Genetics of NAD metabolism in Salmonella typhimurium and cloning of the nadA and pnuC loci.

The nadA and pnuC loci of S. typhimurium were cloned and found to reside within a 2.2-kilobase region. Two-dimensional O'Farrell gel electrophoresis of the proteins produced after chloramphenicol amplification and subsequent release from chloramphenicol inhibition revealed NadA and PnuC to be 43,000- and 25,000-molecular-weight proteins, respectively. The data indicated that nadA and pnuC represent two distinct genes.     

Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB

The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.     

IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes

Virus replication induces the expression of antiviral type I (IFN-alphabeta) and type III (IFN-lambda1-3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-lambda1 and IFN-lambda3 gene promoters revealed them to be similar to IFN-beta and IFN-alpha genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-kappaB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-lambda1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-beta gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-alpha genes.     

Cloning and nucleic acid sequence of the Salmonella typhimurium pncB gene and structure of nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium, encoding nicotinate phosphoribosyltransferase (NAPRTase), was cloned on a 4.7-kb Sau3A fragment. The gene contains a 1,200-bp open reading frame coding for a 400-residue protein. Amino acid sequencing of the amino-terminal and two interior peptides of the purified protein confirmed the deduced sequence and revealed that the amino-terminal methionine residue was removed, giving a 399-residue mature protein of Mr 45,512. No signal sequence was observed in the predicted NAPRTase primary structure, suggesting that the enzyme is not periplasmic. The protein does not demonstrate clear sequence similarity to the other seven phosphoribosyltransferases of known primary structure and frustrates attempts to define a consensus 5-phosphoribosyl-1-pyrophosphate-binding region. The NAPRTase reaction is ATP stimulated, and the protein contains a carboxy-terminal sequence diagnostic of an ATP-binding site. An inverted repeat of the sequence TAAACAA observed in the proposed promoter region of pncB is also present in the promoter of nadA, which, like pncB, is also regulated by the NadR (NadI) repressor. The sequence may thus define an NadR repressor-binding site.     

Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes.     

Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots

DNA methylation plays an important role in the regulation of gene expression in eukaryotes. In this study, the extent and patterns of DNA methylation were assessed in date palm mother-plants and their off-shoots using the methylation-sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I were present in both ECOR I + HPA II and ECOR I + MSP I lanes; type II were present in ECOR I + HPA II lanes, but not in ECOR I + MSP I lanes; and type III bands were present in ECOR I + MSP I lanes, but not in ECOR I + HPA II lanes. The total numbers of these three types of bands were 782, 55, and 34, respectively. Among these three types of bands, the polymorphic bands were, respectively, 37, 10, and 0. The distribution of polymorphic bands among mother-plants and off-shoots suggests the methylation variation was present in both the mother-plants and off-shoots. Forty- four out of these 47 polymorphic bands show clear difference between mother-plant and off-shoots: 38 were present only in off-shoots and 6 in both mother-plants and off-shoots. Compared to methylation status in mother-plants, the methylation variation during off-shoot growth of date palm can be characterized as a process involving primarily de-methylation. Hypomethylation of DNA in off-shoots, compared with mother-plants, reflects the marked expression of this molecular feature, which may be related to gene expression during off-shoot development. The methylation or de-methylation status of specific loci in the mother-plants and their off-shoots were probably random events.     

Cloning and promoter analysis of the chicken interferon regulatory factor-3 gene

Interferon regulatory factors (IRFs) are a family of DNA-binding proteins involved in mediating the cellular response to interferons (IFNs) and viral infection. Although extensively studied in mammals, IRFs of other vertebrates have been less well characterized. Previously, we cloned chicken interferon regulatory factor-3 (chIRF-3) mRNA, which is rapidly and transiently induced by double-stranded (ds)RNA. The chIRF-3 mRNA encodes a protein distinct from any known mammalian IRF. Here, we show that chIRF-3 is activated additively by type I and type II IFNs. To delineate the sequence elements required to regulate chIRF-3 expression, we cloned chlRF-3 and 0.48 kb of 5' flanking sequence. Computer analysis of the proximal promoter revealed three putative binding sites for nuclear factor (NF)-kappaB, two overlapping interferon-stimulated response elements (ISREs), and an interferon gamma activating sequence (GAS). The presence of both GAS and ISRE consensus sequences in the chIRF-3 promoter is unique among IRF family members. Both type I and II IFNs, as well as dsRNA and IRF-1, trans-activate the promoter in short-term transfection experiments. Mutational analysis of the promoter demonstrated that the putative NF-kappaB binding sites are needed for stimulation by dsRNA but not by either type I or type II IFN and that both the overlapping ISREs and GAS are required for full induction by type I or type II IFN.