Using 3' untranslated sequences to identify differentially expressed genes in Leishmania.
A more sensitive screen for Leishmania major genes differentially expressed as the insect stage develops into an infectious form (metacyclogenesis) has been devised. The screen expolits the observation that in kinetoplastid protozoa differentially expressed genes are often associated with unique 3' untranslated regions (UTRs). To obtain probes encoding this region, cDNA is synthesised using an oligo-dT primer containing the universal vectorette sequence in the first strand reaction and an oligonucleotide comprising the spliced leader sequence in the second strand reaction. The cDNAs are then cleaved with Sau3AI, ligated to the vectorette and the 3' UTRs polymerase chain reaction (PCR) amplified using the universal vectorette sequence as the primer. Differential screening with PCR-amplified 3' UTRs uncovered: (1) previously identified metacyclic-specific expressed genes; (2) cloned genes which had not been shown to be differentially regulated; and (3) a new gene identified only as a match to two identical L. major expressed sequence tags (ESTs) that is upregulated in the infectious stage.
Coulson
R M
RM
Department of Pathology, University of Cambridge, UK.
Connor
V
V
Ajioka
J W
JW
D000818:Animals; D001483:Base Sequence; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan; D017403:In Situ Hybridization / Q000379:methods*; D018320:Leishmania major / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D015854:Up-Regulation