Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.     

Teleconnection between tree growth in the Amazonian floodplains and the El Niño–Southern Oscillation effect

There is a limited knowledge about the El Niño–Southern Oscillation (ENSO) effects on the Amazon basin, the world's largest tropical rain forest and a major factor in the global carbon cycle. Seasonal precipitation in the Andean watershed annually causes a several month‐long inundation of the floodplains along the Amazon River that induces the formation of annual rings in trees of the flooded forests. Radial growth of trees is mainly restricted to the nonflooded period and thus the ring width corresponds to its duration. This allows the construction of a tree‐ring chronology of the long‐living hardwood species Piranhea trifoliata Baill. (Euphorbiaceae). El Niño causes anomalously low precipitation in the catchment that results in a significantly lower water discharge of the Amazon River and consequently in an extension of the vegetation period. In those years tree rings are significantly wider. Thus the tree‐ring record can be considered as a robust indicator reflecting the mean climate conditions of the whole Western Amazon basin. We present a more than 200‐year long chronology, which is the first ENSO‐sensitive dendroclimatic proxy of the Amazon basin and permits the dating of preinstrumental El Niño events. Time series analyses of our data indicate that during the last two centuries the severity of El Niño increased significantly.     

Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli

A new, heterologous, dominant marker for selection of Aspergillus transformants is described. This marker is based on the Escherichia coli hygromycin B (HmB) phosphotransferase gene (hph). Expression of the hph gene is controlled by A. nidulans gpd and trpC expression signals. An Aspergillus transformation vector was constructed which contains this marker and confers HmB resistance to Aspergillus species. With both A. niger and A. nidulans, transformation frequencies of 5-20 transformants per micrograms vector DNA were obtained. Cotransformation with other vectors was shown to be very efficient in both species, when selection for HmB resistance was applied.     

Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods

The side chains of tyrosine, tryptophan and histidine are able to produce CIDNP (Chemically Induced Dynamic Nuclear Polarization) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies surface accessibility of the respective groups to the light-absorbing dye. In principle, this technique allows the monitoring of the effect of ligand binding to a receptor and of site-directed mutagenesis on conformational aspects of any protein if CIDNP-reactive amino acids are involved. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial model study for several N-acetyl-glucosamine-binding lectins of increasing structural complexity as well as for a wild type bacterial sialidase and its mutants. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate is bound, CIDNP signals of side chain protons of tyrosine, tryptophan or histidine residues can be broadened and of reduced intensity. This is the case for hevein, pseudo-hevein, the four hevein domains-containing lectin wheat germ agglutinin (WGA) and the cloned B-domain of WGA 1 (domB) representing one hevein domain. This response indicates either a spatial protection by the ligand or a ligand-induced positioning of formerly surface-exposed side chains into the protein’s interior part, thereby precluding interaction with the photo-activated dye. Some signals of protons from the reactive side chains can even disappear when the lectin-ligand complexes are monitored. The ligand binding, however, can apparently also induce a conformational change in a related lectin that causes the appearance of a new signal, as seen for Urtica dioica agglutinin (UDA) which consists of two hevein domains. Additionally, the three CIDNP-reactive amino acids are used as sensors for the detection of conformational changes caused by pH variations or by deliberate amino acid exchanges, as determined for the isolectins hevein and pseudo-hevein as well as for the cloned small sialidase of Clostridium perfringens and two of its mutants. Therefore, CIDNP has proven to be an excellent tool for protein-carbohydrate binding studies and can be established in glycosciences as a third biophysical method beside X-ray-crystallography and high-resolution multidimensional NMR studies which provides reliable information of certain structural aspects of carbohydrate-binding proteins in solution. This revised version was published online in November 2006 with corrections to the Cover Date.     

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.