Yuko Yoneda

cDNA cloning and primary structure of yellow tail (Seriola quinqueradiata) pregrowth hormone

Full-length cDNA of yellow tail (Seriola quinqueradiata) growth hormone (GH) was cloned from the pituitary gland and nucleotide sequence was analyzed. The cDNA clone contained one open reading frame to encode a preprotein consisting of 204 amino acids. The deduced amino acid sequence shows two possible sites for signal peptide cleavage, suggesting that the mature forms of yellow tail growth hormone consist of 185 or 187 amino acids. Yellow tail growth hormone exhibits a typical structural feature as growth hormone, including four cysteine residues to form two disulfide bonds and other identical amino acids with other vertebrate GHs. Amino acid sequence of yellow tail growth hormone shows homology of approximately 66, 42, 37, and 34% with those of salmon, eel, bovine, and human GHs, respectively. Nucleotide sequence of the coding region of yellow tail growth hormone cDNA shows approximately 58 and 40% homology with those of salmon and human growth hormone cDNAs, respectively.

Recognition Sites of 3′-OH Group by T7 RNA Polymerase and Its Application to Transcriptional Sequencing

When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3′-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3′-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3′-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3′-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3′-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called “transcriptional sequencing.”

The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.