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doi:10.1016/S0166-6851(96)02792-2    
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Copyright © 1997 Elsevier Science B.V. All rights reserved

A survey of the Trypanosoma brucei rhodesiense genome using shotgun sequencing1

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Najib M. A. El-Sayeda, b and John E. Donelsona, b, *

a Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA

b Howard Hughes Medical Institute, Iowa City, IA 52242, USA


Received 9 September 1996;
revised 5 November 1996;
accepted 5 November 1996.
Available online 10 March 1998.

Abstract

A comparison of the efficiency of sequencing random genomic DNA fragments versus random cDNAs for the discovery of new genes in African trypanosomes was undertaken. Trypanosome DNA was sheared to a 1.5–2.5 kb size distribution, cloned into a plasmid and the sequences at both ends of 183 cloned fragments determined. Sequences of both kinetoplast and nuclear DNA were identified. New coding regions were discovered for a variety of proteins, including cell division proteins, an RNA-binding protein and a homologue of the Leishmania surface protease GP63. In some cases, each end of a fragment was found to contain a different gene, demonstrating the proximity of those genes and suggesting that the density of genes in the African trypanosome genome is quite high. Repetitive sequence elements found included telomeric hexamer repeats, 76 bp repeats associated with VSG gene expression sites, 177 bp satellite repeats in minichromosomes and the Ingi transposon-like elements. In contrast to cDNA sequencing, no ribosomal protein genes were detected. For the sake of comparison, the sequences of 190 expressed sequence tags (ESTs) were also determined, and a similar number of new trypanosomal homologues were found including homologues of another putative surface protein and a human leucine-rich repeat-containing protein. We conclude from this analysis and our previous work that sequencing random DNA fragments in African trypanosomes is as efficient for gene discovery as is sequencing random cDNA clones.

Author Keywords: Expressed sequence tag; Genome survey sequence; Trypanosoma brucei rhodesiense

Abbreviations: EST, expressed sequence tag; GSS, genome survey sequence

Article Outline

1. Introduction
2. Materials and methods
2.1. Construction of libraries
2.2. Sequencing and data analysis
2.3. Northern blot analysis
3. Results and discussion
3.1. T. b. rhodesiense genome survey sequences
3.2. Expressed sequence tags
3.3. Comparison of sequencing approaches and concluding remarks
Acknowledgements
References


1Note: Nucleotide sequence data reported in this paper are available in the GenBank™ dbEST and dbGSS databases under accession numbers N45733-N45918 and W88248-W88251 for expressed sequence tags, and B07182-B07505 for genome survey sequences.

*Corresponding author. Tel.: +1 319 3357889; fax: +1 319 3356764; e-mail: jedonels@vaxa.weeg.uiowa.edu


 
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