was detected in 12 to 17% of cells infected with AD169, despite the fact that this strain lacks expression of the UL131A gene as a consequence of a frameshift mutation (26). These results suggested the existence of additional viral genes with products involved in mediating tropisms for specific cell types, such as DC. To identify possible candidates, we compared the amino acid sequence of each ORF found in the genome of TB40-BAC4, a sequenced clone of the TB40/E strain in a bacterial artificial chromosome (BAC) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EF999921","term_id":"157779983","term_text":"EF999921"}}EF999921) (69), to the sequence of each ORF found in AD169 and AD169-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"X17403","term_id":"59591","term_text":"X17403"}}X17403 and {"type":"entrez-nucleotide","attrs":{"text":"AC146999","term_id":"38093733","term_text":"AC146999"}}AC146999) (10, 49), Towne and Towne-BAC (accession numbers {"type":"entrez-nucleotide","attrs":{"text":"FJ616285","term_id":"222354438","term_text":"FJ616285"}}FJ616285, {"type":"entrez-nucleotide","attrs":{"text":"AC146851","term_id":"37654163","term_text":"AC146851"}}AC146851, and {"type":"entrez-nucleotide","attrs":{"text":"AY315197","term_id":"124303521","term_text":"AY315197"}}AY315197) (17, 18, 49), and Toledo-BAC (accession number {"type":"entrez-nucleotide","attrs":{"text":"AC146905","term_id":"37777314","term_text":"AC146905"}}AC146905) (49). The product of a putative ORF, originally identified by Murphy et al. and named conserved ORF 29 (c-ORF29) (49), was considered of particular interest because the amino acid sequence of the putative protein encoded by Toledo and Towne was extended by 79 residues compared to the putative protein encoded by TB40/E and AD169. This led to our speculation that that the extended version might result in a nonfunctional version of the c-ORF29-encoded protein. We thus focused our studies on the products of this ORF.Here, we show for the first time that CMV c-ORF29 encodes a protein expressed at early to late times postinfection (p.i.) and localizes to the nuclear rim in peculiar invaginations of the nuclear lamina and in cytoplasmic vesicular structures at late times p.i. Based on this localization pattern, we named this gene product nuclear rim-associated cytomegaloviral protein, or RASCAL. Surprisingly, no difference was observed in the distributions of RASCAL during infection of HF with TB40/E or Towne, suggesting that the intracellular trafficking of this protein is not affected by the presence of the additional residues at the C terminus of RASCAL from strain Towne (RASCAL_Towne). Ectopic expression of RASCAL in human embryo kidney 293T (HEK293T) cells further revealed that this protein requires the presence of the nuclear egress complex (NEC) member UL50 to reach the nuclear rim, while coimmunoprecipitation (co-IP) assays provided evidence for the existence of an interaction between RASCAL and UL50. These findings suggest that RASCAL may be a new component of the NEC with possible roles in remodeling the nuclear lamina during nucleocapsid egress from the nucleus.     

Gene Regulation of Iron-Deficiency Responses Is Associated with Carbon Monoxide and Heme Oxydase 1 in Chlamydomonas reinhardtii

Carbon monoxide (CO) as an endogenous gaseous molecule regulates a variety of biological processes in animals. However, CO regulating nutrient stress responses in green alga is largely unknown. On the other hand, heme oxydase (HO1 as a rate-limiting enzyme of the first step for heme degration and to catalyze heme into biliverdin (BV), which is concomitant with releasing of CO and ferrous ions, probably participates in the process of CO-regulating response to nutrient stress in green alga. In this paper, we described an observation that CO could regulate iron-homeostasis in iron-starving Chlamydomonas reinhardtii. Exogenous CO at 8 µM was able to prevent the iron deficient-inducing chlorosis and improve chlorophyll accumulation. Expression pattern of FOX1, FTR1 and ferredoxin was up-regulated by CO exposure in iron-deficient mediam. treatment with external CO increasing iron accumulation in iron-deficient C. reinhardtii. Moreover, to get insights into the regulatory role of HO1, we constructed a transgenic alga overexpressing HO1 and HO1 knock-out mutants. The results show that there was no significant influence on chlorosis with HO1 overexpression of C. reinhardtii under iron-deficiency and the chlorophyll accumulation, and gene expression associated with iron deficiency of mutant were greatly improved. Otherwise, those results from HO1 knock-out mutants were opposite to HO1 overexpression mutants. Finally, CO exposure induced NO accumulation in cells. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO/HO1 may play an important role in improving green algae adaptation to iron deficiency or cross-talking with NO under the iron deficiency.     

An AP Endonuclease Functions in Active DNA Demethylation and Gene Imprinting in Arabidopsis

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l^+/−zdp^−/− mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.     

Biphasic Activation of p38MAPK Suggests That Apoptosis Is a Downstream Event in Pemphigus Acantholysis

In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.Pemphigus is a group of related autoimmune diseases characterized by blistering in the skin. The histologic hallmark of these disorders is termed acantholysis, which describes the loss of adhesion between adjacent epithelial cells. The two major variants are pemphigus foliaceus (PF)² and pemphigus vulgaris (PV). In PF, acantholysis is observed beneath the stratum corneum and within the granular layer of epidermal epithelia, whereas in PV, blister formation occurs above the basal layer of epidermal epithelia and mucosal epithelium. Passive transfer of IgG purified from both PV and PF patient sera reproduces the clinical, histological, and immunologic features of the human diseases, demonstrating that these autoantibodies are pathogenic (1, 2). In PF, autoantibodies target the desmosomal cadherin desmoglein (dsg) 1, whereas in PV, autoantibodies initially target dsg3 (3, 4) in mucosal PV and then subsequently target both dsg1 and dsg3 in mucocutaneous PV (5-7).The mechanism by which pemphigus autoantibodies induce blistering has been under investigation. Work from a number of laboratories has suggested that activation of intracellular events is induced by binding of PF or PV IgG to dsg1 and dsg3, respectively (8-14). Previously, we have reported that PV IgG activate p38MAPK and heat shock protein (HSP) 27 in human keratinocyte tissue cultures (15). Significantly, p38MAPK inhibitors blocked PV IgG-induced keratin filament retraction and actin reorganization in human keratinocyte tissue cultures. Furthermore, we have demonstrated that both PV and PF IgG induce phosphorylation of p38MAPK and HSP25, the murine HSP27 homologue, in mouse models and that inhibitors of p38MAPK block blistering in both the PV (16) and the PF (17) passive transfer mouse models. Additionally, in human skin biopsies from both PV and PF patients, phosphorylation of p38MAPK and HSP27 has been observed (18). Collectively, these observations suggest that activation of p38MAPK within the target keratinocyte contributes directly to loss of cell-cell adhesion induced by pemphigus autoantibodies.Both p38MAPK and HSP27 have been implicated in the regulation of the intermediate filament and actin cytoskeletons (19-25); the ability of p38MAPK inhibitors to block both pemphigus IgG-activated cytoskeletal reorganization and pemphigus IgG-activated blistering suggests that p38MAPK may be acting upstream of the cytoskeleton in the mechanism of acantholysis; however, p38MAPK signaling has been implicated in other cellular responses (reviewed in Ref. 26). For example, there is abundant evidence for p38MAPK involvement in apoptosis (27-29); however, the role of p38MAPK in apoptosis seems to be cell type- and stimulus-dependent. Although p38MAPK signaling promotes cell death in some cell lines, it also functions to enhance survival, growth, and differentiation in other cell lines (30). Several reports describe increased apoptosis of keratinocytes in pemphigus (31-35); however, the relationship between PV IgG-mediated p38MAPK signaling, the induction of apoptosis, and the relationship of apoptosis to blistering has not been defined. This study was undertaken to investigate the relationship between p38MAPK activation, apoptosis, and acantholysis.     

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas.     

Hfq Is a Global Regulator That Controls the Pathogenicity of Staphylococcus aureus

The Hfq protein is reported to be an RNA chaperone, which is involved in the stress response and the virulence of several pathogens. In E. coli, Hfq can mediate the interaction between some sRNAs and their target mRNAs. But it is controversial whether Hfq plays an important role in S. aureus. In this study, we found that the deletion of hfq gene in S. aureus 8325-4 can increase the surface carotenoid pigments. The hfq mutant was more resistant to oxidative stress but the pathogenicity of the mutant was reduced. We reveal that the Hfq protein can be detected only in some S. aureus strains. Using microarray and qRT-PCR, we identified 116 genes in the hfq mutant which had differential expression from the wild type, most of which are related to the phenotype and virulence of S. aureus. Among the 116 genes, 49 mRNAs can specifically bind Hfq protein, which indicates that Hfq also acts as an RNA binding protein in S. aureus. Our data suggest that Hfq protein of S. aureus is a multifunctional regulator involved in stress and virulence.     

Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.     

Species and Population Level Molecular Profiling Reveals Cryptic Recombination and Emergent Asymmetry in the Dimorphic Mating Locus of C. reinhardtii

Heteromorphic sex-determining regions or mating-type loci can contain large regions of non-recombining sequence where selection operates under different constraints than in freely recombining autosomal regions. Detailed studies of these non-recombining regions can provide insights into how genes are gained and lost, and how genetic isolation is maintained between mating haplotypes or sex chromosomes. The Chlamydomonas reinhardtii mating-type locus (MT) is a complex polygenic region characterized by sequence rearrangements and suppressed recombination between its two haplotypes, MT+ and MT−. We used new sequence information to redefine the genetic contents of MT and found repeated translocations from autosomes as well as sexually controlled expression patterns for several newly identified genes. We examined sequence diversity of MT genes from wild isolates of C. reinhardtii to investigate the impacts of recombination suppression. Our population data revealed two previously unreported types of genetic exchange in Chlamydomonas MT—gene conversion in the rearranged domains, and crossover exchanges in flanking domains—both of which contribute to maintenance of genetic homogeneity between haplotypes. To investigate the cause of blocked recombination in MT we assessed recombination rates in crosses where the parents were homozygous at MT. While normal recombination was restored in MT+×MT+ crosses, it was still suppressed in MT−×MT− crosses. These data revealed an underlying asymmetry in the two MT haplotypes and suggest that sequence rearrangements are insufficient to fully account for recombination suppression. Together our findings reveal new evolutionary dynamics for mating loci and have implications for the evolution of heteromorphic sex chromosomes and other non-recombining genomic regions.