Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12

Summary The trmD gene, which governs the formation of 1-methyl-guanosine (m¹G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA ⁺ hybrid plasmid was isolated, which carried the trmD ⁺ gene and was shown to over-produce the tRNA (m¹G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD ⁺ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA ⁺ gene. The mutation trmD1, which renders the tRNA (m¹G) methyltransferase temperaturesensitive both in vivo and in vitro could be complemented by trmD ⁺ plasmids. These results suggest that the gene trmD ⁺ is the structural gene for the tRNA (m¹G)methyltransferase (EC 2.1.1.3.1).     

Characterization and partial purification of <Emphasis Type="Italic">Candida albicans</Emphasis> Secretory IL-12 Inhibitory Factor

Background  

We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.     

<Superscript>15</Superscript>N natural abundance of fossil peat reflects the influence of animal-derived nitrogen on vegetation

'¹⁵N signatures of fossil peat were used to interpret past ecosystem processes on tectonically active subantarctic Macquarie Island. By comparing past vegetation reconstructed from the fossil record with present-day vegetation analogues, our evidence strongly suggests that changes in the '¹⁵N signatures of fossil peat at this location reflect mainly past changes in the proportion of plant nitrogen derived from animal sources. Associated with uplift above sea level over the past 8,500 years, fossil records in two peat deposits on the island chronicle a change from coastal vegetation with fur and elephant seal disturbance to the existing inland herbfield. Coupled with this change are synchronous changes in the '¹⁵N signatures of peat layers. At two sites ¹⁵N-enriched peat '¹⁵N signatures of up to +17‰ were associated with a high abundance of pollen of the nitrophile Callitriche antarctica (Callitrichaceae). At one site fossil seal hair was also associated with enriched peat '¹⁵N. Less ¹⁵N enriched '¹⁵N signatures (e.g. -1.9‰ to +3.9‰) were measured in peat layers which lacked animal associated C. antarctica and Acaena spp. Interpretation of a third peat profile indicates continual occupation of a ridge site by burrowing petrels for most of the Holocene. We suggest that ¹⁵N signatures of fossil peat remained relatively stable with time once deposited, providing a significant new tool for interpreting the palaeoecology.     

The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the alpha-subunit and nine in the beta-subunit), with the C terminus of the alpha-subunit and the N terminus of the beta-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.     

Simulation methods with extended stability for stiff biochemical Kinetics

Background  

With increasing computer power, simulating the dynamics of complex systems in chemistry and biology is becoming increasingly routine. The modelling of individual reactions in (bio)chemical systems involves a large number of random events that can be simulated by the stochastic simulation algorithm (SSA). The key quantity is the step size, or waiting time, τ, whose value inversely depends on the size of the propensities of the different channel reactions and which needs to be re-evaluated after every firing event. Such a discrete event simulation may be extremely expensive, in particular for stiff systems where τ can be very short due to the fast kinetics of some of the channel reactions. Several alternative methods have been put forward to increase the integration step size. The so-called τ-leap approach takes a larger step size by allowing all the reactions to fire, from a Poisson or Binomial distribution, within that step. Although the expected value for the different species in the reactive system is maintained with respect to more precise methods, the variance at steady state can suffer from large errors as τ grows.     

Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.     

Predicted impact of exotic vines on an endangered ecological community under future climate change

Potential interactions between climate change and exotic plant invasions may affect areas of high conservation value, such as land set aside for the protection of endangered species or ecological communities. We investigated this issue in eastern Australia using species distribution models for five exotic vines under climate regimes for 2020 and 2050. We examined how projected changes in the distribution of climatically suitable habitat may coincide with the remaining remnants of an endangered ecological community—littoral rainforests—in this region. The number of known infestations of each weed in tropical, subtropical and temperate areas was used to assess the likelihood of further expansion into areas projected to provide suitable habitat under future conditions. Littoral rainforest reserves were consistently predicted to provide bioclimatically suitable habitat for the five vines examined under both current and future climate scenarios. We explore the consequences and potential strategies for managing exotic plant invasions in these protected areas in the coming decades.