[1]
RECOMBINANT D N A TECHNIQUES
[1] Recombinant By
DNA
3
Techniques
JOHN F. MORROW
T h e r e c o m b i n a n t D N A m e t h o d c o n s i s t s o f j o i n i n g D N A m o l e c u l e s in v i t r o a n d i n t r o d u c i n g t h e m into living cells w h e r e t h e y r e p l i c a t e . R e s e a r c h u s i n g this m e t h o d is r e l a t i v e l y n e w a n d f a s t - m o v i n g . I n o n l y 6 y e a r s o f rec o m b i n a n t D N A r e s e a r c h , a n u m b e r o f significant a c c o m p l i s h m e n t s h a v e b e e n m a d e . T w o m a m m a l i a n h o r m o n e s h a v e b e e n p r o d u c e d in b a c t e r i a b y m e a n s o f s y n t h e t i c D N A . m P o l y p e p t i d e s s i m i l a r o r i d e n t i c a l to s e v e r a l f o u n d in e u k a r y o t e s h a v e b e e n s y n t h e s i z e d in E s c h e r i c h i a c o l i . 3-~° T h e s e achievements promise a new, inexpensive means of large-scale production o f s e l e c t e d p e p t i d e s o r p r o t e i n s . F u r t h e r m o r e , u s i n g r e c o m b i n a n t DNA, somatic recombination of immunoglobulin genes has been establ i s h e d , 11 a n d a large n u m b e r o f v a r i a b l e - r e g i o n g e n e s h a v e b e e n f o u n d . TM I n t e r v e n i n g s e q u e n c e s ( i n t r o n s ) h a v e b e e n f o u n d in t h e D N A o f euk a r y o t i c cells. 1a-t6 I w o u l d like to m e n t i o n t h e origins o f this v e r s a t i l e n e w t e c h n o l o g y b e fore describing recent advances. The isolation of mutant E. coli strains u n a b l e to r e s t r i c t f o r e i g n D N A ( c l e a v e it s p e c i f i c a l l y a n d d e g r a d e it) laid i K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F, Bolivar, and H. W. Boyer, Science 198, 1056 (1977). 2 D. V. Goeddel, D. G. Kleid, F. Bolivar, H. L. Heyneker, D. G. Yansura, R. Crea, T. Hirose, A. Kraszewski, K. Itakura, and A. D. Riggs, Proc. Natl. Acad. Sci. U.S.A. 76, 106 (1979). K. Struhl, J. R. Cameron, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 1471 (1976). 4 B. Ratzkin and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 74, 487 (1977). D. Vapnek, J. A. Hautala, J. W. Jacobson, N. H. Giles, and S. R. Kushner, Proc. Natl. Acad. Sci. U.S.A. 74, 3508 (1977). e R. C. Dickson and J. S. Markin, Cell 15, 123 (1978). L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). s A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen, Nature (London) 275, 617 (1978). 9 0 . Mercereau-Puijalon, A. Royal, B. Carol, A. Garapin, A. Krust, F. Gannon, and P. Kourilsky, Nature (London) 275, 505 (1978). 1oT. H. Fraser and B. J. Bruce, Proc. Natl. Acad. Sci. U.S.A. 75, 5936 (1978). 11C. Brack, M. Hirama, R. Lenhard-Schuller, and S. Tonegawa, Cell 15, 1 (1978). n j. G. Seidman, A. Leder, M. Nau, B. Norman, and P. Leder, Science 202, 11 (1978). 13 D. M. Glover and D. S. Hogness, Cell 10, 167 (1977). x4 R. L. White and D. S. Hogness, Cell 10, 177 (1977). 15 p. K. Wellauer and I. B. Dawid, Cell 10, 193 (1977). 1, S. M. Tilghman, D. C. Tiemeier, J. G. Seidman, B. M. Peterlin, M. Sullivan, J. V. Maizel, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 75, 725 (1978).
METHODS IN ENZYMOJ.OGY, VOL. 68
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-12-181968-X
4
INTRODUCTION
[1]
part of the foundation. 17 The discovery of site-specific restriction endonucleases lsa9 also contributed (see Nathans and Smith, ~° Roberts, 21 and this volume [2], for review). Two general methods for joining DNA molecules from different sources were found. 2z-24 Particularly useful was the first enzyme found to create self-complementary, cohesive termini on DNA molecules by specific cleavage at staggered sites in the two D N A strands, the E c o R I restriction endonuclease. 25-2r It was used in the first in vitro construction of recombinant molecules that subsequently replicated in vivo. 2s
What can be done by the recombinant DNA method? Principally three sorts of things: 1. Isolation of a desired sequence from a complex mixture of DNA molecules, such as a eukaryotic genome, and replication of it to provide milligram quantities for biochemical study. 2. Alteration of a DNA molecule. One can insert restriction endonuclease recognition sites, or other DNA segments, at random or predetermined locations. One can also delete restriction sites, or DNA segments between such sites, by techniques that permit joining any two D N A termini after their appropriate modification. Such an alteration can be helpful in determining the functions performed by various parts of a DNA sequence. This is attractive where efficient means of fine-structure genetic analysis of random mutations are lacking, as in animals and plants. 3. Synthesis in bacteria of large amounts o f peptides or proteins that are of interest to science, medicine, or commerce. Before indicating specifically the most useful methods for obtaining each of the above goals, we look at recent advances in the basic techniques. The essential ingredients of a recombinant DNA experiment are: 17 W. B. Wood, J. Mol. Biol. 16, 118 (1966). is H. O. Smith and K. W. Wilcox, J. Mol. Biol. 51, 379 (1970). ~9 T. J. Kelly, Jr. and H. O. Smith, J. Mol. Biol. 51, 393 (1970). 2o D. N a t h a n s and H. O. Smith, Annu. Rev. Biochem. 44, 273 (1975). 21 R. J. Roberts, Gene 4, 183 (1978). 22 p. E. L o b b a n and A. D. Kaiser, J. Mol. Biol. 78, 453 (1973). 23 D. A. Jackson, R. H. S y m o n s , and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 69, 2904 (1972). 24 V. Sgaramella, J. H. van de Sande, and H. G. K h o r a n a , Proc. Natl. Acad. Sci. U.S.A. 67, 1468 (1970). 25 j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972). 2e j. Hedgpeth, H. M. G o o d m a n , and H. W. Boyer, Proc. Natl. Acad. Sci. U.S.A. 69, 3448 (1972). z7 V. Sgaramella, Proc. Natl. Acad. Sci. U.S.A. 69, 3389 (1972). 2s S. N. Cohen, A. C. Y. Chang, H. W. Boyer, and R. B. Helling, Proc. Natl. Acad. Sci. U.S.A. 70, 3240 (1973).
[1]
RECOMBINANT D N A
TECHNIQUES
5
1. A DNA vehicle (vector, replicon) which can replicate in living cells after foreign DNA is inserted into it. 2. A DNA molecule to be replicated (passenger), or a collection of them. 3. A method of joining the passenger to the vehicle. 4. A means of introducing the joined DNA molecule into a host organism in which it can replicate (DNA transformation or transfection). 5. A means of screening or genetic selection for those cells that have replicated the desired recombinant molecule. This is necessary since transformation and transfection methods are inefficient, so that most members of the host cell population have no recombinant DNA replicating in them. This selection or screening for desired recombinants provides a route to recovery of the recombinant DNA of interest in pure form.
Since a thorough review of recombinant DNA was completed in 1976, 29 I will concentrate on progress since then. Cloning Vehicles Plasmids
Many bacterial plasmids have been used as cloning vehicles. Currently, E. coli and its plasmids constitute the most versatile type of host-vector system for DNA cloning. A number of derivatives of natural plasmids have been developed for cloning. Most of these new plasmid vehicles were made by combining DNA segments, and desirable qualities, of older vehicles (Table I). All those listed have a "relaxed" mode of replication, such that plasmid DNA accumulates to make up about one-third of the total cellular DNA when protein synthesis is inhibited by chloramphenicol or spectinomycin. ~0 pBR322 is now the most widely used plasmid for cloning of DNA. One of its virtues is that it has six different types of restriction cleavage termini at which foreign DNA can be inserted. A very detailed restriction enzyme cleavage map and DNA sequence information are also important. 31a2 The PstI site in the Ap (penicillinase) gene has further advantages. If dG homopolymer tails are added to Pst-cleaved pBR322 DNA, and dC homopolymer tails to the DNA to be inserted, the PstI sites are reconstituted in 29 R. L. Sinsheimer, A n n u . Rev. Biochem. 46, 415 (1977). 3o A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978). at j. G. Sutcliffe, Proc. Natl. Acad. Sci. U.S.A. 75, 3737 (1978). 32 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978).
6
INTRODUCTION
[ 1]
o
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RECOMBINANT D N A TECHNIQUES
[1] Recombinant By
DNA
3
Techniques
JOHN F. MORROW
T h e r e c o m b i n a n t D N A m e t h o d c o n s i s t s o f j o i n i n g D N A m o l e c u l e s in v i t r o a n d i n t r o d u c i n g t h e m into living cells w h e r e t h e y r e p l i c a t e . R e s e a r c h u s i n g this m e t h o d is r e l a t i v e l y n e w a n d f a s t - m o v i n g . I n o n l y 6 y e a r s o f rec o m b i n a n t D N A r e s e a r c h , a n u m b e r o f significant a c c o m p l i s h m e n t s h a v e b e e n m a d e . T w o m a m m a l i a n h o r m o n e s h a v e b e e n p r o d u c e d in b a c t e r i a b y m e a n s o f s y n t h e t i c D N A . m P o l y p e p t i d e s s i m i l a r o r i d e n t i c a l to s e v e r a l f o u n d in e u k a r y o t e s h a v e b e e n s y n t h e s i z e d in E s c h e r i c h i a c o l i . 3-~° T h e s e achievements promise a new, inexpensive means of large-scale production o f s e l e c t e d p e p t i d e s o r p r o t e i n s . F u r t h e r m o r e , u s i n g r e c o m b i n a n t DNA, somatic recombination of immunoglobulin genes has been establ i s h e d , 11 a n d a large n u m b e r o f v a r i a b l e - r e g i o n g e n e s h a v e b e e n f o u n d . TM I n t e r v e n i n g s e q u e n c e s ( i n t r o n s ) h a v e b e e n f o u n d in t h e D N A o f euk a r y o t i c cells. 1a-t6 I w o u l d like to m e n t i o n t h e origins o f this v e r s a t i l e n e w t e c h n o l o g y b e fore describing recent advances. The isolation of mutant E. coli strains u n a b l e to r e s t r i c t f o r e i g n D N A ( c l e a v e it s p e c i f i c a l l y a n d d e g r a d e it) laid i K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F, Bolivar, and H. W. Boyer, Science 198, 1056 (1977). 2 D. V. Goeddel, D. G. Kleid, F. Bolivar, H. L. Heyneker, D. G. Yansura, R. Crea, T. Hirose, A. Kraszewski, K. Itakura, and A. D. Riggs, Proc. Natl. Acad. Sci. U.S.A. 76, 106 (1979). K. Struhl, J. R. Cameron, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 1471 (1976). 4 B. Ratzkin and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 74, 487 (1977). D. Vapnek, J. A. Hautala, J. W. Jacobson, N. H. Giles, and S. R. Kushner, Proc. Natl. Acad. Sci. U.S.A. 74, 3508 (1977). e R. C. Dickson and J. S. Markin, Cell 15, 123 (1978). L. Villa-Komaroff, A. Efstratiadis, S. Broome, P. Lomedico, R. Tizard, S. P. Naber, W. L. Chick, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 3727 (1978). s A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N. Cohen, Nature (London) 275, 617 (1978). 9 0 . Mercereau-Puijalon, A. Royal, B. Carol, A. Garapin, A. Krust, F. Gannon, and P. Kourilsky, Nature (London) 275, 505 (1978). 1oT. H. Fraser and B. J. Bruce, Proc. Natl. Acad. Sci. U.S.A. 75, 5936 (1978). 11C. Brack, M. Hirama, R. Lenhard-Schuller, and S. Tonegawa, Cell 15, 1 (1978). n j. G. Seidman, A. Leder, M. Nau, B. Norman, and P. Leder, Science 202, 11 (1978). 13 D. M. Glover and D. S. Hogness, Cell 10, 167 (1977). x4 R. L. White and D. S. Hogness, Cell 10, 177 (1977). 15 p. K. Wellauer and I. B. Dawid, Cell 10, 193 (1977). 1, S. M. Tilghman, D. C. Tiemeier, J. G. Seidman, B. M. Peterlin, M. Sullivan, J. V. Maizel, and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 75, 725 (1978).
METHODS IN ENZYMOJ.OGY, VOL. 68
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN O-12-181968-X
4
INTRODUCTION
[1]
part of the foundation. 17 The discovery of site-specific restriction endonucleases lsa9 also contributed (see Nathans and Smith, ~° Roberts, 21 and this volume [2], for review). Two general methods for joining DNA molecules from different sources were found. 2z-24 Particularly useful was the first enzyme found to create self-complementary, cohesive termini on DNA molecules by specific cleavage at staggered sites in the two D N A strands, the E c o R I restriction endonuclease. 25-2r It was used in the first in vitro construction of recombinant molecules that subsequently replicated in vivo. 2s
What can be done by the recombinant DNA method? Principally three sorts of things: 1. Isolation of a desired sequence from a complex mixture of DNA molecules, such as a eukaryotic genome, and replication of it to provide milligram quantities for biochemical study. 2. Alteration of a DNA molecule. One can insert restriction endonuclease recognition sites, or other DNA segments, at random or predetermined locations. One can also delete restriction sites, or DNA segments between such sites, by techniques that permit joining any two D N A termini after their appropriate modification. Such an alteration can be helpful in determining the functions performed by various parts of a DNA sequence. This is attractive where efficient means of fine-structure genetic analysis of random mutations are lacking, as in animals and plants. 3. Synthesis in bacteria of large amounts o f peptides or proteins that are of interest to science, medicine, or commerce. Before indicating specifically the most useful methods for obtaining each of the above goals, we look at recent advances in the basic techniques. The essential ingredients of a recombinant DNA experiment are: 17 W. B. Wood, J. Mol. Biol. 16, 118 (1966). is H. O. Smith and K. W. Wilcox, J. Mol. Biol. 51, 379 (1970). ~9 T. J. Kelly, Jr. and H. O. Smith, J. Mol. Biol. 51, 393 (1970). 2o D. N a t h a n s and H. O. Smith, Annu. Rev. Biochem. 44, 273 (1975). 21 R. J. Roberts, Gene 4, 183 (1978). 22 p. E. L o b b a n and A. D. Kaiser, J. Mol. Biol. 78, 453 (1973). 23 D. A. Jackson, R. H. S y m o n s , and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 69, 2904 (1972). 24 V. Sgaramella, J. H. van de Sande, and H. G. K h o r a n a , Proc. Natl. Acad. Sci. U.S.A. 67, 1468 (1970). 25 j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972). 2e j. Hedgpeth, H. M. G o o d m a n , and H. W. Boyer, Proc. Natl. Acad. Sci. U.S.A. 69, 3448 (1972). z7 V. Sgaramella, Proc. Natl. Acad. Sci. U.S.A. 69, 3389 (1972). 2s S. N. Cohen, A. C. Y. Chang, H. W. Boyer, and R. B. Helling, Proc. Natl. Acad. Sci. U.S.A. 70, 3240 (1973).
[1]
RECOMBINANT D N A
TECHNIQUES
5
1. A DNA vehicle (vector, replicon) which can replicate in living cells after foreign DNA is inserted into it. 2. A DNA molecule to be replicated (passenger), or a collection of them. 3. A method of joining the passenger to the vehicle. 4. A means of introducing the joined DNA molecule into a host organism in which it can replicate (DNA transformation or transfection). 5. A means of screening or genetic selection for those cells that have replicated the desired recombinant molecule. This is necessary since transformation and transfection methods are inefficient, so that most members of the host cell population have no recombinant DNA replicating in them. This selection or screening for desired recombinants provides a route to recovery of the recombinant DNA of interest in pure form.
Since a thorough review of recombinant DNA was completed in 1976, 29 I will concentrate on progress since then. Cloning Vehicles Plasmids
Many bacterial plasmids have been used as cloning vehicles. Currently, E. coli and its plasmids constitute the most versatile type of host-vector system for DNA cloning. A number of derivatives of natural plasmids have been developed for cloning. Most of these new plasmid vehicles were made by combining DNA segments, and desirable qualities, of older vehicles (Table I). All those listed have a "relaxed" mode of replication, such that plasmid DNA accumulates to make up about one-third of the total cellular DNA when protein synthesis is inhibited by chloramphenicol or spectinomycin. ~0 pBR322 is now the most widely used plasmid for cloning of DNA. One of its virtues is that it has six different types of restriction cleavage termini at which foreign DNA can be inserted. A very detailed restriction enzyme cleavage map and DNA sequence information are also important. 31a2 The PstI site in the Ap (penicillinase) gene has further advantages. If dG homopolymer tails are added to Pst-cleaved pBR322 DNA, and dC homopolymer tails to the DNA to be inserted, the PstI sites are reconstituted in 29 R. L. Sinsheimer, A n n u . Rev. Biochem. 46, 415 (1977). 3o A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978). at j. G. Sutcliffe, Proc. Natl. Acad. Sci. U.S.A. 75, 3737 (1978). 32 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978).
6
INTRODUCTION
[ 1]
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O'r'
E O
'r" o
O
e~
e~ r~
,4
"4
¢N
0o
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