Structure of human phenylethanolamine n-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672–7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5′ terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.     

Nucleotide sequence of Suncus murinus immunoglobulin mu gene and comparison with mouse and human mu genes

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [-³³P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly⁸⁴³ and Met⁸⁹⁵ of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.     

Induction of Cytoplasmic Streaming and Movement of Chloroplast Induced by L-Histidine and its Derivatives in Leaves of Egeria densa

Rotational cytoplasmic streaming in leaves of Egeria densa wasinduced by light as well as by L-histidine (L-His). During bothtreatement with light and with L-His chloroplasts on the periclinalface were dislodged and moved to the anticlinal face where rotationalcytoplasmic streaming occurred. The effective concentrationof L-His was about 0.01 mM and the effect was almost saturatedat 0.1 mM. A derivative of L-His, 3-methyl-L-histidine, wasslightly less effective than L-His. By contrast, 1-methyl-L-histidinewas almost ineffective for induction of streaming, not onlyin Egeria but also in Vallisneria. Our resutlts are in markedcontrast to Fitting's result (1936) that 1-M-L-His is more effectivethan L-His. In Egeria, 1-methyl-L-His counteracted the stimulativeeffect of L-His. 1-Methyl-L-His penetrated into leaf cells ofEgeria to the same extent as 3-methyl-L-His and to a greaterextent than L-His. This observation excludes the possibilitythat the impermeability of leaves to 1-M-L-His might be responsiblefor its ineffectiveness. 1-M-L-His did not interfere with photodinesis.Differences in the mechanism of induction of rotational streamingby L-His and by light are discussed. ⁴ Present address: Fukui Institute of Technology, Gakuen, Fukui,910 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Expression of four types of human tyrosine hydroxylase in COS cells

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1-4), which have structural diversity only in the N-terminal region. We attempted expression of the type 1-4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. (c) 1993 John Wiley & Sons, Inc.