Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short,identical exon at their 5′ end

The nucleotide sequence of the 5′ end of the mRNA for variant surface glycoprotein (VSG) 117 has been determined and compared with the sequence of the unexpressed basic copy (BC) of the VSG 117 gene. This shows the existence of an exon 35 nucleotides long at the 5′ end of the mRNA. The evidence suggests that this ‘mini-exon’ is derived from the expression site into which the VSG 117 BC is transposed during activation. The nucleotide sequence of this mini-exon is indistinguishable from that recently found for a different VSG, 118 (Van der Ploeg et al., Nucl. Acids Res. 10 (1982) 3591–3604). Analysis of the 5′ end of the mRNA for another VSG (221) whose gene is thought to be activated by a different mechanism to that of VSGs 117 and 118 indicates that the 5′- most 35 nucleotides of the VSG 221 mRNA are identical to the 117/118 mini-exon sequence. The implications of these results for the mechanism of VSG gene expression are discussed.     

Size fractionation of Trypanosoma brucei DNA: localization of the 177-bp repeat satellite DNA and a variant surface glycoprotein gene in a mini-chromosomal DNA fraction

We have size-fractionated intact DNA from Trypanosoma brucei into a major large DNA fraction (greater than 350S) and minor middle-sized (60-250S) and small (less than 60S) DNA fractions. Large DNA contains the rRNA genes, the basic copy genes for several variant surface glycoproteins (VSGs), including one which lies near a telomer, and the expression-linked copies of the two VSG genes. The middle-sized DNA contains at least one VSG gene, but the hybridization of this fraction with probes for the conserved repetitive sequences that mark the edges of the transposed segments of VSG genes, suggests that it may contain many VSG genes. The 177-bp repeat satellite DNA is also exclusively found in this fraction.     

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: Coding region hypervariability and downstream recombinogenic repeats

The surface of the parasitic protozoanTrypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome ofT. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 by downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy. Correspondence to: J.C. Boothroyd     

Frequent independent duplicative transpositions activate a single VSG gene.

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.     

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.     

Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei

The genome of Trypanosoma brucei carries over a hundred genes coding for different variants of the major surface glycoprotein. Activation of some of these genes is accompanied by a duplication and transposition of the gene (the basic copy) to another region in the genome where it is transcribed. We present here physical maps of the basic and transposition-activated genes for two surface glycoproteins of Trypanosoma brucei, stock 427. In both cases the transposed segment starts 1-2 kb in front of the coding region and ends within the 3'-terminal region of the gene. The DNA segments flanking both transposed genes are indistinguishable and share a 6-kb stretch upstream and a 8-kb stretch downstream of the transposed segment not cut by several restriction endonucleases. The 5' borders of the two transposed segments are homologous and contain sequences present in many copies in the genome. A different repeated sequence has previously been found at the 3' edge of the transposed segment. The replicative transposition may, therefore, involve a unidirectional gene conversion initiated by base pairing between the edges of the transposed sequence and a single expression site elsewhere in the genome.     

Characterization of the expression-linked gene copies of variant surface glycoprotein 118 in two independently isolated clones of Trypanosoma brucei.

We have analysed the gene for variant surface glycoprotein 118 in eight independent clones of Trypanosoma brucei, two of which express the 118 gene. Expression of this gene is strictly coupled to the presence of an extra copy of the gene. In both clones the expression-linked copy is transposed to the same (or a very similar) expression site elsewhere in the genome, but the length of the sequences flanking the transposed segment in the expression site differs markedly. By means of S1 nuclease protection experiments we demonstrate that the 3'-ends of the messenger RNAs for variant surface glycoproteins 118a and 118b are different, in agreement with the hypothesis that the generation of an expression-linked copy involves a recombination between the 3' segment of the basic gene copy and a homologous region present in the expression site.     

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules

Pulsed field gradient gel electrophoresis fractionates chromosome-sized DNA molecules from T. brucei. About 60% of the DNA remains in or close to the gel slot (large DNA). There are about three chromosomes of approximately 2 Mb, at least six chromosomes of 200-700 kb, and roughly a hundred mini-chromosomes of 50-150 kb. The basic copy genes for VSGs 118 and 221 reside in large DNA. Their activation by duplicative transposition leads to the appearance of an additional copy in the 2 Mb DNA, showing that activation involves an interchromosomal gene transposition. When gene 221 is activated without duplication, it remains in large DNA, proving that at least two sites for expression of VSG genes exist. In support of this, the mini-exons encoding the 5' 35 nucleotides of VSG messenger RNAs are in large and 2 Mb DNA. The mini-chromosomes hybridize strongly to VSG gene probes and are absent in C. fasciculata. We suggest that their main function is to provide a large pool of telomeric VSG genes.     

The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene.

The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

Genomic organization and context of a trypanosome variant surface glycoprotein gene family.

We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.