Masanori Watahiki

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.”

Isolation of a novel prolactin-like cDNA clone from bovine placenta: Occurrence of new family members

A novel cDNA clone that hybridized to bovine prolactin cDNA was isolated from a bovine (Bos taurus) placental cDNA library and the nucleotide sequence was analyzed. The cDNA clone, named bPLP-III, contained one open reading frame encoding a protein consisting of 239 amino acids. The amino-acid sequence of bPLP-III is 43 and 57% homologous to bovine preprolactin and a bovine prolactin-related protein, bPRC-I, respectively, but only 23% homologous to bovine pregrowth hormone. The predicted mature protein of bPLP-III is distinct from bovine placental lactogens in amino-acid composition, suggesting that bPLP-III is the clone for a new member of prolactin-related mRNA expressed in bovine placenta.

Nucleotide sequence of cDNA and primary structure for hard tail growth hormone

Full-length cDNA for hard tail growth hormone (htGH) has been cloned, and the nucleotide and deduced amino acid sequences have been analyzed. htGH is composed of 188 amino acid residues, and it shows 79, 74, 72, 59, 56, 37, 33 and 30% identity of amino acid with yellow tail, tuna, sea bream, flounder, salmon, blue shark, bullfrog and human GHs, respectively.