High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements.
We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales. (+info)
The chemistry of the reaction determines the invariant amino acids during the evolution and divergence of orotidine 5'-monophosphate decarboxylase.
Orotidine 5'-phosphate (OMP) decarboxylase has the largest rate enhancement for any known enzyme. For an average protein of 270 amino acids from more than 80 species, only 8 amino acids are invariant, and 7 of these correspond to ligand-binding residues in the crystal structures of the enzyme from four species. It appears that the chemistry required for catalysis determines the invariant residues for this enzyme structure. A motif of three invariant amino acids at the catalytic site (DXKXXD) is also found in the enzyme hexulose-phosphate synthase. Although the core of OMP decarboxylase is conserved, it has undergone a variety of changes in subunit size or fusion to other protein domains, such as orotate phosphoribosyltransferase, during evolution in different kingdoms. The phylogeny of OMP decarboxylase shows a unique subgroup distinct from the three kingdoms of life. The enzyme subunit size almost doubles from Archaea (average mass of 24.5 kDa) to certain fungi (average mass of 41.7 kDa). These observed changes in subunit size are produced by insertions at 12 sites, largely in loops and on the exterior of the core protein. The consensus for all sequences has a minimal size of <20 kDa. (+info)
Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.
We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. (+info)
Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa.
Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis. (+info)
Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set.
We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution. (+info)
Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene.
So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described. (+info)
Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius.
Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection. (+info)
Molecular typing of IberoAmerican Cryptococcus neoformans isolates.
A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism analysis with HhaI and Sau96I in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gatii serotypes B and C isolates. (+info)