ANALYSIS OF TRYPANOSOME MRNA SYNTHESIS BY GENE TRANSFER

Project Details

Description

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.
StatusFinished
Effective start/end date1/1/918/31/12

Funding

  • National Institutes of Health: $443,340.00
  • National Institutes of Health: $405,360.00
  • National Institutes of Health: $353,617.00
  • National Institutes of Health: $364,227.00
  • National Institutes of Health: $463,458.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $466,883.00
  • National Institutes of Health
  • National Institutes of Health: $466,172.00
  • National Institutes of Health: $430,427.00
  • National Institutes of Health: $386,409.00
  • National Institutes of Health: $375,154.00
  • National Institutes of Health: $39,900.00
  • National Institutes of Health: $106,894.00
  • National Institutes of Health: $405,360.00
  • National Institutes of Health: $173,685.00
  • National Institutes of Health: $24,545.00
  • National Institutes of Health: $284,977.00
  • National Institutes of Health: $243,770.00

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)

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