Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification

<p>Abstract</p> <p>Background</p> <p>Many structural biology- and high-throughput laboratories experience the acquisition of multiple cDNAs from different sources as a rather time- and resource-consuming procedure. The techniques presented here solve these problems.</p> <p>Results</p> <p>An advanced target cDNA amplification procedure employing RNA- or cDNA-derived pseudolibraries circumvents the usual DNA transfection during library establishment. A small sample of reverse transcribed ss- or ds-cDNA or DNA from a pre-existing library is multiplied by in vitro rolling circle ramification amplification. The resulting cDNA pseudolibrary serves as a template for numerous highly efficient PCR amplifications and permits production and analysis of target cDNAs on an automated liquid handling workstation.</p> <p>Conclusion</p> <p>The overall efficiency of the simple protocol collection approaches 100% for targets from libraries with low complexity such as Drosophila and yields >80% of amplicons up to 3 kb size in the case of human cDNA.</p

Climate-trait relationships exhibit strong habitat specificity in plant communities across Europe

Ecological theory predicts close relationships between macroclimate and functional traits. Yet, global climatic gradients correlate only weakly with the trait composition of local plant communities, suggesting that important factors have been ignored. Here, we investigate the consistency of climate-trait relationships for plant communities in European habitats. Assuming that local factors are better accounted for in more narrowly defined habitats, we assigned > 300,000 vegetation plots to hierarchically classified habitats and modelled the effects of climate on the community-weighted means of four key functional traits using generalized additive models. We found that the predictive power of climate increased from broadly to narrowly defined habitats for specific leaf area and root length, but not for plant height and seed mass. Although macroclimate generally predicted the distribution of all traits, its effects varied, with habitat-specificity increasing toward more narrowly defined habitats. We conclude that macroclimate is an important determinant of terrestrial plant communities, but future predictions of climatic effects must consider how habitats are defined

3′-cyclic phosphorylation of U6 snRNA leads to recruitment of recycling factor p110 through LSm proteins

Pre-mRNA splicing proceeds through assembly of the spliceosome complex, catalysis, and recycling. During each cycle the U4/U6.U5 tri-snRNP is disrupted and U4/U6 snRNA base-pairing unwound, releasing separate post-spliceosomal U4, U5, and U6 snRNPs, which have to be recycled to the splicing-competent tri-snRNP. Previous work implicated p110—the human ortholog of the yeast Prp24 protein—and the LSm2-8 proteins of the U6 snRNP in U4/U6 recycling. Here we show in vitro that these proteins bind synergistically to U6 snRNA: Both purified and recombinant LSm2-8 proteins are able to recruit p110 protein to U6 snRNA via interaction with the highly conserved C-terminal region of p110. Furthermore, the presence of a 2′,3′-cyclic phosphate enhances the affinity of U6 snRNA for the LSm2-8 proteins and inversely reduces La protein binding, suggesting a direct role of the 3′-terminal phosphorylation in RNP remodeling during U6 biogenesis

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.

The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies

Production of amplified DNA pseudolibraries and high-throughput cDNA target amplification-0

<p><b>Copyright information:</b></p><p>Taken from "Production of amplified DNA pseudolibraries and high-throughput cDNA target amplification"</p><p>BMC Biotechnology 2007;7():31-31.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1894961.</p><p></p>nspected and bands with suitable DNA size were scored (cf. fig. 2). Species identifiers: Hs, Homo sapiens; Gg, Gallus gallus (chicken); Dm, Drsosophila melanogaster; At, Arabidopsis thaliana. Domain abbreviations: Hel, helicase; ATP, ATPase, dsrm, double-stranded RNA-binding motiv. Designations: Species identifier, protein name, short designation of target domain(s)

LSm1-7 complexes bind to specific sites in viral RNA genomes and regulate their translation and replication

LSm1-7 complexes promote cellular mRNA degradation, in addition to translation and replication of positive-strand RNA viruses such as the Brome mosaic virus (BMV). Yet, how LSm1-7 complexes act on their targets remains elusive. Here, we report that reconstituted recombinant LSm1-7 complexes directly bind to two distinct RNA-target sequences in the BMV genome, a tRNA-like structure at the 3′-untranslated region and two internal A-rich single-stranded regions. Importantly, in vivo analysis shows that these sequences regulate the translation and replication of the BMV genome. Furthermore, both RNA-target sequences resemble those found for Hfq, the LSm counterpart in bacteria, suggesting conservation through evolution. Our results provide the first evidence that LSm1-7 complexes interact directly with viral RNA genomes and open new perspectives in the understanding of LSm1-7 functions

Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes.

The survival of motor neurons (SMN) complex mediates the assembly of small nuclear ribonucleoproteins (snRNPs) involved in splicing and histone RNA processing. A crucial step in this process is the binding of Sm proteins onto the SMN protein. For Sm B/B', D1, and D3, efficient binding to SMN depends on symmetrical dimethyl arginine (sDMA) modifications of their RG-rich tails. This methylation is achieved by another entity, the PRMT5 complex. Its pICln subunit binds Sm proteins whereas the PRMT5 subunit catalyzes the methylation reaction. Here, we provide evidence that Lsm10 and Lsm11, which replace the Sm proteins D1 and D2 in the histone RNA processing U7 snRNPs, associate with pICln in vitro and in vivo without receiving sDMA modifications. This implies that the PRMT5 complex is involved in an early stage of U7 snRNP assembly and hence may have a second snRNP assembly function unrelated to sDMA modification. We also show that the binding of Lsm10 and Lsm11 to SMN is independent of any methylation activity. Furthermore, we present evidence for two separate binding sites in SMN for Sm/Lsm proteins. One recognizes Sm domains and the second one, the sDMA-modified RG-tails, which are present only in a subset of these proteins

Lsm proteins bind and stabilize RNAs containing 5 poly(A) tracts

Many orthopoxvirus messenger RNAs have an unusual nontemplated poly( A) tract of 5 to 40 residues at the 5 end. The precise function of this feature is unknown. Here we show that 5 poly( A) tracts are able to repress RNA decay by inhibiting 3 - to- 5 exonucleases as well as decapping of RNA substrates. UV cross- linking analysis demonstrated that the Lsm complex associates with the 5 poly( A) tract. Furthermore, recombinant Lsm1 - 7 complex specifically binds 5 poly( A) tracts 10 to 21 nucleotides in length, consistent with the length of 5 poly( A) required for stabilization. Knockdown of Lsm1 abrogates RNA stabilization by the 5 poly( A) tract. We propose that the Lsm complex simultaneously binds the 3 and 5 ends of these unusual messenger RNAs and thereby prevents 3 - to-5 decay. The implications of this phenomenon for cellular mRNA decay are discussed