Phylogenetic placement and lectotypification of Pseudotryblidium neesii (Helotiales, Leotiomycetes).

A phylogenetic analysis of combined rDNA LSU and ITS sequence data was carried out to determine the phylogenetic placement of specimens identified as Pseudotryblidium neesii. The species forms a distinct clade within Dermateaceae (Helotiales, Leotiomycetes) with Rhizodermea veluwiensis and two Dermea species. The geographical distribution of this species, previously known only from Europe on Abies alba, is extended to north-western North America where it grows exclusively on A. grandis. The name P. neesii is lectotypified in order to disentangle the complicated nomenclature of the species. A new, detailed description of P. neesii with illustrations is provided after comparison of sequenced specimens with the type material. Furthermore, the new combination Pseudographis rufonigra (basionym Peziza rufonigra) is made for a fungus previously known as Pseudographis pinicola.

First results from the Cryogenic Dark Matter Search in the Soudan Underground Laboratory.

We report the first results from a search for weakly interacting massive particles (WIMPs) in the Cryogenic Dark Matter Search experiment at the Soudan Underground Laboratory. Four Ge and two Si detectors were operated for 52.6 live days, providing 19.4 kg d of Ge net exposure after cuts for recoil energies between 10 and 100 keV. A blind analysis was performed using only calibration data to define the energy threshold and selection criteria for nuclear-recoil candidates. Using the standard dark-matter halo and nuclear-physics WIMP model, these data set the world's lowest exclusion limits on the coherent WIMP-nucleon scalar cross section for all WIMP masses above 15 GeV/c2, ruling out a significant range of neutralino supersymmetric models. The minimum of this limit curve at the 90% C.L. is 4 x 10(-43) cm2 at a WIMP mass of 60 GeV/c2.

Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element.

Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase, is a member of the T-box family.

The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase (IleRS), contains a long mRNA leader region. This region exhibits many of the features of the gram-positive synthetase gene family, including the T box and leader region terminator and antiterminator. The terminator was shown to be functional in vivo, and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity. The S. aureus ileS leader structure includes several critical differences from the other members of the T-box family, suggesting that regulation of this gene in S. aureus may exhibit unique features.

Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation.

A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction of the E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.

Dynamics of ubiquitin conjugation during erythroid differentiation in vitro.

To gain insight into the role of ubiquitin-mediated proteolysis in erythroid differentiation, levels of ubiquitin conjugating enzymes (E2s) and ubiquitin conjugates were analyzed during in vitro differentiation of murine erythroleukemic (MEL) cells. After 4 days of culture in the presence of the inducer dimethyl sulfoxide, MEL cells expressed high levels of the erythroid-specific proteins, globin, and band 3. During the same interval, cellular contents (mol/cell) of E2-14K, E2-25K, and E2-35K decreased up to approximately 5-fold; as suggested by results obtained with E2-25K, this reflected a lower level of mRNA in differentiating cells. Concentrations of these E2s changed more modestly during in vitro differentiation, since cellular volume also decreased. Comparison of levels of the three E2s in undifferentiated MEL cells and reticulocytes suggests that their concentrations remain fairly constant during in vivo differentiation of proerythroblasts into reticulocytes. Thus, these components of the ubiquitin-mediated proteolytic pathway are likely to function constitutively during this interval. Two-dimensional Western blots showed a broad spectrum of ubiquitin conjugates, including free multiubiquitin chains, in undifferentiated MEL cells. As seen for several E2s, the concentration of ubiquitin conjugates (including free chains) decreased modestly during in vitro differentiation. E2-20K and E2-230K, which are abundant in reticulocytes, were low or absent in undifferentiated and differentiated MEL cells. In erythroid cells these two E2s are reticulocyte-specific; apparently MEL cells do not differentiate far enough to allow induction of their expression.

Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K).

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.