Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10 -7 per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

We have developed a procedure for the affinity purification of small nuclear ribonucleoproteins (snRNPs) of Trypanosoma brucei (U2 and U4/U6 snRNPs), which are essential for trans splicing. Each of these snRNPs can be specifically and efficiently selected from T. brucei extracts through biotinylated antisense 2'-0-methylated RNA oligonucleotides immobilized on streptavidin-agarose. Protein analysis revealed a set of five low molecular weight polypeptides common to the U2 and U4/U6 snRNPs and the spliced leader RNP. In addition, several U2 and U4/U6 snRNP-specific protein components were identified. Using monoclonal antibodies against human snRNP proteins, we could not detect any significant cross-reaction with the trypanosomal U2 snRNP proteins. Thus, the trypanosomal snRNPs exhibit principal differences from the higher eukaryotic snRNPs not only in their RNA but also in their protein components.trans splicing in trypanosomatids and nematodes follows a two-step mechanism of cleavage-ligation reactions analogous to nuclear pre-mRNA splicing (cis splicing) of other eukaryotes. In trypanosomes, a mini-exon derived from the 5' end of the spliced leader (SL) RNA is trans-spliced onto every protein-coding exon of long, polycistronic pre-mRNAs (reviewed in refs. 1 and 2). As in cis splicing, small nuclear RNAs (snRNAs) function as essential splicing factors in trans splicing (3): trypanosomal U2, U4, and U6 RNAs have been identified (4-6) that are in the form of U2, U4/U6, and U6 small nuclear ribonucleoproteins (snRNPs) (7, 27); however, no U5 RNA homologue is known in trypanosomes. The SL RNP can be considered a chimeric molecule that possesses characteristics of an snRNP and carries the mini-exon sequence at the SL RNA 5' terminus, thereby possibly performing the role of the U1 snRNP (8-10). In contrast to the mammalian snRNPs (reviewed in ref. 11), we know little about the RNA-protein structure of the trypanosomal snRNPs and their mechanism of action during trans splicing. The Trypanosoma brucei snRNAs deviate in many aspects from their strongly conserved eukaryotic counterparts (reviewed in ref.2). Thus, through the characterization and comparison of cis-and trans-spliceosomal snRNPs, we expect to gain more information about the specific requirements of trans splicing. We have developed procedures for the affinity purification of SL, U2, and U4/U6 snRNPs on the basis of biotinylated 2'-O-methyl (2'-OMe) RNA oligonucleotides, resulting in the identification of a set of five common proteins and additional, snRNP-specific proteins. Interestingly, no immunological relationship of these proteins with mammalian snRNP proteins could be detected.

β-d-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. InTrypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of theT. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.