We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductasethymidylate synthase (DHFR-TS; EC 1.5

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Trypanosomes cannot synthesize sialic acids. Infectious stages of the life cycle of the human pathogen Trypanosoma cruzi express a cell-surface glycolipid-anchored trans-sialidase, which can transfer sialic acid between glyco-conjugates. Sialic acid is transferred from host cell-surface and serum sialylglycoproteins to trypanosome cell-surface glycoconjugates. The transfer reaction is specific for donors with terminal alpha-2,3-linked sialic acid, and terminal beta-1,4-linked galactose is the preferred acceptor. In the absence of an acceptor, the enzyme acts as a hydrolase, but cleavage is less efficient than transfer. Trans-sialidase activity is attributable to a few members of a large family of T. cruzi surface glycoproteins, many of which are simultaneously expressed. The functions of the trans-sialidase surface glycoprotein family are unknown but may be important for adhesion, invasion, virulence, or pathogenicity. A trans-sialidase is also expressed in the procyclic forms of Trypanosoma brucei.

Cells of the insect (procyclic) stage of the life cycle of the African trypanosome, Trypanosoma brucei, express an abundant stage‐specific glycosylated phosphatidylinositol (GPI) anchored glycoprotein, the procyclic acidic repetitive protein (PARP). The anchor is insensitive to the action of bacterial phosphatidylinositol‐specific phospholipase C (PI‐PLC), suggesting that it contains an acyl‐inositol. We have recently described the structure of a PI‐PLC resistant glycosylphosphatidylinositol, PP1, which is specific to the procyclic stage, and have presented preliminary evidence that the phosphatidylinositol portion of the protein‐linked GPI on PARP has a similar structure. In this paper we show, by metabolic labelling with [3H]fatty acids, that the PARP anchor contains palmitate esterified to inositol, and stearate at sn‐1, in a monoacylglycerol moiety, a structure identical to PP1. Using pulse‐chase labelling, we show that both fatty acids are incorporated into the GPI anchor from a large pool of metabolic precursors, rather than directly from acyl‐CoA. We also demonstrate that the addition of the GPI anchor moiety to PARP is dependent on de novo protein synthesis, excluding the possibility that incorporation of fatty acids into PARP can occur by a remodelling of pre‐existing GPI anchors. Finally we show that the phosphatidylinositol (PI) species that are utilized for GPI biosynthesis are a subpopulation of the cellular PI molecular species. We propose that these observations may be of general validity since several other eukaryotic membrane proteins (e.g. human erythrocyte acetylcholine esterase and decay accelerating factor) have been reported to contain palmitoylated inositol residues.

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This report describes a method for growing both bloodstream-and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HM-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 106 cells at maturity. Tanzania (19) and was cloned as a metacyclic taken directly from an infected tsetse salivary gland (kindly provided by L. Jenni, Swiss Tropical Institute, Basel). It has since been propagated through a total of eight animals (mice or rats) with intermittent cryopreservation. Prior to use in this study, all of these cell lines were recloned by limiting dilution in vitro and checked by immunofluorescence using variant-specific antibodies to ensure expression of the correct variant surface glycoprotein (VSG; except STIB 366E, for which variant-specific antibodies are unavailable).Adaptation of Bloodstream Forms to Liquid Culture. Trypanosomes were grown in either mice or rats to subsaturation density (1-5 x 108 cells per ml of blood) and adapted to grow in HMI-9 medium essentially as described (12). Briefly, 1 ml of infected blood [collected with 0.2 vol of citrate glucose anticoagulant (0.1 M sodium citrate/0.04 M glucose, pH 7.7)] was diluted in 9 ml of HMI-9 medium and then centrifuged at 200 x g for 5 min at room temperature. The supernatant (containing trypanosomes) was transferred to a T-25 culture flask and incubated upright at 370C for 2 hr to allow the majority ofthe remaining blood cells to settle. Trypanosomes in the supernatant were then used to initiate 5-ml cultures in HMI-9 at a density of 1.2 x 106 cells per ml. Cultures were fed daily by adding 2.5 ml of fresh medium after removing an equal volume. In the initial stages, many cells died. Once the trypanosomes began to divide rapidly (when densities recovered to 106 cells per ml), cultures were routinely diluted 1:10 to 1:20 daily to maintain densities in the range of 105-106 cells per ml.Preparation of Agarose Plates and Growth of Colonies.Agarose plates were prepared using 0.65% agarose as suggested by Lee and Van der Ploeg (17). To achieve the desired final concentration ofingredients, it was necessary to prepare and then mix 2x solutions ofmedium and agarose. 2x HMI-9 medium was prepared by dissolving 5.31 g of powdered Iscove's modified Dulbecco's medium (IMDM, GIBCO/ BRL) and 0.907 g of sodium bicarbonate in 92 ml of lowconductivity water followed by the addition of 40 ml of Abbreviation: VSG, variant surface glycoprotein.

SummaryClones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 102–103/well on days 4–16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2–5 × 105 trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1–2 ml of the trypanosome suspension to a new culture flask at 4–5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1·4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8·7, 14·5 and 15·5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8–32 days from cloning. One cloned population of TC52 consisted of 99·8% VAT 052 and 0·2% VAT 221 at the time when the first VAT test was made on day 18.