High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have modified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fragment encoding for mouse ornithine decarboxylase (ODC) proline-glutamate-serine-threonine-rich (PEST) sequence in order to target the protein to the proteasomes and achieved an unprecedentedly fast GFP turnover in permanently transfected human cells. As early as 1 h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fold. Concordantly, treatments with transcription inhibitors α-amanitin and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) resulted in progressive depletion of the destabilized GFP, detected as fluorescence decline, while the stable protein levels were not affected under the same conditions. Moreover, fluorescence of the destabilized but not of normal GFP decreased strongly and in a dose-dependent manner following an instant transcription block induced by ultraviolet-C (UVC) irradiation. In agreement with the transient nature of the transcriptional block due to transcription-coupled DNA repair, the GFP fluorescence fully recovered after several hours.
Cessation of transcription occurs very rapidly when genotoxic lesions that act as roadblock for RNA polymerases are induced in the template DNA strand (1,2,3,4). This transcription block is physiologically important since it triggers a specialized transcription-coupled DNA repair (TCR) pathway (5,6). Hence the mechanism underlying the establishment of the block and its subsequent release are intensively studied at the present. Reporter proteins can be highly useful to monitor the generation and processing of the transcription-blocking DNA modifications in specific genes. Green fluorescent protein (GFP) is a widely used reporter for gene expression studies because of its bright and stable fluorophore, low toxicity, and ease of quantitative detection without disrupting the cell (7). Since GFP is very stable in cells (8), it accumulates to high levels that facilitate detection. At the same time, this high stability makes the protein unsuitable for the tasks when the dynamical changes in gene expression are to be studied.
To overcome this problem, some destabilized variants of fluorescent proteins have been engineered during the last decade. Different groups succeeded in considerably reducing the protein lifetimes by the fusion of natural protein degradation signals. The most promising results were obtained with the use of an ornithine decarboxylase (ODC) proline-glutamate-serine-threonine-rich (PEST) sequence fused to the C terminus of the reporter proteins. The PEST domain is well-known to be responsible for fast proteasomal degradation of ODC in many animal species (9,10) and commits rapid turnover when transferred to naturally stable proteins (11). Wild-type GFP has a half-life of about 26 h. However, when PEST-containing sequences from mouse Odc gene were fused to GFP, the protein half-life shortened to 5.5 h in permanent transfection experiments (8) and even 2 h (12) in transiently transfected cells. To accelerate turnover, these proteins were further destabilized with a mouse cyclin B destruction box in addition to the ODC fragment (8) or by engineering of the PEST sequence optimized for the most efficient degradation (12). Firefly luciferase is intrinsically less stable than GFP and its half-life could be further reduced to somewhat shorter than 1 h by a similar fusion approach (13). Yet, even when a reporter protein with a sufficiently high clearance rate is used, the stability of its messenger RNA (mRNA) might still interfere with sensitive and fast detection of changes in gene expression. Indeed, improved responses to a transcriptional repressor were achieved with double-destabilized reporter constructs that contained both protein and RNA decay determinants (14,15).
Here, we used an episomal expression vector carrying a strong constitutive promoter and the signal elements for a rapid decay of GFP mRNA and further destabilized the protein by fusion with a mouse ODC fragment. The goal of this work was to create stably transfected cell lines in which reporter protein expression could be monitored in nearly real-time without harmful effects on the host cells. We describe a sufficiently sensitive reporter system to allow the detection of the short-lasting transcription block caused by ultra-violet-C (UVC) in mammalian cells.Materials and Methods Vectors
pMARS episomal vector (16) contains a modified GFP gene from Aequorea victoria (17) that is expressed under the control of the human cytomegalovirus (CMV) promoter and harbors in its 3′ untranslated region (UTR) a nuclear scaffold/matrix attachment region (MAR) core element from the human interferon gene repeated four times. pMARS vector was linearized by using an available BglII site between the GFP and MAR sequences, and the produced overhangs were filled in with T4 DNA polymerase (New England Biolabs, Frankfurt am Main, Germany) for a subsequent bluntend cloning of the ODC fragment. The 3′ portion of Odc gene exon 10 (Mouse Genome Informatics; accession no. MGI:97402) was produced from mouse genomic DNA by PCR using PfuTurbo® DNA Polymerase (Stratagene, Amsterdam, The Netherlands). 5′-Phosphorylated primers (Operon, Cologne, Germany) were the following: 5′-TCTCATGAAGCAGATCCAGA-3′ (forward) and 5′-TAGTACTCATCTACACATTGATCCTAG-3′ (reverse) and amplified the fragment encoding for the 46 C-terminal amino acid residues of ODC. Underlined portions of the oligonucleotides correspond to Odc gene sequence, and bold letters correspond to stop-codons for the two possible insert orientations. PCR products were purified from the agarose gel and ligated into the linearized blunted vector in random orientations. To pick the clones containing the ODC fragment in sense (designated AZ) and antisense (ZA) directions, 30 clones were screened by three-primer PCR with primers: 5′-GACCACTACCAGCAGAACAC-3′ (GFP), 5′-TCTCCTGGGCACAAGACAT-3′ (AZ-ODC), and 5′-GCCTGTGCTTCTGCTAGGAT-3′ (ZA-ODC). After sequencing of selected positive clones, we named them pMAZ-ODC (for the correct in-frame insertion of ODC fragment) and pMAZ-ODC (for the inverted ODC fragment, preceded by the PCR-introduced translation stop-codon).