Comparative Genomics of Phylogenetically Diverse Unicellular Eukaryotes Provide New Insights into the Genetic Basis for the Evolution of the Programmed Cell Death Machinery

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes—from ligands and receptors to executors of PCD—was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae—used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for myxobacterial origin of eukaryotic defensins

Antimicrobial defensins with the cysteine-stabilized α-helical and β-sheet (CSαβ) motif are a large family of ancient, evolutionarily related innate immunity effectors of multicellular organisms. Although the widespread distribution in plants, fungi, and invertebrates suggests their uniqueness to Eukarya, it is unknown whether these eukaryotic defensins originated before or posterior to the emergence of eukaryotes. In this study, we provide evidence in support of the existence of defensin-like peptides (DLPs) in myxobacteria based on structural bioinformatics analysis, which recognized two bacterial peptides with a conserved cysteine-stabilized α-helical motif, a nested structural unit of the CSαβ motif. Similarity in sequence and structure to fungal DLPs together with restricted distribution to the myxobacteria as well as central role of the myxobacteria in the origin of eukaryotes suggest that the bacterial DLPs represent the ancestor of the eukaryotic defensins and could mediate immune defense of early eukaryotes after gene transfer to the proto-eukaryotic genome. Our work thus offers a basis for further investigation of prokaryotic origin of eukaryotic immune effector molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Occurrence and phylogenetic significance of cytokinesis-related callose in green algae, bryophytes, ferns and seed plants

 In order to investigate the occurrence of callose in dividing cells, we cultivated a selection of 30 organisms (the prokaryotic cyanobacterium Anabaena and eukaryotic green algae, bryophytes, ferns and seed plants) under defined conditions in the laboratory. Samples from these photoautotrophs, which are members of the evolutionary 'green lineage' leading from freshwater algae to land plants, were analysed by fluorescence microscopy. The β-1,3-glucan callose was identified by its staining properties with aniline blue and sirofluor. With the exception of the prokaryotic cyanobacterium, all of the eukaryotic organisms studied were capable of producing wound-induced callose. No callose was detected during cytokinesis of dividing cells of unicellular green algae (and Anabaena). However, in all of the multicellular green algae and land plants (embryophytes) investigated, callose was identified in newly made septae by an intense yellow fluorescence. The formation of wound callose was never detected in cells with callose in the newly formed septae. Additional experiments verified that no fixation-induced artefacts occurred. Our results show that callose is a regular component of developing septae in juvenile cells during cytokinesis in multicellular green algae and embryophytes. The implications of our results with respect to the evolutionary relationships between extant charophytes and land plants are discussed. Received: 15 September 2000 / Revision received: 23 October 2000 / Accepted: 23 October 2000     

Structure and molecular phylogeny of sasA genes in cyanobacteria: insights into evolution of the prokaryotic circadian system

Cyanobacteria are the simplest organisms known to have a circadian system. In addition to the three well-studied kai genes, kaiA, kaiB, and kaiC, an important element of this system is a two-component sensory transduction histidine kinase sasA. Using publicly available data of complete prokaryotic genomes, we performed structural and phylogenetic analyses of the sasA genes. Results show that this gene has a triple-domain structure, and the domains are under different selective constraints. The sasA gene originated in cyanobacteria probably through the fusion of the ancestral kaiB gene with a double-domain, two-component sensory transduction histidine kinase. The results of the phylogenetic analyses suggest that sasA emerged before the kaiA gene, about 3,000-2,500 MYA, and has evolved in parallel with the evolution of the kaiBC cluster. The observed concordant patterns of the sasA and kaiBC evolution suggest that these genes might compose an ancient KaiBC-SasA-based circadian system, without the kaiA gene, and that such a system still exists in some unicellular cyanobacteria.     

Actin on disease--studying the pathobiology of cell motility using Dictyostelium discoideum

The actin cytoskeleton in eukaryotic cells provides cell structure and organisation, and allows cells to generate forces against membranes. As such it is a central component of a variety of cellular structures involved in cell motility, cytokinesis and vesicle trafficking. In multicellular organisms these processes contribute towards embryonic development and effective functioning of cells of all types, most obviously rapidly moving cells like lymphocytes. Actin also defines and maintains the architecture of complex structures such as neuronal synapses and stereocillia, and is required for basic housekeeping tasks within the cell. It is therefore not surprising that misregulation of the actin cytoskeleton can cause a variety of disease pathologies, including compromised immunity, neurodegeneration, and cancer spread. Dictyostelium discoideum has long been used as a tool for dissecting the mechanisms by which eukaryotic cells migrate and chemotax, and recently it has gained precedence as a model organism for studying the roles of conserved pathways in disease processes. Dictyostelium's unusual lifestyle, positioned between unicellular and multicellular organisms, combined with ease of handling and strong conservation of actin regulatory machinery with higher animals, make it ideally suited for studying actin-related diseases. Here we address how research in Dictyostelium has contributed to our understanding of immune deficiencies and neurological defects in humans, and briefly discuss its future prospects for furthering our understanding of neurodegenerative disorders.     

Homologous Protein Domains in Superkingdoms Archaea,Bacteria, and Eukaryota and the Problem of the Origin of Eukaryotes

The distribution of protein domains was analyzed in superkingdoms Archaea, Bacteria, and Eukaryota. About a half of eukaryotic domains have prokaryotic origin. Many domains related to information processing in the nucleocytoplasm were inherited from archaea. Sets of domains associated with metabolism and regulatory and signaling systems were inherited from bacteria. Many signaling and regulatory domains common for bacteria and eukaryotes were responsible for the cellular interaction of bacteria with other components of the microbial community but were involved in coordination of the activity of eukaryotic organelles and cells in multicellular organisms. Many eukaryotic domains of bacterial origin could not originate from ancestral mitochondria and plastids but rather were adopted from other bacteria. An archaeon with the induced incorporation of alien genetic material could be the ancestor of the eukaryotic nucleocytoplasm.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 389–400.Original Russian Text Copyright © 2005 by Markov, Kulikov.     

Sigma 1- and mu 1-Adaptin homologues of Leishmania mexicana are required for parasite survival in the infected host

The sorting of membrane-bound proteins from the trans-Golgi network to lysosomal/endosomal compartments is achieved by preferential inclusion into clathrin-coated vesicles. Contained within the cytoplasmic domains of such proteins, specific sequence motifs have been identified (tyrosine-based and/or di-leucine-based) that are essential for targeting and are recognized by a family of heterotetrameric adaptor complexes, which then recruit clathrin. These cytosolic protein complexes, which have been found in a wide variety of higher eukaryotic organisms, are essential for the development of multicellular organisms. In trypanosomatids, the adaptin-mediated sorting of proteins is largely uncharacterized. In order to identify components of the adaptor-complex machinery, this study reports the cloning and characterization of sigma 1- and mu 1-adaptin gene homologues from the eukaryotic protozoan parasite, Leishmania mexicana. Generation of sigma 1- and mu 1-adaptin gene deletion mutants shows that these promastigote parasites are viable in culture, but are unable to establish infection of macrophages or mice, indicating that adaptin function is crucial for pathogenesis in these unicellular organisms.     

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.     

The evolution of immune mechanisms

From early on in evolution, organisms have had to protect themselves from pathogens. Mechanisms for discriminating "self" from "non-self" evolved to accomplish this task, launching a long history of host-pathogen co-evolution. Evolution of mechanisms of immune defense has resulted in a variety of strategies. Even unicellular organisms have rich arsenals of mechanisms for protection, such as restriction endonucleases, antimicrobial peptides, and RNA interference.In multicellular organisms, specialized immune cells have evolved, capable of recognition, phagocytosis, and killing of foreign cells as well as removing their own cells changed by damage, senescence, infection, or cancer. Additional humoral factors, such as the complement cascade, have developed that co-operate with cellular immunity in fighting infection and maintaining homeostasis. Defensive mechanisms based on germline-encoded receptors constitute a system known as innate immunity. In jaw vertebrates, this system is supplemented with a second system, adaptive immunity, which in contrast to innate immunity is based on diversification of immune receptors and on immunological memory in each individual.Usually, each newly evolved defense mechanism did not replace the previous one, but supplemented it, resulting in a layered structure of the immune system. The immune system is not one system but rather a sophisticated network of various defensive mechanisms operating on different levels, ranging from mechanisms common for every cell in the body to specialized immune cells and responses at the level of the whole organism. Adaptive changes in pathogens have shaped the evolution of the immune system at all levels.     

Evolution and adaptation of single-pass transmembrane proteins

A comparative analysis of 6039 single-pass (bitopic) membrane proteins from six evolutionarily distant organisms was performed based on data from the Membranome database. The observed repertoire of bitopic proteins is significantly enlarged in eukaryotic cells and especially in multicellular organisms due to the diversification of enzymes, emergence of proteins involved in vesicular trafficking, and expansion of receptors, structural, and adhesion proteins. The majority of bitopic proteins in multicellular organisms are located in the plasma membrane (PM) and involved in cell communication. Bitopic proteins from different membranes significantly diverge in terms of their biological functions, size, topology, domain architecture, physical properties of transmembrane (TM) helices and propensity to form homodimers. Most proteins from eukaryotic PM and endoplasmic reticulum (ER) have the N-out topology. The predicted lengths of TM helices and hydrophobic thicknesses, stabilities and hydrophobicities of TM α-helices are the highest for proteins from eukaryotic PM, intermediate for proteins from prokaryotic cells, ER and Golgi apparatus, and lowest for proteins from mitochondria, chloroplasts, and peroxisomes. Tyr and Phe residues accumulate at the cytoplasmic leaflet of PM and at the outer leaflet of membranes of bacteria, Golgi apparatus, and nucleus. The propensity for dimerization increases from unicellular to multicellular eukaryotes, from enzymes to receptors, and from intracellular membrane proteins to PM proteins. More than half of PM proteins form homodimers with a 2:1 ratio of right-handed to left-handed helix packing arrangements. The inverse ratio (1:2) was observed for dimers from the ER, Golgi and vesicles.     

Microcystin biosynthesis in planktothrix: genes,evolution, and manipulation

Microcystins represent an extraordinarily large family of cyclic heptapeptide toxins that are nonribosomally synthesized by various cyanobacteria. Microcystins specifically inhibit the eukaryotic protein phosphatases 1 and 2A. Their outstanding variability makes them particularly useful for studies on the evolution of structure-function relationships in peptide synthetases and their genes. Analyses of microcystin synthetase genes provide valuable clues for the potential and limits of combinatorial biosynthesis. We have sequenced and analyzed 55.6 kb of the potential microcystin synthetase gene (mcy) cluster from the filamentous cyanobacterium Planktothrix agardhii CYA 126. The cluster contains genes for peptide synthetases (mcyABC), polyketide synthases (PKSs; mcyD), chimeric enzymes composed of peptide synthetase and PKS modules (mcyEG), a putative thioesterase (mcyT), a putative ABC transporter (mcyH), and a putative peptide-modifying enzyme (mcyJ). The gene content and arrangement and the sequence of specific domains in the gene products differ from those of the mcy cluster in Microcystis, a unicellular cyanobacterium. The data suggest an evolution of mcy clusters from, rather than to, genes for nodularin (a related pentapeptide) biosynthesis. Our data do not support the idea of horizontal gene transfer of complete mcy gene clusters between the genera. We have established a protocol for stable genetic transformation of Planktothrix, a genus that is characterized by multicellular filaments exhibiting continuous motility. Targeted mutation of mcyJ revealed its function as a gene coding for a O-methyltransferase. The mutant cells produce a novel microcystin variant exhibiting reduced inhibitory activity toward protein phosphatases.     

SWEET蛋白家族研究进展

SWEET是新发现的一类具有7次跨膜?-螺旋的糖运输蛋白,它们由2个重复的具有3次跨膜?-螺旋的MtN3 motif和一个起连接作用的跨膜?-螺旋组成.SWEET广泛存在于真核单细胞生物、高等植物以及动物中.它们在生殖发育、植物与微生物的相互作用、植物的逆境反应及衰老等许多方面起重要作用.最近的研究显示,原核生物中存在与真核生物SWEET类似的、只含有一个3次跨膜?-螺旋的蛋白,这些蛋白属于MtN3或PQ-Loop家族.从慢生根瘤菌中克隆的SWEET同源蛋白BjSemiSWEET1和已经鉴定的部分真核生物SWEET蛋白一样具有运输蔗糖的能力,这个结果与其他相关研究一起暗示真核生物7次跨膜?-螺旋的糖或氨基酸运输蛋白可能由原核生物中3次跨膜?-螺旋的小分子蛋白通过复制或横向基因转移融合进化而来,并且它们在行使功能时可能形成和其他许多膜转运蛋白相似的、具有12次跨膜结构的功能单位.对SWEET的研究将为揭示多种生命现象提供重要线索.     

Protein Thermodynamics Can Be Predicted Directly from Biological Growth Rates

Life on Earth is capable of growing from temperatures well below freezing to above the boiling point of water, with some organisms preferring cooler and others hotter conditions. The growth rate of each organism ultimately depends on its intracellular chemical reactions. Here we show that a thermodynamic model based on a single, rate-limiting, enzyme-catalysed reaction accurately describes population growth rates in 230 diverse strains of unicellular and multicellular organisms. Collectively these represent all three domains of life, ranging from psychrophilic to hyperthermophilic, and including the highest temperature so far observed for growth (122°C). The results provide credible estimates of thermodynamic properties of proteins and obtain, purely from organism intrinsic growth rate data, relationships between parameters previously identified experimentally, thus bridging a gap between biochemistry and whole organism biology. We find that growth rates of both unicellular and multicellular life forms can be described by the same temperature dependence model. The model results provide strong support for a single highly-conserved reaction present in the last universal common ancestor (LUCA). This is remarkable in that it means that the growth rate dependence on temperature of unicellular and multicellular life forms that evolved over geological time spans can be explained by the same model.     

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.     

Diatoms: A Novel Source for the Neurotoxin BMAA in Aquatic Environments

Amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease is a neurological disorder linked to environmental exposure to a non-protein amino acid, β-N-methylamino-L-alanine (BMAA). The only organisms reported to be BMAA-producing, are cyanobacteria – prokaryotic organisms. In this study, we demonstrate that diatoms – eukaryotic organisms – also produce BMAA. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry revealed the occurrence of BMAA in six investigated axenic diatom cultures. BMAA was also detected in planktonic field samples collected on the Swedish west coast that display an overrepresentation of diatoms relative to cyanobacteria. Given the ubiquity of diatoms in aquatic environments and their central role as primary producers and the main food items of zooplankton, the use of filter and suspension feeders as livestock fodder dramatically increases the risk of human exposure to BMAA-contaminated food.     

Programmed cell death in trypanosomatids and other unicellular organisms

In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms. However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms. The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway. Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.     

Global orchestration of gene expression by the biological clock of cyanobacteria

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a central clock. Recent studies shed light on the mechanisms governing circadian rhythms in cyanobacteria and highlight key differences between prokaryotic and eukaryotic clocks.     

Comparative analyses of fundamental differences in membrane transport capabilities in prokaryotes and eukaryotes

Whole-genome transporter analyses have been conducted on 141 organisms whose complete genome sequences are available. For each organism, the complete set of membrane transport systems was identified with predicted functions, and classified into protein families based on the transporter classification system. Organisms with larger genome sizes generally possessed a relatively greater number of transport systems. In prokaryotes and unicellular eukaryotes, the significant factor in the increase in transporter content with genome size was a greater diversity of transporter types. In contrast, in multicellular eukaryotes, greater number of paralogs in specific transporter families was the more important factor in the increase in transporter content with genome size. Both eukaryotic and prokaryotic intracellular pathogens and endosymbionts exhibited markedly limited transport capabilities. Hierarchical clustering of phylogenetic profiles of transporter families, derived from the presence or absence of a certain transporter family, showed that clustering patterns of organisms were correlated to both their evolutionary history and their overall physiology and lifestyles.