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Genetics and Molecular Biology |
Departamento de Inmunologí a, Instituto de Investigaciones Biomé dicas, UNAM, Apartado Postal 70228, 04510 México DF, Mexico1
GBF, German National Research Center for Biotechnology, 38124 Braunschweig, Germany2
Hospital General de México, SSA , Mexico3
MRC Tuberculosis and Related Infections Unit, Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London, UK 4
Author for correspondence: Clara Espitia. Tel: +52 5 6223884. Fax: +52 5 6223369. e-mail: espitia{at}servidor.unam.mx
A clone was isolated by screening of a cosmid library of Mycobacterium tuberculosis with an oligonucleotide designed from the N-terminal sequence of a previously reported proline-rich protein. Characterization of the 4481 bp insert showed the presence of polymorphic CG-repetitive sequences (PGRSs) with an ORF of 2·7 kb, encoding a 81·3 kDa protein (PE- PGRS81). Southern blot analysis and BLAST-p searches revealed several homologous sequences in the genome of M. tuberculosis. The deduced amino acid sequence was highly similar to a stretch of about 98 residues in the N-terminus present in several members of the PE-PGRS family available in the GenBank database, including 100% identity with the partial amino acid sequence of the potential protein encoded by orf3' as well as with the Rv0278c sequence. A neighbour-joining analysis of the 99 PE-PGRS sequences available in the database indicated that PE-PGRS81 is included in a group where its closest relatives are the sequences orf3', Rv0278c, Rv0279c, Rv1759c, Rv3652 and Rv0747. Probing with the complete coding regions of PE- PGRS81 and Rv1759c in Southern blot assays, on samples of genomic DNA from M. tuberculosis H37Rv, Mycobacterium bovis BCG and M. tuberculosis clinical isolates, showed a complex hybridization pattern for all strains. This shows the existence of intrastrain PGRS variability as reported for other PGRS members. In contrast, probing with the short conserved N-terminal region of Rv1759c reduced the hybridization to a single band. This marker allowed identification of M. tuberculosis clinical strains that lack Rv1759c. A recombinant C-terminal fragment of Rv1759c showed fibronectin-binding properties and was recognized by sera from patients infected with M. tuberculosis, suggesting that at least this member of thePE-PGRS is expressed in tuberculosis infection.
Keywords: Mycobacterium tuberculosis, PGRS family genes
Abbreviations: Fn, fibronectin; PGRS, polymorphic GC-repetitive sequence
The GenBank accession number for the sequence reported in this paper is AF071081.
a Present address: Dept of Molecular Microbiology and Immunology, Johns Hopkins University, School of Hygiene and Public Health, 615 N Wolfe Street, Baltimore, MD 21205, USA.
b Present address: Dept of Immunology, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK.
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