Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.     

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular genetics of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is a gram-negative, highly acidophilic (pH 1.5 to 2.0), autotrophic bacterium that obtains its energy through the oxidation of ferrous iron or reduced inorganic sulfur compounds. It is usually dominant in the mixed bacterial populations that are used industrially for the extraction of metals such as copper and uranium from their ores. More recently, these bacterial consortia have been used for the biooxidation of refractory gold-bearing arsenopyrite ores prior to the recovery of gold by cyanidation. The commercial use of T. ferrooxidans has led to an increasing interest in the genetics and molecular biology of the bacterium. Initial investigations were aimed at determining whether the unique physiology and specialized habitat of T. ferrooxidans had been accompanied by a high degree of genetic drift from other gram-negative bacteria. Early genetic studies were comparative in nature and concerned the isolation of genes such as nifHDK, glnA, and recA, which are widespread among bacteria. From a molecular biology viewpoint, T. ferrooxidans appears to be a typical member of the proteobacteria. In most instances, cloned gene promoters and protein products have been functional in Escherichia coli. Although T. ferrooxidans has proved difficult to transform with DNA, research on indigenous plasmids and the isolation of the T. ferrooxidans merA gene have resulted in the development of a low-efficiency electroporation system for one strain of T. ferrooxidans. The most recent studies have focused on the molecular genetics of the pathways associated with nitrogen metabolism, carbon dioxide fixation, and components of the energy-producing mechanisms.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Appropriate characters for racial classification in maize

To determine the relative importance of the genotype, the environment, and their interaction on the expression of morphological characters in maize races, 50 Mexican races and 24 races from Central America, South America, and the U.S. were grown in several locations and seasons in México and 47 characters were measured directly. Estimates of the ratio of variance components, rc = [Vc/(Ve+VCs/], were used as criteria to determine the appropriate characters for racial classification. Twenty-four useful variables were identified. Analysis of the structure of the data matrix facilitated visual examination of correlations among the variables and of the variability represented by each variable. Based on these analyses, a minimum list of 9 characters was suggested to be appropriate variables for racial classification: number of leaves per plant, branched part length/tassel length, central spike internode length, male glume length, kernel width, rachis segment length, pith diameter, ear diameter/length, and kernel width/length.     

Measures of nematode community structure and sources of variability among and within agricultural fields

Whole nematode communities, extracted from soil samples taken from agricultural fields, were enumerated by taxonomic family and trophic group (i.e., bacterivores, fungivores, omnivores, plant-parasites, and predators) to evaluate nematode community structure as an indicator for monitoring ecological condition of soil. No differences were found in mixing treatments or methods of packing or shipping samples. However, extraction using Cobb's sifting and gravity method, followed by sucrose centrifugation, gave greater recovery of free-living nematodes than elutriation followed by sucrose centrifugation. Population means and variance of the sampled area were similar when sampled using different strategies for collecting soil samples within fieds, including several patterns, directions and repetitions of transects. Components of variation associated with ratios among the five trophic groups of nematodes and selected indices of community structure were quantified as variation among regions, among counties, among agricultural fields (2-ha area), among transects within agricultural fields, and within composite soil samples. The variance component for'within composite soil samples' was relatively large compared to the other components of variance. Variation within composite soil samples was less for maturity indices (based on life-history strategy characteristics), ratio of bacterivores to plant-parasites, sum of bacterivores and fungivores, populations of plant-parasites, and populations of bacterivores than for trophic diversity indices, populations of fungivores, populations of omnivores, populations of predators, or the ratio of fungivores to bacterivores. With a single composite sample per field, the ability to differentiate ecological condition of soils among fields within a region improved if the variance among and within fields exceeded the variance within composite samples. Given the variance components, power curves indicated that detection of a 10% change (with 0.8 power) in the ecological condition of soils within a region between two time periods would require sampling a minimum of 25 and 50 fields with one composite soil sample analyzed per field for the maturity and trophic diversity index, respectively. More than 100 fieldsper region would be required to detect temporal change in populations of individual trophic groups. Biplots of maturity indices, but not of trophic diversity or populations of individual trophic groups, identified clear differences among fields. Thus, maturity indices, which differentiated among sampling sites better and more efficiently than trophic diversity indices or measures based on populations of individual trophic groups, may be appropriate for use in a regional and/or national monitoring program.     

The poison–antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison–antidote plasmid stability system. The three genes were named pasA , pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison–antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison–antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.     

Duration of the breeding season and response to reproductive manipulation in five breeds of sheep under northern prairie conditions

The breeding season was 157, 154, <126, 210 and 217 days for Rambouillet, Columbia, Suffolk, Rambouillet x Finnish Landrace and Columbia x Finnish Landrace ewes respectively. Treatment of cyclic ewes with pregnant mare serum gonadotropin (PMSG) (500 IU), following a 12-day treatment with progestin-containing intravaginal sponges, did not affect fertility, but did decrease the time from sponge removal to estrus, (control 48.0 +/- 3.1 hr; PMSG 39.4 +/- 1.8 hr) to the preovulatory surge of LH (control 52.7 +/- 2.8 hr; PMSG 39.0 +/- 1.7 hr) and FSH (control 52.3 +/- 2.9 hr; PMSG 42.8 +/- 1.6 hr) and caused an elevation of serum LH levels prior to the preovulatory surge (control 1.25 +/- 0.18 ng/ml; PMSG 2.31 +/- 0.22 ng/ml). Exposure of the purebred ewes to 18 hours of daylight in January, decreasing by 30 minutes a week subsequently, counteracted the seasonal reduction in the number of ewes lambing following induced breeding under natural daylight in May. Prolificacy was greatest in crossbred ewes and their fertility was not affected by season. Gestation period was longer for fall-bred ewes and varied with breed.