The human and animal trypanosomiases have major medical and veterinary consequence throughout equatorial Africa. The salivarian trypanosomes have developed a unique mechanism for antigenic variation, which enables them to avoid elimination by the host's immune responses. Each trypanosome is covered by a surface coat consisting of a closely packed layer of about 10 million molecules of, usually, a single member of a large family of variant surface glycoproteins (VSGs). Antigenic variation is generally accompanied by gene rearrangements, which somehow regulate the expression of new VSGs; either allowing the expression of a pre- existing VSG gene (in situ, or by duplication and transposition, or by chromosomal translocations), or generating a new one by segmental gene conversion. Evidence suggests that individual trypanosomes may contain 1,000 VSG genes (including pseudogenes), but that the capacity for generating antigenic diversity is unlimited, due to a combination of genetic factors and the molecular topology of the surface coat. During the past four years, several laboratories have accumulated substantial data on VSG gene structure, and on chromosomal alterations accompanying antigenic switches. However, we seem little closer to understanding what aspects of VSG gene organization are crucial for the regulation of transcription. Three factors primarily account for this situation. They are, first, the large number of VSG genes involved, and the number of sites in which they can apparently be transcriptionally activated. Secondly, when VSG genes are transcribed, so is a large upstream region, apparently containing co-expressed genes. The third problem is the lack of a system for conventional genetic analysis in trypanosomes. A significant additional problem is the unprecedented manner in which mature mRNAs are formed in trypanosomatids, which may involve dissection of large polycistronic primary RNA transcripts by a trans-splicing reaction, in which a 39-nucleotide mini-exon, or leader sequence, is fused to form the 5' end of the mature mRNA. The primary objectives of this renewal proposal are twofold. First, to continue characterizing VSG gene expression sites, and especially to explore the structure, cellular location and function, of expression site associated genes (ESAGs). Secondly, to develop systems for introducing and perpetuating recombinant molecules in trypanosomatids, which will open several routes of analysis that will be essential to fully understand the regulation of gene expression in trypanosomes.
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