- Email: [email protected]
Mapping 3′ Termini of mRNA on DNA Templates with Taq Polymerase 3′-End-Labeled Probes
NOTES & TIPS
much more prominent than the m/z 872 peak indicates that, for the most part, those cells that begin to fix nitrogen from air use air as their main or sole nitrogen source. These two examples lead us to conclude that MALDI–TOF MS can be used as a simple and conclusive method for monitoring isotope incorporation. REFERENCES 1. Patterson, W., B., Carraro, F., and Wolfe, R., R. (1993) Measurement of 15N enrichment in multiple amino acids and urea in a single analysis by gas chromatography/mass spectrometry. Biol. Mass Spectrom. 22, 518 –523. 2. Fogg, G. E., Steward, W. D. P., Fay, P., and Walsby, A. E. (1973) The Blue–Green Algae, Academic Press, London and New York. 3. Bothe, H. (1982) in The Biology of Cyanobacteria (Carr, N. G., and Whitlon, B. A., Eds.), pp. 87–104, Univ. California Press, Berkeley and Los Angeles. 4. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63, 1193–1203. 5. Whittal, M. R., and Li, L. (1995) High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal. Chem. 67, 1950 –1954. 6. Peschek, G. A., Vilgrater, K., and Wastyn, M. (1991) Respiratory protection of nitrogenase in dinitrogen-fixing cyanobacteria. Plant Soil 137, 17–24. 7. Walsby, A. E., and Buckland, B. (1969) Isolation and purification of intact gas vesicles from a blue– green algae. Nature 224, 716 –717. 8. Erhard, M., Von Doehren, H., and Jungblut, P. (1998) MALDI– TOF mass spectrometry. Fast-screening and structure analysis of secondary metabolites. Biospectrum 4, 42– 46.
Mapping 3⬘ Termini of mRNA on DNA Templates with Taq Polymerase 3⬘-End-Labeled Probes Julio C. Pareja and Antonio Jime´nez-Ruiz 1 Departamento de Bioquı´mica y Biologı´a Molecular, Campus Universitario, Universidad de Alcala´, 28871 Alcala´ de Henares, Madrid, Spain Received October 12, 1999
The location of the 3⬘ termini of the mRNAs can be mapped by using nuclease S1, which digests DNA that has not formed duplexes with RNA. The length of the 3⬘-end-labeled DNA fragment protected from digestion allows precise mapping of the 3⬘ termini of the mRNA. In this paper we show that Taq polymerase can be used to add a single radioactive nucleotide to the 3⬘ end of 1 To whom correspondence should be addressed. Fax: 34-1-885 4585. E-mail: [email protected].
Analytical Biochemistry 280, 185–186 (2000) doi:10.1006/abio.2000.4499 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
185
the DNA probe and that the specific activity obtained is sufficient for the S1 protection assay. In other methods, the 3⬘ termini of double-stranded DNA is labeled by using the Klenow fragment of Escherichia coli DNA polymerase I or bacteriophage T4 DNA polymerase. Both enzymes can be used to label ends created by cleavage of DNA with restriction enzymes (1). Restriction sites with 5⬘ protruding ends are easily labeled by filling the recessed 3⬘ termini with radiolabeled nucleotides. However, this method depends on the presence of a restriction site in a position which allows fine mapping of the 3⬘ end of the gene. Moreover, which of the four [␣- 32P]dNTPs is added to the reaction depends on the sequence of the protruding 5⬘ termini at the ends of the DNA. Terminal transferase can also be used for 3⬘ endlabeling, but this enzyme incorporates a long tail of nucleotides at the 3⬘ end of the DNA strands. To obtain a labeled fragment of a known size, the substrate for terminal transferase must be a labeled dideoxynucleotide, which is not a common reagent in the laboratory. The method described in this paper takes advantage of the ability of Taq polymerase to add a single nucleotide after the end of the template. This terminal transferase activity is currently used to prepare vectors designed to clone PCR products (2) and, as it will be shown, can also be employed to add any labeled deoxynucleotide to the 3⬘ end of any amplified fragment. Even though the specific activity of the probes obtained by this method is not as high as, for instance, that obtained by filling the recessed 3⬘ termini of the restriction sites, the widespread use of highly sensitive methods for detecting radioactive signals makes this method highly recommended for 3⬘ mRNA end-mapping. Moreover, the primers used for amplification can also be used in a parallel sequencing reaction to determine the exact position of the 3⬘ end. Labeling protocol. DNA obtained after standard PCR amplification is purified with Geneclean (Bio 101, Inc. U.S.A.) and eluted in a final volume of 15 l. The nucleotide added by Taq polymerase to the 3⬘ end of the amplified products is removed with 4 units of Klenow fragment of E. coli DNA polymerase I (Boehringer GmbH, Germany) in Klenow buffer (15 mM MgCl 2, 3 mM DTT, 0.75 mM dATP, 0.75 mM dGTP, 0.75 mM dCTP, 0.75 mM dTTP, 135 mM Hepes, pH 6.6) in a final volume of 50 l for 30 min at 37°C. After incubation, DNA is purified again with Geneclean (Bio 101, Inc., U.S.A.) to remove free nucleotides. One hundred nanograms of the blunt-ended DNA obtained after Klenow treatment is labeled with 2.5 units of Taq polymerase (Perkin–Elmer Cetus Instruments, U.S.A.) and 100 Ci of [␣- 32P]dCTP (3000 Ci/mmol) in a final volume of 50 l in labeling buffer (50 mM KCl, 1.5 mM MgCl 2, 10 mM Tris–HCl, pH 8.3) at 72°C for 30 min.
186
NOTES & TIPS
REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154 3. Jime´nez-Ruiz, A., Boceta, C., Bonay, P., Requena, J. M., and Alonso, C. (1998) Cloning, sequencing and expression of the PSA genes from Leishmania infantum. Eur. J. Biochem. 251, 389 –397.
Measurement of Apoptosis by the TUNEL Method Using Scintillating Microplates FIG. 1. Location of the 3⬘ termini of the mRNA on a DNA template from a novel transcription unit located in the PSA gene cluster of Leishmania infantum. A parallel sequencing reaction primed with the appropriate oligonucleotide from those used for PCR amplification of the DNA probe is shown at the left. (A) Nuclease S1 protection assay using total RNA from culture parasites at the logarithmic growth phase. (B) Nuclease S1 protection assay using total RNA from culture parasites at the stationary growth phase. Nuclease S1 incubation periods are indicated at the top of each lane. After drying, the polyacrylamide/urea gel was exposed to a phosphor screen for 24 h and analyzed using the Fuji BAS 1000 system. The arrow indicates the protected DNA fragment after complete nuclease S1 digestion of the DNA that has not formed duplexes with RNA. The upper bands indicate the presence of DNA fragments which were not digested to completion due to the proximity of the DNA:RNA hybrid. As indicated in the sequencing ladder, the 3⬘ termini of this mRNA is CATCGT-3⬘.
The labeling reaction continues for another 15 min after addition of cold dCTP to a final concentration of 0.12 mM. The labeled DNA is purified with Geneclean (Bio 101, Inc., U.S.A.) and eluted in 25 l of water. The protocol described was employed to label a PCR product that was used in a S1 protection assay to detect the 3⬘ end of a transcription unit located in the PSA gene cluster of Leishmania infantum (3). The specific activity of the probe after purification was 5 ⫻ 10 6 cpm/g. S1 mapping was performed according to the method described by Sambrook et al. (1). As shown in Fig. 1, this procedure allowed fine mapping of the 3⬘ end of a novel transcription unit from the PSA gene cluster in L. infantum. In conclusion, because any radiolabeled dNTP can be used, no restriction site close to the 3⬘ end of the gene is needed and only enzymes frequently used in the laboratory such as Klenow polymerase and Taq polymerase are employed; the method described in this paper can be considered a simple and convenient protocol for 3⬘ end-labeling. Acknowledgments. We thank Dr. M. McAndrew for critical reading of the manuscript. This work was supported by Grant PB97-0767 from Direccio´n General de Ensan˜anza Superior e Investigacio´n Cientı´fica.
Viggo Linde,* ,† ,1 Hans Flodgaard,* Jette Sandholm Kastrup,† and Søren Bjørn* *Novo Nordisk A/S, Novo Alle, DK-2880 Bagsværd, Denmark; and †Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received October 22, 1999
One of the hallmarks of apoptosis is the fragmentation of nuclear DNA. However, when DNA fragmentation is measured in adherent cells, the number of samples is often limited, due to a high complexity of the available assays. In this report, a new TUNEL-based assay is described for measuring DNA fragmentation. The assay is simple, quick, and nonseparational and is performed in a 96-well format. Apoptosis is a genetically programmed form of cell suicide producing distinct morphologically and biochemically changes of the cell (reviewed in 1). In order to analyze apoptosis, a number quantifiable markers have been found. Due to changes in cell membrane morphology, phosphatidylserine (PS) 2 is translocated from the interior side of the plasma membrane to the exterior side, which can be detected by means of Annexin-V binding. Most often this is performed by FACS analysis requiring a substantial amount of cells in suspension, making the procedure tedious and nonefficient for adherent cells. However, a homogenous scintillating microplate-based assay for measuring PS exposure has recently been developed (2). Staining nuclear DNA with selective fluorescent dyes (e.g., propidium iodide and Hoechst dyes) is another means of investigating the integrity of the cell membrane. So 1
To whom correspondence should be addressed at Novo Nordisk A/S, Novo Alle, 6A1.014, 2880 Bagsværd, Denmark. Fax: ⫹45 4444 4565. E-mail: [email protected]. 2 Abbreviations used: PS, phosphatdylserine; Tdt, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase nucleic acid end labeling; DMEM, Dulbecco’s minimal essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline. Analytical Biochemistry 280, 186 –188 (2000) doi:10.1006/abio.2000.4512 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
much more prominent than the m/z 872 peak indicates that, for the most part, those cells that begin to fix nitrogen from air use air as their main or sole nitrogen source. These two examples lead us to conclude that MALDI–TOF MS can be used as a simple and conclusive method for monitoring isotope incorporation. REFERENCES 1. Patterson, W., B., Carraro, F., and Wolfe, R., R. (1993) Measurement of 15N enrichment in multiple amino acids and urea in a single analysis by gas chromatography/mass spectrometry. Biol. Mass Spectrom. 22, 518 –523. 2. Fogg, G. E., Steward, W. D. P., Fay, P., and Walsby, A. E. (1973) The Blue–Green Algae, Academic Press, London and New York. 3. Bothe, H. (1982) in The Biology of Cyanobacteria (Carr, N. G., and Whitlon, B. A., Eds.), pp. 87–104, Univ. California Press, Berkeley and Los Angeles. 4. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63, 1193–1203. 5. Whittal, M. R., and Li, L. (1995) High-resolution matrix-assisted laser desorption/ionization in a linear time-of-flight mass spectrometer. Anal. Chem. 67, 1950 –1954. 6. Peschek, G. A., Vilgrater, K., and Wastyn, M. (1991) Respiratory protection of nitrogenase in dinitrogen-fixing cyanobacteria. Plant Soil 137, 17–24. 7. Walsby, A. E., and Buckland, B. (1969) Isolation and purification of intact gas vesicles from a blue– green algae. Nature 224, 716 –717. 8. Erhard, M., Von Doehren, H., and Jungblut, P. (1998) MALDI– TOF mass spectrometry. Fast-screening and structure analysis of secondary metabolites. Biospectrum 4, 42– 46.
Mapping 3⬘ Termini of mRNA on DNA Templates with Taq Polymerase 3⬘-End-Labeled Probes Julio C. Pareja and Antonio Jime´nez-Ruiz 1 Departamento de Bioquı´mica y Biologı´a Molecular, Campus Universitario, Universidad de Alcala´, 28871 Alcala´ de Henares, Madrid, Spain Received October 12, 1999
The location of the 3⬘ termini of the mRNAs can be mapped by using nuclease S1, which digests DNA that has not formed duplexes with RNA. The length of the 3⬘-end-labeled DNA fragment protected from digestion allows precise mapping of the 3⬘ termini of the mRNA. In this paper we show that Taq polymerase can be used to add a single radioactive nucleotide to the 3⬘ end of 1 To whom correspondence should be addressed. Fax: 34-1-885 4585. E-mail: [email protected].
Analytical Biochemistry 280, 185–186 (2000) doi:10.1006/abio.2000.4499 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
185
the DNA probe and that the specific activity obtained is sufficient for the S1 protection assay. In other methods, the 3⬘ termini of double-stranded DNA is labeled by using the Klenow fragment of Escherichia coli DNA polymerase I or bacteriophage T4 DNA polymerase. Both enzymes can be used to label ends created by cleavage of DNA with restriction enzymes (1). Restriction sites with 5⬘ protruding ends are easily labeled by filling the recessed 3⬘ termini with radiolabeled nucleotides. However, this method depends on the presence of a restriction site in a position which allows fine mapping of the 3⬘ end of the gene. Moreover, which of the four [␣- 32P]dNTPs is added to the reaction depends on the sequence of the protruding 5⬘ termini at the ends of the DNA. Terminal transferase can also be used for 3⬘ endlabeling, but this enzyme incorporates a long tail of nucleotides at the 3⬘ end of the DNA strands. To obtain a labeled fragment of a known size, the substrate for terminal transferase must be a labeled dideoxynucleotide, which is not a common reagent in the laboratory. The method described in this paper takes advantage of the ability of Taq polymerase to add a single nucleotide after the end of the template. This terminal transferase activity is currently used to prepare vectors designed to clone PCR products (2) and, as it will be shown, can also be employed to add any labeled deoxynucleotide to the 3⬘ end of any amplified fragment. Even though the specific activity of the probes obtained by this method is not as high as, for instance, that obtained by filling the recessed 3⬘ termini of the restriction sites, the widespread use of highly sensitive methods for detecting radioactive signals makes this method highly recommended for 3⬘ mRNA end-mapping. Moreover, the primers used for amplification can also be used in a parallel sequencing reaction to determine the exact position of the 3⬘ end. Labeling protocol. DNA obtained after standard PCR amplification is purified with Geneclean (Bio 101, Inc. U.S.A.) and eluted in a final volume of 15 l. The nucleotide added by Taq polymerase to the 3⬘ end of the amplified products is removed with 4 units of Klenow fragment of E. coli DNA polymerase I (Boehringer GmbH, Germany) in Klenow buffer (15 mM MgCl 2, 3 mM DTT, 0.75 mM dATP, 0.75 mM dGTP, 0.75 mM dCTP, 0.75 mM dTTP, 135 mM Hepes, pH 6.6) in a final volume of 50 l for 30 min at 37°C. After incubation, DNA is purified again with Geneclean (Bio 101, Inc., U.S.A.) to remove free nucleotides. One hundred nanograms of the blunt-ended DNA obtained after Klenow treatment is labeled with 2.5 units of Taq polymerase (Perkin–Elmer Cetus Instruments, U.S.A.) and 100 Ci of [␣- 32P]dCTP (3000 Ci/mmol) in a final volume of 50 l in labeling buffer (50 mM KCl, 1.5 mM MgCl 2, 10 mM Tris–HCl, pH 8.3) at 72°C for 30 min.
186
NOTES & TIPS
REFERENCES 1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154 3. Jime´nez-Ruiz, A., Boceta, C., Bonay, P., Requena, J. M., and Alonso, C. (1998) Cloning, sequencing and expression of the PSA genes from Leishmania infantum. Eur. J. Biochem. 251, 389 –397.
Measurement of Apoptosis by the TUNEL Method Using Scintillating Microplates FIG. 1. Location of the 3⬘ termini of the mRNA on a DNA template from a novel transcription unit located in the PSA gene cluster of Leishmania infantum. A parallel sequencing reaction primed with the appropriate oligonucleotide from those used for PCR amplification of the DNA probe is shown at the left. (A) Nuclease S1 protection assay using total RNA from culture parasites at the logarithmic growth phase. (B) Nuclease S1 protection assay using total RNA from culture parasites at the stationary growth phase. Nuclease S1 incubation periods are indicated at the top of each lane. After drying, the polyacrylamide/urea gel was exposed to a phosphor screen for 24 h and analyzed using the Fuji BAS 1000 system. The arrow indicates the protected DNA fragment after complete nuclease S1 digestion of the DNA that has not formed duplexes with RNA. The upper bands indicate the presence of DNA fragments which were not digested to completion due to the proximity of the DNA:RNA hybrid. As indicated in the sequencing ladder, the 3⬘ termini of this mRNA is CATCGT-3⬘.
The labeling reaction continues for another 15 min after addition of cold dCTP to a final concentration of 0.12 mM. The labeled DNA is purified with Geneclean (Bio 101, Inc., U.S.A.) and eluted in 25 l of water. The protocol described was employed to label a PCR product that was used in a S1 protection assay to detect the 3⬘ end of a transcription unit located in the PSA gene cluster of Leishmania infantum (3). The specific activity of the probe after purification was 5 ⫻ 10 6 cpm/g. S1 mapping was performed according to the method described by Sambrook et al. (1). As shown in Fig. 1, this procedure allowed fine mapping of the 3⬘ end of a novel transcription unit from the PSA gene cluster in L. infantum. In conclusion, because any radiolabeled dNTP can be used, no restriction site close to the 3⬘ end of the gene is needed and only enzymes frequently used in the laboratory such as Klenow polymerase and Taq polymerase are employed; the method described in this paper can be considered a simple and convenient protocol for 3⬘ end-labeling. Acknowledgments. We thank Dr. M. McAndrew for critical reading of the manuscript. This work was supported by Grant PB97-0767 from Direccio´n General de Ensan˜anza Superior e Investigacio´n Cientı´fica.
Viggo Linde,* ,† ,1 Hans Flodgaard,* Jette Sandholm Kastrup,† and Søren Bjørn* *Novo Nordisk A/S, Novo Alle, DK-2880 Bagsværd, Denmark; and †Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark Received October 22, 1999
One of the hallmarks of apoptosis is the fragmentation of nuclear DNA. However, when DNA fragmentation is measured in adherent cells, the number of samples is often limited, due to a high complexity of the available assays. In this report, a new TUNEL-based assay is described for measuring DNA fragmentation. The assay is simple, quick, and nonseparational and is performed in a 96-well format. Apoptosis is a genetically programmed form of cell suicide producing distinct morphologically and biochemically changes of the cell (reviewed in 1). In order to analyze apoptosis, a number quantifiable markers have been found. Due to changes in cell membrane morphology, phosphatidylserine (PS) 2 is translocated from the interior side of the plasma membrane to the exterior side, which can be detected by means of Annexin-V binding. Most often this is performed by FACS analysis requiring a substantial amount of cells in suspension, making the procedure tedious and nonefficient for adherent cells. However, a homogenous scintillating microplate-based assay for measuring PS exposure has recently been developed (2). Staining nuclear DNA with selective fluorescent dyes (e.g., propidium iodide and Hoechst dyes) is another means of investigating the integrity of the cell membrane. So 1
To whom correspondence should be addressed at Novo Nordisk A/S, Novo Alle, 6A1.014, 2880 Bagsværd, Denmark. Fax: ⫹45 4444 4565. E-mail: [email protected]. 2 Abbreviations used: PS, phosphatdylserine; Tdt, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase nucleic acid end labeling; DMEM, Dulbecco’s minimal essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline. Analytical Biochemistry 280, 186 –188 (2000) doi:10.1006/abio.2000.4512 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.