Homology Cloning of GTP-cydohydrolase I from Fungi and Plants by Reverse-transcription PCR Using a General Set of Degenerate Primers

Abstract

Unconjugated pteridines are present in fungi, algae and plants. However, the functions of pteridines in these organisms are not thoroughly investigated. The biosynthesis follows the same steps as were shown for animals and eubacteria. One possible function of pteridines in these organisms is participation in blue-light reception. To analyze this or other functions of pteridines it would be useful to inhibit pteridine synthesis specifically by genetic engineering. GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis. A comparison of amino acid sequences of GTP-cyclohydrolase I (EC 3.5.4.16) previously known from various species allowed the construction of degenerate primers, based on highly conserved regions. The same consensus primers are able to bind to cDNAs of unrelated eukaryotes. By reverse-transcriptase PCR cDNAs of the conserved C-terminal part of the fungi Neurospora and Phycomyces, the phytoflagellate Euglena and the higher plant Mucuna hassjoo were amplified and cloned. Similarities between the sequences agreed with the evolutionary relationship of the investigated organisms. Various regions strictly conserved between unrelated eukaryotes and bacteria were observed, which could be essential for the function of GTP-cyclohydrolase I.