[Antigenic variation of African trypanosomes: molecular aspects].
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Animals↗
Biomedical subjects
M Steinert
Publications and source records attributed to M Steinert.
[Laboratory studies of long-term photochemotherapy of psoriasis vulgaris].
The study of 40 laboratory parameters from a total of 40,000 individual samples taken from patients who had been treated by means of systemic PUVA therapy for a period up to three years did not reveal any evidence for negative influences on selected peripheral blood enzymes, substrates, metabolites, proteins, lipid fractions, electrolytes, antistreptolysin titer, rheumatoid factors and coagulation values, or urinalysis values. The changes observed were either statistically insignificant, remained within normal ranges, or could not be classified. Under the conditions described, no evidence for chronic toxic organic manifestations could be found, even after several years of PUVA treatment.
Blood Cells↗
Inactivation and reactivation of a variant-specific antigen gene in cyclically transmitted Trypanosoma brucei.
In Trypanosoma brucei, the activation of the variant-specific antigen gene AnTat 1.1 proceeds by the synthesis of an additional gene copy, the AnTat 1.1 ELC, which is transposed to a new location, the expression site, where it is transcribed. Using the AnTat 1.1 variant to infect flies, we investigated the fate of the AnTat 1.1 ELC during cyclic transmission of T. brucei. We show here that the AnTat 1.1 ELC is conserved in procyclic trypanosomes, obtained either from the midgut of infected Glossina or from cultures, and in metacyclic trypanosomes, although the AnTat 1.1 serotype is not detected among metacyclic antigen types. This same AnTat 1.1 ELC, which is thus silent as the parasite develops in the insect vector, can be reactivated without duplication during the first parasitemia wave following cyclical transmission. This re-expression of the conserved ELC accounts for the early appearance of the 'ingested' antigenic type after passage through the fly.
Animals↗
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.
Animals↗
Genetic control of antigenic variation in trypanosomes.
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Animals↗
Translocation alters the activation rate of a trypanosome surface antigen gene.
We report here the characterization of the gene coding for AnTat 1.13, a very late variable antigen type (VAT) from Trypanosoma b. brucei. This gene is chromosome-internal and it is activated by the duplicative mechanism. Like in another case of late VAT expression (1), its expression-linked copy (ELC) is flanked by "companion" sequences. It was possible to convert the late expression of this VAT into an early one, by changing the location of the gene in the genome. This has been achieved by selecting an AnTat 1.6 clone among heterotypes arising in the AnTat 1.13 cloned population. Indeed, this particular derivation leads to the conservation of the AnTat 1.13 ELC as a new telomeric member of the gene family, and this conserved ELC (or ex-ELC) appears to be preferentially activable. The telomeric position and other factors possibly involved in early or late antigen gene expression are discussed; in this respect, we propose that some antigen genes are rarely activated because their duplicative transposition requires the presence, in the expression site, of "companion" sequences only shared by a limited number of other genes.
Animals↗
Possible DNA modification in GC dinucleotides of Trypanosoma brucei telomeric sequences; relationship with antigen gene transcription.
Polymorphism in restriction site cleavage (PstI, SphI, PvuII, HindIII) has been noticed in several occasions in the telomeric sequences harbouring trypanosome variant-specific antigen genes (1, 2, 3). This polymorphism has been further investigated and seems best interpreted as due to partial DNA modification in GC dinucleotides. The actively transcribed telomeric genes do not exhibit such a polymorphism; furthermore, in at least three independent cases, gene inactivation is linked to the appearance of polymorphism. It could thus be hypothesized that DNA modification prevents antigen gene transcription, or vice-versa. We report however that at least some telomeric antigen-specific sequences of the procyclic trypanosomes (in vitro culture form) are not polymorphic, although they do not synthesize any variant-specific antigen mRNA. There is thus no absolute relationship between the absence of polymorphism and antigen gene transcription.
Animals↗
Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei.
We have studied the genes coding for the variant-specific surface antigen (VSA) in a series of seven trypanosome clones derived from AnTat 1.1: 1.1 leads to 1.3 leads to 1.6 leads to 1.16 leads to 1.1C leads to 1.3B leads to 1.18 These genes are all telomeric (1-5), and their surrounding, although sometimes similar, differs in each case. The length between these antigen genes and the corresponding DNA end appears to increase at each antigenic switch, with however occasional sharp size reductions, often linked to the involvement of the telomere in gene expression. This increase is due to a constant "growth" of the telomeres, at a rate of about 28 bp per day in at least four cases and probably linked to chromosome duplication. The telomere harbouring the transcribed VSA gene is growing slightly faster (about 36 bp per day), and it is the only one whose size reduction is progressive, leading to a terminal length heterogeneity within a clone. As a result, the active VSA gene is found in a population of telomeres which, as the trypanosomes divide, becomes increasingly heterogeneous, with however a preferred discrete size class about 1.4 kb smaller. The fact that the "active" telomere is the only one in a chromatin conformation highly sensitive to DNAaseI (1-4, 6), suggests that chromatin structure influences the rate and extent of both size increase and shortening of telomeres.
Animals↗
Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein.
The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.
Animals↗
Modifications of a Trypanosoma b. brucei antigen gene repertoire by different DNA recombinational mechanisms.
In the Trypanosoma b. brucei AnTat 1.1C clone, the gene coding for the variant-specific surface antigen is telomeric and appears as a hybrid sequence, partially modified by gene conversion. This conversion is very similar to that observed in another AnTat 1.1-expressor clone (AnTat 1.1B). This sequence is not activated by duplicative transposition, although it could be activated by duplication in another clone (AnTat 1.10). Instead activation of the AnTat 1.1C gene seems operated by reciprocal recombination between its own telomere and the telomere carrying the previous (AnTat 1.16) ELC. Indeed, from the switch to AnTat 1.1C onward, the AnTat 1.16 ELC becomes a new silent member of its gene family, whereas in the variant directly derived from AnTat 1.1C (AnTat 1.3B), the AnTat 1.1C-containing telomere is lost, probably replaced by a large duplicate, at least 40 kb long, of the AnTat 1.3 gene-containing telomere. Different DNA rearrangement mechanisms used by the trypanosome to change its antigenic type thus contribute, by gain and loss of genes, to the evolution of the repertoire for surface antigens.
Animals↗
At least two transposed sequences are associated in the expression site of a surface antigen gene in different trypanosome clones.
The expression of several trypanosome surface antigen genes proceeds by duplication of a basic copy (BC) of the gene and transposition of the expression-linked copy (ELC) into an expression site. This site, which seems to be the same for different genes of the same repertoire, is located near a chromosome end. In the AnTat 1.1 antigen gene expression site, the ELC is found associated with another sequence that we have called the "companion." We found that this companion is the transposed copy of another sequence also located in an unstable DNA terminus, and that it is conserved in the expression site of AnTat 1.10 and AnTat 1.1B, two clones successively derived from AnTat 1.1. The companion sequence is not part of the surface antigen gene, but we may infer from extensive homologies with another ELC sequence (IoTat 1.3, J. E. Donelson, personal communication) that it represents a 5' residual fragment of a former ELC. In three other AnTat 1.1-like clones, the companion sequence was not found associated with the ELC. It is concluded that the expression-linked duplicative transposition of variable antigen genes is a flexible mechanism, which can apply to variably sized stretches of the same BC.
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Gene conversion as a mechanism for antigenic variation in trypanosomes.
Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs--AnTat 1.1, 1.10, and 1.1B--are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.
Amino Acid Sequence↗
Comparative analysis of a Trypanosoma brucei gambiense antigen gene family and its potential use in epidemiology of sleeping sickness.
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Animals↗
Structure and expression of a Trypanosoma brucei gambiense variant specific antigen gene.
The expression-linked copy of the T. b. gambiense variant specific antigen gene LiTat 1.6 is transposed in a 20 kb DNA region devoid of restriction sites, located near a chromosome end. This expression site is very similar to that of T. b. brucei variants 117, 118 (1) and AnTat 1.8. In the basic copy, the transposable element (TE) is flanked by repetitive sequences; it includes the gene copy as well as a sequence of 0.9 to 2.1 kb (probably around 1.1 kb) long, upstream from the gene. Probes derived from the 5' part of the TE specifically reveal three polyadenylated transcripts of 4.2, 1.45 and 0.85 kb, respectively, distinct from the 2.1 kb mRNA. The amount of the 4.2 kb sequence is probably less than 0.01% of total trypanosome RNA. Whereas the mRNAs coding for the three isotypic antigens AnTat 1.8 (T. b. brucei), 12.2 (T. b. rhodesiense) and 3.3 (T. evansi) are recognized by LiTat 1.6 probes extending into the 3' half of the transposed sequence, the 5' genomic probes do not hybridize with any of these RNAs. These observations suggest that the LiTat 1.6 gene could be first transcribed in a large precursor molecule. This precursor would be rapidly processed, loosing a large portion of less conserved sequence from its 5' half. Our data are compatible with a model in which the promoter would be provided by the expression site.
Animals↗
Conservation of a variant-specific surface antigen gene in different trypanosome species and sub-species.
In Trypanosoma brucie brucie, T. b. rhodesiense, T. b. gambiense and T. evansi, the variant-specific antigen (VSA) genes are organized in families of related sequences, one of which is duplicated when expressed. Some of these VSA sequences appear to be conserved in the different species and sub-species: restriction mapping of isotypic genes of AnTat 1.8 VSA (from T. b. brucei) reveals extensive homology in T. b. rhodesiense, T. b. gambiense and T. evansi, although the genetic surrounding differs in each case. By contrast, the AnTat 1.1 sequence (also from T. b. brucei) appears to be absent from T. b. gambiense DNA.
Animals↗
Analysis of the DNA and RNA changes associated with the expression of isotypic variant-specific antigens of trypanosomes.
Using specific (32P) labelled cDNA probes, we compared the mRNAs and the genomic DNA sequences coding for the synthesis of two pairs of serologically related variant-specific antigens (VSAs) of trypanosomes: AnTat 1.1 and AnTat 1.1b, both from the strain 1125 of T.b.brucei and AnTat 1.8 and LiTat 1.6 from T.b.brucei and T.b. gambiense, respectively. Within each pair, large similarities were observed in the coding sequence, except in the 3' region which appears to be highly variable. However, a low level of cross-hybridization can be detected between all sequences, in the 3' region only. The expression of these VSAs is linked to a similar duplication-transposition mechanism. The insertion locus of the transposition unit is the same both in AnTat 1.1 and AnTat 1.1b DNAs. In both pairs, the transposition unit seems to comprise at least about 200 bp upstream of the 5' extremity of the coding sequence. The significance of these results, regarding the structure and synthesis of the VSAs, is discussed.
Animals↗
The expression-linked copy of a surface antigen gene in Trypanosoma is probably the one transcribed.
The antigenic specificity of the living trypanosome seems to be determined by the protein component of a unique glycoprotein species covering the whole surface of the parasite. During chronic infection, a single clone of trypanosomes may successively express a large repertoire of different variable antigen types (VATs). There are probably as many genes as variant-specific antigens (VSAs) (see refs 1-3 for reviews). The expression of the genes coding for the synthesis of these antigens is linked to genomic rearrangements involving duplication of the coding sequence and transposition of the additional copy. The regulation of the expression of the VSA genes is operated at the transcriptional level. It can thus be supposed that their transcription depends on the presence of the additional, transposed copy. We report here that this additional copy is in a chromatin configuration highly sensitive to pancreatic deoxyribonuclease, suggesting that it is the transcribed one.
Animals↗
Gene duplication and transposition linked to antigenic variation in Trypanosoma brucei.
DNA sequence complementary to Trypanosoma brucei mRNAs coding for the synthesis of the variant-specific antigens AnTat 1.1 and AnTat 1.8 have been cloned and characterized. These sequences have been used as probes to analyze the corresponding genes in the nuclear DNA. The two genes seem to be represented in several (three to six) copies, some of which are incomplete. Transcription of one or the other of these two genes is linked to a genetic rearrangement implying duplication and transposition of the "basic" coding sequence. There is probably one additional copy of each gene, and it seems to be complete. The 3' end of each cloned sequence contains, within a 300-base-pair fragment, a genetic element that seems to be repeated and widely distributed in the genome. This repetitive sequence is variant specific. The expression-linked copy of the gene is lost in the culture (procyclic) form of the trypanosome, where the synthesis of variant-specific antigens is shut down. Comparison of two different cloned populations expressing the same serotype (AnTat 1) showed that the recurrence of a given antigenic type may be accompanied by the production of the same additional copy.
Animals↗