Suzanne Van Assel

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.

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.

Kinetoplast DNA. A unique macromolecular structure of considerable size and mechanical resistance

Abstract Highly purified k-DNA has been prepared from Crithidia luciliae by a two step procedure, involving differential sedimentation in sucrose followed by banding in a CsCl density gradient. This k-DNA is isolated as compact particles of homogeneous size and DNA content, each one corresponding to the whole DNA complement of individual kinetoplasts (3.72 × 10−14g).

Comparative analysis of Trypanosoma brucei gambiense antigen gene family and its potential use in epidemiology of sleeping sickness

The identification of African trypanosomes infective for man and the characterization of their repertoire of variable antigen types (VATs) is a major challenge to epidemiologists. The present work, based on previous studies, brings evidence that DNA analysis may provide a molecular approach to these problems. Two cloned cDNAs coding for Trypanosoma brucei brucei variant-specific antigens have been used as probes to evaluate, by hybridization on DNA blots, the relatedness of different trypanosome clone stocks, mainly of T. b. gambiense. It appears that, in all T. b. gambiense stocks, the AnTat 1.8-like gene family produces a typical pattern of bands which is discussed in terms of gene evolution and of possible use as a means to discriminate T. b. gambiense from the other sub-species. A deviation from this common pattern concerns the basic copy of the gene, the presence or absence of which is correlated with the ability of the stocks to express AnTat 1.8 isotypes: this observation suggests that only one member of the AnTat 1.8 DNA sequence family might be involved in the expression of the AnTat 1.8 isotype, and that useful information on serodemes and their VAT repertoires could be obtained from DNA analysis. Among the silent sequences of this family, one appears remarkably similar to the basic copy. Its apparent inability to be expressed could be linked to alterations of the repetitive sequences flanking the transposable element. This observation supports the hypothesis that these repeats are involved in the transposition leading to expression of the gene.

Specific detection of kinetoplast DNA in cytological preparations of trypanosomes by hybridization with complementary RNA

Abstract We have used the method of cytological hybridization with complementary RNA to study the base sequence homology between kinetoplast DNAs of different hemoflagellates. 3 H-labelled complementary RNA (spec. act. 20 × 10 6 dpm/μg) was synthesized on purified kinetoplast DNA with RNA polymerase of Escherichia coli . Smears of hemoflagellates were fixed with a formaldehyde-containing fixative, the DNA was heat-denatured in situ in the presence of 50% formamide and hybridization with complementary RNA was carried out with 20 μl RNA at 0.6 μg/ml. Unspecifically bound RNA was removed by treatment with ribonucleases and hybridization was quantitatively evaluated on autoradiograms with a microphotometer. In intraspecific hybridization reactions with Crithidia luciliae and Trypanosoma mega , RNA was bound only to the kinetoplast and not to the nucleus. In the interspecific hybridization reaction no cross-hybridization was found at all, either on cytological preparations or with kinetoplast DNA fixed to nitrocellulose filters. This shows that the evolution of a major part of kinetoplast DNA is not highly restricted. Cytological hybridization may, therefore, provide a useful method to differentiate closely related species of pathogenic hemoflagellates.

Large circular mitochondrial DNA in Crithidia luciliae

A novel circular DNA, 11.3 μm in contour length, has been found in a pure kinetoplast DNA fraction of Crithidia luciliae. The mitochondrial nature of the kinetoplast and the absence of these large circular molecules in the nuclear fraction of DNA suggest that they constitute the mitochondrial genome of this species.

Étude, par autoradiographie, des effets du bromure d'éthidium sur la synthèse des acides nucléiques de Crithidia luciliae

Abstract The effects of ethidium bromide upon 3 H-thymidine and 3 H-uridine incorporation by Crithidia luciliae have been studied by autoradiography. The drug, at the concentration of 20 μg/ml inhibits kinetoplast DNA synthesis up to 90% after a few minutes. Nuclear DNA synthesis is also very rapidly affected, but the inhibition does not exceed 28% in the same experimental conditions. Some hypotheses relevant to this specificity toward kinetoplast DNA are discussed. 3 H-Uridine pulse experiments have revealed that the kinetoplast is the site of RNA syntheses. EB inhibits 3 H-uridine incorporation into kinetoplast and nuclear RNA, but this effect on RNA synthesis is slower than the very quick process which affects DNA replication.

Maxi-circles in the kinetoplast DNA of Trypanosoma mega.

Abstract We have analysed the kinetoplast DNA (kDNA) of Trypanosoma mega for the presence of DNA circles other than mini-circles and mini-circle oligomers. Degradation of networks with restriction endonucleases EcoRI, HhaI or PstI or S 1 nuclease yields minor bands on gels, in addition to the mini-circle bands. The combined molecular weight of the minor bands is 15–17 × 10 6 . Electron micrographs of kinetoplast DNA spread in a protein monolayer contain occasional circles longer than 3 μm. The majority class of these circles has a contour length corresponding to a mass of 16.1 × 10 6 D. We conclude that these maxi-circles, catenated into mini-circle networks, are an essential component of intact kinetoplast DNA of Trypanosoma mega .

Base composition heterogeneity in kinetoplast DNA from four species of hemoflagellates

Summary Evidence for intramolecular base composition heterogeneity in kinetoplast DNA has been found by high resolution melting analysis. The specific melting profiles, obtained for the kinetoplast DNA isolated from four different species, indicate that extensive variations in base sequence occurred in this DNA during evolution of the trypanosomatidae.