Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Laboratory investigations of mars: Chemical and spectroscopic characteristics of a suite of clays as Mars Soil Analogs

Two major questions have been raised by prior explorations of Mars. Has there ever been abundant water on Mars? Why is the iron found in the Martian soil not readily seen in the reflectance spectra of the surface? The work reported here describes a model soil system of Mars Soil Analog Materials, MarSAM, with attributes which could help resolve both of these dilemmas. The first set of MarSAM consisted of a suite of variably iron/calcium-exchanged montmorillonite clays. Several properties, including chemical composition, surface-ion composition, water adsorption isotherms, and reflectance spectra, of these clays have been examined. Also, simulations of the Viking Labeled Release Experiment using the MarSAM were performed. The results of these studies show that surface iron and adsorbed water are important determinants of clay behavior as evidenced by changes in reflectance, water absorption, and clay surface reactions. Thus, these materials provide a model soil system which reasonably satisfies the constraints imposed by the Viking analyses and remote spectral observations of the Martian surface, and which offers a sink for significant amounts of water. Finally, our initial results may provide insights into the mechanisms of reactions that occur on clay surfaces as well as a more specific approach to determining the mineralogy of Martian soils.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Clonal Analysis of Cell Division in the Bacillus subtilis div IV-B1 Minicell-Producing Mutant

Spores of the Bacillus subtilis minicell-producing mutant div IV-B1 were germinated and grown to microcolonies in chambers which facilitate continuous observation of the developing clones with a phase-contrast microscope. Time lapse photographs were taken of 46 clones, covering the period from the beginning of outgrowth until at least two rounds of cell division had been completed. Cell lineages were constructed from contour length measurements of the photographs. These data include cell lengths, division site locations, and cell numbers in clones of various ages. From these data we have determined that the probability of a minicell being produced at any division by the div IV-B1 mutant is 0.31. The location of the abnormal division site which generates the first minicell produced in the outgrowing clone appears to be random with respect to the existing cell poles. In contrast, the location of the second abnormal division site, and hence the second minicell, is not random but rather occurs preferentially in proximity to the first minicell. This clustering of abnormal events suggests that division site location is related to pole age (generations), although other influences on minicell clustering cannot be ruled out at present.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.