Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: twin N-terminal PCR.
The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae.
Wilton
J
J
Microbial Pathogenicity Research Group, Clinical Research Centre, Harrow, Middlesex, UK.
Ala'Aldeen
D
D
Palmer
H M
HM
Borriello
S P
SP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D033862:Iron-Binding Proteins; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins