Trypanosomes which are the etiological agents of devastating diseases both in man and in cattle display many unusual mechanisms of gene expression. At present little is known about their transcriptional and post-transcriptional control of gene expression, about the specific DNA sequences and protein factors involved in transcription, and about the mechanism of the trans-splicing reaction. The long term goal of this proposal is to unravel the physiology of transcription and trans-splicing in Trypanosoma brucei and to identify parasite-specific functions which might be exploited against the parasites themselves. We plan to: 1) identify what regulatory circuits determine the final output of stable RNAs from the calmodulin gene locus. Transcriptional control and the presence of unique or multiple promoters will be assayed by run-on transcription experiments. At the same time the stability of the various transcripts will be determined by measuring RNA decay rates and we will investigate the possibility that trans-splicing might be coupled to transcription. 2) establish an in vitro system capable of accurately transcribing trypanosome genes. We will initially use Crithidia fasciculata as a model system, because this organism is ideally suited for biochemical studies. Only the availability of an in vitro transcription system will allow the functional analysis of trypanosome promoter architecture and of the factors involved in the transcription of various genes. The information obtained with the Crithidia system will then be applied to establish a T.brucei in vitro extract. The T.brucei DNA templates to be used for in vitro transcription will be designed utilizing the information obtained by in vitro run-on assays and by in vivo footprinting experiments. 3) clone cDNAs coding for the immunodominant antigens of T.Brucei small nuclear ribonucleoprotein particles to obtain information about the primary structure of the proteins and to be able to generate polyclonal monospecific antibodies. These antibodies will be used to investigate the structure of snRNPs and their binding to synthetic pre-mRNA substrates. 4) identify the molecular defect(s) which preclude the utilization of T.brucei pre-mRNA 3' splice sites in a mammalian splicing extract. It will be of particular interest to determine whether the malfunctioning of trypanosome splicing substrates is sequence specific. If we succeed in correcting the defect(s), it will become possible to reconstitute trans-splicing using a mammalian extract. Together, these experiments will provide the basis for a detailed understanding of the mechanism of gene expression and trans-splicing in T.brucei cells.
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