The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.

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A time-resolved optical imaging system using near-infrared light has been developed. The system had three pulsed light sources and total 64 channels of detection, working simultaneously for acquisition of the time-resolved data of the pulsed light transmitted through scattering media like biological tissues. The light sources were provided by high power picosecond pulsed diode lasers, and optical switches directed one of the light sources to the object through an optical fiber. The light signals reemitted from the surface of the object were collected by optical fibers, and transmitted to a time-resolved detecting system. Each of the detecting channels consisted of an optical attenuator, a fast photomultiplier, and a time-correlated single photon counting circuit which contained a miniaturized constant fraction discriminator/time-to-amplitude converter module, and a signal acquisition unit with an A/D converter. The performance and potentiality of the imaging system have been examined by the image reconstruction from the measured data using solid phantoms.

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