Derivation of the complete msp4 gene sequence of Anaplasma marginale without cloning.
The gene encoding MSP4, a 31-kDa surface protein of the rickettsia Anaplasma marginale, was completely sequenced without prior cloning of the gene. Degenerate oligodeoxyribonucleotides (oligos) corresponding to the N-terminal amino-acid sequence of MSP4 were used as primers in the polymerase chain reaction (PCR) to amplify a 65-bp fragment of which the central 32 bp was nondegenerate. The 32-bp probe was hybridized to restriction enzyme-digested, genomic A. marginale DNA in Southern blots, and the hybridizing fragment was size selected from agarose gels and ligated into the Bluescript II SK (+/-) plasmid. The ligation reaction mixture was used as a template for PCR, amplified from the 32-bp oligo or its inverse complement to plasmid sequences. The PCR products were sequenced to obtain the entire msp4 gene and surrounding regions. This method of sequencing a gene without previously obtaining a clone for that gene could be beneficial in situations where conventional cloning and screening strategies are inefficient or ineffective.
Oberle
S M
SM
Department of Infectious Diseases, University of Florida, Gainesville 32611-0880.
Barbet
A F
AF
D000595:Amino Acid Sequence; D000709:Anaplasma / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D001426:Bacterial Proteins; D008565:Membrane Proteins; C085498:Msp4 protein, Anaplasma marginale; D009838:Oligodeoxyribonucleotides