Poly(ADP-ribose) polymerase-1 (PARP-1 1 ; EC 2.4.2.30) is an abundant nuclear protein that is activated by DNA strand breakage and that catalyzes the covalent attachment of poly-(ADP-ribose) (PAR) from NAD ϩ to numerous nuclear proteins and transcription factors, including histones; DNA polymerase ␣ and ␤; p53; and PARP-1, itself being the major target, via its automodification domain (1, 2). Besides PARP-1, another six PARPs have been identified: short PARP, PARP-2, PARP-3, tankylase-1/2, and vault PARP (2, 3). However, the physiological roles of poly(ADP-ribosyl)ation of nuclear proteins and transcription factors induced by PARPs are not completely understood. The initially identified subtype of the enzyme, PARP-1, has been thought to play a central role in the process of poly(ADP-ribosyl)ation because poly(ADP-ribosyl)ation is markedly reduced in most tissues of PARP-1 null mice (4). Transient poly(ADP-ribosyl)ation by PARP-1 can be induced by a wide variety of environmental stimuli, including reactive oxygen, ionizing radiation, and genotoxic stress (1, 2). Thus, PARP-1 has been suggested to regulate DNA repair (5). On the other hand, overactivation of PARP-1 by massively damaged DNA consumes NAD ϩ and consequently ATP, resulting in necrotic cell death by energy failure (3, 6).There are many reports suggesting that PARP-1 is also involved in regulation of gene expression at the transcriptional step (2, 3). PARP-1 seems to play dual roles in transcription. Poly(ADP-ribosyl)ation of transcription factors such as YinYang 1 (7), RNA polymerase II-associated factors (8), and p53 (9) results in reversible silencing of transcription by impairing the DNA binding of these proteins. In other instances, PARP-1 was found to have only one function, stimulating the DNA binding activity of transcription factors such as Oct-1 (10) and B-Myb (11). Recent reports have also shown that PARP-1 is required for specific nuclear factor-B (NF-B)-dependent gene expression and acts as a coactivator for 14). Indeed, the NF-B-dependent transcription of some inflammatory mediators in response to endotoxin (13) or pro-inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤) (12-14) is almost completely abrogated in PARP-1 null mice. Thus, anti-inflammatory effects of PARP-1 inhibitors have been extensively discussed in relation to various inflammation-related diseases (15, 16). However, the exact biochemical mechanism by which PARP-1 regulates NF-B-dependent transcription is obscure. To date, some groups have reported that the enzyme activity of PARP-1 might directly influence NF-B-dependent transcription. Kameoka et al. (17) showed that poly(ADP-ribosyl)ation markedly suppresses the DNA binding activity of NF-B via direct modification in vitro. demonstrated that the DNA binding activity of NF-B p50 is NAD ϩ -dependent and reversibly regulated by the automodification of PARP-1 under cell-free conditions. In contrast, Hassa et al. (14) concluded that neither the enzyme activity nor the DNA binding

Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'-flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Spl binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Spl could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts.The enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes transformation of arachidonic acid to (5S)-hydroperoxy-6-trans-6,11,14-ciseicosatetraenoic acid (5-HPETE) and further to leukotriene A4 [(5S)-6-oxido-7,9,11-trans-14-cis-eicosatetraenoic acid]. The enzymes participating in leukotriene biosynthesis have been the subject for several recent studies, and cDNA clones corresponding to 5-lipoxygenase have been isolated (for review, see ref. 1). Also, the human 5-lipoxygenase gene has been isolated and characterized (2). Several features of the putative promoter region were characteristic for so-called housekeeping genes (3) (no TATAA or CCAAT, G+C-rich, multiple GGGCGG sequences). This paper describes further studies regarding the 5-lipoxygenase gene promoter.t In particular, a sequence containing five GGGCGG repeats was required for efficient transcription of chloramphenicol acetyltransferase (CAT) gene constructs, and gel-shift assays showed that transcription factor Spl could bind to DNA containing this G+C-rich region.MATERIALS AND METHODS Plasmid Constructions. Plasmid pUCOCAT was constructed from pCAT3M (4) and pUC19 (5). 5-Lipoxygenase gene promoter DNA was excised from recombinant phage 1x12A [5.9 kilobase (kb) Kpn I-BstEII fragment] (2). Insertion into pUCOCAT provided 5LO5900CAT, from which several deletion derivatives were prepared (compare Fig. 2). A detailed description of the plasmid constructions can be obtained from the authors.Cell Culture and DNA Transfection. HeLa and HeLaS3 cells (maintained in this institute) were cultivated in Dulbecco's modified Eagle's medium supplemented with 5% (vol/ vol) fetal calf serum. HL-60 cells (American Type Culture Collection) and K-562 were maintained in RPMI 1640 medium containing 10% (vol/vol) fetal calf serum. HepG2 and RBL1 cells were maintained in Eagle's medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% (vol/vol) fetal calf serum. Plasmids for DNA transfection were prepared by alkaline lysis and purified by CsCI centrifugation. For tr...

The gene for human 5-lipoxygenase has been isolated from three different bacteriophage genomic libraries and a genomic cosmid library. The gene spans >82 kilobases and consists of 14 exons. The size range for the exons is 82-613 base pairs, whereas that for the introns is =z2OO bp to >26 kb. A major site of transcription initiation in leukocytes was mapped to a thymidine residue 65 base pairs upstream of the ATG initiation codon by nuclease S1 protection and primer extension experiments. Other potential minor initiation sites were found. The putative promoter region contains no TATA and CCAAT sequences in the expected positions upstream of the major transcription initiation site but contains multiple GC boxes within a (G + C)-rich region, as does the immediate 5' region of the first intron. Characteristics common to the 5' end of the human 5-lipoxygenase gene and the promoter regions of the housekeeping genes raise important questions concerning the regulation of 5-lipoxygenase gene expression.

A full-length cDNA done encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.

Horizontal gene transfer has been identified in only a small number of genes in Haemophilus influenzae, an organism which is naturally competent for transformation. This report provides evidence for the genetic transfer of the ftsI gene, which encodes penicillin-binding protein 3, in H. influenzae. Mosaic structures of the ftsI gene were found in several clinical isolates of H. influenzae. To identify the origin of the mosaic sequence, complete sequences of the corresponding gene from seven type strains of Haemophilus species were determined. Comparison of these sequences with mosaic regions identified a homologous recombination of the ftsI gene between H. influenzae and Haemophilus haemolyticus. Subsequently, ampicillin-resistant H. influenzae strains harboring identical ftsI sequences were genotyped by pulsed-field gel electrophoresis (PFGE). Divergent PFGE patterns among ␤-lactamase-nonproducing ampicillin-resistant (BLNAR) strains from different hospitals indicated the potential for the genetic transfer of the mutated ftsI gene between these isolates. Moreover, transfer of the ftsI gene from BLNAR strains to ␤-lactamase-nonproducing ampicillin-susceptible (BLNAS) H. influenzae strains was evaluated in vitro. Coincubation of a BLNAS strain (a rifampin-resistant mutant of strain Rd) and BLNAR strains resulted in the emergence of rifampin-and cefdinir-resistant clones at frequencies of 5.1 ؋ 10 ؊7 to 1.5 ؋ 10 ؊6 . Characterization of these doubly resistant mutants by DNA sequencing of the ftsI gene, susceptibility testing, and genotyping by PFGE revealed that the ftsI genes of BLNAR strains had transferred to BLNAS strains during coincubation. In conclusion, horizontal transfer of the ftsI gene in H. influenzae can occur in an intraspecies and an interspecies manner.