Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei.
Journal - The EMBO journal (ENGLAND )
Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.
|ISSN : ||0261-4189|
|Mesh Heading : ||Animals Anti-Bacterial Agents Cloning, Molecular Drug Resistance Fluorescent Antibody Technique Gene Expression Regulation Genetic Markers Hygromycin B Telomere Time Factors Transcription, Genetic Trypanosoma brucei brucei Variant Surface Glycoproteins, Trypanosoma pharmacology genetics analogs & derivatives pharmacology physiology cytology|
|Mesh Heading Relevant : ||Cinnamates Genes, Protozoan genetics genetics metabolism|
Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei.
Journal - Proceedings of the National Academy of Sciences of the United States of America (UNITED STATES )
In Trypanosoma brucei, transcription by RNA polymerase II and 5' capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.
|ISSN : ||0027-8424|
|Mesh Heading : ||Animals Cell Nucleus DNA Polymerase II DNA, Ribosomal Genetic Markers Immunohistochemistry In Situ Hybridization, Fluorescence Poly(ADP-ribose) Polymerases Trypanosoma brucei brucei Variant Surface Glycoproteins, Trypanosoma metabolism genetics genetics enzymology|
|Mesh Heading Relevant : ||Gene Expression metabolism genetics genetics|