The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.     

Establishment of murine cell lines by constitutive and conditional immortalization

Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells.     

Thermostable RNase P RNAs lacking P18 identified in the Aquificales

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up- and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and delta/epsilon proteobacterial branches.     

Evaluating effects of weed cutting on water level and ecological status in Danish lowland streams

相似文献   

Secondary dispersal mechanisms of winged seeds: a review

Winged seeds, or samaras, are believed to promote the long‐distance dispersal and invasive potential of wind‐dispersed trees, but the full dispersive potential of these seeds has not been well characterised. Previous research on the ecology of winged seeds has largely focussed on the initial abscission and primary dispersal of the samara, despite it being known that the primary wind dispersal of samaras is often over short distances, with only rare escapes to longer distance dispersal. Secondary dispersal, or the movement of the seeds from the initial dispersal area to the site of germination, has been largely ignored despite offering a likely important mechanism for the dispersal of samaras to microhabitats suitable for establishment. Herein, we synthesise what is known on the predation and secondary dispersal of winged seeds by multiple dispersive vectors, highlighting gaps in knowledge and offering suggestions for future research. Both hydrochory and zoochory offer the chance for samaroid seeds to disperse over longer distances than anemochory alone, but the effects of the wing structure on these dispersal mechanisms have not been well characterised. Furthermore, although some studies have investigated secondary dispersal in samaroid species, such studies are scarce and only rarely track seeds from source to seedling. Future research must be directed to studying the secondary dispersal of samaras by various vectors, in order to elucidate fully the invasive and colonisation potential of samaroid trees.     

Organ correlations inchenopodium rabrum l. shoots studied by Means of32P Distribution

21-day old plants ofChenopodium rubrum L. ecotype 374 were used. Organ relationships in the shoots were investigated by³²P distribution, which indicated different organ correlations in plants grown in continuous light and in plants treated with flower-inducing and non-inducing dark periods. Dark periods were associated with a low³²P distribution in young leaves and a high one in axillary buds. In the following light period the high³²P distribution in axillary buds continued whereas the³²P distribution in the leaves on the main axis increased and was similar to that in plants grown in continuous light. The high³²P distribution in axillary buds was brought about by both, flower-inducing and non-inducing dark treatments. Decapitation resulted in a high³²P distribution in buds, in continuous light an increased³²P distribution was also found in leaves. These effects were not fully cancelled by IAA application. The results are discussed with respect to an assumption that decrease of apical dominance represents a step in a sequence of events leading to flowering.     

Diversity in hapten recognition: structural study of an anti-cocaine antibody M82G2

Antibodies against cocaine and other drugs of abuse are the basis for diagnostic tests for the presence of those drugs in human serum. The 1.7A resolution crystal structure of the anti-cocaine monoclonal antibody M82G2 in complex with cocaine is presented. This structure determination was undertaken to establish the stereochemical features in the antibody binding site that confer specificity for cocaine, and as part of an ongoing project to understand the rules that govern molecular recognition. The cocaine-binding site can be characterized topologically as a narrow groove on the protein surface. The antibody utilizes water-mediated hydrogen bonding, and cation-pi and stacking (pi-pi) interactions to provide specificity. Comparison with the previously published structure of the anti-cocaine antibody GNC92H2 shows that binding of a small ligand can be achieved in diverse ways, both in terms of a binding site structure/topology and protein-ligand interactions.