Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.target location | establishment of lysogeny | viral transduction T he search for specific sequences along genomic DNA plays a key role in the location of specific sites by transcription factors (1), the repair of DNA lesions (2), and horizontal gene transfer (3). Common to these processes is a search through a very large number of possible sequences because of the long genomes involved. A fundamental question is how specific target sequences can be located with high efficiency, within physiologically relevant times. This question is crucial to understand viral transduction, one of the fundamental mechanisms of horizontal gene transfer driving the evolution of prokaryotes (3, 4). In transduction, a viral genome integrates at a unique site on a bacterial genome following infection, conferring new traits such as pathogenicity (5). A classic example of transduction is furnished by the infection of Escherichia coli cells by bacteriophage λ.Infection of an E. coli host by the temperate bacteriophage λ begins with the binding of the phage to the E. coli maltose pore LamB (6, 7). The phage then injects its DNA into the cell, a process that lasts about 5 min (8). Infection can lead to two possible outcomes, lysis or lysogeny, which reflect alternative pathways of gene expression (9-11). In the lytic pathway, execution of a viral gene expression cascade leads to the replication of the viral DNA, resulting in cell death and lysis to release about 100 phage progeny (12). Alternatively, by establishing lysogeny, the phage shuts off the lytic cycle and locates with high efficiency (13) a unique sequence along the cellular genome where it integrates i...

Abstract:The Technion autonomous underwater vehicle (TAUV) is an ongoing project aiming to develop and produce a small AUV to carry on research missions, including payload dropping, and to demonstrate acoustic communication. Its navigation system is based on an inertial navigation system (INS) aided by a Doppler velocity log (DVL), magnetometer, and pressure sensor (PS). In many INSs, such as the one used in TAUV, only the velocity vector (provided by the DVL) can be used for aiding the INS, i.e., enabling only a loosely coupled integration approach. In cases of partial DVL measurements, such as failure to maintain bottom lock, the DVL cannot estimate the vehicle velocity. Thus, in partial DVL situations no velocity data can be integrated into the TAUV INS, and as a result its navigation solution will drift in time. To circumvent that problem, we propose a DVL-based vehicle velocity solution using the measured partial raw data of the DVL and additional information, thereby deriving an extended loosely coupled (ELC) approach. The implementation of the ELC approach requires only software modification. In addition, we present the TAUV six degrees of freedom (6DOF) simulation that includes all functional subsystems. Using this simulation, the proposed approach is evaluated and the benefit of using it is shown.

Single nucleotide polymorphisms (SNPs) are amenable to automation and therefore have become the marker of choice for DNA profiling. SNaPshot, a primer extension-based method, was used to multiplex 25 SNPs that have been previously validated as useful for identity control. Detection of extended products was based on four different fluorochromes and extension primers with oligonucleotide tails of differing lengths, thus controlling the concise length of the entire chromatogram to 81 bases. Allele frequencies for Holstein, Simmental, Limousin, Angus, Charolais and Tux Cattle were estimated and significant positive Pearson-correlation coefficients were obtained among the analysed breeds. The probability that two randomly unrelated individuals would share identical genotypes for all 25 loci varied from 10(-8) to 10(-10) for these breeds. For parentage control, the exclusion power was found to be 99.9% when the genotypes of both putative parents are known. A traceability test of duplicated samples indicated a high genotyping precision of >0.998. This was further corroborated by analysis of 60 cases of parent-sib pairs and trio families. The 25-plex SNaPshot assay is adapted for low- and high-throughput capacity and thus presents an alternative for DNA-based traceability in the major commercial cattle breeds.