Silent point mutation in DsRed resulting in enhanced relative fluorescence intensity
2004; Future Science Ltd; Volume: 36; Issue: 2 Linguagem: Inglês
10.2144/04362bm06
ISSN1940-9818
Autores Tópico(s)Molecular Biology Techniques and Applications
ResumoBioTechniquesVol. 36, No. 2 BenchmarksOpen AccessSilent point mutation in DsRed resulting in enhanced relative fluorescence intensityMaik Klasen & Matthias WablMaik Klasen*Address correspondence to: Maik Klasen, Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0670, USA. e-mail: E-mail Address: mkl@itsa.ucsf.eduUniversity of California, San Francisco, CA, USA & Matthias WablUniversity of California, San Francisco, CA, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04362BM06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail The commercially available red fluorescence protein DsRed2 (Living Colors™; BD Biosciences Clontech, Palo Alto, CA, USA) is a useful tool in multicolor tagging experiments. Particularly, its emission maximum at 587 nm makes it easy to separate from the green fluorescent protein (GFP), with an emission maximum at 508 nm. DsRed was originally cloned from a coral of the Discosoma genus (1). For expression in mammalian systems, the gene was codon-optimized according to the human codon usage table for highly expressed genes (2) to have all serines encoded by the predominant UCC codon. In human cells, UCC recruits an abundant serine tRNA, with a frequency of 17.8 times per 1000 codons (http://www.kazusa.or.jp/codon/). The UCA codon, which also incorporates serine, is less frequent (12.3 times per 1000 codons). Furthermore, between human and mouse, the UCC to UCA tRNA ratios differ only slightly, with 1.46:1 and 1.57:1, respectively.In a cloning strategy to make constructs for a project using mutation indicator plasmids, we mutated the serine codon UCC at amino acid position 179 to a UCA codon in the DsRed gene. To obtain this mutant, the pDsRed2-1 served as PCR template for site-directed PCR mutagenesis using the oligomer 5′-TGGTGGAGTTCAAGTCAATCTACATGGCCAAGAAGCC-3′. Because the procedure replaced the cytidine by an adenosine at nucleotide position 537, we designated the mutated gene DsRed 537. The sequence of the mutated gene was verified before and after cloning into the mammalian vectors. The mutated and original DsRed genes were cloned into the XhoI and NotI restriction sites of the CRU5-IRES-GFP vector (J.B. Lorens, personal communication), replacing the IRES-GFP fragment. The CRU5-IRES-GFP vector is based on the CRU5-96-dsGFP vector (3); it allows for DsRed to be either transiently expressed directly from the plasmid, driven by the cytomegalovirus (CMV) promoter, or by the retroviral Moloney murine leukemia virus (MMLV) promoter after the stable integration of the provirus into the host cell genome. To produce retroviral particles, 1 × 106 Phoenix E packaging cells (4) were transfected with 6 µg plasmid DNA in 18 µL FuGENE™ 6 reagent (Roche Applied Science, Indianapolis, IN, USA). For transduction, 5 ×105 NIH3T3 cells were spin-infected with 2 mL of the virus containing supernatant from the transfected Phoenix E cells (4,5). For this procedure, we used Polybrene® (Sigma, St. Louis, MO, USA) at a concentration of 4 µg/mL and centrifuged the cells at 800×g for 1 h at room temperature. For the transduction of the murine 18-81 lymphocyte line (6), we used 6 mL of virus containing supernatant to transduce 5 ×105 cells. The transduced cells were analyzed after 3 and 5 days by flow cytometry on a FACSCalibur™ (BD Biosciences, San Jose, CA, USA). Because the construct copy number per cell influences relative fluorescence intensity, samples with identical transduction rates were compared.The direct correlation between expression rate and the representation of rare versus common tRNAs has been extensively documented (7,8). According to these reports, if the switch of the UCC codon to the slightly less favorable UCA codon would directly translate into gene expression levels, one could predict a slight reduction of relative fluorescence intensity for the mutated DsRed protein. However, surprisingly, the single base pair substitution resulted in an increase, rather than in a decrease, in relative fluorescence intensity (Figure 1). The relative fluorescence intensity of DsRed 537 was increased 3.6-fold in the NIH3T3 line (Figure 1A) and 4.1-fold in the 18-81 line (Figure 1B). Even in DsRed-Express1 (Living Colors; BD Biosciences Clontech), which is an improved variant of DsRed2 because it shows a much higher fluorescence intensity from the beginning, the same single base pair substitution resulted in a 1.5-fold increase (data not shown). Because the single base pair substitution presumably has no effect on the transcription of the gene and the amino acid sequence has not changed, the effect should be at the mRNA or translational level. Initiation of mRNA translation is the same for both transcripts, but the elongation at the mutated site might be affected. The point mutation could have altered tertiary mRNA structure and, therefore, have improved processivity of the ribosomes. However, the mRNA prediction software mfold (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/) did not show any changes in mRNA structure. Another possibility could be an increase in mRNA stability; but, theoretically, the less frequently used codons decrease it (9). Other explanations include the influence of flanking codons on codon usage at particular sites. The so-called codon-context effect has been found in various species; it describes the finding that some major codons are not used preferentially at all sites (10). The effect is presumably due to codon-codon interaction during translation (11). A more complex explanation for our findings would be decreased differences in the energy of codon-anticodon pairing during the elongation phase (12).Figure 1. Flow cytometry analysis of DsRed-expressing cells.Fluorescent positive cells were gated, and transduction rates and relative y-means were determined. (A) NIH3T3 cells and (B) 18-81 cells transduced with the retroviral CRU5-DsRed2 and CRU5-DsRed537, respectively.It is also possible that tRNA depletion occurs during the translation of overexpressed genes. To date, the tRNA drop-off phenomenon has been shown only under starvation conditions for aminoacylatable tRNA in Escherichia coli and in vitro (13), but it seems to be an attractive explanation for the results of our study. Because all serines in the DsRed gene are encoded by the UCC codon, substituting one UCC with a UCA codon makes available an entirely unused tRNA pool. 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Chem. 253:6808–6813.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByIntroduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells24 August 2013 | Biotechnology Letters, Vol. 35, No. 12Retroviral vectors to monitor somatic hypermutationJournal of Immunological Methods, Vol. 300, No. 1-2Codon bias and heterologous protein expressionTrends in Biotechnology, Vol. 22, No. 7 Vol. 36, No. 2 Follow us on social media for the latest updates Metrics Downloaded 254 times History Received 30 October 2003 Accepted 24 November 2003 Published online 6 June 2018 Published in print February 2004 Information© 2004 Author(s)AcknowledgmentsWe thank J.B. Lorens (Rigel Inc., San Francisco, CA, USA) for his kind gift of the CRU5-IRES-GFP vector. This study was supported by a stipend of the Boehringer Ingelheim Fonds to M.K. and by National Insitutes of Health grant no. AG20684 to M.W. This work has been performed as part of a requirement for the Ph.D. degree at the Ludwig-Maximilians-University, Munich (supervisor Dr. Elizabeth Weiss).PDF download
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