~QMMENT
but a number of hexamer sequences are found in both species' promoter regions to which trans-acting factors may bind 16,17. Occasionally in interspecific transformation experiments novel patterns of expression arise that do not appear in either species. This was observed when the D. affinidisjuncta alcohol dehydrogenase (Adh) gene was transformed into D. melanogaster is. This unusual result suggests that within each species, the transcription factors and cis regulatory sequences have coevolved and, when one is mixed with the other, a transcriptional 'error' occurs, revealing a novel phenotype. This in turn implies that different species may reach the same regulatory end point - getting gene X expressed in tissue Y - by using different sequence information within the same gene. Thus, diverged cis regulatory regions of a gene could be functionally important too. These types of experiment provide tantalizing glimpses of the
complexities of the evolution of transcription factors and their target sites, as well as assessing the species-specific potential of diverged coding sequences. The remarkable evolutionary flexibility of the tra gene, while still providing normal function in a heterologous host, will provide a yardstick against which the diverged and conserved DNA sequences of other genes will be compared. Finally, interested readers should read the reviews by Dickinson 19 and Cavener 2° on interspecific gene comparisons and their functional implications.
References 1 Hodgkin, J. (1992) BioEssays 14, 253-261 2 Koopman, P. et aL (1991) Nature 351, 117-121 3 Baker, B.S. (1989) Nature 340, 521-524 4 Bridges, C.B. (1925) Am. Nat. 59, 127-137 5 Bell, L.R., Maine, E.M., Schedl, P. and Cline, T.W. (1988) Cell 55, 1037-1046 6 Belote, J.M. et aL (1989) Dev.
Genet. 10, 143-154 7 Hoshijima, K. et al. (1991) Science 252, 833466 8 Healey, M.L. and Maniatis, T. (1991) Cell 65, 579-588 9 Burtis, K.C. and Baker, B.S. (1989) Cell 56, 997-1010 10 O'Neil, M.T. and Belote, J. (1992) Genetics 131, 113-128 11 Lebedeff, G.A. (1939) Genetics 24, 553-586 12 Li, H. and Bingham, P.M. (1991) Cell 67, 335-342 13 Colot, H.V., Hall, J.C. and Rosbash, M. (1988) EMBOJ. 7, 3929-3937 14 Wheeler, D.A. et aL (1991) Science 251, 1082-1085 15 Petersen, G., Hall, J.C. and Rosbash, M. (1988) E~BOJ. 7, 3939-3947 16 Mitsialis, S.A., Spoerel, N., Leviten. M. and Kafatos, F.C. (1987) Proc. Natl Acad. ScL USA 84, 7987-7989 17 Mitsialis, S.A., Veletza, S. and Kafatos, F.C. (1989) J. Mol. EvoL 22, 486-495 I 8 Brennan, M.S. and Dickinson, W.J. (1989) Dev. BioL 125, 64-74 I9 Dickinson, w.J. (1990) Evol. Biol. 25, 127-172 20 Cavener, D.R. (1992) BioEssays 14, 237-244
~'--~ECHNICAL UlPS
Detection o f rare mRNAs via quantitative RT-PCR The detection of rare mRNAs and their quantitation h,-ts been the goal of many RT-PCR (reverse transcripiase-p0Iymemse chain reaction) BLOCK THERMOCYCLER ~IIR THERMOCYCLER I 2 3 4 5 M I 2 3 4 5 protocols 1,2. Our objective was to design an RT~PCR assay to detect an alternatively spliced form of transcript from the mouse Cr2 gene; a lowabundance transcript 3. We chose to reverse transcribe 5 ~g of total RNA using random primers (hexamers) instead of the PCR primers for two reasons. First, when the PCR primers are used at 37°C, they can prime reverse transcription of RNA inappropriately, creating an undesirably high background in the PCR analysis. Second, this approach produces 5-10 ~tg of total cDNA which can then be used, at 100-200 ng of cDNA per reaction, with more than 50 different oligo sets (i.e. more than 50 different gene sequences). When this cDNA was amplified in the standard heat block instruments (94°C denaturation for 15 s, 59°C annealing for 30 s, 72°C extension for 30 s) for 4-7 min per cycle (depending upon the thermocycler), and the products analysed by denaturing sequencing gel electrophoresis, the amount of material amplified varied from experiment to experiment, presumably due to limiting enzyme activity. To avoid losing enzyme activity because of the length of the total PCR amplification, we used an air-driven thermocyeler that allows a single cycle time of 24 s (94°C denaturation for 1 s, 59°C annealing for FIGR 1 s, 72°C extension for 4 s)4. As shown in Fig. 1, when five different PCR amplification using heat block thermal cycler spleen cDNA samples were set up for PCR amplification and equally (left five lanes) and air cycler (right five lanes). split between the standard heat block instrument (first five lanes) and M, markers. the air cycler (last five lanes) for the same number of cycles, substantially more product was formed with the air cycler than with the heat block machine. Quantitation (cutting the bands out of the gel and counting incorporated P2p]dCTP) indicated that there was at least 100-fold more product.
TIG AUGUST1992 VOL. 8 NO. 8
-~ECHNICAL
By this assay, a single cDNA sample can be analysed for the expression of many different transcripts by PCR amplifying gene sequences of 90-400 bp, Figure 2 illustrates amplification using [8-actin primers (actin) and the Cr2-specific primers (giving products of 3 kbp and 5 kbp) with 200 ng of cDNA (S.S. Tan. C.B, Kurtz. E.M. O'Toole and J,H. Weis. submitted). Since actin transcripts are much more abundant than Cr2 mRNA. it is important to analyse the actin products after 13 cydes and the Cr2 gene products after 20 cycles to ensure that the respective products are still within the linear range of amplification. For quantitative purposes, it is crucial that only the cDNA is limiting and that the amount of product is directly proportional to the amount of input cDNA. The sensitivity and linearity of this assay were measured by PCR analysis of tenfold dilutions of a Cr2 cDNA plasmid and spleen cDNA (Fig. 3). Comparison of reactions done with 1 lag ptasmid per amplification (1), 0,1 lag per reaction (-1~. 0.01 lag per reaction (-2), etc.. with those derived from mouse BALB/c spleen cDNA (0.9, 9, 90 and 900 ng of cDNA) indicated that approximately the same signal was obtained from 90 ng of spleen cDNA and 100 fg of plasmid (~1.2× 107 plasmid molecules) (16 h exposure; 20 cycles). Quantitation of the spleen cDNA PCR products by scintillation analysis indicated nearly a tenfold increase in product was obtained with a tenfold increase in the amount of starting cDNA. Although an air-driven PCR thermocycler was used in these experiments, presumably any machine or protocol that allows similarly fast reaction rates would provide the same level of sensitivity. A detailed protocol for this procedure can be obtained from J.H.W.
E~IPS
I000001 I00001 1ooo1 =~
100 t
lO1
!
•
FIGM PCR amplification of ~-actin and Cr2 cDNAs.
,, -,-2, _3, _, -5, _6 -,, -, -g, ,,
0, g, ,0, ,00,
REFERENCES 1 Chelly, J. et al. (1990) Eur.J. Biochem. 187, 691-698 2 Robinson. M.O. and Simon. M.I. (1991) NucteicAcids Res. 19, 1557-1562 3 Kurtz, C.B., O'Toole. E.. Christensen. S.M. and Weis, J.H. (1990) J. Immunol. 144, 3581-3592 4 Wittwer, C.T. and Garling, D.J. (1991) Biotechniques 10, 76--83 Contributed by John H. Weis. Sally S. Tan. Brian K. Martin and Carl T. Wittwer, Department o f Pathology, University of Utah School of Medicine. Salt Lake City, UT84132, USA.
FIGI! Amplification with varying amounts of Cr2 plasmid (left ten lanes) and BALB/c spleen cDNAs (right four lanes~. R. RNA-negative control.
Regulation of mit0ch0ndrial gene expression: transcriptionversus replicati0n., In a Comment article published in T/G, regulation of the copy n u m b e r of mitochondrial DNA (mtDNA) in animal tissues was p r o p o s e d to be an important mechanism for coordinating the a b u n d a n c e of mitochondrial transcripts with the expression of nuclear genes encoding proteins targeted for the mitochondrionL
This hypothesis was b a s e d on the finding that long-term stimulation of skeletal muscle leads to simultaneous increases in both mtDNA and mitochondrial mRNAs 2. It is apparently s u p p o r t e d by the recent characterization of a trans-acting factor, NRF-1, that binds to promoters of such nuclear genes as well as to the gene encoding TIG AUGUST1992 VOL. 8 NO. 8
m
the RNA moiety of a ribonucleoprotein involved in mtDNA replication 3. However, several arguments can be raised against this hypothesis. (1) Currently available data rely on determination of mtDNA levels relative to total DNA by hybridization techniques. Most of the mtDNA is found within the cell type occupying the bulk of the tissue, while each cell, no matter h o w large, contributes significantly to tissue DNA. If the n u m b e r of cells changes as a result of a
but a number of hexamer sequences are found in both species' promoter regions to which trans-acting factors may bind 16,17. Occasionally in interspecific transformation experiments novel patterns of expression arise that do not appear in either species. This was observed when the D. affinidisjuncta alcohol dehydrogenase (Adh) gene was transformed into D. melanogaster is. This unusual result suggests that within each species, the transcription factors and cis regulatory sequences have coevolved and, when one is mixed with the other, a transcriptional 'error' occurs, revealing a novel phenotype. This in turn implies that different species may reach the same regulatory end point - getting gene X expressed in tissue Y - by using different sequence information within the same gene. Thus, diverged cis regulatory regions of a gene could be functionally important too. These types of experiment provide tantalizing glimpses of the
complexities of the evolution of transcription factors and their target sites, as well as assessing the species-specific potential of diverged coding sequences. The remarkable evolutionary flexibility of the tra gene, while still providing normal function in a heterologous host, will provide a yardstick against which the diverged and conserved DNA sequences of other genes will be compared. Finally, interested readers should read the reviews by Dickinson 19 and Cavener 2° on interspecific gene comparisons and their functional implications.
References 1 Hodgkin, J. (1992) BioEssays 14, 253-261 2 Koopman, P. et aL (1991) Nature 351, 117-121 3 Baker, B.S. (1989) Nature 340, 521-524 4 Bridges, C.B. (1925) Am. Nat. 59, 127-137 5 Bell, L.R., Maine, E.M., Schedl, P. and Cline, T.W. (1988) Cell 55, 1037-1046 6 Belote, J.M. et aL (1989) Dev.
Genet. 10, 143-154 7 Hoshijima, K. et al. (1991) Science 252, 833466 8 Healey, M.L. and Maniatis, T. (1991) Cell 65, 579-588 9 Burtis, K.C. and Baker, B.S. (1989) Cell 56, 997-1010 10 O'Neil, M.T. and Belote, J. (1992) Genetics 131, 113-128 11 Lebedeff, G.A. (1939) Genetics 24, 553-586 12 Li, H. and Bingham, P.M. (1991) Cell 67, 335-342 13 Colot, H.V., Hall, J.C. and Rosbash, M. (1988) EMBOJ. 7, 3929-3937 14 Wheeler, D.A. et aL (1991) Science 251, 1082-1085 15 Petersen, G., Hall, J.C. and Rosbash, M. (1988) E~BOJ. 7, 3939-3947 16 Mitsialis, S.A., Spoerel, N., Leviten. M. and Kafatos, F.C. (1987) Proc. Natl Acad. ScL USA 84, 7987-7989 17 Mitsialis, S.A., Veletza, S. and Kafatos, F.C. (1989) J. Mol. EvoL 22, 486-495 I 8 Brennan, M.S. and Dickinson, W.J. (1989) Dev. BioL 125, 64-74 I9 Dickinson, w.J. (1990) Evol. Biol. 25, 127-172 20 Cavener, D.R. (1992) BioEssays 14, 237-244
~'--~ECHNICAL UlPS
Detection o f rare mRNAs via quantitative RT-PCR The detection of rare mRNAs and their quantitation h,-ts been the goal of many RT-PCR (reverse transcripiase-p0Iymemse chain reaction) BLOCK THERMOCYCLER ~IIR THERMOCYCLER I 2 3 4 5 M I 2 3 4 5 protocols 1,2. Our objective was to design an RT~PCR assay to detect an alternatively spliced form of transcript from the mouse Cr2 gene; a lowabundance transcript 3. We chose to reverse transcribe 5 ~g of total RNA using random primers (hexamers) instead of the PCR primers for two reasons. First, when the PCR primers are used at 37°C, they can prime reverse transcription of RNA inappropriately, creating an undesirably high background in the PCR analysis. Second, this approach produces 5-10 ~tg of total cDNA which can then be used, at 100-200 ng of cDNA per reaction, with more than 50 different oligo sets (i.e. more than 50 different gene sequences). When this cDNA was amplified in the standard heat block instruments (94°C denaturation for 15 s, 59°C annealing for 30 s, 72°C extension for 30 s) for 4-7 min per cycle (depending upon the thermocycler), and the products analysed by denaturing sequencing gel electrophoresis, the amount of material amplified varied from experiment to experiment, presumably due to limiting enzyme activity. To avoid losing enzyme activity because of the length of the total PCR amplification, we used an air-driven thermocyeler that allows a single cycle time of 24 s (94°C denaturation for 1 s, 59°C annealing for FIGR 1 s, 72°C extension for 4 s)4. As shown in Fig. 1, when five different PCR amplification using heat block thermal cycler spleen cDNA samples were set up for PCR amplification and equally (left five lanes) and air cycler (right five lanes). split between the standard heat block instrument (first five lanes) and M, markers. the air cycler (last five lanes) for the same number of cycles, substantially more product was formed with the air cycler than with the heat block machine. Quantitation (cutting the bands out of the gel and counting incorporated P2p]dCTP) indicated that there was at least 100-fold more product.
TIG AUGUST1992 VOL. 8 NO. 8
-~ECHNICAL
By this assay, a single cDNA sample can be analysed for the expression of many different transcripts by PCR amplifying gene sequences of 90-400 bp, Figure 2 illustrates amplification using [8-actin primers (actin) and the Cr2-specific primers (giving products of 3 kbp and 5 kbp) with 200 ng of cDNA (S.S. Tan. C.B, Kurtz. E.M. O'Toole and J,H. Weis. submitted). Since actin transcripts are much more abundant than Cr2 mRNA. it is important to analyse the actin products after 13 cydes and the Cr2 gene products after 20 cycles to ensure that the respective products are still within the linear range of amplification. For quantitative purposes, it is crucial that only the cDNA is limiting and that the amount of product is directly proportional to the amount of input cDNA. The sensitivity and linearity of this assay were measured by PCR analysis of tenfold dilutions of a Cr2 cDNA plasmid and spleen cDNA (Fig. 3). Comparison of reactions done with 1 lag ptasmid per amplification (1), 0,1 lag per reaction (-1~. 0.01 lag per reaction (-2), etc.. with those derived from mouse BALB/c spleen cDNA (0.9, 9, 90 and 900 ng of cDNA) indicated that approximately the same signal was obtained from 90 ng of spleen cDNA and 100 fg of plasmid (~1.2× 107 plasmid molecules) (16 h exposure; 20 cycles). Quantitation of the spleen cDNA PCR products by scintillation analysis indicated nearly a tenfold increase in product was obtained with a tenfold increase in the amount of starting cDNA. Although an air-driven PCR thermocycler was used in these experiments, presumably any machine or protocol that allows similarly fast reaction rates would provide the same level of sensitivity. A detailed protocol for this procedure can be obtained from J.H.W.
E~IPS
I000001 I00001 1ooo1 =~
100 t
lO1
!
•
FIGM PCR amplification of ~-actin and Cr2 cDNAs.
,, -,-2, _3, _, -5, _6 -,, -, -g, ,,
0, g, ,0, ,00,
REFERENCES 1 Chelly, J. et al. (1990) Eur.J. Biochem. 187, 691-698 2 Robinson. M.O. and Simon. M.I. (1991) NucteicAcids Res. 19, 1557-1562 3 Kurtz, C.B., O'Toole. E.. Christensen. S.M. and Weis, J.H. (1990) J. Immunol. 144, 3581-3592 4 Wittwer, C.T. and Garling, D.J. (1991) Biotechniques 10, 76--83 Contributed by John H. Weis. Sally S. Tan. Brian K. Martin and Carl T. Wittwer, Department o f Pathology, University of Utah School of Medicine. Salt Lake City, UT84132, USA.
FIGI! Amplification with varying amounts of Cr2 plasmid (left ten lanes) and BALB/c spleen cDNAs (right four lanes~. R. RNA-negative control.
Regulation of mit0ch0ndrial gene expression: transcriptionversus replicati0n., In a Comment article published in T/G, regulation of the copy n u m b e r of mitochondrial DNA (mtDNA) in animal tissues was p r o p o s e d to be an important mechanism for coordinating the a b u n d a n c e of mitochondrial transcripts with the expression of nuclear genes encoding proteins targeted for the mitochondrionL
This hypothesis was b a s e d on the finding that long-term stimulation of skeletal muscle leads to simultaneous increases in both mtDNA and mitochondrial mRNAs 2. It is apparently s u p p o r t e d by the recent characterization of a trans-acting factor, NRF-1, that binds to promoters of such nuclear genes as well as to the gene encoding TIG AUGUST1992 VOL. 8 NO. 8
m
the RNA moiety of a ribonucleoprotein involved in mtDNA replication 3. However, several arguments can be raised against this hypothesis. (1) Currently available data rely on determination of mtDNA levels relative to total DNA by hybridization techniques. Most of the mtDNA is found within the cell type occupying the bulk of the tissue, while each cell, no matter h o w large, contributes significantly to tissue DNA. If the n u m b e r of cells changes as a result of a
