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Differentiation of African trypanosomes from replicating slender bloodstream forms to nondividing stumpy forms limits the parasite population size, allowing survival of the mammalian host and establishment of a stable host-parasite relationship. Using a novel in vitro culture system we have shown that slender to stumpy differentiation is induced by parasite density alone and thus is independent of host cues. Here we investigate the density sensing mechanism and show that trypanosomes release a soluble activity of low relative molecular mass, termed stumpy induction factor (SIF), which accumulates in conditioned medium. SIF activity triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and rapid kinetics. Membrane-permeable derivates of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity. Furthermore, SIF activity elicits an immediate two- to threefold elevation of intracellular cAMP content upon addition to slender forms. We conclude that SIF and hence density sensing operate through the cAMP signalling pathway. Temporal correlation of markers indicates that cell cycle arrest invariably precedes differentiation. Thus, our results indicate that the cell cycle regulation of bloodstream forms is under dominant control of cAMP signalling. Irreversible commitment to the quiescent state is elicited by a cAMP agonist within a period shorter than one complete cell cycle.

It has been reported that selenocysteine tRNA from bovine liver is completely edited to two isoacceptor species, called tRNA([Ser]SecNCA) and tRNA([Ser]SecCmCa), which differ from the gene sequence. We used direct tRNA sequencing, mobility shift analyses, primer extension, restriction enzyme digestion and single strand conformational polymorphism (SSCP) analyses of products from reverse transcription coupled with polymerase chain reaction (RT/PCR), sequencing of RT/PCR products and HPLC-coupled mass spectrometry to reproduce this result and show here that editing of these tRNAs does not occur.

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