Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.     

Pollination biology of two species ofKielmeyera (Guttiferae) from Brazilian cerrado vegetation

The pollination biology and breeding systems ofKielmeyera coriacea andK. speciosa, two sympatric woody species common in the cerrado vegetation of C. Brazil, were studied. Both species have similar nectarless, polystemonous Papaver-type flowers which are visited by a similar spectrum of insects, though they bloom in different seasons and are thus phenologically isolated. Large carpenter bees seem to be the most important pollinators and these and other bees effect buzz pollen retrieval despite the fact that anthers are not poricidal. Both species ofKielmeyera possess strong xenogamous breeding systems. The presence of staminate flowers and andromonoecy inK. coriacea, as well as the longevity ofK. speciosa flowers are discussed as alternative strategies to improve pollination success and reproductive efficacy.     

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.     

Paracoccidioidomycosis in nude mice: Presence of filamentous forms of the fungus

Congenitally athymic nude mice (nu/nu) and their phenotypically normal littermates (nu/+) were intraperitoneally infected with yeast cells of a strain of Paracoccidioides brasiliensis. The nude mice developed a severe and generalized infection with an intense parasitism of several organs, accompanied by a low-grade of tissue reaction. The lesions were characterized by abundant yeast-like cells of the fungus, and in some animals, numerous hyphal forms could be well visualized. In control animals, infection was moderate, almost exclusively restricted to the area of inoculation, and the lesions presented few parasites surrounded by an inflammatory response. Filamentous forms of the fungus were never encountered in these animals.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Singlet oxygen induced mutation spectrum in mammalian cells.

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.     

A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.     

Contrasting breeding systems in twoEriotheca (Bombacaceae) species of the Brazilian cerrados

The pollination biology and breeding systems ofEriotheca pubescens andE. gracilipes have been studied. These two species occur as trees in cerrado vegetation, the neotropical savannas of Central Brazil, with partially sympatric distributions. They have similar phenology and floral structure, although the flowers ofE. pubescens are larger. Both species have nectar flowers pollinated by largeAnthophoridae bees but the main pollinators of each species differ in size. The species have markedly different breeding systems: late-acting self-incompatibility inE. gracilipes and apomixis stimulated by pollination inE. pubescens.     

Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Summary The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 6-dimethylallyl aminopurine - IAA indole-3-acetic acid - NOA naphthoxyacetic acid - SEM Scanning Electron Microscopy