Memories of dr. Josef Konforti

U okviru brojnih manifestacija i komemoracija kojima je u toku 1971. godine obeležena trideseta godišnjica ulaska Jugoslavije u Drugi svetski rat i narodnih ustanaka protiv fašističkih okupatora, javila se i želja bivših logoraša, Jevreja koji su preživeli užase jednog od najzloglasnijih logora – Jasenovca, da obelodane svoja sećanja na teške dane logorskog života; teške dane svoje, a poslednje dane mnogih članova njihovih porodica kao i brojnih žrtava drugih naroda: Srba, Hrvata i Roma. Savez jevrejskih opština Jugoslavije, u želji da udovolji nastojanjima preživelih logoraša iz Jasenovca, publikovao je njihova sećanja u knjizi "Sećanja Jevreja na logor Jasenovac". Jedno od tih svedočanstava predstavlja priča "Sjećanja dr Josefa Konfortija".Within the framework of numerous manifestations and commemorations marking the thirtieth anniversary of Yugoslavia's entry into the Second World War and the national uprisings against fascist invaders in 1971, the desire of former inmates, Jews who survived the horrors of one of the most notorious camps - Jasenovac, to be revealed their memories of the difficult days of camp life; their hard days, and the last days of many members of their families as well as numerous victims of other nations: Serbs, Croats, and Roma. The Association of Jewish Communities of Yugoslavia, wishing to satisfy the efforts of the surviving Jasenovac camp inmates, published their memoirs in the book "Sećanja Jevreja na logor Jasenovac“ (Memories of Jews at the Jasenovac Camp). One of these testimonies is the story of "Sjećanja dr. Josefa Konfortija"

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5′ splice site (5′ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5′ free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5′ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells

DNA End Resection Controls the Balance between Homologous and Illegitimate Recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3′ ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD’s RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3′ tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3′ ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis

Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5′ and 3′ Patches

BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism

Shaping and Structuring Supramolecular Gels

Supramolecular gels assemble via non-covalent interactions between low-molecular-weight gelators (LMWGs). The gels form a solid-like nanoscale network spanning a liquid-like continuous phase, translating molecular-scale information into materials performance. However, gels based on LMWGs are often difficult to manipulate, easily destroyed and have poor rheological performance. The recurring image of newly-discovered supramolecular gels is that of an inverted vial showing that the gel can support its own weight against gravity. Such images reflect the limitation that these gels simply fill the vessel in which they are made, with limited ability to be shaped. This property prevents supramolecular gels from having the same impact as polymer gels, despite greater synthetic tunability, reversibility and bio/environmental compatibility. In this Review, we evaluate strategies for imposing different shapes onto supramolecular gels and for patterning structures within them. We review fabrication methods including moulding, self-healing, 3D printing, photopatterning, diffusion and surface-mediated patterning. We discuss gelator chemistries amenable to each method, highlighting how a multi-component approach can aid shaping and structuring. Supramolecular gels with defined shapes, or patterned structures with precisely-controlled compositions, have the potential to intervene in applications such as tissue engineering and nanoscale electronics, as well as opening-up new technologies