Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas.
We have shown previously that the HSP70A (A) promoter, when fused upstream of other promoters, significantly improves their performance in driving transgene expression in Chlamydomonas. Here, we employed the bacterial resistance gene ble, driven by the RBCS2 (R) promoter or an AR promoter fusion, to determine, by which mechanism(s) the A promoter may exert its enhancing effect. We observed that transformation rates of AR-ble constructs were significantly higher than those of R-ble constructs. However, ble mRNA levels in pools of transformants generated with either construct type were the same. Co-transformation experiments revealed that the R-ble transgene was silenced in 80% of the transformants, whereas this fraction was reduced to 36% in transformants harbouring the AR-ble transgene. We conclude that the A promoter acts by decreasing the probability that a transgene becomes transcriptionally silenced. We mapped two elements within the A promoter that are responsible for this effect. The core of the first element appears to be located between nucleotides - 7 and + 67 relative to the HSP70A transcriptional start site. Its activity is strongly dependent on its spatial setting with respect to the R promoter and is increased by upstream sequences (- 196 to - 8). The second element is independent of the first and is located to the region from - 754 to - 197. Its activity is spacing-independent and additive to the first element.
Institut de Biologie Physico-Chimique UPR 1261, 13, rue Pierre et Marie Curie, 75005 Paris, France. michael.schroda@biologie.uni-freiburg.de
Michael
M
Schroda
Christoph F
CF
Beck
Olivier
O
Vallon
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002874:Chromosome Mapping; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins