Isolating high-quality RNA from mangroves without liquid nitrogen

Mangroves form unique communities in tropical coastal regions and tidal lowlands. Isolating RNA from mangrove leaves is difficult because of high amounts of secondary metabolites and polysaccharides. Conventional extraction methods produce poor-quality mangrove RNA. We present a simple, fast, and convenient protocol for isolating RNA from 5 mangrove species:Aegiceras corniculatum, Bruguiera gymnorrhiza, Ceriops tagal, Kandelia candel, andSonneratia apetala. Isolating RNA from other mangrove species is also possible. Obtained RNA was of high quality and used in an RT-PCR reaction that amplified 0.6 kb of theA. corniculatum CPI-1 gene.     

Toxicity of two type II ribosome-inactivating proteins (cinnamomin and ricin) to domestic silkworm larvae

Cinnamomin and ricin are two type II ribosome-inactivating proteins. They exhibited a different toxicity to domestic silkworm (Bombyx mori) larvae by oral feeding bioassay. The LC50 of ricin to the silkworm larvae at third instar was much lower than that of cinnamomin. When the isolated 80S ribosome from domestic silkworm pupae was treated separately with the reduced cinnamomin or the reduced ricin, a specific RNA fragment (R-fragment) was produced as characterized by 8 M urea-denatured polyacrylamide gel (3.5%) electrophoresis. The purified A-chains of both cinnamomin and ricin showed a slightly different RNA N-glycosidase activity to the domestic silkworm pupal ribosome. It was proposed that the difference of their toxicity to domestic silkworm larvae was not related to their A-chains but to the properties of their B-chains. It was also found that the vomit obtained from the midgut of domestic silkworm larvae could hydrolyze these two proteins apparently to a similar extent.     

Cloning and expression of a sialic acid-binding lectin from the snail Cepaea hortensis

Highly degenerated gene-specific oligonucleotide primers (GSPs) were constructed from the amino acid sequence of tryptic fragments produced from the purified sialic acid-specific lectin of the garden snail Cepaea hortensis. From the albumin glands, the total RNA or the mRNA was prepared. Combination of a universal primer with the GSPs delivered gene-specific fragments of about 650, 620 and 280 bp by polymerase chain reaction (PCR). These fragments were cloned into the vector pDrive (Qiagen) and sequenced. The resulting cDNA sequence consisted of 744 bp, including an open reading frame of 480 bp. The encoded protein consists of 159 amino acids, including the putative signal sequence peptide. The mature protein should comprise 141 amino acid residues with a calculated molecular mass of 15,529 Da. The expression of the recombinant lectin in Escherichia coli resulted in a soluble protein reacting specifically with rabbit antiserum raised against the native lectin.     

Evidence for ditopic coordination of phosphate diesters to [Mg(15-crown-5)]<Superscript>2+</Superscript>. Implications for magnesium biocoordination chemistry

The interaction of a series of phosphate diesters and triesters (1=diphenyl phosphate, 2=dimethyl phosphate, 3=bis(2-ethylhexyl) phosphate, 4=trimethyl phosphate, 5=methyldiphenyl phosphate, 6=triphenyl phosphate) with [Mg(15-crown-5)]²⁺ (15-crown-5=1,4,7,10,13-pentaoxocyclopentadecane) was studied as a simplified model for the interaction of aqueous Mg²⁺ ion with phosphate-containing biomolecules such as RNA. Using electrospray mass spectrometry, we confirm the formation of 1:1 adducts in the gas phase. Proton and ³¹P NMR titration data were used to construct binding isotherms, and a 1:1 binding equilibrium was fit to the isotherms at room temperature to estimate the binding affinities. The binding affinity data are consistent with ditopic coordination of neutral dialkyl phosphate ligands to the [Mg(15-crown-5)]²⁺ unit. This involves inner-sphere coordination to the Mg²⁺ via an oxygen atom, which is complemented by a weak hydrogen-bonding interaction with the crown ether ligand. Ditopic interaction is consistent with low-temperature NMR spectra showing four different configurations for 1 coordinated to [Mg(15-crown-5)]²⁺, which are interpreted in terms of hindered rotation around the Mg–O_phos bond. Thermochemical analysis of the binding affinity data suggests that the second-shell interaction contributes only about 1 kcal/mol to the binding free energy, so additional factors, such as steric constraints, must be operative to give a preferred phosphate orientation in this system. However, the experimental data do suggest that second-shell interactions contribute as much as 40% of the total binding energy, consistent with the pronounced ability of aqueous Mg²⁺ to form salt-bridges linking secondary and tertiary elements of RNA structure.Abbreviations OTf trifluoromethanesulfonate - ESI-MS electrospray mass spectrometry     

Helical packaging of semiflexible polymers in bacteriophages

We investigate multilayered helical packaging of double-stranded DNA, or of a general polymer chain with persistence length l_b, into an ideal, inert cylindrical container, reaching densities slightly below close packaging. We calculate the free energy as a function of the packaged length, based on the energies for bending, twisting, the suffered entropy loss, and the electrostatic energy in a Debye–Hückel model. In the absence of charges on the packaged polymer, a critical packaging force can be determined, similar to the mechanism involved in DNA unzipping models. When charges are taken into consideration, in the final packaging state the charges which are chemically distant become geometrically close, and therefore a steep rise is seen in the free energy. We argue that due to the extremely ordered and almost closely packaged final state the actual packaging geometry does not influence the behaviour of the free energy, pointing towards a certain universality of this state of the polymer. Our findings are compared to a recent simulations study, showing that the model is sensitive to the screening length.