Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups.

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12.

The pyrC and pyrD genes of Escherichia coli K-12 encode the pyrimidine biosynthetic enzymes dihydroorotase and dihydroorotate dehydrogenase, respectively. A highly conserved sequence in the promoter regions of these two genes is similar to the pur operator, which is the binding site for the purine repressor (PurR). In this study, we examined the role of PurR in the regulation of pyrC and pyrD expression. Our results show that pyrC and pyrD expression was repressed approximately twofold in cells grown in the presence of adenine [corrected] through a mechanism requiring PurR. A mutation, designated pyrCp926, which alters a 6-base-pair region within the conserved sequence in the pyrC promoter eliminated PurR-mediated repression of pyrC expression. This result indicates that PurR binds to the pyrC (and presumably to the pyrD) conserved sequence and inhibits transcriptional initiation. We also demonstrated that the pyrCp926 mutation had no effect on pyrimidine-mediated regulation of pyrC expression, indicating that pyrimidine and purine effectors act through independent mechanisms to control the expression of the pyrC and pyrD genes.

Molecular cloning and transcript mapping of the dihydroorotate dehydrogenase dhod locus of Drosophila melanogaster.

The dhod locus encodes dihydroorotate dehydrogenase, the fourth enzymatic step of de novo pyrimidine biosynthesis. This locus was cloned previously by a chromosome walk in cytogenetic region 85A. The location of dhod within 85A DNA has been determined by mapping two rearrangement mutations to a small DNA region. A nearly full-length cDNA clone of the dhod transcript was isolated and partially sequenced, to confirm its identity. The cDNA clone was also used to map the transcribed DNA. A 1.5 kb dhod RNA is described which is most abundant in embryos and displays minor length heterogeneity in pupae and adults. The developmental expression of this transcript is discussed relative to the expression of dihydroorotate dehydrogenase activity and other genes of the pyrimidine biosynthetic pathway.

Genetic and molecular mapping of chromosome region 85A in Drosophila melanogaster.

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.

Developmental control of the expression of the dihydroorotate dehydrogenase and UMP synthase genes in Dictyostelium discoideum.

Developmental variations in the expression of two genes of the de novo pyrimidine biosynthetic pathway have been examined in Dictyostelium discoideum. One gene, DdPYR4, encodes the dihydroorotate dehydrogenase (EC 1.3.3.1); the other, DdPYR5-6, encodes the UMP synthase which in D. discoideum is a bifunctional enzyme harboring both the orotate phosphoribosyl transferase activity (EC 2.4.2.10) and the OMP decarboxylase activity (EC 4.1.1.23). The relative amount of mRNA for both genes has been estimated by hybridization with the previously cloned DNAs and compared with the amount of actin mRNA. The level of both mRNAs is dramatically reduced after 4 h of development and remains at a low level later in development. In contrast to these variations, the specific activity of the enzymes encoded by these genes during development is similar to that measured during exponential growth. These results lead us to propose that DdPYR4 and DdPYR5-6 genes encode for relatively stable proteins and that their synthesis is reduced to maintain a constant level of enzymes in non-growing cells. This mode of regulation could apply to a large number of housekeeping genes.

Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase.

Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Escherichia coli carrying the pyrD gene on a multicopy plasmid. The preparation had a specific activity of 120 mumol min-1 mg-1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr = 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors. The complete sequence of 1357 base pairs of an EcoRI-EcoRI DNA fragment containing the pyrD gene was established. Dihydroorotate dehydrogenase is encoded by a 336-triplets open reading frame. The molecular mass (Mr = 36732), the amino acid composition and the N-terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292-303, i.e. Ile-Ile-Gly-Val-Gly-Gly-Ile-Asp-Ser-Val-Ile-Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed. By the S1-nuclease technique an mRNA start was mapped 34 +/- 2 nucleotide residues upstream of the beginning of the coding frame of pyrD. The leader region contains no similarity to the attenuators of the pyrB and pyrE genes of E. coli.

Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast.

A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity. The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000. The gene shows preferential use of codons with weak pairing forces. Eleven codons, mainly those with a G in the third position, are absent. The flanking sequences are unusually rich in A + T (80%). Several direct and inverted repeats exist in the 5' flanking sequence.

The Dhod locus of Drosophila: mutations and interrelationships with other loci controlling de novo pyrimidine biosynthesis.

Mutations at the Dhod locus have been isolated following ethylmethanesulfonate mutagenesis. These mutants express those phenotypes common to other mutations of the de novo pyrimidine pathway: specific wing and leg defects and female sterility. Dihydroorotate dehydrogenase activity is severely reduced in all Dhod mutants, whereas levels of the other pathway enzymes are largely unaffected. The twelve Dhod mutations described here comprise a single complementation group. All of these mutations are nonlethal and the collection includes apparent amorphic as well as hypomorphic alleles. These results are discussed relative to the properties of the complex loci that encode the other steps of de novo pyrimidine biosynthesis.

Expression of a cloned Saccharomyces cerevisiae gene (URA1) is controlled by a bacterial promoter in E. coli and by a yeast promoter in S. cerevisiae.

The expression of a cloned yeast URA1 gene in Escherichia coli and in Saccharomyces cerevisiae was studied. In E. coli, only one orientation of the cloned yeast DNA segment inserted into the bacterial vector (pBR322) allows URA1 expression. Moreover, the permissive orientation changes with the cloning site. The absence of URA1 expression in E. coli can be corrected by the spontaneous integration into the cloned yeast DNA of a 0.9-kb bacterial DNA. Several copies of such a bacterial IS element have been detected in the host E. coli genome. The results strongly suggest that, in E. coli, transcription of the yeast URA1 needs a prokaryotic promoter for its initiation. In S. cerevisiae, the expression of non-chromosomally cloned URA1 does not depend on the orientation of the cloned fragment. Furthermore, it remains under the control of a nuclear regulatory gene (pprX-1) which constitutively enhances the expression of URA1 as well as URA3 at the transcriptional level. Therefore, in S. cerevisiae, transcription of non-chromosomally cloned URA1 involves a physiological yeast promoter cloned along with the structural part of the gene.

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster.

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.

Cloning of a eukaryotic regulatory gene.

From a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast (S. cerevisiae) genome, a DNA fragment containing the regulatory gene PPRI was cloned by complementation of a non-inducible ppr1 mutation which confers to the cells an increased sensitivity to 6-azauracil. Cells containing the cloned DNA regained the ability to induce the synthesis of URA1 and URA3 gene products controlled by PPR1. A physical map has been constructed and the study of subcloned restriction endonuclease fragments from the original yeast DNA fragment allowed us to localize the wile-type PPR1 regulatory gene within a 3 kilobase-pair region. The ppr1 RNA level was measured and the hybridization data indicate in a wild-type strain a low efficiency of transcription of PPR1 as compared to the structural URA3 gene, without effect of inducing conditions.

Characterization of an autosomal rudimentary-shaped wing mutation in Drosophila melanogaster that affects pyrimidine synthesis.

A new autosomal mutation, rudimental (ral), which causes rudimentary-shaped wings in Drosophila melanogaster, has been isolated following ethyl methanesulfonate (EMS) mutagenesis. The wing phenotype of rudimental is identical to that of the X-linked rudimentary (r) mutation, which affects the first three enzymes in the pyrimidine biosynthetic pathway. The autosomal mutant maps very close to ebony (3-70.7) at 70.42 on the right arm of chromosome 3. Analysis of the enzyme activities of orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) indicates that the rala26a allele has less than wild-type activity for both enzymes. This result is discussed in light of the fact that the OPRTase and ODCase activities are part of an enzyme complex, as are the carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which are encoded by the complex rudimentary locus. We suggest that rudimental is also a complex lucus.

Cloning of the URA1 gene of Saccharomyces cerevisiae.

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of Chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity.

A mutant (A204) of Chinese hamster ovary cells (CHO-K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3,3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild-type cells. Analysis of cell-free extracts of mutant and wild-type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild-type activity. this evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.