Phylogeny of conserved adenines in linkers of Group-I introns

We have analyzed the linkers in group-I introns, a characteristic region that is crucial to the folding and splicing process of the folded RNA, in seventy sequences spreading across r-RNA, t-RNA and organelle genes from various organisms including algae, fungi and protozoa. The study revealed a high degree of consensus of specific adenine residues in J3/4, J6/7 and J8/7 stems of the linker regions that were required to stabilize the local orientation, either as single residue or by forming unusual base pairs along with divalent metal ions. Conservation of these residues in the Group-I intron linkers suggests their significant contribution to the folded structure whose bonding and geometry recruit metal ions to interact in stabilizing the folded nature of RNA

Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy

Changes in morphology and growth curve of Candida albicans in response to treatment by Cisplatin has been studied using fluorescence staining with ethidium bromide. Treatment with Cisplatin was found to markedly inhibit hyphae and ovoid growth as revealed by ethidium bromide staining of drug treated cells. These changes were concomitant with inhibitory effects on the growth curve with respect to untreated cells Presence of Cisplatin not only caused suppression in the limiting values in the growth curve, but also caused a slight left shift in the EC50 values. Some of the ovoid cells undergoing poisoning with cisplatin were found to be unusually enlarged before undergoing their natural fate thus suggesting formation of similar cytotoxic end products with DNA

Crystallization of \u3ci\u3eChlorella\u3c/i\u3e deoxyuridine triphosphatase

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant. In the presence of 2\u27-deoxyuridine-5\u27-[(α,β)-imido]triphosphate and magnesium, the enzyme produced die-shaped hexagonal R3 crystals with unit-cell parameters a = b = 66.9, c = 93.6 A, ƴ = 120°. X-ray diffraction data for chdUTPase were collected to 1.6 A resolution. The crystallization of chdUTPase with manganese resulted in very fragile clusters of needles

Epistasis Among Adaptive Mutations in Deer Mouse Hemoglobin

Epistatic interactions between mutant sites in the same protein can exert a strong influence on pathways of molecular evolution. We performed protein engineering experiments that revealed pervasive epistasis among segregating amino acid variants that contribute to adaptive functional variation in deer mouse hemoglobin (Hb). Amino acid mutations increased or decreased Hb-O2 affinity depending on the allelic state of other sites. Structural analysis revealed that epistasis for Hb-O2 affinity and allosteric regulatory control is attributable to indirect interactions between structurally remote sites. The prevalence of sign epistasis for fitness-related biochemical phenotypes has important implications for the evolutionary dynamics of protein polymorphism in natural populations

The mechanistic basis of hemoglobin adaptation in the high-flying barheaded goose: insights from ancestral protein resurrection

The bar-headed goose (‘BHG’, Anser indicus) is renowned for its trans-Himalayan migratory flights, and the elevated hemoglobin (Hb)-O2 affinity of this species is thought to make a key contribution to its capacity for powered flight at elevations of ~9000 m. Here we revisit the molecular basis of this text-book example of biochemical adaptation. Previous hypotheses about the molecular basis of the evolved increase in Hb-O2 affinity were tested by engineering BHGspecific mutations into recombinant human Hb. This approach can provide important insights, but one problem with such ‘horizontal’ comparisons – swapping residues between proteins of contemporary species – is that the focal mutations are introduced into a sequence context that may not be evolutionarily relevant. If mutations have context-dependent effects, then introducing BHG-specific substitutions into human Hb may not recapitulate the functional effects of causative mutations on the genetic background in which they actually occurred during evolution (i.e., in the BHG ancestor). An alternative ‘vertical’ approach is to reconstruct and resurrect ancestral proteins to test the effects of historical mutations on the genetic background in which they actually occurred. We used this approach to measure the independent and joint effects of amino acid substitutions that occurred in the reconstructed BHG ancestor. Measuring the additive and nonadditive effects of these substitutions enabled us to address several important evolutionary questions about molecular adaptation: (1) Do each of the substitutions contribute to the increased Hb-O2 affinity? If so, what are their relative effects? (2) Does the sequential order in which they occur make a difference? In other words, do the functional effects of mutations depend on which other substitutions have already occurred

The mechanistic basis of hemoglobin adaptation in the high-flying barheaded goose: insights from ancestral protein resurrection

The bar-headed goose (‘BHG’, Anser indicus) is renowned for its trans-Himalayan migratory flights, and the elevated hemoglobin (Hb)-O2 affinity of this species is thought to make a key contribution to its capacity for powered flight at elevations of ~9000 m. Here we revisit the molecular basis of this text-book example of biochemical adaptation. Previous hypotheses about the molecular basis of the evolved increase in Hb-O2 affinity were tested by engineering BHGspecific mutations into recombinant human Hb. This approach can provide important insights, but one problem with such ‘horizontal’ comparisons – swapping residues between proteins of contemporary species – is that the focal mutations are introduced into a sequence context that may not be evolutionarily relevant. If mutations have context-dependent effects, then introducing BHG-specific substitutions into human Hb may not recapitulate the functional effects of causative mutations on the genetic background in which they actually occurred during evolution (i.e., in the BHG ancestor). An alternative ‘vertical’ approach is to reconstruct and resurrect ancestral proteins to test the effects of historical mutations on the genetic background in which they actually occurred. We used this approach to measure the independent and joint effects of amino acid substitutions that occurred in the reconstructed BHG ancestor. Measuring the additive and nonadditive effects of these substitutions enabled us to address several important evolutionary questions about molecular adaptation: (1) Do each of the substitutions contribute to the increased Hb-O2 affinity? If so, what are their relative effects? (2) Does the sequential order in which they occur make a difference? In other words, do the functional effects of mutations depend on which other substitutions have already occurred

Predictable convergence in hemoglobin function has unpredictable molecular underpinnings

To investigate the predictability of genetic adaptation, we examined the molecular basis of convergence in hemoglobin function in comparisons involving 56 avian taxa that have contrasting altitudinal range limits. Convergent increases in hemoglobin-oxygen affinity were pervasive among high-altitude taxa, but few such changes were attributable to parallel amino acid substitutions at key residues.Thus, predictable changes in biochemical phenotype do not have a predictable molecular basis. Experiments involving resurrected ancestral proteins revealed that historical substitutions have context-dependent effects, indicating that possible adaptive solutions are contingent on prior history. Mutations that produce an adaptive change in one species may represent precluded possibilities in other species because of differences in genetic background

Predictable convergence in hemoglobin function has unpredictable molecular underpinnings

To investigate the predictability of genetic adaptation, we examined the molecular basis of convergence in hemoglobin function in comparisons involving 56 avian taxa that have contrasting altitudinal range limits. Convergent increases in hemoglobin-oxygen affinity were pervasive among high-altitude taxa, but few such changes were attributable to parallel amino acid substitutions at key residues.Thus, predictable changes in biochemical phenotype do not have a predictable molecular basis. Experiments involving resurrected ancestral proteins revealed that historical substitutions have context-dependent effects, indicating that possible adaptive solutions are contingent on prior history. Mutations that produce an adaptive change in one species may represent precluded possibilities in other species because of differences in genetic background

Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa