Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum

Abstract

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.

GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing

“…However, the genomic structure of GTP cyclohydrolase I varies considerably between these species : whereas the Saccharomyces cere isiae gene consists of only one exon [22] and the Dictyostelium discoideum gene of two exons interrupted by a 101 bp intron [19], the exon-intron structure of Physarum resembles those found for Drosophila [5] and mammals [20]. As can be seen from Figure 2, Physarum GTP cyclohydrolase I is structured into seven exons.…”

“…Physarum intron 4 is at a homologous position to the only Dictyostelium intron (Figure 2). Physarum introns 2 and 5 have no corresponding introns in the other species studied so far (for a summary of the exon-intron structures of various species see [19]). …”

“…The first DNA sequence available was isolated from E. coli [14]. Subsequently, cDNA sequences were identified from various species, including rat [15], human [16,17], mouse [18] and the cellular slime mould Dictyostelium discoideum [19]. Genomic structures were characterized for D. melanogaster [5], human and mouse [20,21], Dictyostelium discoideum [19] and Saccharomyces cere isiae [22].…”

“…Genomic structures were characterized for D. melanogaster [5], human and mouse [20,21], Dictyostelium discoideum [19] and Saccharomyces cere isiae [22]. The mammalian gene is organized in six exons containing introns of several kilobases [20] and the Drosophila gene in five exons [5], whereas the Saccharomyces gene consists only of one exon [22] and the Dictyostelium gene of two exons separated by a 101 bp intron [19].…”

D-erythro-neopterin biosynthesis in the methanogenic archaea Methanococcus thermophila and Methanobacterium thermoautotrophicum deltaH

“…In the case of E. coli, the required four steps of this transformation are carried out by the folE gene product (6). Genes encoding highly conserved sequences homologous to the sequence of this enzyme have been identified from a wide range of different Eucarya and Bacteria (12), and in a few cases, the proteins have been cloned, sequenced, overexpressed, and shown to catalyze the expected reaction (8,25). Early labeling experiments using archaea indicated that GTP cyclohydrolase I also catalyzed the first committed step in the biosynthesis of the pterin portion of the modified folate, methanopterin (9,20).…”