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Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190‐Kilodalton Surface Antigen Gene Primers
Author(s) -
Yan Yansheng,
Uchiyama Tsuneo,
Uchida Takahiro
Publication year - 1994
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1994.tb02139.x
Subject(s) - biology , polymerase chain reaction , nucleic acid sequence , gene , rickettsia rickettsii , inverse polymerase chain reaction , microbiology and biotechnology , dna , virology , rickettsia , genetics , multiplex polymerase chain reaction , spotted fever , virus
The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190‐kDa antigen gene was cloned into M13mp19 RF DNA at the Eco RI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer‐flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii , demonstrating 35 nucleotide substitutions in R. japonica . The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR‐amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease Afa I but two Pst I sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii . The cleavage sites for endonucleases Alu I, Hin fI, and Mun I that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did Pst I. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.