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PMID: 3294797 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping.

Journal of bacteriology ·Vol. 169 ·No. 6 ·1987-06-00 ·Pages 2561-9

Buysse JM, Stover CK, Oaks EV, Venkatesan M, Kopecko DJ

Abstract

Tn5-tagged invasion plasmid DNA (pWR110) from Shigella flexneri serotype 5 (strain M90T) was cloned into the expression vector lambda gt11. Recombinant phage (lambda gt11Sfl) expressing pWR110-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M90T invasion plasmid antigens. Antigens encoded by lambda gt11Sfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitrocellulose-bound plaques of lambda gt11Sfl recombinants, with virulent, wild-type S. flexneri M90T polypeptides in Western blot analyses. lambda gt11Sfl clones directing the synthesis of complete, truncated, and beta-galactosidase fusion versions of three previously identified outer membrane polypeptides (57-, 43-, and 39-kilodalton [kDa] antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M90T invasion plasmid DNA hybridized with lambda gt11Sfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), 43-kDa (ipaC), and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb HindIII fragments contained within a larger (23-kb) BamHI fragment. The ipaH gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.

MeSH Terms
Antigens, Bacterial/genetics Chromosome Mapping Cloning, Molecular Genes Genes, Bacterial Molecular Weight Nucleic Acid Hybridization Plasmids Recombinant Fusion Proteins/genetics Shigella flexneri/genetics,pathogenicity
Chemicals
Antigens, Bacterial Recombinant Fusion Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Buysse J M
Stover C K
Oaks E V
Venkatesan M
Kopecko D J
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29 references, click to expand
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.169.6.2561-2569.1987
Published
1987-06-00
Pages
2561-9
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC212123
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.169.6.2561-2569.1987 PMC Full Text View journal details
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