Transcription Of Variant Surface Glycoprotein Genes Research Articles

Biological Variation Among African Trypanosomes: I. Clonal Expression of Virulence Is Not Linked to the Variant Surface Glycoprotein or the Variant Surface Glycoprotein Gene Telomeric Expression Site

The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.

Co-duplication of a Variant Surface Glycoprotein Gene and Its Promoter to an Expression Site in African Trypanosomes

Activation of the metacyclic variant antigen type 7 (MVAT7) variant surface glycoprotein (VSG) gene in bloodstream Trypanosoma brucei rhodesiense involves a duplicative transposition of the gene. The DNA transposition unit extends from a site approximately 3.0 kilobases upstream of the VSG gene through the coding region and includes a 73-base pair sequence that possesses promoter activity in transient transfections. This MVAT7 promoter has 80% identity to a previously characterized promoter for the MVAT4 VSG gene. Nuclear run-on assays demonstrate that the MVAT7 promoter is active in MVAT7 bloodstream organisms and that its transcript is synthesized by an RNA polymerase resistant to alpha-amanitin, consistent with previously published reports regarding VSG gene transcription. The transcription start site was identified by primer extension studies and a modified rapid amplification of cDNA ends protocol. Selective mutational analysis of the MVAT7 promoter showed that two conserved trinucleotide regions are important for full promoter function. This study demonstrates that the MVAT7 VSG gene is co-duplicated with its promoter and transcribed into a monocistronic precursor RNA.

Trypanosoma brucei variant-specific glycoprotein gene chromatin is sensitive to single-strand-specific endonuclease digestion

Active variant surface glycoprotein (VSG) gene chromatin is preferentially digested by the restriction enzyme HinfI in nuclei of bloodstream variants of Trypanosoma brucei. HinfI sensitivity of VSG gene chromatin is not observed in nuclei of relapse variants in which the VSG gene has been inactivated in situ. Active VSG gene chromatin is preferentially degraded by the single-strand-specific endonucleases S 1 and Bal31. This sensitivity is not the result of pre-existing single-strand breaks or a detectably altered nucleosomal organization. Trypanosome nuclei in which the run-on transcription of VSG genes has been specifically shut down have been used to show that Hinfl and Bal31 sensitivity is not dependent upon continued transcription of the VSG gene. The presence of single-stranded DNA regions within VSG gene chromatin is consistent with a model in which VSG genes are activated by increased torsional stress.

RNA Turnover in Trypanosoma brucei

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

RNA turnover in Trypanosoma brucei.

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.

Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei.

The gene encoding variant surface glycoprotein 221 in Trypanosoma brucei is located adjacent to a chromosome end and can be activated with or without a concomitant gene duplication. To test whether transcription initiates within the cloned segment of the 221 gene, we analyzed nascent and stable transcripts. We show here that the 221 coding region and 8.5 kilobases of adjacent upstream DNA are transcribed into nascent RNA at a similar rate when gene 221 is activated without duplication. Since only part of this transcribed upstream segment is transferred with the coding region to another telomere upon duplicative activation of gene 221, we infer that initiation of variant surface glycoprotein gene transcription occurs outside the gene segment that moves into an expression site by gene conversion. Our analysis shows that part of the variant surface glycoprotein 221 transcription unit consists of an unusual 3.5-kilobase tandem array of ca. 50 repeat segments and that a rearrangement in this array accompanies the nonduplicative activation of gene 221. A variant surface glycoprotein pseudogene is located within the transcription unit of gene 221, and we discuss models that account for this unusual situation.

Tandem repetition of the 5′ mini-exon of variant surface glycoprotein genes: A multiple promoter for VSG gene transcription?

Activation of some variant surface glycoprotein (VSG) genes involves a duplicative transposition to an expression site, which completes the gene by addition of a mini-exon coding for the 5′ 35 nucleotides of VSG mRNAs. Using a 22 nucleotide probe we have found some 200 copies of the mini-exon on a tandemly arranged 1.35 kb repetitive element. This repeat is highly conserved in three trypanosome species. The mini-exon on the repeat is flanked by a 5′ splice site that resembles the consensus sequence. We have not found a single mini-exon within 10 kb of the transposed VSG gene exon in the expression site. We propose a model in which the arrays of mini-exon repeats function as a repetitive promoter for efficient transcription of VSG genes.