Identification and characterization of the kinetoplastid membrane protein-11 (KMP-11) gene locus from trypanosoma brucei and its genetic deletion
Date
1998
Authors
Bridge, Michael Adrian Anthony Bridge
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Abstract
The kinetoplastid membrane protein (KMP)-11 is an amphipathic membrane associated molecule that was first identified in Leishmania donovani and was subsequently found to have a ubiquitous distribution among kinetoplastid parasites, including African trypanosomes. KMP-11 has been identified, by immunofluorescence, along the length of the flagellum and in the flagellar pocket. A 3500 bp genomic DNA fragment containing the KMP-11 sequence was identified, isolated and subcloned from a Pl bacteriophage library containing T.brucei strain TREU 927 /4 genomic DNA. A comprehensive library for automated DNA sequencing was produced by nebulizing the 3500 bp P1 fragment containing the KMP-11 locus. Seventy-two individual sequencing reads averaging 550bp per read were aligned and gave an 11.5 fold sequence redundancy. This allowed accurate assembly of the highly repetitive KMP-11 sequence. The KMP-11 locus contains a tightly packed array of four highly conserved KMP-11 repeats found within a 2200 bp region. The repeated genes contain only two positions at which the sequences diverge. In both cases transitions have occurred in the third position of the codon and do not alter the predicted amino acid sequence of the protein. The four genes are arranged in two sets of larger repeats that are separated by an intergenic region of 200 bp. This intergenic region is the only non-duplicated portion of the entire KMP-11 locus and was the key to assembling the sequence. Upstream of each of the four genes is a conserved 5' untranslated region containing long poly-pyrimidine rich elements and putative splice acceptor sites that may play an important role in accurate splicing of the KMP-11 polycistronic transcript.
Once the KMP-11 locus had been fully characterized, gene deletion studies were pursued in the hope of elucidating the function of KMP-11. The deletion cassettes contained genomic flanking sequences from the KMP-11 locus surrounding either the neomycin or phleomycin drug resistance gene. The first round of knockouts produced the expected null/+ genotype having replaced one of the two KMP-11 loci with the neomycin drug resistance deletion cassette. Subsequent attempts to produce complete null/null mutants, under a wide variety of conditions, have been unsuccessful and suggest that the full deletion of KMP-11 in Trypanosoma brucei is lethal.