Plant mRNA 3′-end formation

Our understanding of how the 3 ends of mRNAs are formed in plants is rudimentary compared to what we know about this process in other eukaryotes. The salient features of plant pre-mRNAs that signal cleavage and polyadenylation remain obscure, and the biochemical mechanism is as yet wholly uncharacterised. Nevertheless, despite the lack of universally conserved cis-acting motifs, a common underlying architecture is emerging from functional analyses of plant poly(A) signals, allowing meaningful comparison with components of poly(A) signals in other eukaryotes. A plant poly(A) signal consists of one or more near-upstream elements (NUE), each directing processing at a poly(A) site a short distance downstream of it, and an extensive far-upstream element (FUE) that enhances processing efficiency at all sites. By analogy with other systems, a model for a plant 3-end processing complex can be proposed. Plant poly(A) polymerases have been isolated and partially characterised. These, together with hints that some processing factors are conserved in different organisms, opens promising avenues toward initial characterisation of the trans-acting factors involved in 3-end formation of mRNAs in higher plants.     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli

Analysis of Tn1725 insertions in the Pif+ plasmid pRS2496 showed the maximum limits of the F pif region to be between 43.7 and 47.15 on the 100-kb map of the F plasmid. The effect of these insertions on the expression of pif polypeptides indicated that two of the pif genes, pifA and pifC, lie within a polycistronic operon.     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

The effect of melanins on oxidation of lecithin in liposomal membranes

Lecithin peroxidation in liposomal membranes induced by UV light was studied in the presence of natural eye melanin and synthetic melanins prepared from various precursors. It was shown that melanins inhibited lecithin photooxidation, and that the extent of this effect strongly depended on the type and concentration of melanin. Comparative study indicated that melanin obtained from adrenolutin was the most effective antioxidant. The ability to inhibit lipid peroxidation depends both on the concentration of paramagnetic centers in the melanin polymer and the accessibility of these centers for free radicals formed during irradiation of liposomes.