Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Transferability,development of simple sequence repeat (SSR) markers and application to the analysis of genetic diversity and population structure of the African fan palm (Borassus aethiopum Mart.) in Benin

Biological pathways play an important role in the occurrence, development and recovery of complex diseases, such as cancers, which are multifactorial complex diseases that are generally caused by mutation of multiple genes or dysregulation of pathways. We propose a path-specific effect statistic (PSE) to detect the differential specific paths under two conditions (e.g. case VS. control groups, exposure Vs. nonexposure groups). In observational studies, the path-specific effect can be obtained by separately calculating the average causal effect of each directed edge through adjusting for the parent nodes of nodes in the specific path and multiplying them under each condition. Theoretical proofs and a series of simulations are conducted to validate the path-specific effect statistic. Applications are also performed to evaluate its practical performances. A series of simulation studies show that the Type I error rates of PSE with Permutation tests are more stable at the nominal level 0.05 and can accurately detect the differential specific paths when comparing with other methods. Specifically, the power reveals an increasing trends with the enlargement of path-specific effects and its effect differences under two conditions. Besides, the power of PSE is robust to the variation of parent or child node of the nodes on specific paths. Application to real data of Glioblastoma Multiforme (GBM), we successfully identified 14 positive specific pathways in mTOR pathway contributing to survival time of patients with GBM. All codes for automatic searching specific paths linking two continuous variables and adjusting set as well as PSE statistic can be found in supplementary materials.  The proposed PSE statistic can accurately detect the differential specific pathways contributing to complex disease and thus potentially provides new insights and ways to unlock the black box of disease mechanisms.     

Distribution and content of calcium and potassium in eucalyptus leaves infected with Calonectria pteridis

Calonectria leaf spot, caused by Calonectria pteridis, is a serious problem in Eucalyptus crops in both nursery and the field. Under ideal conditions, the disease can cause severe defoliation. It is known that calcium and potassium are directly related to the plant's resistance to pathogens. Thus, the knowledge of how a balanced fertilization of Ca and K interferes in the distribution of these nutrients at the infection site would contribute to elucidate the resistance of the plant related to its nutrition. This study investigated the effect of calcium and potassium fertilizer application on the content and distribution of these nutrients in the symptomatic leaf area, transition zone and asymptomatic leaf area over time. Eucalyptus seedlings were grown in nutrient solution under different Ca and K treatments (6 mmol/L K + 4 mmol/L Ca, 6 mmol/L K + 8 mmol/L Ca and 9 mmol/L K + 12 mmol/L Ca) and inoculated with C. pteridis. Leaves were removed at 24, 48 and 72 hr after inoculation (hai) and evaluated by X-ray microanalysis. The highest calcium content among the different leaf areas was observed in the symptomatic area, and the levels in this area increased over time, with the highest mean value observed at 72 hai in the 6K + 8Ca treatment. In the other treatments, the mean calcium content peaked at 48 hai and then decreased. A similar pattern in asymptomatic tissue was observed for potassium in the 6K + 8Ca treatment. Fertilization with calcium and potassium directly affected the demand and availability of nutrients at different times during infection. These results demonstrate that plant defence responses and their continuity over time during infection rely on balanced calcium and potassium fertilization because these nutrients are directly involved in plant resistance to the pathogen.