Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA fingerprinting.
RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei, as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5' mini-exon sequence, to ensure that most of the products contained the 5' end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.
Mathieu-Daudé
F
F
Sidney Kimmel Cancer Center, San Diego, CA 92121, USA.
Welsh
J
J
Davis
C
C
McClelland
M
M
D000818:Animals; D002453:Cell Cycle / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D018869:Heat-Shock Response; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D016053:RNA, Protozoan / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D016053:RNA, Protozoan