Part:BBa_K404103

p5 promoter

p5 promotor
BioBrick Nr.	BBa_K404103
RFC standard	RFC 10
Requirement	pSB1C3
Source	pAAV_RC from Stratagene
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

The p5 promotor, located downstream of the rep and cap ORF (Figure 1: The p5 promotor of the wtAAV-2 is located upstream of the rep and cap ORF and contains several elements, which interact with Rep and endogenous proteins.)of the wtAAV-2, regulates gene expression of the two larger non-structural proteins Rep 78 and Rep 68 that are essential in genome replication and viral genome integration into several hotspots of the human chromosome.
Several binding elements for cellular and viral proteins involved in regulation can be found in the p5 promotor (Figure 1) therefore playing an important role in gene transcription, integration and replication, dependent on the presence or absence of helper viruses such as adenovirus (Ad) or herpes simplex virus (HSV) (Murphy et al. 2007). Besides regulation of gene expression, the p5 integration efficient element (p5IEE) containing the rep binding element (RBE) and a terminal resolution site (trs) is responsible for mediating site specific integration into the human genome (Philpott et al. 2002).

Figure 1: The p5 promotor of the wtAAV-2 is located upstream of the rep and cap ORF and contains
several elements, which interact with Rep and endogenous proteins .

Containing two consensus sequences for binding immediate early E1A gene product from adenoviruses (Chang et al. 1989), p5 promotor is transactivated in the presence of helper viruses whereas suppression occurs in absence of adenoviral proteins by low levels of Rep proteins (Beaton et al. 1989). Regulating of Rep78/68 by its negative feedback loop is critical since overexpression leads to cell cycle arrest in the S-phase (Berthet et al. 2005) and suppression of cellular promotor (Jing et al. 2001).

Characterization

P5 promotor activity was characterized by using the measurement for eukaryotic promotors proposed by the iGEM team Heidelberg 2009. Since we wanted to compare the promotor strength of a viral promotor we used the production cell line AAV-293 derived from the human embryonic kidney cells (HEK 293). Further aberrations are that we are comparing the natural p5 promotor with the CMV promotor and the p40 promotor driving cap transcription. AAV-293 cells were transfected with plasmids containing the p5, p40 and CMV promotor respectively and harvested after 48 hours. Total mRNA was isolated and reverse transcribed. Promotor strength was determined by quantitative real-time PCR.