RESUMEN
Current understanding of expression-site transcription in Trypanosoma brucei, has been refined by recent results of promoter manipulations at vsg expression sites (ES) and examination of the behavior of ES promoters in ectopic locations both within the ES and at other loci. In summary, ES promoter sequences inserted into non-transcribed rRNA spacers are generally inactive, or have low activity, in bloodstream and procyclic forms. Some mechanism apparently operates to ensure full activation of a single ES in bloodstream-form trypanosomes and the inactivity of all ES promoters in procyclic forms. As previously shown, a rRNA promoter can replace an ES promoter. In bloodstream forms, the replacement rRNA promoter was down-regulated in a 'silent' ES but it was active in procyclic forms. In addition to manipulations of endogenous promoters, we have recently shown that, when an ES promoter is replaced by a T7 promoter, the T7 promoter is unregulated but transcription is attenuated before the vsg, and another ES switches on to maintain cell viability. However, T7 transcription is repressed in the context of core ES-promoter sequences in both stages, particularly in procyclic forms. These observations strongly argue that sequences in the vicinity of the ES core promoter play a role in ES control by nucleating critical events in silencing as well as in activation. Deletions of sequences surrounding the ES core promoter, in situ, did not affect its activity or regulation. In bloodstream forms, rRNA or ES promoters inserted adjacent to silent telomeres or to a non-telomeric 'basic-copy' vsg were > 98% repressed. After transformation to procyclic forms, the sub-telomeric rRNA promoter regained about 10% of its maximal activity but the 'basic-copy' rRNA promoter was fully active. Similarly-positioned ES promoters remained silent in procyclic forms. These results suggest that telomere-proximal or vsg-proximal sequences might mediate suppression of transcription via position-effects that could be sufficient to suppress the expression of chromosome-internal vsgs or telomeric metacyclic vsgs, in bloodstream-form trypanosomes. Recent experiments with T7 promoters indicate that sequences within the ES core promoter might be responsible for silencing ES promoters in procyclic forms. Precedents for regulatory mechanisms that modulate transcription over large chromatin domains are reviewed and possible models for ES regulation are presented.
Asunto(s)
Variación Antigénica , Regulación de la Expresión Génica , Transcripción Genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Reordenamiento Génico , Regiones Promotoras Genéticas , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaRESUMEN
We previously described a system for exogenous control of gene expression in procyclic trypanosomes which depends upon the binding of a tetracycline-inducible repressor to operators situated at the transcriptional start site of the PARP promoter. The recombinant constructs are introduced into non-transcribed spacers of the ribosomal RNA repeat, in an orientation opposite to that of rRNA transcription. Using this system, gene expression could be regulated over four orders of magnitude, but it was not possible to express toxic gene products because selection of recombinant trypanosomes depended on the activity of the inducible promoter. We describe here the characteristics of vectors that include two promoters: a tetracycline-inducible one to drive expression of the toxic products, and a constitutive one to drive transcription of the selectable marker. Relatively high levels of non-induced (non-tetracycline-dependent) expression were seen in some trypanosome clones; this was not usually due to read-through of multiple tandemly-integrated plasmids or tet operator mutations. A variety of constructs differing in resistance marker, 3'-untranslated region (3'-UTR) and the nature of the constitutive promoter was tested. Vectors allowing the successful expression of toxic and other genes in both life cycle stages with regulation factors of up to 700 fold were obtained.
Asunto(s)
Clonación Molecular/métodos , Regulación de la Expresión Génica , Vectores Genéticos , Proteínas Protozoarias , Toxinas Biológicas/biosíntesis , Trypanosoma brucei brucei/genética , Animales , ADN Ribosómico/genética , Genes Reporteros , Marcadores Genéticos , Luciferasas/biosíntesis , Luciferasas/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas , Toxinas Biológicas/genéticaRESUMEN
African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.