Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Incorporating the speciation process into species delimitation

The “multispecies” coalescent (MSC) model that underlies many genomic species-delimitation approaches is problematic because it does not distinguish between genetic structure associated with species versus that of populations within species. Consequently, as both the genomic and spatial resolution of data increases, a proliferation of artifactual species results as within-species population lineages, detected due to restrictions in gene flow, are identified as distinct species. The toll of this extends beyond systematic studies, getting magnified across the many disciplines that rely upon an accurate framework of identified species. Here we present the first of a new class of approaches that addresses this issue by incorporating an extended speciation process for species delimitation. We model the formation of population lineages and their subsequent development into independent species as separate processes and provide for a way to incorporate current understanding of the species boundaries in the system through specification of species identities of a subset of population lineages. As a result, species boundaries and within-species lineages boundaries can be discriminated across the entire system, and species identities can be assigned to the remaining lineages of unknown affinities with quantified probabilities. In addition to the identification of species units in nature, the primary goal of species delimitation, the incorporation of a speciation model also allows us insights into the links between population and species-level processes. By explicitly accounting for restrictions in gene flow not only between, but also within, species, we also address the limits of genetic data for delimiting species. Specifically, while genetic data alone is not sufficient for accurate delimitation, when considered in conjunction with other information we are able to not only learn about species boundaries, but also about the tempo of the speciation process itself.     

Influence of Species of Vesicular-Arbuscular Mycorrhizal Fungi and Phosphorus Nutrition on Growth,Development, and Mineral Nutrition of Potato (Solanum tuberosum L.)

Growth, development, and mineral physiology of potato (Solanum tuberosum L.) plants in response to infection by three species of vesicular-arbuscular mycorrhizal (VAM) fungi and different levels of P nutrition were characterized. P deficiency in no-P and low-P (0.5 mM) nonmycorrhizal plants developed between 28 and 84 d after planting. By 84 d after planting, P deficiency decreased plant relative growth rate such that no-P and low-P plants had, respectively, 65 and 45% less dry mass and 76 and 55% less total P than plants grown with high P (2.5 mM). A severe reduction in leaf area was also evident, because P deficiency induced a restriction of lateral bud growth and leaf expansion and, also, decreased the relative plant allocation of dry matter to leaf growth. Root growth was less influenced by P deficiency than either leaf or stem growth. Moreover, P-deficient plants accumulated a higher proportion of total available P than high-P plants, indicating that P stress had enhanced root efficiency of P acquisition. Plant P deficiency did not alter the shoot concentration of N, K, Mg, or Fe; however, the total accumulation of these mineral nutrients in shoots of P-stressed plants was substantially less than that of high-P plants. P uptake by roots was enhanced by each of the VAM symbionts by 56 d after planting and at all levels of abiotic P supply. Species differed in their ability to colonize roots and similarly to produce a plant growth response. In this regard, Glomus intraradices (Schenck and Smith) enhanced plant growth the most, whereas Glomus dimorphicum (Boyetchko and Tewari) was least effective, and Glomus mosseae ([Nicol. and Gerd.] Gerd. and Trappe) produced an intermediate growth response. The partial alleviation of P deficiency in no-P and low-P plants by VAM fungi stimulated uptake of N, K, Mg, Fe, and Zn. VAM fungi enhanced shoot concentrations of P, N, and Mg by 28 d after planting and, through a general improvement of overall plant mineral nutrition, promoted plant growth and development.     

Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato (Solanum tuberosum L.)

Planta - Tolerance to heat stress for retention of low-temperature sweetening-resistant phenotype in potato is conferred by insensitivity of acid invertase activity to cold induction. Heat stress...     

Age of A2 Horizon Charcoal and Forest Structure near Porto Trombetas,Pará, Brazil1

To study the structure and composition of old‐growth forest in the Saracá‐Taquera National Forest near Porto Trombetas, Brazil, we established 36 0.25 ha plots and described the vegetation. We collected charcoal from the A2 soil horizon of each plot for radiocarbon dating. Although fires have been very rare in this forest during historic times, the presence of charcoal in these soils indicates fire at some earlier period. The ages (conventional radiocarbon age adjusted to 1997) of the charcoal ranged from 177 to 1547 years. These ages, however, did not correlate significantly with any of several measures of biodiversity or stand characteristics. The relative uniformity of the current old‐growth forest indicates that either the prehistoric fires were of such low intensity that they had little long‐term effect on the vegetation or that the present stands have progressed to near steady state.     

<Emphasis Type="Italic">Strboh A</Emphasis> homologue of NADPH oxidase regulates wound-induced oxidative burst and facilitates wound-healing in potato tubers

During 30-months of storage at 4°C, potato (Solanum tuberosum L.) tubers progressively lose the ability to produce superoxide in response to wounding, resist microbial infection, and develop a suberized wound periderm. Using differentially aged tubers, we demonstrate that Strboh A is responsible for the wound-induced oxidative burst in potato and aging attenuates its expression. In vivo superoxide production and NADPH oxidase (NOX) activity from 1-month-old tubers increased to a maximum 18–24 h after wounding and then decreased to barely detectable levels by 72 h. Wounding also induced a 68% increase in microsomal protein within 18 h. These wound-induced responses were lost over a 25- to 30-month storage period. Superoxide production and NOX activity were inhibited by diphenylene iodonium chloride, a specific inhibitor of NOX, which in turn effectively inhibited wound-healing and increased susceptibility to microbial infection and decay in 1-month-old tubers. Wound-induced superoxide production was also inhibited by EGTA-mediated destabilization of membranes. The ability to restore superoxide production to EGTA-treated tissue with Ca⁺² declined with advancing tuber age, likely a consequence of age-related changes in membrane architecture. Of the five homologues of NOX (Strboh A-D and F), wounding induced the expression of Strboh A in 6-month-old tubers but this response was absent in tubers stored for 25–30 months. Strboh A thus mediates the initial burst of superoxide in response to wounding of potato tubers; loss of its expression increases the susceptibility to microbial infection and contributes to the age-induced loss of wound-healing ability.     

Mutagenesis of lysine 62, asparagine 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2)

The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.     

Shifting distributions and speciation: species divergence during rapid climate change

Questions about how shifting distributions contribute to species diversification remain virtually without answer, even though rapid climate change during the Pleistocene clearly impacted genetic variation within many species. One factor that has prevented this question from being adequately addressed is the lack of precision associated with estimates of species divergence made from a single genetic locus and without incorporating processes that are biologically important as populations diverge. Analysis of DNA sequences from multiple variable loci in a coalescent framework that (i) corrects for gene divergence pre-dating speciation, and (ii) derives divergence-time estimates without making a priori assumptions about the processes underlying patterns of incomplete lineage sorting between species (i.e. allows for the possibility of gene flow during speciation), is critical to overcoming the inherent logistical and analytical difficulties of inferring the timing and mode of speciation during the dynamic Pleistocene. Estimates of species divergence that ignore these processes, use single locus data, or do both can dramatically overestimate species divergence. For example, using a coalescent approach with data from six loci, the divergence between two species of montane Melanoplus grasshoppers is estimated at between 200,000 and 300,000 years before present, far more recently than divergence estimates made using single-locus data or without the incorporation of population-level processes. Melanoplus grasshoppers radiated in the sky islands of the Rocky Mountains, and the analysis of divergence between these species suggests that the isolation of populations in multiple glacial refugia was an important factor in promoting speciation. Furthermore, the low estimates of gene flow between the species indicate that reproductive isolation must have evolved rapidly for the incipient species boundaries to be maintained through the subsequent glacial periods and shifts in species distributions.