Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2-oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis-aconitate, succinate, proline or 2-oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected.Addition of citratelcis-aconitate at 37 "C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an 'abortive' transformation to non-dividing procyclic-like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms.At 27°C and in the presence of citratelcis-aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2-oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases.The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.The parasitic protozoan, Trypanosoma brucei, undergoes a series of differentiation steps during its cyclical development in the mammalian host and the arthropod vector, the tsetse fly [l]. Differentiation of bloodstream forms to procyclic cells, a process called transformation, is initiated in the mammal by the transition of dividing slender forms to intermediate and non-dividing stumpy forms, giving rise to a pleomorphic population. After uptake with the blood meal into the midgut of the fly, cells with stumpy morphology are considered to transform most readily to dividing procyclic cells.Transformation of pleomorphic as well as monomorphic populations of rodent-adapted strains which have a uniform slender morphology [2] can be studied in various in vitro systems [3 -91. Synchronous transformation requires two external signals, a temperature change from 37°C to 27°C and the addition of cis-aconitate and/or citrate as inducers Some of the complex morphological, ultrastructural and metabolic changes which characterize transformation have been studied in detail. First, the repression of the synthesis of the variant surface glycoprotein (VSG) in the coated bloodstream forms is an early event which is followed by coat [lo-131. release to form coatless procyclic cells [7, 8, 111. Second, transformation involves profound changes in energy metabolism [14-161. In bloodstream forms glucose is degraded solely by glycolysis to form two mol pyruvate/mol glucose. Reducing equivalents are transferred to oxygen via a unique mitochondrial glycerol-3-phosphate oxidase [17,18]. The promitochondrion lacks a functional citric acid cycle as well as a cytochrome-linked respiratory chain. Stumpy forms contain some mitochondrial enzymes, such as 2-oxoglutarate dehydrogenase and proline oxidase [19]. During transformation to procyclic cells a functional respiratory ...
The biosynthesis of the variant surface glycoprotein (VSG) and its release from the surface of Trypanosoma brucei 427 variant clone MITat 1.4 (117) during in vitro transformation of bloodstream trypomastigotes to procyclic trypomastigotes was investigated. After transfer to the transformation medium at 27 degrees C, VSG synthesis is repressed with a half‐time, t1/2 = 30 min. Concomitantly VSG‐specific mRNA is lost suggesting that repression operates at the transcriptional level. The expression‐linked extra gene copy, which codes for VSG, is retained during and after completion of transformation. After repression of VSG synthesis, surface VSG is shed from the cells into the culture medium. During release part of VSG (apparent mol. wt. 61 000) is proteolytically cleaved to a product (apparent mol. wt. 51 000) which represents the N‐terminal domain of the protein as judged by the absence of the carbohydrate moiety normally linked to the C terminus.
Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procycic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27°C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, ,-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of ,B-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (tj/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t012, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37°C caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while f8-tubulin mRNA was stabilized 2-to 3-fold and medRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.The regulation of mRNA synthesis in the parasitic protozoan Trypanosoma brucei represents an interesting problem. First, throughout its complex life cycle in the mammalian host and the tsetse fly, this unicellular flagellate experiences dramatic changes in environment, to which it adapts by regulating the expression of numerous genes (42); second, it has the unique feature of discontinuous mRNA synthesis (4). In an as yet undefined mechanism, a 140-nucleotide (nt) transcript, designated mini-exon-derived RNA (medRNA), donates a 35-nt sequence located at its 5' end (mini-exon) to the 5' end of all mRNAs coding for proteins (8,9,12,21,26,44 (17,36,40). The mini-exon has been explicitly demonstrated at the 5' end of the VSG and tubulin mRNAs (3,17,36,41). medRNA is transcribed from mini-exon genes present in about 200 copies per nucleus. Most of these genes are clustered in the genome in tandemly linked 1.35-kilobase (kb) repeats (11,27 bloodstream forms to procyclic cells. In a second, related part, mRNA synthesis and decay in the absence of protein synthesis were investigated. MATERIALS AND METHODSTrypanos...
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