The gene for Selenoprotein A (SPA), designated grdA, of the glycine reductase complex from Clostridium purinolyticum (C.p.)had recently been isolated and characterized. The grdA gene from C.p. was used as a probe for the selection of the homologous gene from Clostridium sticklandii (C.s.). Oligonucleotide primers to the corresponding N-terminus and C-terminus of the gene for C.p. were synthesized and used to amplify the gene by the polymerase chain reaction (PCR) technique. The specific DNA product was used to select a 4,500 base pair fragment from C.s. genomic DNA digested to completion with restriction enzyme Hind HI. The fragment was inserted into the Hind HI site of pUC - 13 and cloned into Escherichia coli. The entire SPA gene of C.s. was found in the 4,500 base pair fragment. The gene was sequenced from both strands. The gene consists of 474 bp and encodes a protein of 158 amino acids with a calculated MW of 16,400. A putative ribosome binding site is located -6 bases upstream from an initiating methionine. A single TGA codon located at 282 bp corresponds exactly to the location of the single selenocysteine determined in a previously identified internal peptide sequence. The internal peptide sequence of 20 amino acids was 100% homologous with that predicted from the gene sequence. The previously identified C-terminal aspartate was also correctly predicted. The gene has two TAA tandem termination codons.
