MethDB—a public database for DNA methylation data

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Phosphatidylcholine Synthesis Influences the Diacylglycerol Homeostasis Required for Sec14p-dependent Golgi Function and Cell Growth

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20–40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

Distinct Cyclin D Genes Show Mitotic Accumulation or Constant Levels of Transcripts in Tobacco Bright Yellow-2 Cells1

The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle. In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls. Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown. We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1). To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle. In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells. This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins. In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins. This could suggest a novel role for plant D-type cyclins during mitosis.

Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Horizontal transfer of oncogenes by uptake of apoptotic bodies

Tumor formation involves the accumulation of a series of genetic alterations that are required for malignant growth. In most malignancies, genetic changes can be observed at the chromosomal level as losses or gains of whole or large portions of chromosomes. Here we provide evidence that tumor DNA may be horizontally transferred by the uptake of apoptotic bodies. Phagocytosis of apoptotic bodies derived from H-rasV12- and human c-myc-transfected rat fibroblasts resulted in loss of contact inhibition in vitro and a tumorigenic phenotype in vivo. Fluorescence in situ hybridization analysis revealed the presence of rat chromosomes or of rat and mouse fusion chromosomes in the nuclei of the recipient murine cells. The transferred DNA was propagated, provided that the transferred DNA conferred a selective advantage to the cell and that the phagocytotic host cell was p53-negative. These results suggest that lateral transfer of DNA between eukaryotic cells may result in aneuploidy and the accumulation of genetic changes that are necessary for tumor formation.

Molecular and cell biology aspects of plague

A 70-kb virulence plasmid (sometimes called pYV) enables Yersinia spp. to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, a system consisting of secreted proteins called Yops and their dedicated type III secretion apparatus called Ysc. The Ysc apparatus forms a channel composed of 29 proteins. Of these, 10 have counterparts in almost every type III system. Secretion of some Yops requires the assistance, in the bacterial cytosol, of small individual chaperones called the Syc proteins. These chaperones act as bodyguards or secretion pilots for their partner Yop. Yop proteins fall into two categories. Some are intracellular effectors, whereas the others are “translocators” needed to deliver the effectors across the eukaryotic plasma membrane, into eukaryotic cells. The translocators (YopB, YopD, LcrV) form a pore of 16–23 Å in the eukaryotic cell plasma membrane. The effector Yops are YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT. YopH is a powerful phosphotyrosine phosphatase playing an antiphagocytic role by dephosphorylating several focal adhesion proteins. YopE and YopT contribute to antiphagocytic effects by inactivating GTPases controlling cytoskeleton dynamics. YopP/YopJ plays an anti-inflammatory role by preventing the activation of the transcription factor NF-κB. It also induces rapid apoptosis of macrophages. Less is known about the role of the phosphoserine kinase YopO/YpkA and YopM.

Isolation and Characterization of Effector-Loop Mutants of CDC42 in Yeast

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of “effector-loop” mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

Molecular kinesis in cellular function and plasticity

Intracellular transport and localization of cellular components are essential for the functional organization and plasticity of eukaryotic cells. Although the elucidation of protein transport mechanisms has made impressive progress in recent years, intracellular transport of RNA remains less well understood. The National Academy of Sciences Colloquium on Molecular Kinesis in Cellular Function and Plasticity therefore was devised as an interdisciplinary platform for participants to discuss intracellular molecular transport from a variety of different perspectives. Topics covered at the meeting included RNA metabolism and transport, mechanisms of protein synthesis and localization, the formation of complex interactive protein ensembles, and the relevance of such mechanisms for activity-dependent regulation and synaptic plasticity in neurons. It was the overall objective of the colloquium to generate momentum and cohesion for the emerging research field of molecular kinesis.

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Kinetics and mechanism of DNA uptake into the cell nucleus

Gene transfer to eukaryotic cells requires the uptake of exogenous DNA into the cell nucleus. Except during mitosis, molecular access to the nuclear interior is limited to passage through the nuclear pores. Here we demonstrate the nuclear uptake of extended linear DNA molecules by a combination of fluorescence microscopy and single-molecule manipulation techniques, using the latter to follow uptake kinetics of individual molecules in real time. The assays were carried out on nuclei reconstituted in vitro from extracts of Xenopus eggs, which provide both a complete complement of biochemical factors involved in nuclear protein import, and unobstructed access to the nuclear pores. We find that uptake of DNA is independent of ATP or GTP hydrolysis, but is blocked by wheat germ agglutinin. The kinetics are much slower than would be expected from hydrodynamic considerations. A fit of the data to a simple model suggests femto-Newton forces and a large friction relevant to the uptake process.

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase1

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V0 sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated V0 subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans EmbryosV⃞

γ-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that γ-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans γ-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::γ-tubulin or GFP::β-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of γ-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of γ-tubulin. γ-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that γ-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.

The architecture of the human Rad54–DNA complex provides evidence for protein translocation along DNA

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54–DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins.

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.