Evolution of gene families: the multidrug resistance transporter genes in five related yeast species

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.     

microRNAs – kleine Moleküle ganz groß

microRNAs, regulators of complex phenotypes microRNAs (miRNAs) are small, non‐coding RNAs that regulate a number of biological processes, including development. Due to their mode of action some miRNAs are also causally involved in diseases like cancer. miRNAs bind base‐complementary mRNAs and lead to either degradation of the bound mRNAs or translational repression. Both result in decreased protein levels of the particular target. miRNA selectivity is governed via a very short, just six to seven nucleotides long, ”seed" sequence which is likely to exist in many mRNAs. miRNAs are, therefore, believed to target a larger number of mRNAs, each, leading to the concerted regulation of functionally connected proteins and to thus substantially contribute to phenotypes. We have performed several screenings which suggest that, indeed, many miRNAs regulate a larger number of proteins. However, we also showed that the proteins we tested were regulated by a larger number of miRNAs each. The complexity of miRNA regulation opens new avenues towards reaching a molecular understanding of disease phenotypes via the integrated consideration of coordinated regulations.     

Untersuchungen zur Tiergesundheit,Leistung und zum Rückstandsverhalten beim Schwein bei gleichzeitiger Aufnahme der Mykotoxine Ochratoxin A,Deoxynivalenol, Zearalenon über das Futter im 90-Tage-Test

The effect of the administration of the mycotoxins OTA, ZEA and DON alone resp. in combination on animal health and the residue behavior of pigs from 50 – 60 kg living weight over 90 days was investigated in 4 separate studies. Due to its fast metabolisation the administration of 1000 µg DON resp. 250 µg ZEA per kg feed alone or in combination with other mycotoxins does not lead to detectable residues of these mycotoxins in organs and tissues. Therefore these mycotoxins should not be relevant to the consumer.There is an effect of the simultaneous administration of ZEA resp. DON on the metabolisation resp. secretion of OTA. OTA is of relevance from the point of view of residue toxicology.     

Coordinating migratory neuron polarization by numb-ing communication

An interplay between intrinsic polarity and extracellular cues guides neuronal migration during cerebellar development. In this issue of Developmental Cell, Zhou et al. (2011) demonstrate that Numb is the focal point in mediating the chemotactic response of migrating cerebellar granule cells to BDNF through its regulation of cell polarity.     

The unique hexokinase of Kluyveromyces lactis. Molecular and functional characterization and evaluation of a role in glucose signaling

The Crabtree-negative yeast Kluyveromyces lactis is capable of adjusting its glycolytic flux to the requirements of respiration by tightly regulating glucose uptake. RAG5 encoding the only glucose and fructose phosphorylating enzyme present in K. lactis is required for the up-regulation of glucose transport and also for glucose repression. To understand the significance of the molecular identity and specific function(s) of the corresponding kinase to glucose signaling, RAG5 was overexpressed and its gene product KlHxk1 (Rag5p) isolated and characterized. Stopped-flow kinetics and sedimentation analysis indicated a monomer-homodimer equilibrium of KlHxk1 in a condition of catalysis, i.e. in the presence of substrates and products. The kinetic constants of ATP-dependent glucose phosphorylation identified a 53-kDa monomer as the high affinity/high activity form of the novel enzyme for both glycolytic substrates suggesting a control of glucose phosphorylation at the level of dimer formation and dissociation. In contrast to the highly homologous hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2), KlHxk1 was not inhibited by free ATP in a physiological range of nucleotide concentration. Mass spectrometric sequencing of tryptic peptides of KlHxk1 identified unmodified serine at amino acid position 156. The corresponding amino acid in ScHxk2 is serine 157, which represents the autophosphorylation-inactivation site. KlHxk1 did not display, however, the typical pattern of inactivation under the respective in vitro conditions and maintained a high residual glucose phosphorylating activity. The biophysical and functional data are discussed with respect to a possible regulatory role of KlHxk1 in glucose metabolism and signaling in K. lactis.     

Analysis of a eukaryotic beta-galactosidase gene: the N-terminal end of the yeast Kluyveromyces lactis protein shows homology to the Escherichia coli lacZ gene product.

The LAC4 gene of Kluyveromyces lactis, encoding the enzyme beta-galactosidase was mapped on a cloned DNA fragment and the sequence of the 5' end was determined. This sequence includes the 5' regulatory region involved in the induction by lactose and the N-terminal end of the protein coding region. Comparison of the deduced amino acid sequence of this eukaryotic enzyme with the N-terminal end of the Escherichia coli beta-galactosidase revealed substantial homology. Two major RNA initiation sites were mapped at -115 and -105. A number of structural peculiarities of the 5'non-coding region are discussed as in comparison to Saccharomyces cerevisiae genes.