Reporter plasmids utilizing the cat gene for the analysis of promoter and enhancer sequences in vertebrate cells, were constructed. These plasmids minimize the background of transcription derived from cryptic promoters or cryptic regulatory elements within the vecto

Andrea Schmidt

Boshart

Bruno Luckow

Buchman

Günther Schütz

Heard

Luckow

Michael Boshart

Michael Klüppel

Ruppert

Schüle

Seamon

English

Boshart, Michael

Klüppelberg, Michael

Schmidt, Andrea

Schütz, Günther

Luckow, Bruno

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Gene, 110 (1992) 129-130 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00 129 GENE 06238 Brief Notes Reporter constructs with low background activity utilizing the cat gene (Recombinant DNA; chloramphenicol acetyltransferase assay; gene transfer; promoter/enhancer analysis; transcriptional regulation; cryptic promoter/enhancer) Michael Boshart, Michael Ki0ppel*, Andrea Schmidt, GUnther Schtitz and Bruno Luckow Institute of Cell and Tumor Biology, German Cancer Research Center, W-6900 Heidelberg I (F,R.G.) Received by H.G. Zachau: 12 September 1991 Revised/Accepted: 22September/23 September 1991 Received at publishers: 22 October 1991 SUMMARY Reporter plasmids utilizing the cat gene for the analysis of promoter and enhancer sequences in vertebrate cells, were constructed. These plasmids minimize the background of transcription derived from cryptic promoters or cryptic regula- tory elements within the vector. Reporter genes allowing functional analysis of eukary- otic promoters and regulatory cis-elements by a simple en- zymatic assay (CAT, luciferase,/~-galactosidase, etc.)are widely used. A major caveat of these assays is that expres- sion of enzymatic activity may result from transcription initiation within the vector. Also, vector sequences may affect the activity of the promoter under test. The former situation can be controlled by RNA start point mapping whereas exclusion of the latter possibility requires prepar- ative removal of vector sequences before gene transfer. Both methods are impractical for routine transient trans- fection assays. We have previously described the reporter plasmids Correspondence to: Dr. G. SchOtz, Institute of Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld 280, W-6900 Heidelberg 1 (F.R.G.) Tel. (49.6221)42-3411; Fax(49-6221)40-2598. * Present address: Mount Sinai Hospital, Samuel Lunenfeld Research Institute, 600 University Ave., Toronto, Ontario M5G IX5 (Canada) Tel. (416)586-8272. Abbreviations: bla, gene ncoding ~-lactamase; bp,base pair(s); CAT, Cm acetyltransferase; cat, gene encoding CAT; C/EBP, CCAAT box/ enhancer-binding protein; Cm, chloramphenicol; HSV, herpes implex virus; kb, kilobase(s) or1000 bp; nt, nucleotide(s); SV40, simian virus 40; tk, H SV gene ncoding thymidine kinase. pBLCAT2 and pBLCAT3 with convenient restriction sites 5' and 3' of the cat transcription unit (Luckow and Sch0tz, 1987). In a few cell lines we noted cat expression even from the promoterless construct pBLCAT3 and strong activa- tion of expression after cAMP induction. Also, cotransfec- tion of an expression vector encoding the transcription factor C/EBP led to strong activation of cat expression from pBLCAT3. We and others have attempted to identify and delete the respective prokaryotic sequences (SchOle et al., 1988) giving rise to high background of cat expres- sion in some cell lines. Also, insertion of 'terminator' se- quences has been suggested to prevent initiation of cat transcripts at cryptic promoters in the vector (Heard et al., 1987). To improve pBLCAT2 and pBLCAT3, we have combined both strategies: 467 bp of vector sequence im- mediately upstream from the polylinker were deleted and replaced by two fragments, each containing the polyade- nylation signal of the SV40 large T encoding ene. As a result, transcripts derived from vector starts should be 3' processed efficiently and no mature mRNA encoding CAT should be generated. The resulting cat reporter plasmids pBLCAT5 (with HSV tk promoter) and pBLCAT6 (pro- moterless) have polylinkers 5' and 3' to the cat transcrip- tion unit (Fig. 1) and give high plasmid yields. 'Vector ef- fects' on cat expression are abolished or strongly reduced. 130 = I .~  ~m e'~ ml  ~ AAUAAA S~sll clai Smal Kpnl ~c+ a .~ ×- ' , , , ' ,x  I AAUAJ~A _~ ~ . . ~  AAUAAA Fig. 1. Maps of pBLCAT5 and pBLCAT6. The plasmids contain SV40 polyadenylation signals (black boxes, AAUAAA), the cat gone, the SV40 small t intron sequences (SV40), the ~lactamase encoding one (bla) and pBLCAT5 contains the tk promoter (open box, tk). For construction of pBLCATS, the sequence from nt 4428 (Aatll site) to nt 398 (5' of HindIll site) of pBLCAT2 (Luckow and Sch(ltz, 1987) was deleted and replaced by two fragments from SV40 (Buchman et al., ~981) inserted in tandem (Hpai-BamHI, nt 2668-2534 and BclI-BamHI, nt 2770-2534). Plasmid pBLCAT6 was similarly derived from pBLCAT3. The modified parts of the vectors wore verified by sequencing. Compiled sequences for pBLCAT5 and pBLCAT6 are available (GenBank accession Nos. M80483 and M80484, respectively). As an example, Fig. 2 shows that in PC12 cells high basal expression (even from the promoterless construct) and re- sponse to cAMP induction is abolished. In FT02B rat hepatoma ceils, transactivation of vector derived cat ex- pression by cotransfected C/EBP is reduced about eight- fold with the modified versions pBLCAT5 and pBLCAT6 (Ruppert et ai., 1990) (data not shown). In human mela- noma cells (SKMeI25 and SKMeI28), vector derived ex- pression from pBLCAT6 is more than fivefold reduced compared to pBLCAT3 (data not shown), In conclusion, a substantial improvement of the pBLCAT reporter con- structs, now used in many laboratories, has been achieved. REFERENCES Boshart, M., Weih, F., Schmidt, A., Fournier, R.E.K. and Sch(ltz, G.: A cyclic AMP response lement mediates repression of tyrosine ami- notransferase g ne transcription by the tissue specific extinguisher locus Tse-l. Cell 61 (1990) 905-916. pBLCAT 2 tk 4- m + + lm + ,-. .. i i i  i l l  ? ? rel. CAT act. ( t  a B 3 1 7.12 0.09 • e t o oql - O ~ ~O 0.24 ® • Fig. 2. Comparison of pB LCAT reporter constructs in PC 12 cells. PC 12 cells were electroporated as described (Boshart etai., 1990) with pBLCAT2 and 3 (Luckow and Schtltz, 1987) and pBLCAT5 and 6 (see Fig. 1) and induced 4h later with forskolin (forsk., which activates ade- nylate cyclase; Seamen et al., 1981) as indicated by + symbols. After 24 h of expression, extracts were prepared and assayed for CAT activity and protein as described (Boshart et al., 1990). CAT activity was quantified, corrected for protein and is given as relative activity, setting the value of uninduced pBLCAT2 to 1. An autoradiogram of the thin layer chromate- gram used to separate chloramphenicol from its acetylated erivatives (l-ac and 3-ac) is shown. Buchman, A.R., Burnett, L. and Berg, P.: Appendix A/The SV40 nucle- otide sequence. In: Tooze, J. (Ed.), DNA Tumor Viruses, Molecular Biology of Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981, pp. 799-841. Heard, J.-M., Herbomel, P., Ott, M.-O., Mottura-Roller, A., Weiss, M,C. and Yaniv, M.: Determinants of rat albumin promoter tissue speci- ficity analysed by an improved transient expression system. Mol. Cell. Biol. 7 (1987) 2425-2434. Luckow, B. and Schlitz, G.: CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic A,.~ids Res. 15 (1987) 5490. Ruppert, S., Boshart, M., Bosch, F.X., Schmid, W., Fournier, R.E.K. and SchOtz, G.: Two genetically defined trans-acting loci coordinately regulate overlapping sets of liver-specific genes. Cell 61 (1990) 895- 904. SchOle, R., Muller, M., Otsuka-Murakami, H. and Renkawitz R.: Coop- erativity of the glucocorticoid receptor and the CACCC-box binding factor. Nature 322 (1988) 87-90. Seamen, K.B., Padgett, W. and Daly, J.W.: Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Prec. Natl. Acad. Sci. USA 78 (1981) 3363-3367. 

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue specific extinguisher locus Tse-l.

Reporter constructs with low background activity utilizing the cat gene

Crossref

Universität München: Elektronischen Publikationen

Gene, 110 (1992) 129-130 
© 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00 129 
GENE 06238 
Brief Notes 
Reporter constructs with low background activity utilizing the ca t  gene 
(Recombinant DNA; chloramphenicol acetyltransferase assay; gene transfer; promoter/enhancer analysis; transcriptional 
regulation; cryptic promoter/enhancer) 
Michael Boshart, Michael Ki0ppel*, Andrea Schmidt, GUnther Schtitz and Bruno Luckow 
Institute of Cell and Tumor Biology, German Cancer Research Center, W-6900 Heidelberg I (F,R.G.) 
Received by H.G. Zachau: 12 September 1991 
Revised/Accepted: 22 September/23 September 1991 
Received at publishers: 22 October 1991 
SUMMARY 
Reporter plasmids utilizing the cat gene for the analysis of promoter and enhancer sequences in vertebrate cells, were 
constructed. These plasmids minimize the background of transcription derived from cryptic promoters or cryptic regula- 
tory elements within the vector. 
Reporter genes allowing functional analysis of eukary- 
otic promoters and regulatory cis-elements by a simple en- 
zymatic assay (CAT, luciferase,/~-galactosidase, etc.) are 
widely used. A major caveat of these assays is that expres- 
sion of enzymatic activity may result from transcription 
initiation within the vector. Also, vector sequences may 
affect the activity of the promoter under test. The former 
situation can be controlled by RNA start point mapping 
whereas exclusion of the latter possibility requires prepar- 
ative removal of vector sequences before gene transfer. 
Both methods are impractical for routine transient trans- 
fection assays. 
We have previously described the reporter plasmids 
Correspondence to: Dr. G. SchOtz, Institute of Cell and Tumor Biology, 
German Cancer Research Center, Im Neuenheimer Feld 280, W-6900 
Heidelberg 1 (F.R.G.) Tel. (49.6221)42-3411; Fax (49-6221)40-2598. 
* Present address: Mount Sinai Hospital, Samuel Lunenfeld Research 
Institute, 600 University Ave., Toronto, Ontario M5G IX5 (Canada) 
Tel. (416)586-8272. 
Abbreviations: bla, gene encoding ~-lactamase; bp, base pair(s); CAT, Cm 
acetyltransferase; cat, gene encoding CAT; C/EBP, CCAAT box/ 
enhancer-binding protein; Cm, chloramphenicol; HSV, herpes simplex 
virus; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); SV40, simian virus 40; 
tk, H SV gene encoding thymidine kinase. 
pBLCAT2 and pBLCAT3 with convenient restriction sites 
5' and 3' of the cat transcription unit (Luckow and Sch0tz, 
1987). In a few cell lines we noted cat expression even from 
the promoterless construct pBLCAT3 and strong activa- 
tion of expression after cAMP induction. Also, cotransfec- 
tion of an expression vector encoding the transcription 
factor C/EBP led to strong activation of cat expression 
from pBLCAT3. We and others have attempted to identify 
and delete the respective prokaryotic sequences (SchOle 
et al., 1988) giving rise to high background of cat expres- 
sion in some cell lines. Also, insertion of 'terminator' se- 
quences has been suggested to prevent initiation of cat 
transcripts at cryptic promoters in the vector (Heard et al., 
1987). To improve pBLCAT2 and pBLCAT3, we have 
combined both strategies: 467 bp of vector sequence im- 
mediately upstream from the polylinker were deleted and 
replaced by two fragments, each containing the polyade- 
nylation signal of the SV40 large T encoding gene. As a 
result, transcripts derived from vector starts should be 3' 
processed efficiently and no mature mRNA encoding CAT 
should be generated. The resulting cat reporter plasmids 
pBLCAT5 (with HSV tk promoter) and pBLCAT6 (pro- 
moterless) have polylinkers 5' and 3' to the cat transcrip- 
tion unit (Fig. 1) and give high plasmid yields. 'Vector ef- 
fects' on cat expression are abolished or strongly reduced. 
130 
= 
I . ~  ~ m  e'~ m l  
~ AAUAAA 
S~sll claiSmal Kpnl 
~ c +  a . ~  × - ' , , , ' , x  I 
AAUAJ~A _~ 
~ . . ~  AAUAAA 
Fig. 1. Maps of pBLCAT5 and pBLCAT6. The plasmids contain SV40 
polyadenylation signals (black boxes, AAUAAA), the cat gone, the SV40 
small t intron sequences (SV40), the ~lactamase encoding gone (bla) and 
pBLCAT5 contains the tk promoter (open box, tk). For construction of 
pBLCATS, the sequence from nt 4428 (Aatll site) to nt 398 (5' of HindIll 
site) of pBLCAT2 (Luckow and Sch(ltz, 1987) was deleted and replaced 
by two fragments from SV40 (Buchman et al., ~981) inserted in tandem 
(Hpai-BamHI, nt 2668-2534 and BclI-BamHI, nt 2770-2534). Plasmid 
pBLCAT6 was similarly derived from pBLCAT3. The modified parts of 
the vectors wore verified by sequencing. Compiled sequences for 
pBLCAT5 and pBLCAT6 are available (GenBank accession Nos. 
M80483 and M80484, respectively). 
As an example, Fig. 2 shows that in PC12 cells high basal  
expression (even from the promoterless construct)  and re- 
sponse to c A M P  induction is abolished. In FT02B rat 
hepatoma ceils, t ransactivation of  vector derived cat ex- 
pression by cotransfected C /EBP is reduced about eight- 
fold with the modified versions pBLCAT5  and pBLCAT6 
(Ruppert  et ai., 1990) (data  not shown). In human mela- 
noma cells (SKMeI25 and SKMeI28),  vector derived ex- 
pression from p B L C A T 6  is more than fivefold reduced 
compared to p B L C A T 3  (data  not shown),  In conclusion, 
a substantial improvement  of the p B L C A T  reporter con- 
structs, now used in many laboratories,  has  been achieved. 
REFERENCES 
Boshart, M., Weih, F., Schmidt, A., Fournier, R.E.K. and Sch(ltz, G.: A 
cyclic AMP response element mediates repression of tyrosine ami- 
notransferase gene transcription by the tissue specific extinguisher 
locus Tse-l. Cell 61 (1990) 905-916. 
pBLCAT 
2 tk 
4- 
m 
+ 
+ 
lm  
+ 
,-. . .  
i i i  i l l  
? ? rel. CAT act. 
( t  a 
B 3 
1 
7.12 
0.09 
• e t 
o oql 
- O 
~ ~ O  0.24 
® • 
Fig. 2. Comparison of pB LCAT reporter constructs in PC 12 cells. PC 12 
cells were electroporated as described (Boshart etai., 1990) with 
pBLCAT2 and 3 (Luckow and Schtltz, 1987) and pBLCAT5 and 6 (see 
Fig. 1) and induced 4 h later with forskolin (forsk., which activates ade- 
nylate cyclase; Seamen et al., 1981) as indicated by + symbols. After 24 h 
of expression, extracts were prepared and assayed for CAT activity and 
protein as described (Boshart et al., 1990). CAT activity was quantified, 
corrected for protein and is given as relative activity, setting the value of 
uninduced pBLCAT2 to 1. An autoradiogram of the thin layer chromate- 
gram used to separate chloramphenicol from its acetylated derivatives 
(l-ac and 3-ac) is shown. 
Buchman, A.R., Burnett, L. and Berg, P.: Appendix A/The SV40 nucle- 
otide sequence. In: Tooze, J. (Ed.), DNA Tumor Viruses, Molecular 
Biology of Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratory, 
Cold Spring Harbor, NY, 1981, pp. 799-841. 
Heard, J.-M., Herbomel, P., Ott, M.-O., Mottura-Roller, A., Weiss, M,C. 
and Yaniv, M.: Determinants of rat albumin promoter tissue speci- 
ficity analysed by an improved transient expression system. Mol. Cell. 
Biol. 7 (1987) 2425-2434. 
Luckow, B. and Schlitz, G.: CAT constructions with multiple unique 
restriction sites for the functional analysis of eukaryotic promoters 
and regulatory elements. Nucleic A,.~ids Res. 15 (1987) 5490. 
Ruppert, S., Boshart, M., Bosch, F.X., Schmid, W., Fournier, R.E.K. and 
SchOtz, G.: Two genetically defined trans-acting loci coordinately 
regulate overlapping sets of liver-specific genes. Cell 61 (1990) 895- 
904. 
SchOle, R., Muller, M., Otsuka-Murakami, H. and Renkawitz R.: Coop- 
erativity of the glucocorticoid receptor and the CACCC-box binding 
factor. Nature 322 (1988) 87-90. 
Seamen, K.B., Padgett, W. and Daly, J.W.: Forskolin: unique diterpene 
activator of adenylate cyclase in membranes and in intact cells. Prec. 
Natl. Acad. Sci. USA 78 (1981) 3363-3367. 


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Reporter constructs with low background activity utilizing the cat gene

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