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To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the "oligo-capping" method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA + /Inr + PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and the TM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

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Determination of the mRNA start site is the first step in identifying the promoter region, which is of key importance for transcriptional regulation of gene expression. The 'oligo-capping' method enabled us to introduce a sequence tag to the first base of an mRNA by replacing the cap structure of the mRNA. Using cDNA libraries made from oligo-capped mRNAs, we could identify the transcriptional start site of an individual mRNA just by sequencing the 5'-end of the cDNA. The fine mapping of transcriptional start sites was performed for 5880 mRNAs in 276 human genes. Contrary to our expectations, the majority of the genes showed a diverse distribution of transcriptional start sites. They were distributed over 61.7 bp with a standard deviation of 19.5. Our finding may reflect the dynamic nature of transcriptional initiation events of human genes in vivo.

The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol · min ؊1 · mg of protein ؊1 . The apparent K m values for PCE and TCE were 105.7 and 535.3 M, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.Recently, a number of studies have described reductive dehalogenation by bacteria which occurs during the initial attack of halogenated hydrocarbons. Such bacteria can grow by anaerobic respiration, a process that has been referred to as halorespiration or dehalorespiration (6, 9, 11). Several dehalorespiring bacteria have been reported to catalyze the reductive dehalogenation of tetrachloroethene (also referred to perchloroethene [PCE]). Most of these bacteria reduce PCE or trichloroethene (TCE) to cis-1,2-dichloroethene (cis-DCE) (5,10,18,19). Dehalococcoides ethenogenes strain 195 is able to catalyze the reductive dehalogenation of PCE to ethene (16,17).PCE dehalogenases have been purified, and their genes were cloned from some anaerobic bacteria such as Dehalospirillum multivorans (21-23), Desulfitobacterium sp. strain PCE-S (20), Dehalobacter restrictus (26, 27), and D. ethenogenes 195 (13). The molecular characterization of the chloroethene reductive dehalogenases from phylogenetically distinct bacteria has revealed significant similarities in molecular masses (51 to 65 kDa) and functional domains, but it is also true that similarities of the entire amino acid sequences are surprisingly low among these enzymes. The PCE dehalogenase of Clostridium bifermentans DPH-1 was recently purified and sequenced (24). This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of DCE isomers along with PCE and TCE. All the PCE dehalogenase genes except pceC from C. bifermentans DPH-1 (24) characterized to date are preceded by twin arginine signal sequences (2,3,25). This class of proteins contains corrinoid and two Fe/S clusters as prosthetic groups (11, 31). The PCE dehalogenase genes were found to be linked with open reading frames (ORFs) coding for small hydrophobic proteins containing two or three transmembrane helices (14,23,28,30). It has been proposed that the PceB protein in D. multivorans might act as a membrane anchor that functionally links the dehalogenase to the respiratory c...

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despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphAI and todCl) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todCIC2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todCl into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway.Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into tolueneutilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli

Background: Serine residues in extensin, a cell wall protein in plants, are glycosylated with O-galactose. Results: Genes encoding the serine O-␣-galactosyltransferase were isolated from Chlamydomonas reinhardtii and plants and were characterized. Conclusion: The serine O-␣-galactosyltransferases belong to a novel glycosyltransferase class existing only in the plant kingdom. Significance: The identification of novel glycosyltransferases contributes to elucidation of how protein glycosylation has evolved in plants.