ABSTRACT
Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.
Subject(s)
Recombination, Genetic , Transposases/metabolism , Amino Acid Sequence , Aspartic Acid/genetics , Aspartic Acid/metabolism , Base Sequence , Catalytic Domain , Conserved Sequence , DNA Mutational Analysis , Models, Molecular , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid , Surface Properties , Transposases/geneticsABSTRACT
This study was undertaken to objectively compare healing rates in wounds produced by the Nd:YAG (Neodymium:Yttrium-Aluminium-Garnet) laser to those caused by steel scalpel, in rabbits. Rates of contraction and epithelialisation in standard wounds were determined by daily measurement of the wound area from the time of wounding until epithelialisation was complete. The wounds were of three types: superficial to the panniculus carnosus, deep to it and ear wounds. Significant delays in both wound contraction (p < 0.0001) and epithelialisation (p < 0.0001) were demonstrated in all Nd:YAG laser wounds. Normal epithelial regenerative capacities were observed. The final appearance of the healed areas was comparable across the laser and scalpel groups.
Subject(s)
Lasers , Wound Healing/radiation effects , Aluminum , Animals , Ear, External , Epithelium/physiology , Epithelium/radiation effects , Laser Therapy , Male , Neodymium , Rabbits , Regeneration/physiology , Wound Healing/physiology , YttriumABSTRACT
The ability of exogenous lysophosphatidylcholine (LPC) to produce electrophysiological abnormalities in cardiac tissues and cardiac arrhythmias in isolated hearts has been well documented. In this study, the arrhythmogenic nature of LPC in the rat, rabbit, and guinea pig hearts was studied. The rat heart was found to be the most susceptible to LPC-induced arrhythmias, while the guinea pig heart was the least susceptible. Perfusion with labelled LPC revealed that the severity of arrhythmias correlates well with the amount of labelled LPC found in the microsomal membrane. The biochemical basis for the differences in the accumulation of LPC in the microsomal membrane of different animal species was investigated. Our results strongly indicate that the LPC level in the microsomal membrane may be regulated by the activity of microsomal lysophospholipase.
