Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent ampli®cation characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR iden-ti®es up to 80% of the differentially expressed genes identi®ed by direct labelling using 30-fold less input RNA for the ampli®cation, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identi®cation of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the ®delity of ampli®cation and show that the ampli®ed material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identi®cation of differentially expressed genes, but it is also faster and less labour intensive than other ampli®cation protocols.

Swarming is a form of collective bacterial motion enabled by flagella on the surface of semisolid media. Swarming populations exhibit non-genetic or adaptive resistance to antibiotics, despite sustaining considerable cell death. Here, we show that antibiotic-induced death of a sub-population benefits the swarm by enhancing adaptive resistance in the surviving cells. Killed cells release a resistance-enhancing factor that we identify as AcrA, a periplasmic component of RND efflux pumps. The released AcrA interacts on the surface of live cells with an outer membrane component of the efflux pump, TolC, stimulating drug efflux and inducing expression of other efflux pumps. This phenomenon, which we call 'necrosignaling', exists in other Gram-negative and Gram-positive bacteria and displays species-specificity. Given that adaptive resistance is a known incubator for evolving genetic resistance, our findings might be clinically relevant to the rise of multidrug resistance.

Transfer RNAs (tRNAs) have been important in shaping biomolecular evolution. Initiator tRNAs (tRNA i ), a special class of tRNAs, carry methionine (or its derivative, formyL-methionine) to ribosomes to start an enormously energy consuming but a highly regulated process of protein synthesis. The processes of tRNA i evolution, and selection of methionine as the universal initiating amino acid remain an enigmatic problem. We constructed phylogenetic trees using the whole sequence, the acceptor-TcC arm ('minihelix'), and the anticodon-dihydrouridine arm regions of tRNA i from 158 species belonging to all 3 domains of life. All the trees distinctly assembled into 3 domains of life. Large trees, generated using data for all the tRNAs of a vast number of species, fail to reveal the major evolutionary events and identity of the probable elongator tRNA sequences that could be ancestor of tRNA i . Therefore, we constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNA Pro , tRNA Glu , or tRNA Thr (but surprisingly not elongator tRNA Met ) as probable ancestors of tRNA i . We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the 3 domains of life. Our correlation analysis shows that its high metabolic cost is independent of many physicochemical properties of the side chain. Our results indicate that selection of methionine, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation.

Two coupled nonlinear evolution equations correct to fourth order in wave steepness are derived for a three-dimensional wave packet in the presence of a thin thermocline. These two coupled equations are reduced to a single equation on the assumption that the space variation of the amplitudes takes place along a line making an arbitrary fixed angle with the direction of propagation of the wave. This single equation is used to study the stability of a uniform wave train. Expressions for maximum growth rate of instability and wave number at marginal stability are obtained. Some of the results are shown graphically. It is found that a thin thermocline has a stabilizing influence and the maximum growth rate of instability decreases with the increase of thermocline depth.

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