A. W. C. A. Cornelissen

Chromosome rearrangements in trypanosoma brucei

We have studied chromosome rearrangements in T. brucei using pulsed field gradient gel electrophoresis to separate chromosome-sized DNA molecules. We detect size changes in a set of small chromosomes (200-700 kb) at a frequency of 10(-5) to 10(-6) per trypanosome division; this results in a radical difference in the size distribution of these chromosomes in different T. brucei isolates. Several of these chromosome rearrangements can be related to a change in the expression of surface antigen genes. Such rearrangements may be undetectable by standard gel electrophoresis and Southern blot analysis because the DNA segment transferred is too large to detect the breakpoint with the antigen gene probe. We also provide additional evidence for the notion that transcription of protein-coding genes in T. brucei and related flagellates is discontinuous. The possibility that gene rearrangements are essential for all changes in variant surface gene expression remains open.

Chromosomes of kinetoplastida.

We have compared chromosome‐sized DNA molecules (molecular karyotypes) of five genera (nine species) of kinetoplastida after cell lysis and deproteinization of DNA in agarose blocks and size fractionation of the intact DNA molecules by pulsed field gradient (PFG) gel electrophoresis. With the possible exception of Trypanosoma vivax and Crithidia fasciculata, all species have at least 20 chromosomes. There are large differences between species in molecular karyotype and in the chromosomal distribution of the genes for alpha‐ and beta‐tubulin, rRNA and the common mini‐exon sequence of kinetoplastid mRNAs. In all cases, the rRNA genes are in DNA that is larger than 500 kb. Whereas T. brucei has approximately 100 mini‐chromosomes of 50‐150 kb, only few are found in T. equiperdum; T. vivax has no DNA smaller than 2000 kb. As all three species exhibit antigenic variation, small chromosomes with telomeric variant surface glycoprotein genes cannot be vital to the mechanism of antigenic variation. The apparent plasticity of kinetoplastid genome composition makes PFG gel electrophoresis a potentially useful tool for taxonomic studies.

Characteristics of trypanosome variant antigen genes active in the tsetse fly

Trypanosoma brucei contains a repertoire of more than 100 different genes for Variant Surface Glycoproteins (VSGs). A small and strain-specific fraction of these genes is expressed in the salivary glands of the tsetse fly (M-genes), giving rise to metacyclic Variable Antigen Types (M-VATs). Antibodies produced in a chronic trypanosome infection initiated by syringe inoculation of bloodstream forms into mammals (i.e. against B-VATs), will react with most of the M-VATs suggesting that these B-VATs express VSG genes that are similar or identical to M-genes. We have cloned DNA complementary to the VSG mRNA of four of such B-VATs and used this to characterize the corresponding VSG genes. In three of the four VATs we find a single VSG gene hybridizing with the cDNA probe and we provide supporting evidence that this gene is expressed as an M-gene. In the bloodstream repertoire these genes appear to be activated by duplicative translocation to another telomere. In all four variants the putative M-genes are telomeric and in the three cases where the location of the genes on chromosome-sized DNA molecules could be determined, the genes were located in large DNA, whereas the majority of the telomeric VSG genes are in chromosomes less than 1000 kb. Our results are best explained by models for M-gene activation involving telomeric expression sites for these genes which are separate from those used by bloodstream forms. The implications of these results for vaccination are discussed.

Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III.

To analyze transcription in Typanosoma brucei, we have characterized the trypanosomal RNA polymerases. Here we present our results, which allow a discrimination between the different classes of RNA polymerases in nuclear run‐on experiments by polymerase inhibitors and Mn2+ dependence. We also describe the separation of trypanosomal RNA polymerases by chromatography, demonstrating that T. brucei contains RNA polymerases I‐III. The outcome of our experiments suggests that the VSG genes of T. brucei are not transcribed by RNA polymerase I, as previously reported, but by RNA polymerase II. We propose that an additional factor modifies RNA polymerase II, resulting in the alpha‐amanitin‐resistant transcription of VSG genes. Our data also suggest that the mini‐exon genes, which encode the 5′ end of each trypanosomal mRNA, are probably transcribed by RNA polymerase III.

Trypanosoma brucei: a surface antigen mRNA is discontinuously transcribed from two distinct chromosomes

The mRNAs for variant surface glycoproteins (VSGs) and many other proteins in Trypanosoma brucei start with the same sequence of 35 nucleotides, encoded by a separate mini‐exon. There are approximately 200 mini‐exon genes per trypanosome and these are highly clustered on large chromosomes. We have found two trypanosome variants that express a VSG gene located on a small, 225‐kb chromosome. Each gene yields a mRNA containing the 35‐nucleotide sequence even though the 225‐kb chromosome does not contain a complete mini‐exon gene. These results provide a strong support for the hypothesis that transcription of protein‐coding genes in trypanosomes is discontinuous.

Effect of 3-aminobenzamide on antigenic variation of Trypanosoma brucei

African trypanosomes, like Trypanosoma brucei, depend on antigenic variation to evade the immune response of the vertebrate host. An antigenic switch corresponds to the activation of a variable surface glycoprotein (VSG) gene from a large silent repertoire. Most switches require the duplicative transposition of a VSG gene, which involves strand breaks in DNA and subsequent repair. The nuclear enzyme adenosine-diphosphoribosyl transferase (ADPRT), which is dependent on the presence of DNA strand breaks for its activity, might be involved in this process because it has a regulatory role in DNA repair in all eukaryotic cells studied so far. In previous work, the presence of ADPRT activity was demonstrated in T. brucei. Moreover, it was also shown in isolated trypanosomes the ADPRT activity, which is stimulated by the induction of DNA strand breaks, could be blocked by the competitive inhibitor 3-aminobenzamide. Here we report experiments using rats which were infected with small numbers of T. brucei expressing VSG gene 118. After two days, the rats were coupled to a continuous intraperitoneal infusion system administrating 3-aminobenzamide in 0.9% NaCl (81.4 mM) at a rate of 0.65 ml/hr/rat for a period of up to five days. Control rats received only a 0.9% NaCl infusion. At days 1, 3 and 5, 250 microliters blood was obtained from a tail artery. Plasma 3-aminobenzamide was determined using a new high performance liquid chromatography method, developed for these experiments. In most rats the plasma concentrations were maintained between 0.8 and 1.2 mM. The rate of antigenic switching was determined by quantitating the fraction of trypanosomes that had lost their VSG 118 coat, using antibody against VSG 118 and a limiting dilution in mice. The average switching rate found was 2.0 X 10(-6) in controls and 1.3 X 10(-7) in drug-treated rats (15-fold reduction). This suggests that ADPRT is required for completing most antigenic switching events. We discuss the possibility that drug-resistant switching only involves non-duplicative VSG gene activation.