Purification and characterization of the m7G(5')pppN-pyrophosphatase from human placenta

A purification scheme has been developed for the m7G(5')pppN-pyrophosphatase from human placenta. The 1400-fold purified placental enzyme exhibited physical and enzymatic properties similar to those previously reported for a crude preparation of the human m7G(5')pppN-pyrophosphatase obtained from HeLa cells. Polyacrylamide gel analysis of enzyme fractions at different stages of purification revealed a Mr = 40,000 polypeptide that increased in relative concentration as the specific activity of the enzyme fractions increased. Copurification of this polypeptide with m7G(5')pppN-pyrophosphatase activity suggests the possibility that the 81,000-dalton native enzyme is a dimer composed of subunits of identical molecular weight. The highly purified placental enzyme, like the crude HeLa enzyme, failed to hydrolyze the cap moiety of intact mRNA even under conditions known to reduce mRNA secondary structure. Moreover, when a series of capped oligonucleotides that differed progressively in chain length by a factor of one nucleotide was tested as substrate, the rate of enzyme-catalyzed cap hydrolysis decreased as the chain length increased. The purified placental enzyme failed to release m7pG from oligonucleotides containing the cap and 3 or more additional nucleotides. These results are discussed in terms of the probable biological function of the m7G(5')pppN-pyrophosphatase.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

Singlet oxygen (¹O₂) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to ¹O₂ formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under ¹O₂ stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards ¹O₂. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to ¹O₂ resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to ¹O₂ exposure in R. sphaeroides was extended.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.