title: A mutagenesis method for the addition and deletion of highly repetitive DNA regions: the paradigm of EPIYA motifs in the cagA gene of Helicobacter pylori.
abstract: BACKGROUND
  CagA protein of Western origin Helicobacter pylori isolates contains at its carboxyl-terminal end repeating types of EPIYA motifs, depending on the surrounding sequence, which dictate hierarchic tyrosine phosphorylation. To produce, in an isogenic background, mutant strains expressing CagA protein with variable numbers of EPIYA-C terminal motifs, we have adopted a mutagenesis assay using a megaprimer approach.
  
  
  METHODS
  The H. pylori P12 reference strain containing two terminal EPIYA-C motifs was utilized. Initially, we cloned, full-length cagA gene, next to the Campylobacter jejuni kanamycin-resistance cassette, followed by the 1200-bp region located immediately after cagA gene (metacagA region). Then, we generated a megaprimer consisting of three consecutive copies of the EPIYA-C coding sequence of cagA gene, followed by the 140-bp region of the cagA genomic sequence present immediately after the second EPIYA-C repeat. We utilized these two products to perform a QuikChange mutagenesis assay and were able to obtain all desired combinations of EPIYA-C motifs, followed by Kan(r) cassette and metacagA region. These constructions were used to perform natural transformation of the P12 parental strain, by directional homologous recombination.
  
  
  RESULTS
  We produced isogenic H. pylori strains that express CagA with variable number of EPIYA-C motifs (AB, ABC, ABCCC) and their phosphorylation-deficient counterparts. They exhibited similar growth characteristics to the parental strain, adhered equally well to gastric cells and successfully translocated CagA, following pilus induction.
  
  
  CONCLUSIONS
  Our method can be used in other cases where highly repetitive sequences need to be reproduced.
authors.affiliation: Laboratory of Medical Microbiology, Hellenic Pasteur Institute, Athens, Greece.
authors.forename: Konstantinos S
authors.initials: KS
authors.lastname: Papadakos
authors.forename: Ioanna S
authors.initials: IS
authors.lastname: Sougleri
authors.forename: Andreas F
authors.initials: AF
authors.lastname: Mentis
authors.forename: Dionyssios N
authors.initials: DN
authors.lastname: Sgouras
mesh_terms: D020816:Amino Acid Motifs; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D003470:Culture Media; D004847:Epithelial Cells / Q000382:microbiology; D005753:Gastric Mucosa / Q000166:cytology / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity; D006801:Humans; D016296:Mutagenesis; D011995:Recombination, Genetic; D020449:Repetitive Sequences, Amino Acid; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003470:Culture Media; C080866:cagA protein, Helicobacter pylori