Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 3 0 -Sphosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC-oligonucleotide complexes could be separated from noncovalently bound protein by SDS-PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18-DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.

Developmental genetics has shown that the Brachyury (T) gene has a key role in mesoderm formation during gastrulation in the mouse. Homozygous embryos have a defective allantois, degenerate or absent notochord and disrupted primitive streak and node. The neural tube is kinked and somite formation interrupted. The T gene has been cloned and is expressed during the early stages of gastrulation, being restricted to the primitive streak region, nascent mesoderm and notochord. Neither the sequence of the gene nor its expression pattern define its developmental function. To study the cell autonomy of the T mutation we have isolated and genetically characterized embryonic stem cell lines and studied their behaviour in chimaeras. T/+ embryonic stem cells form normal chimaeras, whereas T/T in equilibrium with +/+ chimaeras mimic the T/T mutant phenotype. The results indicate that the T gene acts cell autonomously in the primitive streak and notochord but may activate a signalling pathway involved in the specification of other mesodermal tissues.

Light charged-particle emission and neutron emission have been measured for the fusionevaporation and fusion-fission channels in the Ni+' Mo reaction at 550 and 655 MeV bombarding energies. Temperatures, emission barriers, and multiplicities for the particles detected in coincidence with evaporation residues and fission fragments have been determined. For the evaporation residue data, the "first-chance" spectra of particles emitted from the compound nucleus have been isolated and the same initial temperature for the different evaporated particles has been extracted.The inverse level density parameter K = A /a reaches a value of 13.8+0.7 MeV at E,h --236 MeV.A decrease of the apparent emission barriers for the charged-particle emission at high excitation energy suggests dynamical effects on the de-excitation process. Analysis of the fissionlike events shows a saturation of light particle emission from the fission fragments. Using the average energies and multiplicities of the emitted neutrons, and charged particles, it has been found that for both initial excitation energies, 251 and 293 MeV, scission occurs at an excitation energy =140 MeV. For both fusion evaporation and fusion fission, the light charged particles are preferentially emitted during the early part of the de-excitation cascade. Statistical models and dynamical calculations have been used in an attempt to interpret the experimental data.

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Michaelis-Arbuzov reactions of S-aryl disulfide derivatives of 3'-thiothymidine or 5'-thioadenosine with tris(trimethylsilyl) phosphite proceeded in high yields to the corresponding phosphorothiolate monoesters. Subsequent hydrolytic desilylation and phosphate coupling were effected in one-pot using liquid-assisted grinding in a vibration ball mill. Novel 3',5'- and 5',5'-pyrophosphorothiolate-linked dinucleoside cap analogues were thereby prepared.