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At least 19 recordsLinked to original sources
PubMed1979

The use of [14C]eukaryotic initiation factor 2 to measure the endogenous pool size of eukaryotic initiation factor 2 in rabbit reticulocyte lysate.

[14C]Eukaryotic initiation factor 2 (eIF-2), obtained by reductive methylation of the purified initiation factor, was shown to be active in the unfractionated reticulocyte lysate. This allowed a direct measurement of the endogenous pool size of eIF-2 in rabbit reticulocyte lysate according to the principle of isotope dilution. A value of 20 to 30 pmol/ml of lysate was obtained. Although translational inhibition resulting from hemin deficiency appears to be characterized by a change from catalytic to stoichiometric utilization of eIF-2, the pool size of eIF-2 is too small to account for the normal period of protein synthesis before the onset of translation inhibition. This suggests, therefore, that additional events to eIF-2 alpha phosphorylation may be required for translational inhibition.

Animals↗
PubMed2015

Sex is a ubiquitous, ancient, and inherent attribute of eukaryotic life.

Sexual reproduction and clonality in eukaryotes are mostly seen as exclusive, the latter being rather exceptional. This view might be biased by focusing almost exclusively on metazoans. We analyze and discuss reproduction in the context of extant eukaryotic diversity, paying special attention to protists. We present results of phylogenetically extended searches for homologs of two proteins functioning in cell and nuclear fusion, respectively (HAP2 and GEX1), providing indirect evidence for these processes in several eukaryotic lineages where sex has not been observed yet. We argue that (i) the debate on the relative significance of sex and clonality in eukaryotes is confounded by not appropriately distinguishing multicellular and unicellular organisms; (ii) eukaryotic sex is extremely widespread and already present in the last eukaryotic common ancestor; and (iii) the general mode of existence of eukaryotes is best described by clonally propagating cell lines with episodic sex triggered by external or internal clues. However, important questions concern the relative longevity of true clonal species (i.e., species not able to return to sexual procreation anymore). Long-lived clonal species seem strikingly rare. We analyze their properties in the light of meiotic sex development from existing prokaryotic repair mechanisms. Based on these considerations, we speculate that eukaryotic sex likely developed as a cellular survival strategy, possibly in the context of internal reactive oxygen species stress generated by a (proto) mitochondrion. Thus, in the context of the symbiogenic model of eukaryotic origin, sex might directly result from the very evolutionary mode by which eukaryotic cells arose.

Cell Fusion↗
PubMed2026

The archaeal roots of eukaryotic life.

Resolving the biological and geological events that led to the origin of eukaryotes is an ongoing challenge in biology. A major step in the evolution of complex cellular life was the merger between an ancestral host cell and a bacterium (that became the mitochondrion) some two billion years ago. Recently, metagenomics has enabled the reconstruction of a broad diversity of genomes, referred to as the Asgard Archaea. The Asgards are monophyletic with eukaryotes on the tree of life. Asgards have an array of genes, previously thought exclusive to eukaryotes, involved in cellular trafficking, the ubiquitin system, endosomal sorting, and cytoskeleton formation, with growing evidence demonstrating the functions of these proteins mirror those in eukaryotes. This gene repertoire suggests that these Archaea are descendants of the archaeal host from which eukaryotes evolved. Increased sampling has revealed that Asgard lineages are metabolically versatile and play key roles in various ecosystems and uncovered evolutionary transitions between Archaea and eukaryotes, such as innovations in eukaryotic defense systems. The positioning of eukaryotes in the Asgards is debated, but eukaryotes appear to branch within the Heimdallarchaeia. Lineages within this group, particularly Hodarchaeales and Kariarchaeaceae, contain a broad repertoire of eukaryote-like traits, including high-energy yielding metabolisms. Observing and studying Asgard interactions with bacterial descendants of mitochondria in a modern setting will transform our understanding of the origin of complex cellular life.

Archaea↗
PubMedDate not supplied

[Aspects of bacteriophage-eukaryotic cell relationships].

The present work deals with bacteriophage--eukaryotic cell interrelationship, including:--Data on the possible presence of bacteriophages in calf serum and viral vaccines.--Methodological problems of research on phage--eukaryotic cell relations.--Data concerning the effects of phage infection on eukaryotic cells (demonstration of phage infection of the eukaryotic cell; integration of phage genetic material in the genome of the eukaryotic cell; coding of phage proteins in phage-infected eukaryotic cells; modified multiplication parameters and transformed potential of some phage-infected eukaryotic cells).--Indications for solving some of the problems.

Bacteriophages↗
PubMed1978

Implications of RNA-RNA splicing in evolution of eukaryotic cells.

The differences in the biochemistry of messenger RNA formation in eukaryotes compared to prokaryotes are so profound as to suggest that sequential prokaryotic to eukaryotic cell evolution seems unlikely. The recently discovered noncontiguous sequences in eukaryotic DNA that encode messenger RNA may reflect an ancient, rather than a new, distribution of information in DNA and that eukaryotes evolved independently of prokaryotes.

Adenoviruses, Human↗
PubMed2026

A distinct subclade of AlkB family demethylases in ciliates safeguards the positional fidelity of eukaryotic N6-methyladenine (6mA).

DNA N6-methyladenine (6mA) is a newly recognized transcription-associated epigenetic mark in eukaryotes. While its methylation pathway has been well established, the identity of eukaryotic 6mA demethylase(s) responsible for its removal and dynamic regulation has remained elusive. Here, we identify and characterize DMT3 (TtALKBH5), an AlkB family dioxygenase in Tetrahymena thermophila, as a 6mA demethylase in ciliates and potentially several other unicellular eukaryotes with abundant 6mA and a defined AMT1 methyltransferase (MTase) complex, supported by genetic and molecular evidence. DMT3 acts on both fully and hemimethylated ApT dinucleotides, an activity partially facilitated by a naturally occurring cysteine-to-serine substitution. Genome profiling shows that DMT3 is enriched at transcription start sites (TSSs) of 6mA-enriched genes, complementary to the occupancy pattern of the AMT1 complex, where it selectively removes spurious 6mA deposited by AMT1. Genetic disruption of DMT3-mediated demethylation, either by knockout or catalytic inactivation, leads to aberrant 6mA accumulation at TSS regions, transcriptional dysregulation, altered chromatin accessibility, and impaired initiation of sexual reproduction. Notably, simultaneous removal of DMT3 and AMT1 eliminates these defects, indicating that spurious TSS 6mA underlies transcriptional and developmental impairment.

Adenine↗
PubMed2025

High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation.

Noncanonical amino acids (ncAAs) enable diversification of protein functions, but the efficiency of genetic code expansion (GCE) in eukaryotes is hindered by competition between suppressor tRNAs and release factors. Prior work has identified eukaryotic release factor 1 (eRF1) mutants that improve ncAA incorporation, suggesting that screens for improved variants may lead to further enhancements. Here, we developed a high-throughput system to screen eRF1 mutants in Saccharomyces cerevisiae where eRF1 mutants are coexpressed on a plasmid alongside genomically encoded, wild-type eRF1. This strategy enabled recovery of live cells expressing eRF1 variants that enhance ncAA incorporation, even with mutants known to severely affect cell viability in the absence of WT eRF1 expression. We prepared and screened a million-member library of randomly mutated eRF1 variants for clones exhibiting improved ncAA integration phenotypes. Deep sequencing revealed a diverse set of enriched mutations across all three major domains of eRF1. Interestingly, several enriched mutations identified here are also found in naturally occurring eRF1 homologs from species that recode canonical stop codons. When eRF1 variants were combined with yeast knockout strains also known to enhance ncAA incorporation, this resulted in further improvements to efficiency, highlighting the complementarity of release factor engineering to other GCE enhancement strategies. This work demonstrates that high-throughput engineering of the eukaryotic translational apparatus is a powerful approach to identify previously unknown solutions for enhancing ncAA incorporation, with implications for elucidating and precisely manipulating the molecular functions of essential translational machinery.

Noncanonical amino acids↗
PubMed2026

Diversity and evolution of chromatin regulatory states across eukaryotes.

Histone post-translational modifications (hPTMs) are key regulators of chromatin states, influencing gene expression, epigenetic memory and transposable element repression across eukaryotic genomes. While many hPTMs are evolutionarily conserved, the extent to which the chromatin states they define are similarly preserved remains unclear. Here we developed a combinatorial indexing chromatin immunoprecipitation followed by sequencing method to simultaneously profile specific hPTMs across diverse eukaryotic lineages, including amoebozoans, rhizarians, discobans and cryptomonads. Our analyses revealed highly conserved euchromatin states at active gene promoters and gene bodies. In contrast, we observed diverse configurations of repressive heterochromatin states associated with silenced genes and transposable elements, characterized by various combinations of hPTMs such as H3K9me3, H3K27me3 and/or different H3K79 methylations. These findings suggest that, while core hPTMs are ancient and broadly conserved, their functional readout has diversified throughout eukaryotic evolution, shaping lineage-specific chromatin landscapes.

Histones↗
PubMed2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Capsid Proteins↗
PubMed1979

Increased expression of a eukaryotic gene in Escherichia coli through stabilization of its messenger RNA.

The expression of a cloned eukaryotic gene [catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) (qa-2+) from Neurospora crassa] is dramatically increased (as much as 100-fold) in Escherichia coli strains deficient in polynucleotide phosphorylase (pnp) (polynucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) and RNase I (rna). The increased expression is controlled primarily by the absence of polynucleotide phosphorylase and appears to be specific for the eukaryotic gene. No increase in the specific activity of either chromosomal or plasmid-borne prokaryotic genes has been observed. In polynucleotide phosphorylase-deficient strains of E. coli the half-life of plasmid (pVK88, ampr qa-2+)-encoded mRNAs increases from 1.0 to 2.8 min. This increase must be due primarily to stabilization of the aq-2 mRNA because no increase in the half-lives of pBR322 vehicle mRNAs was observed in polynucleotide phosphorylase-deficient strains. These results suggest that there are inherent structural differences between prokaryotic and eukaryotic mRNAs.

DNA, Recombinant↗
PubMed2025

Golgi_traff phylogeny reveals ancient eukaryotic genes with recent surprises: replication and diversification of HID1 domain-containing protein unique to Schizosaccharomyces.

Golgi_traff is a Pfam clan containing two members, Dymeclin (DYM) and HID1 domain-containing protein (HID). Interrogation of over 900 eukaryotic genomes with sequence models showed that both are ancient eukaryotic genes, which have exhibited different paths of gene loss, including from major taxonomic groups. For example, the Metazoa have both genes, whereas the Viridiplantae and Dikarya have lost HID and DYM, respectively. A unique replication event occurred within the genus Schizosaccharomyces in that all sequenced species possess three HID-encoding paralogs, whereas its nearest fungal relatives and other eukaryotes are almost exclusively monogenic. A phylogenetic analysis of yeasts revealed that the Golgi-resident paralog Human ortholog 3 (SPAC17A5.16) is more similar to the HID of other yeasts than to its paralogs. Transmission electron microscopy revealed that the SPAC17A5.16 mutant lacks a stacked Golgi apparatus (GA) form, suggesting a role in maintaining GA structure. Altered proliferation of the SPAC17A5.16 mutant in response to GA disrupting chemical agents indicated a perturbation of GA-related functions. Structural models suggest SPAC17A5.16 has a long, disordered N-terminal region that may facilitate anchoring to GA membranes. A modification to Schizosaccharomyces HID nomenclature is proposed to reflect their evolutionary and functional characteristics. The potential of the Golgi_traff clan to serve as a model for the diversification of protein function according to the concepts of sub/neofunctionalization is discussed.

Schizosaccharomyces↗
PubMed2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We employed protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a supefamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains, and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

adenovirus↗
PubMed1975

Sites of action of fusidic acid in eukaryotes. Inhibition by fusidic acid of a ribosome-independent GTPase from Artemia salina embryos.

1. A ribosome-independent GTPase activity has been isolated from the high-speed supernatant fraction of Artemia salina embryos, and some of its properties have been studied. This activity is inhibited by fusidic acid, an antibiotic generally thought to inhibit only EF-2 in eukaryotes. However, several lines of evidence indicate that the GTPase activity, described here, is distinct from EF-2. The results suggest, therefore, that the inhibitory effect of fusidic acid in eukaryotic systems is not restricted to EF-2 (and ribosome)-dependent functions only. 2. The results of other experiments have revealed that, despite its ability to inhibit the GTPase activity mentioned above, fusidic acid is not a non-specific inhibitor of all ribosome-independent GTPase and ATPase activities present in eukaryotic cells.

Adenosine Triphosphatases↗
PubMed2025

Cytoplasmic mRNA decay and quality control machineries in eukaryotes.

mRNA degradation pathways have key regulatory roles in gene expression. The intrinsic stability of mRNAs in the cytoplasm of eukaryotic cells varies widely in a gene- and isoform-dependent manner and can be regulated by cellular cues, such as kinase signalling, to control mRNA levels and spatiotemporal dynamics of gene expression. Moreover, specialized quality control pathways exist to rid cells of non-functional mRNAs produced by errors in mRNA processing or mRNA damage that negatively impact translation. Recent advances in structural, single-molecule and genome-wide methods have provided new insights into the central machineries that carry out mRNA turnover, the mechanisms by which mRNAs are targeted for degradation and the general principles that govern mRNA stability at a global level. This improved understanding of mRNA degradation in the cytoplasm of eukaryotic cells is finding practical applications in the design of therapeutic mRNAs.

RNA Stability↗
PubMed2024

Foraging mechanisms in excavate flagellates shed light on the functional ecology of early eukaryotes.

The phagotrophic flagellates described as "typical excavates" have been hypothesized to be morphologically similar to the Last Eukaryotic Common Ancestor and understanding the functional ecology of excavates may therefore help shed light on the ecology of these early eukaryotes. Typical excavates are characterized by a posterior flagellum equipped with a vane that beats in a ventral groove. Here, we combined flow visualization and observations of prey capture in representatives of the three clades of excavates with computational fluid dynamic modeling, to understand the functional significance of this cell architecture. We record substantial differences amongst species in the orientation of the vane and the beat plane of the posterior flagellum. Clearance rate magnitudes estimated from flow visualization and modeling are both like that of other similarly sized flagellates. The interaction between a vaned flagellum beating in a confinement is modeled to produce a very efficient feeding current at low energy costs, irrespective of the beat plane and vane orientation and of all other morphological variations. Given this predicted uniformity of function, we suggest that the foraging systems of typical excavates studied here may be good proxies to understand those potentially used by our distant ancestors more than 1 billion years ago.

Flagella↗
PubMed1978

Translational control: recognition of the methylated 5' end and an internal sequence in eukaryotic mRNA by the initiation factor that binds methionyl-tRNAfMet.

Structural analogs of the methylated 5' end (cap) of eukaryotic mRNA, such as 7-methylguanosine 5'-monophosphate, specifically inhibit both GTP-dependent binding of Met-tRNAfMet and binding of globin mRNA to eukaryotic initiation factor 2 (eIF-2). Addition of purified eIF-2 effectively relieves the cap analog-induced inhibition of globin mRNA translation. The analog competitively inhibits the function of eIF-2 and of mRNA in protein synthesis. Binding to eIF-2 of capped mRNA as well as noncapped mRNA, such as Mengo virus RNA, can be inhibited completely by free cap molecules, but much more cap is needed to inhibit binding of Mengo virus RNA. mRNA, whether or not it is capped, competitively inhibits the binding of Met-tRNAfMet to eIF-2. These results provide compelling evidence that eIF-2 recognizes mRNA. It is shown that binding of mRNA to eIF-2 is primarily at an internal sequence, and secondarily through the cap. A model for the function of eIF-2 is presented that can account for all these properties. This model can provide a molecular basis for the differential translation of mRNA species, whether or not they are capped.

Eukaryotic Cells↗
PubMed1978

[Structure of the mRNA of eukaryotic cells].

A review is given of the principal achievements in studying the structure of mRNA in eukaryotic cells. The data are provided on the size and life time, complexity and distribution of different kinds of mRNA by the frequency of repetitions; composition and structure of mRNP. The structures of individual mRNA's and general pattern of the structure of eukaryotic mRNA and mRNP are considered.

Animals↗
PubMed1978

Preparation of projection-less particles from influenza virus and their messenger activities in prokaryotic and eukaryotic systems.

A fraction of projection-less particles was prepared from influenza A/Dunedin/4/73 and A/Victoria/3/75 (X-47) (H3N2) by detergent treatment and extraction into ether at 0 degrees C. The activity of this material in stimulating protein synthesis in vitro was studied and compared with that of isolated virion RNA using a) an RNA-dependent E. coli system, and b) a wheat germ system. In the bacterial system the purified RNA had the highest template activity, while in the eukaryotic system the disrupted particle preparation was by far the most active. Translation products were formed with immunological and electrophoretic properties similar to those of several influenza virion proteins. The experiments indicate that, when added in the form of disrupted projection-less particles, RNA from influenza A2 virus is utilized as a template by eukaryotic ribosomes.

Cell-Free System↗