Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coli and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms. The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms. Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4-dihydroxybenzoic acid was studied in detail. Here, the catalytic number (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The Km values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher Km values for catecholamines than MB-COMT. The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L-methionine (AdoMet). The average dissociation constants Ks were 3.4 and 20.2 microM for MB-COMT and S-COMT, respectively. Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed. Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 [Lundström et al. (1991) DNA Cell. Biol. 10, 181-189] or Met-108 [Bertocci et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1416-1420].(ABSTRACT TRUNCATED AT 250 WORDS)

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Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.

Human genomic DNA fragments containing catechol 0-methyltransferase (COMT) sequences were isolated and the exon-intron structure analysed by sequencing, PCR and comparing to the human COMT cDNA sequences. The gene contains six exons, of which exons 1 and 2 are noncoding. MB-ATG and S-ATG codons, responsible for the initiation of translation of the membranebound (MB) and soluble (S) forms of the enzyme, are located in exon 3. Two distinct COMTspecific transcripts, 1.3 kb and 1.5 kb, were detected in various human tissues and cell lines. Different quantities of the shorter COMT-specific mRNA in the tissues studied suggest a tissue-specific regulation of the COMT gene at transcriptional level. Mapping of the 5' ends of the COMT mRNAs showed that transcription initiates at multiple sites in two separate DNA regions, which are preceded by functional promoter sequences. The proximal promoter (Pl), located between the two translation initiation codons and extending approximately 200 bp upstream of the MB-ATG initiation codon, apparently gives rise to the 1.3-kb S-COMT mRNA (S-mRNA). The distal promoter (P2) is located in a DNA fragment in front of and partly overlapping the transcription-start region of the 1.5-kb transcript, suggesting that it controls the expression of this MB-mRNA. Similarities between the rat and human COMT gene promoters are analyzed.

Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m7GpppNm) structures to generate capped primers 10-13 nu-cleotides long, the initiation of transcription via the incorporation ofa guanosine residue onto the primers, and elongation ofthe viral mRNAs [Plotch, S. [a-32P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both oneand two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription. The unique mechanism by which influenza virus initiates the synthesis ofits mRNA has recently been delineated. Transcription in vitro and in vivo is initiated by a primer derived from RNAs containing a 5'-terminal methylated cap (cap 1) structure m7GpppNm (1)(2)(3)(4)(5). As shown by studies in vitro using the virionassociated transcriptase, these capped RNAs are cleaved 10-13 nucleotides from their 5' ends, preferentially after a purine residue, by a viral endonuclease that requires the presence ofa cap 1 structure in the RNA (6). Most of the capped RNA fragments generated by this endonuclease are most likely the actual primers that initiate viral RNA transcription because they were found to be linked to one or more guanosine residues in transcriptase reactions containing GTP as the only ribonucleoside triphosphate (6). This guanosine incorporation is apparently directed by the penultimate cytosine residue at the 3' end of the eight virion RNA (vRNA) templates (6). In the presence of all four triphosphates, the viral RNA transcripts are then elongated.This entire reaction is catalyzed by purified viral cores (nucleocapsids) (6), which contain four known virus-specific proteins: the nucleocapsid protein (NP), which constitutes the majority (about 92%) of the protein, and the three P proteins (6, 7). Studies with temperature-sensitive virus mutants indicate that at least two of these P proteins are required for transcription (8, 9).We undertook the present study to establish the-actual specific functions of individual P proteins in transcription. Using UV light-induced crosslinking, we found that the P3 protein, the smaller of the two basic P proteins of the WSN strain of influenza A virus, most probably is the protein that recognizes the 5'-terminal cap 1 structure on RNAs, and that the P1 protein, the larger of the two basic P proteins, is the-protein that probably catalyzes the initiation of transcription via the incorporation ofa guanosine residue onto th...