Ribosomal DNA variation and its phylogenetic implication in the genusBrachypodium (Poaceae)

The structure of ribosomal DNA ofBrachypodium and several other grass species was investigated using a heterologous rDNA probe from wheat. Several different rDNA families were present among perennial and annual species within the genus. In contrast to the annual species the perennial species exhibited a very low degree of repeat length variation. An extra Eco RI site and a Hin dIII site were observed in the IGS, which distinguishedBrachypodium from other grass genera. The restriction fragment length polymorphism and length variation of the repeat units have taxonomic value withinBrachypodium and are correlated with the classification ofBrachypodium derived from other data.     

Intraspecific molecular variation inHieracium sect.Alpina (Asteraceae), an apomictic group

Intraspecific variation of four agamospecies ofHieracium sect.Alpina was studied using RAPD and isozyme techniques. No variation in either multiprimer RAPD or multi-enzyme phenotypes was observed withinH. holosericeum, suggesting that this widespread species consists of only a single genotype. A low level of within-population isozyme variation was seen inH. tenuifrons andH. calenduliflorum, the origin of which appears to be consistent with somatic mutation. Most isozyme and all RAPD variation in these two species was partitioned between populations. A strong correlation with geography suggests that its cause may be due to polytopic (-polyphyletic?) origin or perhaps to mutation and dispersal. The most variable species wasH. alpinum, in which isozyme variation occurred mostly within populations rather than between them, suggesting occasional sexual events or that the parents ofH. alpinum were heterozygous. RAPD variation in this species, in contrast, was partitioned between Scottish and Swiss populations, suggesting the existence of geographical races.     

Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p

Ypd1p, a histidine-containing phosphotransfer protein, plays an important role in a branched His-Asp phosphorelay signal transduction pathway that regulates cellular responses to hyperosmotic stress in Saccharomyces cerevisiae. Ypd1p is required for phosphoryl group transfer from the membrane-bound Sln1p sensor histidine kinase to two downstream response regulator proteins, Ssk1p and Skn7p. To investigate the molecular basis for interaction of Ypd1p with these response regulator domains, we used an approach that coupled alanine-scanning mutagenesis of surface-exposed residues in Ypd1p with a yeast two-hybrid interaction screen. Mutated residues that adversely affected the interaction of Ypd1p with the C-terminal response regulator domain of Ssk1p were identified and found to cluster on or near the αA helix in Ypd1p. Our results, supported by analysis of a modeled complex, identify a binding site on Ypd1p for response regulators that is composed of a cluster of conserved hydrophobic residues surrounded by less conserved polar residues. We propose that molecular interactions involving Ypd1p are mediated primarily through hydrophobic contacts, whereas binding specificity and strength of interaction may be influenced by select polar side chain interactions.     

Generic and infrageneric nomenclature of annual Poaceae: Poeae related to Vulpia and Desmazeria

The 26 generic and 63 infrageneric names which have been applied to the approximately 47 species belonging to the Vulpia–Desmazeria group of annual Poaceae: Poeae are listed, together with their bibliographic, nomenclatural and typological details. A synopsis of the classification of this group adopted by the author, involving twelve genera and five extra sections, follows, and includes a new combination at the sectional level: Vulpia sect. Apalochloa (Dumort.) Stace. Although not closely related to this group of grasses, the genus Sclerochloa is included as it has been very widely confused taxonomically, nomenclaturally and typographically with Scleropoa.     

Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG

Background

In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production.

Methodology/Principal Findings

To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli.

Conclusions/Significance

We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG''s ability to protect against pulmonary TB.     

Structure-function relationships among wild-type variants of Staphylococcus aureus beta-lactamase: importance of amino acids 128 and 216.

beta-Lactamases inactivate penicillin and cephalosporin antibiotics by hydrolysis of the beta-lactam ring and are an important mechanism of resistance for many bacterial pathogens. Four wild-type variants of Staphylococcus aureus beta-lactamase, designated A, B, C, and D, have been identified. Although distinguishable kinetically, they differ in primary structure by only a few amino acids. Using the reported sequences of the A, C, and D enzymes along with crystallographic data about the structure of the type A enzyme to identify amino acid differences located close to the active site, we hypothesized that these differences might explain the kinetic heterogeneity of the wild-type beta-lactamases. To test this hypothesis, genes encoding the type A, C, and D beta-lactamases were modified by site-directed mutagenesis, yielding mutant enzymes with single amino acid substitutions. The substitution of asparagine for serine at residue 216 of type A beta-lactamase resulted in a kinetic profile indistinguishable from that of type C beta-lactamase, whereas the substitution of serine for asparagine at the same site in the type C enzyme produced a kinetic type A mutant. Similar bidirectional substitutions identified the threonine-to-alanine difference at residue 128 as being responsible for the kinetic differences between the type A and D enzymes. Neither residue 216 nor 128 has previously been shown to be kinetically important among serine-active-site beta-lactamases.     

Hybridization in the genera Vulpia and Festuca (Poaceae): meiotic behaviour of artificial hybrids

The course of meiosis, including an analysis of chromosome configurations, is described for five diploid × diploid Vulpia crosses, five tetraploid × diploid Vulpia crosses, one hexaploid × diploid Festuca × Vulpia cross, one tetraploid × hexaploid Vulpia × Festuca cross, and one hexaploid × hexaploid Vulpia × Festuca cross. In most cases there was 97.5% or more pollen sterility, but two heptaploid plants obtained (presumably by non-reduction) from a hexaploid × diploid cross had about 60% stainable pollen. In the diploid hybrids pairing was quite extensive, and in V. ligustica × V. geniculata it was more or less as in the parent species (mode 7 bivalents, with regular separation). In the triploid hybrids the modal situation was 7 bivalents + 7 univalents, but evidence concerning the genomes which were pairing was equivocal. Evidence from the crosses at higher ploidy levels shows that both homogenetic and heterogenetic pairing does occur, although the relative amounts are uncertain. The results in general support the current classsification of Vulpia , except that they suggest the removal of V. alopecuros from section Loretia.     

Resurrection of two Eighteenth Century names in American Combretaceae - Studies on the flora of the Guianas 7

Pamea guianensis Aublet and Terminalia nitidissima Rich. are, on the basis of recently collected material, recognized as hitherto overlooked species of Buchenavia Eichler, and the two new combinations are made.