functional cassette is a 237 bp BclI (2770)-BamHI (2533) restriction fragment of the SV40 genome containing the cleavage/polyadenylation signals of the early ...
\.) 1993 Oxford University Press
Nucleic Acids Research, 1993, Vol. 21, No. 21 4987-4988
Mammalian gene expression is improved by longer SV40 early polyadenylation cassette
use
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Maurice J.B.van den Hoff, Wil T.Labruyere, Antoon F.M.Moorman and Wouter H.Lamers* Department of Anatomy and Embryology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Received August 9, 1993; Revised and Accepted September 20, 1993
Expression vectors normally contain sequences that stimulate polyadenylation of the expressed mRNA. These sequences usually include an AATAAA consensus sequence 20- 30 nucleotides 5' of a diffuse GT- or T-rich stretch immediately following the sequences that form the 3' end of the mRNA. The efficiency of the two minimal elements is also influenced by additional upor downstream elements (1). Very often polyadenylation sequences are taken from the viral SV40 genome. The minimal functional cassette is a 237 bp BclI (2770)-BamHI (2533) restriction fragment of the SV40 genome containing the cleavage/polyadenylation signals of the early and the late transcription units, depending on the orientation (2). In commercially available vectors the fragment is frequently used in the early orientation. However, sometimes a longer cassette of the early polyadenylation sequences is used. This cassette contains a 988 bp restriction fragment from BclI (2770) up to EcoRI (1782), i.e. extending into the 3' end of the VP1 coding region (3). The expression efficiency of both cassettes was tested in two pUC-based expression vectors, that are otherwise comparable. In both constructs the respective polyadenylation sequences are fused to the coding sequences of the chloramphenicol acetyltransferase (CAT) gene. The expression of CAT activity was assayed when driven by a weak or by a strong promoter. As weak promoters the first 161 (PstI) or 525 bp (DraI) (4) nucleotides upstream of the transcription initiation site of the rat carbamoylphosphate synthase (CPS) gene were used, while the Rous sarcoma virus long terminal repeat (RSV-LTR) was used as a strong promoter. After CsCl purification, 50 Ag of these constructs was transfected into 1 x 107 FTO2B rat hepatoma cells as described (5). As an internal control 1 /tg pRSVluc was included in each transfection. Two days after electrotransfection, the cells were harvested in ice-cold PBS supplemented with 2 mM EDTA pH 8.0, and lysed in 0.1 % Triton X-100 in 100 mM potassium phosphate buffer pH 7.6. In each lysate the protein concentration (BCA protein reagent kit, Pierce), luciferase (6) and chloramphenicol acetyl transferase activity (7) was determined. Both vectors expressed no detectable CAT activity, when promoter sequences were absent. The activities of the weak promoters when expressed from the vector containing the shorter polyadenylation sequence are below the limit of detection of the *
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assay, whereas when expressed in combination with the longer polyadenylation sequence a low but reproducible activity was detected (data not shown). When expression of the CPS promoter constructs was hormonally stimulated (10-7 M dexamethasone, 10-4 M dibutyryl cAMP and 10-3 M 3-isobutyl-1-methylxanthine), CAT expression became measurable even with the shorter polyadenylation sequence, but calculated activities were more than 100-fold lower than those obtained with the longer sequences for polyadenylation (Table 1). When CAT activity was driven by the strong viral RSV-LTR promoter, a 50-fold enhancement of gene expression was found with the longer polyadenylation sequence compared to the shorter (Table 1). A quantitatively comparable enhancement of expression of these constructs was observed in rat 5123 hepatoma cells, in rat Rat-I fibroblasts and in mouse NIH 3T3 fibroblasts. The shorter polyadenylation sequence in the late orientation has been shown to be more efficient than in the early orientation (10). If the shorter sequences for polyadenylation in the early orientation is extended up to the EcoRI site, gene expression proves to be at least equally if not more efficient than the short one in the late orientation. A convenient cloning vector should contain only the essential sequences necessary for high expression. The plasmid containing the RSV-LTR promoter upstream of the CAT gene and the longer polyadenylation sequence downstream of the CAT gene was used to identify the shortest sequence having the full capacity. The supercoiled plasmid was linearized with restriction enzymes BamHI, ApaI, PstI and NdeI to create sequences for polyadenylation of 237 bp, 512 bp, 782 bp and 988 bp, Table 1. Comparison of the efficiency of two frequently used SV40 sequences for polyadenylation Promoter
SV40 polyadenylation sequences
237 bp
-161 bp CPS -535 bp CPS RSV-LTR
0.4 A 0.1 (3) 0.3 b 0.0 (3) 26.0 A 5.0 (3)
988 bp
134.7 X 8.1 (3) 47.5 + 3.0 (3) 1294.0 + 75.4 (9)
The shorter sequence is a 237 bp Bcll (2770)-BamHI (2533) and the longer one a BclI (2770)-EcoRI (1782) restriction fragment of the SV40 genome. The numbers in the brackets indicate the number of independent transfections, while the other values represent the mean and SEM of the CAT activity in tU/,ug protein, after correction for the internal control (luciferase activity).
4988 Nucleic Acids Research, 1993, Vol. 21, No. 21 Table 2. Effect of the length of the SV40 early polyadenylation sequence on expression efficiency
length of sequence for polyadenylation:
relative transient CAT expression
237 bp (BamHI) 512 bp (ApaI) 782 bp (PstI)
8 1% 28 A 2% 60 A 4% 100 + 7%
988bp(NdeI)
Luc expression
16 X 1% 25 A 1%
100 + 3%
The plasmid containing the RSV-LTR promoter sequence upstream of either the CAT or the Luciferase gene and the BclI (2770)-EcoRI (1782) polyadenylation sequence of SV40 was cut with the restriction enzyme indicated between brackets. The resulting linear plasmids were transfected into FTO2B hepatoma cells. The relative ransient CAT and Luciferase expression, after correction for the internal control, is shown as the mean and SEM of three transfections with the longest sequence being set to 100% (1700 IU CAT/jg protein, 9586 RLU/tg protein).
respectively (EcoRI could not be used, because it also has a restriction site in the CAT gene). To find out if the observed effects were related to the reporter gene used, a plasmid containing the RSV-LTR promoter upstream of the Luciferase (Luc) gene and the longer polyadenylation sequence downstream of the Luc gene was linearized with restriction enzymes BamHI, Apal and NdeI to create sequences for polyadenylation of 237 bp, 512 bp and 988 bp, respectively (EcoRI and PstI could not be used, because both have a restriction site within the Luc gene). The obtained results (Table 2) clearly show that the longest polyadenylation sequence results in the highest level of expression irrespective of the reporter gene used. Our results show that the sequences 3' of the SV40 early polyadenylation signals increase the efficiency of the cleavage and polyadenylation reaction. This claim is supported by the fact that this part of the SV40 genome does not contain sequences that influence the SV40 promoters (7), that the stability of the chimeric mRNAs is not different because only a single polyadenylation signal is present, and that essentially the same results are obtained irrespective of the promoter, reporter gene and cell line used.
REFERENCES 1. Proudfoot,N. (1991) Cell 64, 671-674. 2. Sambrook,J., Fritsch,E.F. and Maniatis,J. (1989) Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, New York,
16, 6-7. 3. Gorman,C.M., Maffat,L.F. and Howard,B.H. (1982) Mol. Cell. Biol. 7, 1044-1051. 4. Lagacd,M., Howell,B.W., Burack,R., Lusty,C.J. and Shore,G.C. (1987) J. Biol. Chepn. 262, 10415-10418. 5. van den Hoff,M.J.B., Labruybre,W.T., Moorman,A.F.M. and Lamers,W.H. (1990) Nucleic Acids Res. 18, 6464. 6. Brasier,A.R., Tata,J.E. and Habener,J.F. (1989) Biotechniques 7, 1116-1121. 7. Seed,B. and Sheen,J.-Y. (1988) Gene 67, 271-277. 8. Carwells,S. and Alwine,J.C. (1989) Mol. Cell. Biol. 9, 4248-4258.
