RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.     

Absence of RT6+ T cells in diabetes-prone biobreeding/Worcester rats is due to genetic and cell developmental defects

Diabetes-prone BB/Wor (DP) rats lack the RT6+ peripheral T cell subset whereas diabetes-resistant BB/Wor rats have normal numbers of RT6+ T cells. Lymphocyte transfusion experiments and in vivo depletion studies have demonstrated that RT6+ T cells have an important regulatory role in the pathogenesis of insulin-dependent diabetes mellitus in BB/Wor rats. In the present study, the results of genetic complementation studies indicate that the DP rat contains an intact RT6 gene, but fails to express the RT6.1 alloantigen in the functional absence of an accessory factor (provided by RT6+ cells). At the cellular level, irradiation chimeras demonstrate that the absence of RT6+ T cells in DP rats is due to an intrinsic defect that results in abnormal development and/or differentiation of prothymocytes into RT6+ T cells. The inability of DP prothymocytes to generate RT6+ T cells is not due to serum autoantibodies, lack of accessory cells, or to the presence of inhibitory cells. Inasmuch as DP bone marrow can transfer the susceptibility for diabetes to irradiated recipients, our present results suggest that an important predisposing factor for insulin-dependent diabetes mellitus in DP rats is the inability of DP prothymocytes to generate RT6+ T cells.     

Rapid homologous up-regulation of binding capacity of androgen receptors in intact cells

Incubation of MCF 7 cells with 5 alpha-dihydrotestosterone (DHT) at 37 degrees C led to a 70% increase in the Bmax of androgen receptor, as compared to the values measured at 2 degrees C, without detectable changes in equilibrium dissociation constants. When MCF 7 cells were incubated with hormone at 2 degrees C, to reach steady-state levels of androgen-receptor complex, a subsequent temperature shift to 37 degrees C induced a rapid (t 1/2 = 3 min) cycloheximide-insensitive increase in DHT binding to androgen receptor. MCF 7 cell treatments at 37 degrees C either before or after incubation with DHT at 2 degrees C showed that up-regulation of binding capacity of androgen receptor could be observed only if hormone is present during incubation at physiological temperature.     

Multiple forms of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol

Summary The investigation on hydrodynamic parameters of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol permitted resolution of four distinct forms. The first one could be detected in concentrated cytosols at low salt concentrations, and had the following properties: sedimentation coefficient = 9 S; R _s = 9.3 nm; M _r = 357,800; f/f _o = 1.83; axial ratio (prolate ellipsoid) = 16. When these cytosol extracts were diluted, a second form could be detected with sedimentation coefficient = 8.3 S; R _s = 9.05 nm; M _r = 320,700;f/f _o = 1.84; axial ratio = 16. Under high salt conditions, glucocorticoid-receptor complexes in concentrated cytosol had the following properties: sedimentation coefficient = 6.4 S; R _s, = 6.7 nm; M _r = 183,100;f/f _o = 1.64; axial ratio = 12. When either these cytosol extracts were diluted, or glucocorticoid-receptor complexes were subjected to repeated analysis, a fourth form was detected with sedimentation coefficient = 3.76 S; R _s = 5.67; M _r = 91,000; f/f _o = 1.75; axial ratio = 14. Besides salt concentration and dilution, the time elapsed between sample dilution and analysis appeared to affect the hydrodynamic properties of glucocorticoid-receptor complexes. On the basis of our findings, it has been concluded that the most likely structure of molybdate-stabilized glucocorticoid-receptor complexes of HeLa cell cytosol can be represented by association of monomers in homodimers, and homotetramers. A homotrimer form could not be deduced from our findings, and the 320,700 glucocorticoid-receptor complex we observed has been suggested to represent an unresolved mixture of trimers and tetramers.     

Biogeographical patterns of endemic diversity and its conservation in Australia's artesian desert springs

Aim

Springs in the Australian arid zone are distinct from other waterways because they house a large number of endemic species. We aimed to assess spatial patterns in endemic diversity at a basin‐wide scale and whether environmental features can help to explain them. In doing so, we take the opportunity to summarize the current state of conservation in the system.

Location

Great Artesian Basin (GAB), arid and semiarid regions of eastern Australia

Methods

We combine data regarding the location of springs with published GIS layers regarding environmental characteristics and a literature review of all species and subspecies documented in the published literature to be endemic to GAB springs.

Results

We found evidence of 96 species and subspecies of fishes, molluscs, crustaceans and plants endemic to these springs. The majority of endemic species are invertebrates with geographical distributions limited to a single spring complex (<61 km²). Endemic taxa are concentrated in 75 of the 326 spring complexes. Spring complexes with a large number of springs, high connectivity via drainage basins and low rainfall were more likely to contain endemic taxa, but environmental models were poor predictors of diversity. Only 24% spring complexes with high conservation value are within conservation reserves, and the majority of endemic species are unassessed under the IUCN and Australian conservation legislation, particularly the invertebrates.

Main conclusions

Diversity in this system is underestimated given the current rate of species discovery and prevailing data deficiency for many taxa. Historical processes and species‐specific environmental requirements may be more important for explaining why diversity is concentrated in particular complexes. Almost a decade after this system was listed as endangered, most complexes of high conservation value remain outside of conservation reserves, and the endangered species status of many taxa, and particularly the invertebrates, remain unassessed.

     

Linkage and association mapping for the slow softening (SwS) trait in peach (<Emphasis Type="Italic">P. persica</Emphasis> L. Batsch) fruit

Fruit texture is a crucial quality factor influencing consumer preference and shelf life. Peach (P. persica L. Batsch) is a highly perishable fruit subjected to a rapid softening after harvest. Improvement of peach shelf life is an important breeding objective, stimulating the characterization and exploitation of texture-related traits. Variants of melting (M) texture have captured an increasing interest, following the economic success of “Big Top” nectarine, one of the most cultivated varieties worldwide. “Big Top” fruit maintains a crispy texture for an extended period before the onset of the melting phase, prolonging its shelf life. Genetic determinants regulating this complex trait, defined as slow softening (SwS), are still unknown, mainly because of limitations in phenotyping methods. In this work, a mechanical approach for measuring SwS fruit texture was used to phenotype offspring derived from a cross between “Rebus028” (SwS texture) and “Max10” (M texture). Mechanical parameters were used in linkage mapping, allowing the identification of a major QTL on chromosome 8 (qSwS8.1). The presence of this QTL was validated by a genome-wide association study (GWAS) in a panel of accessions phenotyped for mechanical properties. Less significant signals were also detected by GWAS in other genomic regions, suggesting that additional loci may regulate the SwS trait, possibly depending on the genetic background. The inheritance pattern of the SwS trait and the presence of additional loci are crucial aspects to be addressed in future studies, along with a better characterization of other important textural attributes.     

Development of microsatellite markers for <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> (F.F. &amp; M.F. Allemão), a highly endangered species from tropical forest based on next-generation sequencing

Myracrodruon urundeuva is a tree species of high economic importance due the strength and durability of its wood. Threatened of extinction in Brazil, it is present only in a few forest remnants, mostly in conservation units. Currently, there is little information on the genetic diversity of natural populations in Brazil and even less information about the genome of this species. Here, new species-specific microsatellite loci were developed based on next-generation sequencing (Illumina). More than 100,000 loci were identified in the run, with di- to hexanucleotides motifs. Of these, 20 loci were selected for validation in 30 individuals, with 15 successfully polymorphic loci detected. The number of alleles ranged among loci from 3 to 16, with an average of 7.73, expected (H _e ) and observed (H _o ) heterozygosity ranged from 0.246 to 0.902 and from 0.103 to 0.867, respectively. These results point out that these new set of markers has a great potential for use in population genetic studies for genetic conservation of the species.     

Long‐distance pollen and seed dispersal and inbreeding depression in Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) in the Brazilian savannah

Hymenaea stigonocarpa is a neotropical tree that is economically important due to its high‐quality wood; however, because it has been exploited extensively, it is currently considered threatened. Microsatellite loci were used to investigate the pollen and seed dispersal, mating patterns, spatial genetic structure (SGS), genetic diversity, and inbreeding depression in H. stigonocarpa adults, juveniles, and open‐pollinated seeds, which were sampled from isolated trees in a pasture and trees within a forest fragment in the Brazilian savannah. We found that the species presented a mixed mating system, with population and individual variations in the outcrossing rate (0.53–1.0). The studied populations were not genetically isolated due to pollen and seed flow between the studied populations and between the populations and individuals located outside of the study area. Pollen and seed dispersal occurred over long distances (>8 km); however, the dispersal patterns were isolated by distance, with a high frequency of mating occurring between near‐neighbor trees and seeds dispersed near the parent trees. The correlated mating for individual seed trees was higher within than among fruits, indicating that fruits present a high proportion of full‐sibs. Genetic diversity and SGS were similar among the populations, but offspring showed evidence of inbreeding, mainly originating from mating among related trees, which suggests inbreeding depression between the seed and adult stages. Selfing resulted in a higher inbreeding depression than mating among relatives, as assessed through survival and height. As the populations are not genetically isolated, both are important targets for in situ conservation to maintain their genetic diversity; for ex situ conservation, seeds can be collected from at least 78 trees in both populations separated by at least 250 m.