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Feasibility of Radioimmunotherapy of Experimental Pneumococcal Infection. E. Dadachova, 2004. Assessment of Production Conditions for Efficient Use of Escherichia coli in High-Yield Heterologous Recombinant Selenoprotein Synthesis. Olle Rengby, 2004.The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon . SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA . Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed . Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes . Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies . Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase . About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture . These findings clearly illustrate the ability of E . coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase . This study also demonstrates that E . coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase . SoxRS Down-Regulation of rob Transcription. Carmen Michán, 2002.Rob is regarded as a constitutively expressed protein, although little is known about how rob gene is regulated . We show here by reverse transcription-PCR that the transcriptional levels of rob are strongly down-regulated in response to the superoxide-generating agent paraquat (PQ) . Repression reached a maximum of 20-fold after 10 min exposure at 10 µM PQ . The magnitude of rob repression was comparable to that of induction quantified for the most sensitive SoxS targets . ß-Galactosidase expression with the rob2::lacZ transcriptional fusion indicates that down-regulation of rob expression takes place, at least in part, at the level of transcription initiation . Moreover, ca . 50% of the rob mRNA was degraded in <1 min after the addition of rifampin to inhibit transcription . This intrinsic short half-life, which is of obvious benefit for a rapid down-regulation after transcription ceases, was unaffected by the addition of PQ . No repression was observed in a soxR-null strain, indicating that the rob transcript level might be negatively modulated by the intracellular amounts of SoxS protein . Gel retardation assays support the idea that in vivo SoxS would block rob transcription directly . cis Elements Necessary for Developmental Expression of a Myxococcus xanthus Gene That Depends on C Signaling. Poorna Viswanathan, 2003.Cell contact-mediated C signaling coordinates morphogenesis and gene expression during development of Myxococcus xanthus . One promoter that depends on C signaling for transcription lies upstream of  4403, the site of a Tn5 lac insertion in the genome . The
4403 promoter has a C-box sequence centered at -49 bp that matches the consensus 5'-CAYYCCY-3', which is found in several C-signal-dependent promoters . Mutational analysis of the
4403 promoter region was performed to test the importance of the C box and to identify other cis-acting elements . A 6-bp change in the -10 region eliminated promoter activity, but a 6-bp change in the -35 region decreased activity only about twofold . Certain single-base-pair changes in the C box centered at -49 bp abolished promoter activity, establishing the importance of this sequence element . Single-base-pair changes in a C-box-like sequence centered at -77 bp also abolished promoter activity, but the pattern of mutational effects was different from that for the C box centered at -49 bp . Additional single-base-pair changes indicated that all 10 bp from -79 to -70 bp are important for
4403 promoter activity . Mutations at -59, -61, -62, and -63 bp also abolished promoter activity, defining a 5-bp element from -63 to -59 bp . This 5-bp element is separated from the 10-bp element (i.e., -79 to -70 bp) by 6 bp that can be changed without loss of promoter activity . Likewise, the 5 bp between the 5-bp element and the C box can be changed without loss of activity, but deletion of these 5 bp abolished activity, indicating that spacing is important . Sequences similar to the 5- and 10-bp elements, as well as the C box, are present in other C-signal-dependent promoters, suggesting some similarity in the regulatory mechanisms, but there are also indications that these cis elements do not function identically in the different promoters .
High Rate of Uptake of Organic Nitrogen Compounds by Prochlorococcus Cyanobacteria as a Key to Their Dominance in Oligotrophic Oceanic Waters. Mikhail V. Zubkov, 2003.Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported . About 33% of the total bacterioplankton turnover of amino acids, determined with a representative [35S]methionine precursor and flow sorting, can be assigned to Prochlorococcus spp . and 3% can be assigned to Synechococcus spp . in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively . This finding may provide a mechanism for Prochlorococcus' competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop .
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