Identification and structural characterization of nucleus-encoded transfer RNAs imported into wheat mitochondria

Despite its large size (200-2400 kilobase pairs), the mitochondrial genome of angiosperms does not encode the minimal set of tRNAs required to support mitochondrial protein synthesis. Here we report the identification of cytosolic-like tRNAs in wheat mitochondria using a method involving quantitative hybridization to distinguish among three tRNA classes: (i) those encoded by mitochondrial DNA (mtDNA) and localized in mitochondria, (ii) those encoded by nuclear DNA and located in the cytosol, and (iii) those encoded by nuclear DNA and found in both the cytosol and mitochondria. The latter class comprises tRNA species that are considered to be imported into mitochondria to compensate for the deficiency of mtDNA-encoded tRNAs. In a comprehensive survey of the wheat mitochondrial tRNA population, we identified 14 such imported tRNAs, the structural characterization of which is presented here. These imported tRNAs complement 16 mtDNA-encoded tRNAs, for a total of at least 30 distinct tRNA species in wheat mitochondria. Considering differences in the set of mtDNA-encoded and imported tRNAs in the mitochondria of various land plants, the import system must be able to adapt relatively rapidly over evolutionary time with regard to the particular cytosolic-like tRNAs that are brought into mitochondria.     

The rescue by phage display of human fabs to gp120 HIV-1 glycoprotein using EBV transformed lymphocytes

Human hybridomas secreting monoclonal antibodies in a stable manner are difficult to develop. The main difficulties are the restricted techniques for B-cell immortalization, the low number of sensitized B cells in peripheral blood, and the impossibility, for ethical reasons, to immunize humans with most antigens. Phage display has proved to be a powerful method for the generation of recombinant antibody fragments. This technology relies on the construction of recombinant Fab or scFv libraries and their display on phage M13. In order to rescue unstable B-cell clones secreting human antibodies we set up a method for the selection by phage display of human IgG fragments from Epstein-Barr virus (EBV)-transformed clones and applied it to the selection by phage display of Fabs directed against HIV-1 gp120, using a seropositive blood sample. The approach combines B-cell transformation by EBV of peripheral blood lymphocytes from a seropositive donor, preselection of specific IgG anti-gp120 producing clones, and the construction of a targeted human antibody library. In this library the percentage of heavy and light chain coding sequences expressed in Escherichia coli, amplified by a set of specific 5′ primers for different antibody germ lines, was similar to that observed with the original untransformed B-cell sample. One round of panning was sufficient for the rescue of three Fabs specific for HIV-1 gp120 protein, which proves the efficiency of this technique.     

RasC plays a role in transduction of temporal gradient information in the cyclic-AMP wave of Dictyostelium discoideum

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC- cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC- cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA-/myoF- myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC- mutant are the immediate result of the absence of RasC function.     

Calcineurin activation influences muscle phenotype in a muscle-specific fashion

Background  

The calcium activated protein phosphatase 2B, also known as calcineurin, has been implicated as a cell signaling molecule involved with transduction of physiological signals (free cytosolic Ca²⁺) into molecular signals that influence the expression of phenotype-specific genes in skeletal muscle. In the present study we address the role of calcineurin in mediating adaptations in myosin heavy chain (MHC) isoform expression and muscle mass using 3-month old wild-type (WT) and transgenic mice displaying high-level expression of a constitutively active form of calcineurin (MCK-CN* mice).     

Rooting phylogenetic trees with distant outgroups: a case study from the commelinoid monocots

Phylogenetic rooting experiments demonstrate that two chloroplast genes from commelinoid monocot taxa that represent the closest living relatives of the pickerelweed family, Pontederiaceae, retain measurable signals regarding the position of that family's root. The rooting preferences of the chloroplast sequences were compared with those for artificial sequences that correspond to outgroups so divergent that their signal has been lost completely. These random sequences prefer the three longest branches in the unrooted ingroup topology and do not preferentially root on the branches favored by real outgroup sequences. However, the rooting behavior of the artificial sequences is not a simple function of branch length. The random outgroups preferentially root on long terminal ingroup branches, but many ingroup branches comparable in length to those favored by random sequences attract no or few hits. Nonterminal ingroup branches are generally avoided, regardless of their length. Comparisons of the ease of forcing sequences onto suboptimal roots indicate that real outgroups require a substantially greater rooting penalty than random outgroups for around half of the least-parsimonious candidate roots. Although this supports the existence of nonrandomized signal in the real outgroups, it also indicates that there is little power to choose among the optimal and nearly optimal rooting possibilities. A likelihood-based test rejects the hypothesis that all rootings of the subtree using real outgroup sequences are equally good explanations of the data and also eliminates around half of the least optimal candidate roots. Adding genes or outgroups can improve the ability to discriminate among different root locations. Rooting discriminatory power is shown to be stronger, in general, for more closely related outgroups and is highly correlated among different real outgroups, genes, and optimality criteria.     

The ATP-operated clamp of human DNA topoisomerase IIalpha: hyperstimulation of ATPase by "piggy-back" binding

We have constructed a series of clones encoding N-terminal fragments of human DNA topoisomerase IIalpha. All fragments exhibit DNA-dependent ATPase activity. Fragment 1-420 shows hyperbolic dependence of ATPase on DNA concentration, whereas fragment 1-453 shows hyperstimulation at low ratios of DNA to enzyme, a phenomenon found previously with the full-length enzyme. The minimum length of DNA found to stimulate the ATPase activity was approximately 10 bp; fragments >or=32 bp manifest the hyperstimulation phenomenon. Molecular mass studies show that fragment 1-453 is a monomer in the absence of nucleotides and a dimer in the presence of nucleotide triphosphate. The results are consistent with the role of the N-terminal domain of topoisomerase II as an ATP-operated clamp that dimerises in the presence of ATP. The hyperstimulation effect can be interpreted in terms of a "piggy-back binding" model for protein-DNA interaction.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.