An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.

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Synthetic c-Ha-ras genes with an analogue of an abasic site in the first or the second position of codon 12, or in the second position of codon 61 were constructed and transfected into NIH3T3 cells. The genes with the lesions in codon 12 exhibited more focus formation than a normal c-Ha-ras gene, while the gene with the lesion in codon 61 did not. Transformed cells were isolated from the foci, and the c-Ha-ras genes present in the transformants were analysed. A point mutation to A in the modified position was found most frequently in the cases of ras genes modified in codon 12. Surprisingly, point mutations in the adjacent position were also detected. These results indicate that dTMP, and not dAMP, was mainly incorporated into the sites opposite to the abasic site analogue, and that incorrect deoxynucleotides were incorporated in the position adjacent to the abasic site analogue.

A highly sensitive enzyme immunoassay of conjugated 1/3-hydroxycholic acid in heterologous combination was developed using horse radish peroxidase labeled antigen. The assay method was optimized for quantitation in double antibodycoated microtiter plates. The assay had a lower limit of sensitivity of 12.5 pg/assay. The cross-reactivity for glyco 1/ihydroxycholic acid was 80.13% and those for conjugated 1/3-hydroxychenodeoxycholic and 1/i-hydroxydeoxycholic acids ranged from 0.13 to 7.58%. The values in dried blood samples obtained by the present method correlated well with those in radioimmunoassay (r=0.964). Concentrations of conjugated l/3-hydroxycholic acid in dried blood spotted onto filter paper in newborns with congenital biliary atresia (737±264 ng/ml) were significantly higher than those of normal newborns (261±190 ng/ml).