Tandem Point Mutations Research Articles

Truncated type IV pilin PilA(108) activates the intramembrane protease AlgW to cleave MucA and PilA(108) itself in vitro.

For alginate production in Pseudomonas aeruginosa, the intramembrane protease AlgW must be activated to cleave the periplasmic domain of anti-sigma factor MucA for release of the sequestered ECF sigma factor AlgU. Previously, we reported that three tandem point mutations in the pilA gene, resulting in a truncated type IV pilin termed PilA(108) with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF), induced mucoidy in strain PAO579. In this study, we purified PilA(108) protein and synthesized a peptide 'SGAGDITFTF' corresponding to C-terminus of PilA(108) and found they both caused the degradation of MucA by AlgW. Interestingly, AlgW could also cleave PilA(108) between alanine(62) and glycine(63) residues. Overexpression of the recombinant FTF motif-bearing MucE protein, originally a small periplasmic polypeptide with the C-terminal motif WVF, could induce mucoid conversion in the PAO1 strain. In all, our results provided a model of activation of AlgW by another protein ending with proper motifs. Our data suggest that in addition to MucA cleavage, AlgW may cleave other substrates.

Mutation Profile of JAK2 Transcripts in Patients with Chronic Myeloproliferative Neoplasias

Here, we describe the JAK2 mutation profile in a series of approximately 20,000 blood samples from patients with clinically suspected myeloproliferative neoplasias. Using a sensitive reverse transcription-PCR and direct sequencing approach on RNA rather than DNA, we detected JAK2 mutations in exons 12-15 in approximately 20% of these patients. We identified new mutations in addition to the known V617F and exon 12 mutations, which were the most common. Most of the novel mutations are located in the pseudokinase domain and therefore are expected to relieve the autoinhibitory function of this domain on JAK2 kinase activity. Our data suggest that molecular testing of JAK2 mutations should not be restricted to the V617F and exon 12 mutations, but perhaps should extend to most of the pseudokinase domain coding region as well. Furthermore, mutation screening using RNA is highly sensitive and could replace DNA-based testing because of the relative abundance of target transcripts and the ease in detecting deletion of the entire exon.