Synthetic polyploids of Tragopogon miscellus and T. mirus (Asteraceae): 60 Years after Ownbey's discovery

In plants, polyploidy has been a significant evolutionary force on both recent and ancient time scales. In 1950, Ownbey reported two newly formed Tragopogon allopolyploids in the northwestern United States. We have made the first synthetic lines of T. mirus and T. miscellus using T. dubius, T. porrifolius, and T. pratensis as parents and colchicine treatment of F(1) hybrids. We also produced allotetraploids between T. porrifolius and T. pratensis, which are not known from nature. We report on the crossability between the diploids, as well as the inflorescence morphology, pollen size, meiotic behavior, and fertility of the synthetic polyploids. Morphologically, the synthetics resemble the natural polyploids with short- and long-liguled forms of T. miscellus resulting when T. pratensis and T. dubius are reciprocally crossed. Synthetic T. mirus was also formed reciprocally, but without any obvious morphological differences resulting from the direction of the cross. Of the 27 original crosses that yielded 171 hybrid individuals, 18 of these lineages have persisted to produce 386 S(1) progeny; each of these lineages has produced S(2) seed that are viable. The successful generation of these synthetic polyploids offers the opportunity for detailed comparative studies of natural and synthetic polyploids within a nonmodel system.     

Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.     

Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bγ, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bγ. We also show that infection by L. pneumophila leads to eEF1A‐mediated activation of the heat shock regulatory protein HSF1 in a virulence‐dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila‐infected cells.     

DNA barcoding Central Asian butterflies: increasing geographical dimension does not significantly reduce the success of species identification

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.     

Decreased Rate of Evolution in Y Chromosome STR Loci of Increased Size of the Repeat Unit

Background

Polymorphic Y chromosome short tandem repeats (STRs) have been widely used in population genetic and evolutionary studies. Compared to di-, tri-, and tetranucleotide repeats, STRs with longer repeat units occur more rarely and are far less commonly used.

Principal Findings

In order to study the evolutionary dynamics of STRs according to repeat unit size, we analysed variation at 24 Y chromosome repeat loci: 1 tri-, 14 tetra-, 7 penta-, and 2 hexanucleotide loci. According to our results, penta- and hexanucleotide repeats have approximately two times lower repeat variance and diversity than tri- and tetranucleotide repeats, indicating that their mutation rate is about half of that of tri- and tetranucleotide repeats. Thus, STR markers with longer repeat units are more robust in distinguishing Y chromosome haplogroups and, in some cases, phylogenetic splits within established haplogroups.

Conclusions

Our findings suggest that Y chromosome STRs of increased repeat unit size have a lower rate of evolution, which has significant relevance in population genetic and evolutionary studies.     

Isoforms of U1-70k Control Subunit Dynamics in the Human Spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B''. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B'' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.     

Flying-patch patch-clamp study of G22E-MscL mutant under high hydrostatic pressure

High hydrostatic pressure (HHP) present in natural environments impacts on cell membrane biophysical properties and protein quaternary structure. We have investigated the effect of high hydrostatic pressure on G22E-MscL, a spontaneously opening mutant of Escherichia coli MscL, the bacterial mechanosensitive channel of large conductance. Patch-clamp technique combined with a flying-patch device and hydraulic setup allowed the study of the effects of HHP up to 90 MPa (as near the bottom of the Marianas Trench) on the MscL mutant channel reconstituted into liposome membranes, in addition to recording in situ from the mutant channels expressed in E. coli giant spheroplasts. In general, against thermodynamic predictions, hydrostatic pressure in the range of 0.1–90 MPa increased channel open probability by favoring the open state of the channel. Furthermore, hydrostatic pressure affected the channel kinetics, as manifested by the propensity of the channel to gate at subconducting levels with an increase in pressure. We propose that the presence of water molecules around the hydrophobic gate of the G22E MscL channel induce hydration of the hydrophobic lock under HHP causing frequent channel openings and preventing the channel closure in the absence of membrane tension. Furthermore, our study indicates that HHP can be used as a valuable experimental approach toward better understanding of the gating mechanism in complex channels such as MscL.