Giuseppe Ianniciello

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

ASulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (λG1–λG6) and one of them (λG4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of theS. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.

The nucleotide sequence of the gene coding for the elongation factor 1α in Sulfolobus solfataricus. Homology of the product with related proteins

The cloning and sequencing of the gene coding for the archaebacterial elongation factor 1 alpha (aEF-1 alpha) was performed by screening a Sulfolobus solfataricus genomic library using a probe constructed from the eptapeptide KNMITGA that is conserved in all the EF-1 alpha/EF-Tu known so far. The isolated recombinant phage contained the part of the aEF-1 alpha gene from amino acids 1 to 171. The other part (amino acids 162-435) was obtained through the amplification of the S. solfataricus DNA by PCR. The codon usage by the aEF-1 alpha gene showed a preference for triplets ending in A and/or T. This behavior was almost identical to that of the S. acidocaldarius EF-1 alpha gene but differed greatly from that of EF-1 alpha/EF-Tu genes in other archaebacteria eukaryotes and eubacteria. The translated protein is made of 435 amino acid residues and contains sequence motifs for the binding of GTP, tRNA and ribosome. Alignments of aEF-1 alpha with several EF-1 alpha/EF-Tu revealed that aEF-1 alpha is more similar to its eukaryotic than to its eubacterial counterparts.

The first nucleotide sequence of an archaeal elongation factor 1β gene

Abstract An archaeal elongation factor 1β gene has been isolated for the first time from a Sulfolobus solfataricus genomic library. The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1β), the other one encoding a nonidentified product (ORF 115). The amino acid sequences of segments at the N-and C-terminal of the translated SsEF-1β were identical to those determined for the native protein. Northern and Southern analyses showed that the SsEF-1β gene is represented in S. solfataricus by a unique sequence. Compared to eubacterial or eukaryal corresponding genes the SsEF-1β is much shorter.

Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus.

The present article is a review of the work done on the elongation factors EF-1 alpha, EF-2 and EF-1 beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The molecular, physical and biochemical properties of the intact, truncated, mutant or chimeric forms are described and compared.