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

Abstract

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.

A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei.

“…In accordance with the data from Northern blots, the results showed that GRESAG 2.1 and ESAG 2 primarily hybridized with transcripts from procyclic and bloodstream nuclei, respectively. Transcription of GRESAG 2.1 appeared to be fully resistant to 1 mg of a-amanitin per ml, while that of ESAG 2 appeared to be relatively sensitive (panel 1, lanes B and B + am), as reported previously (1,27).…”

“…UV irradiation was performed under the conditions defined by Johnson et al (13 (1,27). In procyclic forms, the early data suggested only ot-amanitin-sensitive transcription of ESAG 2-related sequences (27), but it was subsequently found that this transcription also included a-amanitin-resistant components (24 (Fig.…”

“…The procedures for DNA and RNA isolation, Southern and Northern (RNA) blot hybridization, and DNA cloning were as described before (1,25). cDNA libraries were constructed in XgtlO by the method of Gubler and Hoffman (11), with the Amersham cDNA synthesis and cloning kits.…”

“…All ESAGs are members of large multigene families (16,27). Although ESAGs are only expressed in bloodstream forms, at least some members of ESAG families, genes related to ESAGs (GRESAGs), are expressed elsewhere, under separate transcriptional control (1,2,27).…”

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

“…The procedures for DNA and RNA isolation, Southern and Northern (RNA) blot 1ybridization, as well as DNA cloning, were as described (1,18). cDNA libraries were constructed in lambda gtlO according to Gubler and Hoffman (6) homologous sequences from other telomeres show differences ( Fig.…”