ANALYSIS OF TRYPANOSOME MRNA SYNTHESIS BY GENE TRANSFER

点击翻译按钮获取中文摘要

The human and animal trypanosomiases have major medical and veterinary consequences. Two salient and unusual characteristics of trypanosomes are related to expression of their genes. Mature mRNAs of trypanosomatids are formed by a process that may involve dissection of large polycistronic primary RNA transcripts by a reaction in which a 39 nucleotide mini-exon is spliced in trans to form the 5' end of the mature mRNA. The 39-nucleotide mini-exon initially comprises the 5' end of a short, non-polyadenylated RNA that is transcribed from a set of tandemly reiterated genes. Although the genes that encode the mini-function of specific DNA sequences in gene expression have been stymied by the absence of a genetic system. The trypanosomatids Trypanosoma brucei, Leptomonas seymouri, and Leishmania enrietti recently have been shown to be amenable to DNA-mediated transfection; the latter two can be stably transformed by plasmids that contain drug-resistant markers. The availability of these techniques means that for the first time the cis and trans-acting factors involved in the regulation of trypanosome transcription, splicing and translation are accessible to genetic analyses. Two constitutively expressed genes, the mini-exon donor RNA (medRNA) and phosphoglycerate kinase (PGK B and C) genes probably have essential roles in trypanosomes. The mini-exon is the 5' exon on all mRNAs and may be important for mRNA stability and translation. In T. brucei, the PGK genes encode two slightly different proteins, the PGK B gene product resides in the cytoplasm and the PGK C gene product locates to the glycosome, which is the main site of glycolysis in trypanosomes. Although the nascent RNA levels of the PGK B and C genes are equivalent and their transcription probably is derived from a common, upstream promoter, PGK B mRNA levels are much greater than are those from the PGK C gene. This ratio, found in procyclic (insect form) T. brucei is reversed in bloodstream form organisms. Sequences that regulate the expression of these genes will be dissected using marked PGK genes, in which all normally flanking sequences are present on the transfecting constructs. The function of sequences in modulating the efficiency of mini-exon addition will be studied by assaying wild-type and in vitro synthesized mutant sequences for their ability to serve as splice acceptor signals on the marked PGK gene constructs. Splicing signals within the medRNA will be determined by assaying the ability of mutant medRNAs to function as mini-exon donors to a specific mRNA. Trypanosome promoters have never been defined. The medRNA gene promoter will be identified by mutation analysis of a marked medRNA gene present on a transforming DNA construct. Ultimately, a combination of directed mutagenesis and testing of these sequences in trypanosomes will reveal how trypanosomes regulate the expression of medRNA and the maturation, via mini-exon addition and polyadenylation, of pre-mRNA to specific mRNAs.