Mary Gwo-Shu Lee

Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei

Development of methods for the manipulation of the genomes of parasitic protozoa will lead to enhanced understanding of parasite biology and host-parasite relationships. Efficient gene transfer and targeted integration by homologous recombination were achieved in the parasitic protozoan Trypanosoma brucei, the causative agent of sleeping sickness. An expression vector with the neomycin phosphotransferase gene (neo), under the control of a procyclic acidic repetitive protein (PARP) gene promoter, was targeted into an intergenic region in beta alpha-tubulin-gene tandem array. Sixteen copies of neo were found in a tandem array in one of the transfectants where the PARP promoter controlled alpha-amanitin-resistant transcription of neo, whereas transcription of tubulin genes remained alpha-amanitin-sensitive.

Chromosome organization of the protozoan Trypanosoma brucei.

The genome of the protozoan Trypanosoma brucei is known to be diploid. Karyotype analysis has, however, failed to identify homologous chromosomes. Having refined the technique for separating trypanosome chromosomes (L. H. T. Van der Ploeg, C. L. Smith, R. I. Polvere, and K. Gottesdiener, Nucleic Acids Res. 17:3217-3227, 1989), we can now provide evidence for the presence of homologous chromosomes. By determining the chromosomal location of different genetic markers, most of the chromosomes (14, excluding the minichromosomes), could be organized into seven chromosome pairs. In most instances, the putative homologs of a pair differed in size by about 20%. Restriction enzyme analysis of chromosome-sized DNA showed that these chromosome pairs contained large stretches of homologous DNA sequences. From these data, we infer that the chromosome pairs represent homologs. The identification of homologous chromosomes gives valuable insight into the organization of the trypanosome genome, will facilitate the genetic analysis of T. brucei, and suggests the presence of haploid gametes.

Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

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.

A foreign transcription unit in the inactivated VSG gene expression site of the procyclic form of Trypanosoma brucei and formation of large episomes in stably transformed trypanosomes

Transcription of the variant surface glycoprotein (VSG) gene expression site (ES) in Trypanosoma brucei is inactivated upon differentiation from the bloodstream form to the insect-adapted, procyclic form. This paper demonstrates that a foreign transcription unit, containing a procyclic acidic repetitive protein (PARP) gene promoter driving a neomycin phosphotransferase (neo) gene, can be fully active once integrated at the lingering, transcriptionally inactive VSG ES of procyclic trypanosomes. Following targeting into the ES two types of transformants were identified. Type one transformants were generated by integration of the PARP-neo gene into the region downstream of the long 70-bp repeat array of the silent telomeric ES encoding VSG gene 118. alpha-amanitin-resistant transcription at the neo locus proceeded from the PARP promoter to approximately 2.5 kb downstream of the integration site and terminated in front of the VSG 118 gene. Type two transformants contained variously sized large episomes (ranging from 135 kb to 500 kb), consisting of tandemly linked input plasmids. Transcription of the neo gene in the episomes was also resistant to alpha-amanitin. The presence of large amounts of the active episomal PARP promoter did not significantly affect the transcription of most RNA polymerase II transcribed genes, but resulted in a significant and equal reduction of the transcriptional efficiency of the endogenous PARP genes and VSG gene promoter sequences. This observation suggests that transcription of the PARP gene and the VSG gene expression sites in insect form trypanosomes may share a common transcriptional machinery.