SITE-SPECIFIC RECOMBINATION OF THE MYCOBACTERIUM TUBERCULOSISPROPHAGE-LIKE ELEMENT PHIRV1

The site-specific recombination systems of bacteriophages and other mobile elements fall into two categories that are named for the integrase protein that catalyzes integration and excision of the phage genome. These integrases utilize either a tyrosine or a serine residue to carry out the nucleophilic attack of the DNA. In many cases the directionality of the reaction, that is whether the enzyme catalyzes integration or excision, is determined by an additional phage encoded protein referred to as RDF, or recombination directionality factor.Two prophage-like elements, phiRv1 and phiRv2, were found through sequencing Mycobacterium tuberculosis strain H37Rv. These are absent from the vaccine bacillus M. bovis BCG, and are often found in virulent strains of M. bovis and M. tuberculosis. Through the work presented here, one of these elements, phiRv1, was found to encode an active recombination system, with a serine integrase and RDF. The phiRv1 element is found within a degenerate repeated element, REP13E12, which is present in seven non-identical copies in M. tuberculosis and M. bovis BCG. In vivo studies have revealed that four of the seven 13E12 elements can serve as integration sites (attBs) for a plasmid carrying a reconstructed attP and integrase, and that multiple integrations can occur. The fast growing saprophyte, M. smegmatis, also supports integration, although inefficiently. In M. smegmatis, the phiRv1 plasmid integrates into at least two 13E12 repeats that are quite different from those found in M. bovis BCG and M. tuberculosis. Inefficiency is overcome by providing M. smegmatis with an attB site from BCG. These integrated plasmids are stable, and excision occurs in the presence of the phiRv1 RDF encoded by Rv1584c. In vitro assays were developed for both integration and excision. All the substrate site requirements are relatively small. Integration occurs slowly but efficiently in the presence of excess attB or on an intramolecular attP-attB plasmid. In the presence of RDF integration is inhibited and excision is stimulated. The RDF binds to a specific sequence in both attB and attL, although the way in which it functions may be through protein-protein interactions with integrase

Shoot and plantlet regeneration from meristems of Dioscorea rotundata Poir and Dioscorea alata L.

In vitro culture media capable of regenerating moderate to high shoots and/or plantlets from meristems of two yam species - Dioscorea rotundata and Dioscorea alata within comparable duration of 10 weeks as commonly obtained in other monocots and root and tuber crops were investigated. The study comprised 125 phytohormone combinations investigated in three factorial experiments each consisting of an auxin (NAA) and a cytokinin (BAP or kinetin), or two cytokinins only. The frequency of direct plantlet regeneration, though significantly (P < 0.05) higher for D. alata than for D. rotundata, was low and ranged from 0 to 10% at 3 weeks after culture (WAC) and 0 to 35% at 8 WAC. At 8 WAC, shoot regeneration of 42-75% was obtained in D. rotundata in MS medium supplemented with 0.1 M NAA + 0.20 M BAP, and shoot + plantlet regeneration of 60-82% obtained in media containing 0.05 M + 0.20 M BAP or 0.46 M BAP + 0.50 M kinetin in D. alata. Both shoot induction and plantlet regeneration were species-dependent. Induced shoots were successfully rooted in MS medium within 3 to 4 weeks, bringing time taken for plantlet regeneration to 11 to 12 weeks. Regenerants were morphologically similar to the mother plants. Results of the present study will facilitate regeneration of plantlets via meristem in D. rotundata and D. alata

Re-examining sin(2beta) and Delta m(d) from evolution of B(d) mesons with decoherence

In the time evolution of neutral meson systems, a perfect quantum coherence is usually assumed. The important quantities of the B(d) system, such as sin (2beta) and Delta m(d), are determined under this assumption. However, the meson system interacts with its environment. This interaction can lead to decoherence in the mesons even before they decay. In our formalism this decoherence is modelled by a single parameter lambda. It is desirable to re-examine the procedures of determination of sin(2beta) and Delta m(d) in meson systems with decoherence. We find that the present values of these two quantities are modulated by lambda. Re-analysis of B(d) data from B-factories and LHCb can lead to a clean determination of lambda, sin(2beta) and Delta m(d).Comment: Version accepted for publication in Physics Letters