Ultraviolet irradiation inhibits RNA decay and modifies ribosomal RNA processing in Trypanosoma brucei.

A known effect of ultraviolet radiation on transcription is the arrest of RNA elongation. In Trypanosoma brucei, we show that UV also inhibits RNA decay, leading to specific accumulation of transcripts from the beginning of several transcription units. In addition, UV irradiation changes the pattern of ribosomal RNA processing, probably by altering the order in which the non-coding spacers are excised. These effects are still observed on RNA synthesized more than 2 h after irradiation, and do not depend on protein synthesis.

Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters.

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site.

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

Trypanosoma brucei: enrichment by UV of intergenic transcripts from the variable surface glycoprotein gene expression site.

The expression site for the variable surface glycoprotein (VSG) gene AnTat 1.3A of Trypanosoma brucei is 45 kilobases long and encompasses seven expression site-associated genes (ESAGs) (E. Pays, P. Tebabi, A. Pays, H. Coquelet, P. Revelard, D. Salmon, and M. Steinert, Cell 57:835-845, 1989). After UV irradiation, several large transcripts from the putative promoter region were strongly enriched. We report that one such major transcript starts near the poly(A) addition site of the first gene (ESAG 7), spans the intergenic region, and extends to the poly(A) addition site of the second gene (ESAG 6), thus bypassing the normal 3' splice site of the ESAG 6 mRNA. Since this transcript is spliced, we conclude that UV irradiation does not inhibit splicing but stabilizes unstable processing products. This demonstrates that at least some intergenic regions of the VSG gene expression site are continuously transcribed in accordance with a polycistronic transcription model.

The genes and transcripts of an antigen gene expression site from T. brucei.

The AnTat 1.3A antigen gene expression site of T. brucei was cloned from genomic libraries of the 200 kb expressor chromosome. In addition to the antigen gene, it contains seven putative coding regions (ESAGs, for expression site-associated genes), as well as a RIME retroposon. The polypeptide encoded by ESAG 4 shows homology to yeast adenylate cyclase, and possesses structural features of a transmembrane protein. The expression site is transcribed by a pol l-like polymerase in the parasite bloodstream form only, but sequences similar to ESAGs 5, 4, and 2 are also transcribed constitutively elsewhere, by a polymerase sensitive to alpha-amanitin. Ultraviolet irradiation, which seems to block RNA processing, allows the tentative mapping of a transcription promoter about 45 kb upstream of the antigen gene.

Different allele frequencies in Trypanosoma brucei brucei and Trypanosoma brucei gambiense populations.

Restriction fragment length polymorphism (RFLP) has been analysed in Trypanosoma brucei DNA following hybridization with different DNA probes. This polymorphism seems to be due to allelic variation, and not to variation between sequence duplicates, since the genomic environment of the probed polymorphic fragments is conserved over considerable distances. In an analysis of 35 non-gambiense stocks, we found different combinations of homozygotes and heterozygotes for the four RFLP probes used, in keeping with previous observations that genetic reassortment occurs in T. b. brucei. Moreover, the non-gambiense populations from West and East Africa can be differentiated according to their characteristic allele frequencies. In sharp contrast, we found that the 49 T. b. gambiense stocks, analysed with the same probes, share the same single allelic combination and are all homozygous for each one of the four markers. This characteristic gambiense allele combination is very common among Western non-gambiense isolates, but rare or absent among Eastern ones. Two stocks isolated from man in West Africa turned out to be non-gambiense by all molecular criteria examined, including total nuclear DNA content. Taken together, these observations suggest that human serum-resistant variants may appear among the West African T. b. brucei population, and that T. b. gambiense evolved from one of these resistant variants as a man-adapted subspecies that became genetically isolated from the rest of the West African trypanosome population.

Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei.

In a 7-kilobase (kb) sequence upstream from the 5' barren region, the Trypanosoma brucei AnTat 1.3A expression site carries two putative genes, named ESAG 2 and ESAG 3 for expression site-associated genes, as well as a copy of ESAG 1 (D.F. Cully, H.S. Ip, and G.A.M. Cross, Cell 42:173-182, 1985). At least 3 kb of this expression site exhibits a high degree of homology with the silent telomere carrying the AnTat 1.3A basic copy, whose ESAG 1 is interrupted by stop codons. Like the antigen gene, the region containing the ESAGs is transcribed only in the bloodstream forms, although transcription of 5' barren- and ESAG 2-related sequences also occurs in cultured procyclics. Analysis of steady-state and nascent transcripts suggests a continuous transcription of the whole expression site by an RNA polymerase resistant to alpha-amanitin, possibly initiating at a polymerase I-like promoter located about 17 kb upstream from the antigen gene. This polymerase seems prone to becoming inactivated upon incubation of the trypanosomes at low temperature. The putative protein encoded by ESAG 3 may carry a hydrophobic signal peptide, suggesting interaction with a membrane.

Trypanosoma brucei repeated element with unusual structural and transcriptional properties.

The genome of Trypanosoma brucei contains up to 400 copies of a conserved sequence (TRS, trypanosome repeated sequence). The majority of TRS copies (TRS1) are 5.2 X 10(3) base-pairs (kb) and are flanked by different separate halves of the previously described transposable element RIME (ribosomal mobile element), although a variant copy (TRS2) contains only the central 1.45 kb portion and lacks RIME. TRS1 elements can probably undergo transposition, since they are dispersed in all chromosome size classes and are bordered by direct repeats of about four base-pairs. Some TRS1 elements may contain an open reading frame over almost their entire length (1651 codons), encoding a protein showing homology with reverse transcriptase. TRS probes detect poly(A)+ transcripts of 5 to 9 kb, generated by a polymerase moderately sensitive to alpha-amanitin. Transcription is developmentally regulated. Both TRS and RIME sense transcripts are preferentially synthesized compared to anti-sense transcripts, and are much more abundant in bloodstream forms than in cultured procyclics.