Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.     

RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III)

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.     

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.     

Effects of Water and Nutrient Availability on Water Relations, Gas Exchange and Growth Rate of Mature Plants and Resprouts of Arbutus unedo L.

This study examines the effects of water supply and nutritionon the water status, gas exchange and growth of mature plantsand resprouts of Arbutus unedo, a Mediterranean evergreen shrubadapted to drought and poor nutrition. Mature plants of A. unedorespond to irrigation with increased leaf water potential duringsummer drought, but they show a very conservative use of waterand they do not increase leaf conductance. There is also a verysmall increase in net photosynthesis and growth, which doesnot significantly increase productivity. Resprouts of A. unedo increase water potential, leaf conductance,transpiration rate, net photosynthesis and growth rate in responseto watering, showing a less conservative use of water than matureplants. Increased growth rates, both in mature plants and resprouts,are likely to be due to the higher cell turgor caused by improvedleaf water potential, rather than to increased photosynthesis. The only effect of nutrient addition on mature plants is anincrease in leaf nutrient content, and other aspects of thephysiology and growth of resprouts were unaffected. We thereforeconclude that water is a more limiting factor than nutrientsfor mature plants and resprouts of A. unedo growing in the studyarea. These results support previous data which indicate thathigher growth rates in resprouts than in mature plants of A.unedo are mainly the result of a higher water availability.Copyright1994, 1999 Academic Press Arbutus unedo L., strawberry tree, resprouts, water stress, nutrient availability, water relations, gas exchange, growth rate, regeneration     

Phylogeographical analyses of domestic and wild yaks based on mitochondrial DNA: new data and reappraisal

Aim We aimed to examine the phylogeographical structure and demographic history of domestic and wild yaks (Bos grunniens) based on a wide range of samples and complete mitochondrial genomic sequences. Location The Qinghai‐Tibetan Plateau (QTP) of western China. Methods All available D‐loop sequences for 405 domesticated yaks and 47 wild yaks were examined, including new sequences from 96 domestic and 34 wild yaks. We further sequenced the complete mitochondrial genomes of 48 domesticated and 21 wild yaks. Phylogeographical analyses were performed using the mitochondrial D‐loop and the total genome datasets. Results We recovered a total of 123 haplotypes based on the D‐loop sequences in wild and domestic yaks. Phylogenetic analyses of this dataset and the mitochondrial genome data suggested three well‐supported and divergent lineages. Two lineages with six D‐loop haplogroups were recovered for all morphological breeds of domestic yaks across their distributions in the QTP, while one more lineage and more endemic haplogroups or haplotypes were found for wild yaks. Based on the mitochondrial genome data, the divergences of the three lineages were estimated to have occurred around 420,000 and 580,000 years ago, consistent with the geological records of two large glaciation events experienced in the QTP. Main conclusions There are distinct phylogeographical differences between wild and domestic yaks. However, there is no apparent geographical correlation between identified haplogroups and distributions of domestic yaks. Three differentiated lineages of yaks probably evolved allopatrically in different regions during the Pleistocene glaciation events, then reunited into a single gene pool during post‐glacial population expansion and migrations before the start of the domestication of yaks in the Holocene.     

Availability of glucose and light modulates the structure and function of a microbial biofilm

We have studied the differences in the organic matter processing and biofilm composition and structure between autoheterotrophic and heterotrophic biofilm communities. Microbial communities grown on artificial biofilms were monitored, following incubation under light and dark conditions and with or without the addition of glucose as a labile organic compound. Glucose addition greatly affected the microbial biofilm composition as shown by differences in 16S rRNA gene fingerprints. A significant increase in β-glucosidase and peptidase enzyme activities were also observed in glucose-amended biofilms incubated in the dark, suggesting an active bacterial community. Light enhanced the algal and bacterial growth, as well as higher extracellular enzyme activity, thereby indicating a tight algal–bacterial coupling in biofilms incubated under illumination. In these biofilms, organic compounds excreted by photosynthetic microorganisms were readily available for bacterial heterotrophs. This algal–bacterial relationship weakened in glucose-amended biofilms grown in the light, probably because heterotrophic bacteria preferentially use external labile compounds. These results suggest that the availability of labile organic matter in the flowing water and the presence of light may alter the biofilm composition and function, therefore affecting the processing capacity of organic matter in the stream ecosystem.     

Analysis of galanthamine-type alkaloids by capillary gas chromatography-mass spectrometry in plants

Galanthamine, an acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease, and galanthamine-type alkaloids are synthesised in different plants of the family Amaryllidaceae. A capillary gas chromatographic-mass spectroscopic (CGC-MS) method for the separation of 7 galanthamine type alkaloids, including galanthamine and epigalanthamine, is described in the present paper. A simple method for the routine quantification of galanthamine in plants was developed using pre-packed columns with diatomaceous earth (Isolute HM-N), allowing simultaneous preparation of a large number of samples. Galanthamine showed excellent linearity in the range from 50 to 1000 microg/mL and the limit of quantification was 5 microg/mL in total ion current mode and 1.6 ng/mL in selected ion monitoring mode. The recovery of galanthamine was more than 90%. Interday reproducibility (RSD) of the extraction was 2.74%. A method to find and to microextract Amaryllidaceae alkaloids in low-mass plant samples is also described.     

Genetic analyses reveal independent domestication origins of Eurasian reindeer

Although there is little doubt that the domestication of mammals was instrumental for the modernization of human societies, even basic features of the path towards domestication remain largely unresolved for many species. Reindeer are considered to be in the early phase of domestication with wild and domestic herds still coexisting widely across Eurasia. This provides a unique model system for understanding how the early domestication process may have taken place. We analysed mitochondrial sequences and nuclear microsatellites in domestic and wild herds throughout Eurasia to address the origin of reindeer herding and domestication history. Our data demonstrate independent origins of domestic reindeer in Russia and Fennoscandia. This implies that the Saami people of Fennoscandia domesticated their own reindeer independently of the indigenous cultures in western Russia. We also found that augmentation of local reindeer herds by crossing with wild animals has been common. However, some wild reindeer populations have not contributed to the domestic gene pool, suggesting variation in domestication potential among populations. These differences may explain why geographically isolated indigenous groups have been able to make the technological shift from mobile hunting to large-scale reindeer pastoralism independently.