Detection of four oxidation sites in viral prolyl-4-hydroxylase by top-down mass spectrometry

Oxidative inactivation is a common problem for enzymatic reactions that proceed via iron oxo intermediates. In an investigation of the inactivation of a viral prolyl-4-hydroxylase (26 kD), electrospray mass spectrometry (MS) directly shows the degree of oxidation under varying experimental conditions, but indicates the addition at most of three oxygen atoms per molecule. Thus, molecular ion masses (M + nO) of one sample indicate the oxygen atom adducts n = 0, 1, 2, 3, and 4 of 35, 41, 19, 5 +/- 3, and <2%, respectively; "top-down" MS/MS of these ions show oxidation at the sites R(28)-V(31), E(95)-F(107), and K(216) of 22%, 28%, and 34%, respectively, but with a possible (approximately 4%) fourth site at V(125)-D(150). However, for the doubly oxidized molecular ions (increasing the precursor oxygen content from 0.94 to 2), MS/MS showed an easily observable approximately 13% oxygen at the V(125)-D(150) site. For the "bottom-up" approach, detection of the approximately 4% oxidation at the V(125)-D(150) site by MS analysis of a proteolysis mixture would have been very difficult. The unmodified peptide containing this site would represent a few percent of the proteolysis mixture; the oxidized peptide not only would be just approximately 4% of this, but the uniqueness of its mass value (approximately 1-2 kD) would be far less than the 11,933 Dalton value used here. Using different molecular ion precursors for top-down MS/MS also provides kinetic data from a single sample, that is, from molecular ions with 0.94 and 2 oxygens. Little oxidation occurs at V(125)-D(150) until K(216) is oxidized, suggesting that these are competitively catalyzed by the iron center; among several prolyl-4-hydroxylases the K(216), H(137), and D(139) are conserved residues.     

Cyclic nucleotide binding proteins in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis> genomes

Background  

Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules.     

Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base- substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta- galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.      

Effect of phytase supplementation on phosphorus digestibility in low-phytate barley fed to finishing pigs

Forty crossbred barrows (Camborough 15 Line female×Canabred sire) weighing an average of 79.6±8.0?kg were used in a factorial design experiment (5 barleys×2 enzyme levels) conducted to determine the effects of phytase supplementation on nutrient digestibility in low-phytate barleys fed to finishing pigs. The pigs were assigned to one of 10 dietary treatments comprised of a normal 2-rowed, hulled variety of barley (CDC Fleet, 0.26% phytate) or 2 low-phytate hulled genotypes designated as LP422 (0.14% phytate) and LP635 (0.09% phytate). A normal, hulless barley (CDC Dawn, 0.26% phytate) and a hulless genotype designated as LP422H (0.14% phytate) were also included. All barleys were fed with and without phytase (Natuphos 5000 FTU/kg). The diets fed contained 98% barley, 0.5% vitamin premix, 0.5% trace mineral premix, 0.5% NaCl and 0.5% chromic oxide but no supplemental phosphorus. The marked feed was provided for a 7-day acclimatization period, followed by a 3-day faecal collection. In the absence of phytase, phosphorus digestibility increased substantially (P<0.05) as the level of phytate in the barley declined. For the hulled varieties, phosphorus digestibility increased from 12.9% for the normal barley (0.26% phytate) to 35.3 and 39.8% for the two low-phytate genotypes (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 9.2% for the normal barley (0.26% phytate) to 34.7% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In contrast, when phytase was added to the diet, there was little difference in phosphorus digestibility between pigs fed normal barley and those fed the low-phytate genotypes (significant barley×enzyme interaction, P=0.01). For the hulled varieties, phosphorus digestibility was 50.1% for the barley with the normal level of phytate (0.26% phytate) compared with 51.1 and 52.4% for the varieties with 54 and 35% of the normal level of phytate (0.14 and 0.09% phytate respectively). For the hulless varieties, phosphorus digestibility increased from 47.1% for the normal barley (0.26% phytate) to 54.4% for the hulless variety with 54% of the normal level of phytate (0.14% phytate). In conclusion, both supplementation with phytase and selection for low-phytate genotypes of barley were successful in increasing the digestibility of phosphorus for pigs. Unfortunately, the effects did not appear to be additive. Whether or not swine producers will choose low-phytate barley or supplementation with phytase as a means to improve phosphorus utilization, will likely depend on the yield potential of low-phytate barley and the additional costs associated with supplementation with phytase.     

Length variation and secondary structure of introns in the Mlc1 gene in six species of Drosophila

A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.      

Role of cryptic genes in microbial evolution

Cryptic genes are phenotypically silent DNA sequences, not normally expressed during the life cycle of an individual. They may, however, be activated in a few individuals of a large population by mutation, recombination, insertion elements, or other genetic mechanisms. A consideration of the microbial literature concerning biochemical evolution, physiology, and taxonomy provides the basis for a hypothesis of microbial adaptation and evolution by mutational activation of cryptic genes. Evidence is presented, and a mathematical model is derived, indicating that powerful and biologically important mechanisms exist to prevent the loss of cryptic genes. We propose that cryptic genes persist as a vital element of the genetic repertoire, ready for recall by mutational activation in future generations. Cryptic genes provide a versatile endogenous genetic reservoir that enhances the adaptive potential of a species by a mechanism that is independent of genetic exchange.      

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Sequence evolution and phylogenetic signal in control-region and cytochrome b sequences of rainbow fishes (Melanotaeniidae)

The nucleotide sequences of segments of the cytochrome b gene (351 bp), the tRNA(Pro) gene (49 bp), and the control region (approximately 313 bp) of mitochondrial DNA were obtained from 26 fish representing different populations and species of Melanotaenia and one species of Glossolepis, freshwater rainbow fishes confined to Australia and New Guinea. The purpose was to investigate relative rates and patterns of sequence evolution. Overall levels of divergence were similar for the cytochrome b and tRNA control-region sequences, both ranging from < 1% within subspecies to 15%-19% between genera. However, the patterns of sequence evolution differed. For the cytochrome b gene, transitions consistently exceeded transversions, the bias ranging from 4.2:1 to 2:1, depending on the level of sequence divergence. However, in the control-region sequence, a bias toward transitions (2:1) was observed only in comparisons between very similar sequences, and transversions outnumbered transitions in comparisons of divergent sequences. Graphic comparisons suggested that the control region was saturated for transitions at relatively low levels of sequence divergence but accumulated transversions at a greater rate than did the cytochrome b sequence. These distinct patterns of base substitution are associated with differences in A+T content, which is 70% for the tRNA control- region segment versus 50% for cytochrome b. A test for skewness in the distribution of lengths of random trees indicated that both segments contained phylogenetic signal. Parsimony analyses of the data from the two regions, with or without weighting schemes appropriate to the respective patterns of sequence evolution, identified the same five groupings of sequences, but the relationships among the groups differed. However, in most cases the branches uniting different combinations of groups were poorly supported, and the differences among topologies were insignificant. Considering the observed patterns of base substitution and the results of the phylogenetic analyses, we deduce that both the control region and cytochrome b are appropriate for population genetic studies but that the control region is less effective than cytochrome b for resolving relationships among divergent lineages of rainbow fishes.      

Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine

In a 3-year longitudinal study, we examined the relationship between the seasonal prevalence of antimicrobial-resistant (AR) Escherichia coli isolates from human wastewater and swine fecal samples and the following risk factors: the host species, the production type (swine), the vocation (human swine workers, non-swine workers, and slaughter plant workers), and the season, in a multisite, vertically integrated swine and human population representative of a closed agri-food system. Human and swine E. coli (n = 4,048 and 3,429, respectively) isolates from wastewater and fecal samples were tested for antimicrobial susceptibility, using the Sensititre broth microdilution system. There were significant (P < 0.05) differences among AR E. coli prevalence levels of (i) the host species, in which swine isolates were at higher risk for resistance to tetracycline, kanamycin, ceftiofur, gentamicin, streptomycin, chloramphenicol, sulfisoxazole, and ampicillin; (ii) the swine production group, in which purchased boars, nursery piglets, and breeding boars isolates had a higher risk of resistance to streptomycin and tetracycline; and iii) the vocation cohorts, in which swine worker cohort isolates exhibited lower sulfisoxazole and cefoxitin prevalence than the non-swine worker cohorts, while the slaughter plant worker cohort isolates exhibited elevated cefoxitin prevalence compared to that of non-swine workers. While a high variability was observed among seasonal samples over the 3-year period, no significant temporal trends were apparent. There were significant differences in the prevalence levels of multidrug-resistant isolates between host species, with swine at a higher risk of carrying multidrug-resistant strains than humans. Considering vocation, slaughter plant workers were at higher risk of exhibiting multidrug-resistant E. coli than non-swine workers.