Once in a lifetime: strategies for preventing re-replication in prokaryotic and eukaryotic cells

DNA replication is an extremely accurate process and cells have evolved intricate control mechanisms to ensure that each region of their genome is replicated only once during S phase. Here, we compare what is known about the processes that prevent re-replication in prokaryotic and eukaryotic cells by using the model organisms Escherichia coli and Schizosaccharomyces pombe as examples. Although the underlying molecular details are different, the logic behind the control mechanisms is similar. For example, after initiation, crucial molecules required for the loading of replicative helicases in both prokaryotes and eukaryotes are inactivated until the next cell cycle. Furthermore, in both systems the beta-clamp of the replicative polymerase associates with enzymatic activities that contribute to the inactivation of the helicase loaders. Finally, recent studies suggest that the control mechanism that prevents re-replication in both systems also increases the synthesis of DNA building blocks.     

Membrane protein insertion: mixing eukaryotic and prokaryotic concepts

Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.     

The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies

The N-end rule states that the half-life of a protein is determined by the nature of its N-terminal residue. This fundamental principle of regulated proteolysis is conserved from bacteria to mammals. Although prokaryotes and eukaryotes employ distinct proteolytic machineries for degradation of N-end rule substrates, recent findings indicate that they share common principles of substrate recognition. In eukaryotes substrate recognition is mediated by N-recognins, a class of E3 ligases that labels N-end rule substrates via covalent linkage to ubiquitin, allowing the subsequent substrate delivery to the 26S proteasome. In bacteria, the adaptor protein ClpS exhibits homology to the substrate binding site of N-recognin. ClpS binds to the destabilizing N-termini of N-end rule substrates and directly transfers them to the ClpAP protease.     

Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems

Asparagine-linked protein glycosylation is a prevalent protein modification reaction in eukaryotic systems. This process involves the co-translational transfer of a pre-assembled tetradecasaccharide from a dolichyl-pyrophosphate donor to the asparagine side chain of nascent proteins at the endoplasmic reticulum (ER) membrane. Recently, the first such system of N-linked glycosylation was discovered in the Gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate donor to the asparagine side chain of proteins at the bacterial periplasmic membrane. Here we provide a detailed comparison of the machinery involved in the N-linked glycosylation systems of eukaryotic organisms, exemplified by the yeast Saccharomyces cerevisiae, with that of the bacterial system in C. jejuni. The two systems display significant similarities and the relative simplicity of the bacterial glycosylation process could provide a model system that can be used to decipher the complex eukaryotic glycosylation machinery.     

Pathways of arsenic uptake in prokaryotic and eukaryotic cells

Mechanisms of arsenic uptake and detoxification are present in all studied organisms. These mechanisms are considerably well described in unicellular organisms such as bacterium Escherichia coli and baker's yeast Saccharomyces cerevisiae, still leaving much to be revealed in multicellular organisms. Full identification of arsenic uptake and detoxification is of great importance. This knowledge can be very helpful in improving effectiveness of arsenic-containing drugs used in chemotherapy of parasitoses as well as in treatment of acute promielyocytic leukemia. Increased proficiency of bioremediation of arsenic-contaminated soils can be obtained by using plants hyperaccumulating arsenic. This kind of plants can be engineered by modulating expression levels of genes encoding arsenic transporters. The same technique may be used to decrease levels of accumulated arsenic in crops. The aim of this paper is to review current knowledge about systems of arsenic uptake in every studied organism--from bacteria to human.     

Bifunctional reporter genes: construction and expression in prokaryotic and eukaryotic cells

Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli beta-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65 degrees C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C of N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.     

Effects of benzyladenine on prokaryotic and eukaryotic cells.

Comparative studies of the effect of benzyladenine (BA) on the yeast Saccharomyces cerevisiae, the bacterium Salmonella typhimurium, the shallot Allium ascalonicum and Chinese hamster fibroblast cells were performed. The tested substance had no mutagenic activity on yeast, bacteria and cultured fibroblast cells. Changes in mitotic activity and cell division abnormalities were observed after BA treatment in shallot root-tip cells.     

Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant. The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms. There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism. Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions. This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell. In addition, the molecular basis of drug transport by these proteins will be discussed.     

Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant. The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms. There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism. Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions. This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell. In addition, the molecular basis of drug transport by these proteins will be discussed.     

Non-mutagenic and mutagenic post-replicative DNA repair in prokaryotic and eukaryotic cells

The review is devoted to mechanisms of repair gaps in DNA daughter strand, formed during the stall of moving replication forks and restart of replication in cells after the action of DNA damaging agents (predominantly--UV light). The repair of daughter DNA, or postreplication DNA repair (PRR), is realized by error-free (non-mutagenic) and error-prone (mutagenic) pathways. The former is a recombination repair, or recombination between two sister duplexes. By this way the major part of postreplication gaps is eliminated. The second way is related with the induction of SOS-response. In Escherichia coli cells mutagenic SOS-response is realized by proteins RecA, UmuD, UmuC, DNA-polymerase III holoenzyme and others. In E. coli some mutagenic enzymes--DNA-polymerase IV (the product of dinB gene) and DNA-polymerase V (the product of umuDC genes) have been recently discovered. In Saccharomyces cerevisiae cells postreplicative translesion synthesis is realized by newly discovered enzymes deoxycytidilmonophosphatetransferase (encoded by REV1 gene), DNA-polymerase zeta (encoded by REV3 gene), DNA-polymerase eta (encoded by RAD30 gene). All the three enzymes share a great homology with UmuC enzyme of E. coli. DNA polymerase eta correctly inserts adenine residues in the daughter strand opposite noncoded thymine residues in cyclobutane pyrimidine dimer. Based on RAD6 gene of S. cerevisiae, human cells hREV1, hREV3 and hRAD30A have been obtained to encode, respectively, deoxycytidiltransferase, DNA-polymerase zeta and DNA-polymerase eta. It has been shown that the defect of PRR DNA in xeroderma pigmentosum variant is associated with DNA-polymerase eta deficiency. This defect is corrected by the extract of intact HeLa cells. The importance of newly discovered enzymes in the system of mechanisms of DNA repair and replication is discussed.     

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

The origin of eukaryotes: the difference between prokaryotic and eukaryotic cells.

Eukaryotes have long been thought to have arisen by evolving a nucleus, endomembrane, and cytoskeleton. In contrast, it was recently proposed that the first complex cells, which were actually proto-eukaryotes, arose simultaneously with the acquisition of mitochondria. This so-called symbiotic association hypothesis states that eukaryotes emerged when some ancient anaerobic archaebacteria (hosts) engulfed respiring alpha-proteobacteria (symbionts), which evolved into the first energy-producing organelles. Therefore, the intracellular compartmentalization of the energy-converting metabolism that was bound originally to the plasma membrane appears to be the key innovation towards eukaryotic genome and cellular organization. The novel energy metabolism made it possible for the nucleotide synthetic apparatus of cells to be no longer limited by subsaturation with substrates and catalytic components. As a consequence, a considerable increase has occurred in the size and complexity of eukaryotic genomes, providing the genetic basis for most of the further evolutionary changes in cellular complexity. On the other hand, the active uptake of exogenous DNA, which is general in bacteria, was no longer essential in the genome organization of eukaryotes. The mitochondrion-driven scenario for the first eukaryotes explains the chimera-like composition of eukaryotic genomes as well as the metabolic and cellular organization of eukaryotes.     

Evolution of prokaryotic homologues of the eukaryotic SEFIR protein domain

SEF/IL17 receptor (SEFIR) domains are mainly found in IL17 receptors (IL17Rs) and their adaptor proteins CIKS (connection to IKK and SAPK/JNK), which exert a host defense role in numbers of infectious diseases and promote inflammatory pathology in autoimmunity. Exploring the evolutionary pathway of SEFIR domains will provide further insight into their functions. Here, we have identified 84 SEFIR domain-containing proteins from more than 1400 prokaryotic genomes. As most SEFIR domain-containing bacterial genomes possess a single SEFIR encoding gene and the SEFIR protein domain forms homodimeric complexes like the Toll/IL1 receptor (TIR) domain, the single bacterial SEFIR proteins may receive binding partners from other organisms. Through comparative and phylogenetic sequence analyses, we show that bacterial SEFIR domain is more similar to that of vertebrate CIKS than IL17R, and it possibly emerges via a lateral gene transfer (LGT) from animals. In addition, our secondary and three-dimensional structural predictions of SEFIR domains reveal that human and pathogenic bacterial SEFIR domains share similar structural and electrostatic features. Our findings provide important clues for further experimental researches on determining the functions of SEFIR proteins in pathogenic prokaryotes.     

A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.     

牙鲆钙调素基因在原核和真核细胞中的表达

设计特异引物,以SMART cDNA为模板,应用PCR方法扩增牙鲆钙调素基因（Paralichthys olivocew calmodulin,PoCaM）。计算机辅助分析表明,PoCaM基因编码149个氨基酸的推定蛋白,其分子量为17kD,等电点为3．93,含有4个螺旋-环-螺旋样结构,与其它鱼类CaM氨基酸一致性为97．3％-100％。构建原核表达重组质粒pET32a／PoCaM,转化大肠杆菌B121（DE3）,用IPTG进行诱导表达,经SDS—PAGE蛋白电泳,结果显示PoCaM在大肠杆菌中进行了特异性融合表达,融合蛋白分子量约为34kD,与预期分子量大小一致。同时,以绿色荧光蛋白（GFP）为表达标签,构建真核表达重组质粒pEGFP—N3／PoCaM,经Lipofectamine 2000介导转染鲤鱼上皮瘤细胞（ Epithelioma papulosum cyprinid, EPC）,荧光显微镜观察显示,PoCaM在EPC细胞中进行瞬时表达,主要分布于细胞核及胞浆中[动物学报54（6）：1061—1067,2008]。