Osamu Yamamoto

Functional Assessment of Two Vitamin D-responsive Elements in the Rat 25-Hydroxyvitamin D3 24-Hydroxylase Gene

Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5′-upstream region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene at −151/−137 and −259/−245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter −291/+9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat −169/−155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element −169/−155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous β-globin promoter. The fragment −291/−102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element −169/−155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.

16S rRNA gene sequence of Rubrobacter radiotolerans and its phylogenetic alignment with members of the genus Arthrobacter, gram-positive bacteria, and members of the family Deinococcaceae.

The nearly complete sequence of the 16S rRNA gene of an extremely highly radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans based on chemical characteristics), was determined by PCR amplification of the genomic DNA followed by cloning of the amplified gene and sequencing by the dideoxynucleotide method. The sequence was aligned with the sequences of members of the genus Arthrobacter and also with the sequences of representatives of the gram-positive bacteria having high G + C contents and the family Deinococcaceae (radioresistant micrococci and their relatives). The results of our phylogenetic analysis confirmed that R. radiotolerans is not a member of the Arthrobacter group and thus supported the previous reclassification. Moreover, although it is radioresistant and has a high G+C content, R. radiotolerans is more closely related to the gram-positive bacteria with high G+C contents than to the radioresistant members of the Deinococcaceae.

Formation of highly fluorescent adducts between 2′-deoxyguanosine and amino acids by ionizing radiation

Gamma-irradiation of aqueous solutions containing DNA and alcohols is known to generate fluorescent products. In this study, aqueous solutions containing 2′-deoxyguanosine (dG) and amino acids with an aliphatic hydroxyl group [serine (Ser) or threonine (Thr)] were irradiated and analyzed for fluorescence. Both irradiated solutions showed a similar fluorescence profile with the excitation maximum ∼310 nm and the emission maximum ∼370 nm. The fluorescence profiles were very close to that of 2-aminopurine, known as a highly fluorescent base. In the irradiation of dG with Ser, two distinct fluorescent products were observed in liquid chromatographic analysis. The major product was further purified by chromatography. Mass spectrometry (MS) showed that the major fluorescent product was a dG adduct bearing a Ser fragment at the C-6 position. In the case of dG with Thr, only one major fluorescent product was observed. The purified product seemed to be a C-6 adduct of dG with a Thr fragment based on MS and NMR analyses. These structural data for the fluorescent products suggest that C-6 adduct formation of dG was the key to generating fluorescence. This is consistent with our previous finding that the adduct between dG and tert-butanol at C-6 is highly fluorescent.