The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination

The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively. We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis. While mutations at A2451, U2585, and C2063 (E. coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation. This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.     

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.     

AGenDA: homology-based gene prediction

We present a www server for homology-based gene prediction. The user enters a pair of evolutionary related genomic sequences, for example from human and mouse. Our software system uses CHAOS and DIALIGN to calculate an alignment of the input sequences and then searches for conserved splicing signals and start/stop codons around regions of local sequence similarity. This way, candidate exons are identified that are used, in turn, to calculate optimal gene models. The server returns the constructed gene model by email, together with a graphical representation of the underlying genomic alignment.     

Client-server environment for high-performance gene expression data analysis

SUMMARY: We have developed a platform independent, flexible and scalable Java environment for high-performance large-scale gene expression data analysis, which integrates various computational intensive hierarchical and non-hierarchical clustering algorithms. The environment includes a powerful client for data preparation and results visualization, an application server for computation and an additional administration tool. The package is available free of charge for academic and non-profit institutions.     

Making the body plan: precision in the genetic hierarchy of Drosophila embryo segmentation

We quantify fluctuations in protein expression for three of the segmentation genes in the fruit fly, Drosophila melanogaster. These proteins are representative members of the first three levels of a signalling hierarchy which determines the segmented body plan: maternal (Bicoid protein); gap (Hunchback protein); and pair-rule (Even-skipped protein). We quantify both inter-embryo and inter-nucleus (within a single embryo) variability in expression, especially with respect to positional specification by concentration gradient reading. Errors are quantified both early and late in cleavage cycle 14, during which the protein patterns develop, to study the dynamics of error transmission. We find that Bicoid displays very large positional errors, while expression of the downstream genes, Hunchback and Even-skipped, displays far more precise positioning. This is evidence that the pattern formation of the downstream proteins is at least partially independent of maternal signal, i. e. evidence against simple concentration gradient reading. We also find that fractional errors in concentration increase during cleavage cycle 14.     

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells

Antigen-mediated activation of mast cells results in Ca²⁺-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca²⁺ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response .     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.