recombinant dna part a

BAHL 7, Cetus Corporation, Berkeley, Califirnia 94710 RAMAMOORTHY BELAGAJE 8, Lilly Re- search Laboratories, Indianapolis, In- diana 46206 HANS-ULRICH BERNARD 35, Department o['B

P r e f a c e DNA is the genetic material of virtually all living organisms The physical mapping of genes, the sequence analysis of DNA, and the identi- fication of regulatory elements for DNA replication and transcription depend on the availability of pure specific DNA segments The DNA of higher organisms is so complex that it is often impossible to isolate DNA molecules corresponding to a single gene in sufficient amounts for analy- sis at the molecular level However, exciting new developments in re- combinant DNA research make possible the isolation and amplification of specific DNA segments from almost any organism These new develop- ments have revolutionized our approaches in solving complex biological problems

Recombinant DNA technology also opens up new possibilities in medicine and industry It allows the manipulation of genes from different organisms or genes made synthetically for the large-scale production of medically and agriculturally useful products

This volume includes a number of the specific methods employed in recombinant DNA research Other related methods can be found in

"Nucleic Acids," Volume 65, Part I, of this series

I wish to thank the numerous authors who have contributed to this volume, as well as the very capable staff of Academic Press, for their assistance and cooperation I also wish to extend my appreciation to Stanley Cohen and Lawrence Grossman for their advice in planning the contents of this volume

RAY Wu

xiii

C o n t r i b u t o r s to V o l u m e 6 8

Article numbers are in parentheses following the names o f contributors

Affiliations listed are current

DAVID ANDERSON (30), Genex Corporathm,

Rockville, Maryland 20852

JAMES C ALWINE (15), Laboratory of

Molecular Virology, National Cancer In-

stitute, National Institutes gf Health,

Bethesda Maryland 20014

S L AUCKERMAN (38), Department of

Biology, The Johns Hopkins University,

Baltimore, MaiTland 21218

KEITH BACKMAN (16), Department of

Biology, Massachusetts Institute of Tech-

nology Cambridge, Massachusetts 02139

C P BAHL (7), Cetus Corporation,

Berkeley, Califi)rnia 94710

RAMAMOORTHY BELAGAJE (8), Lilly Re-

search Laboratories, Indianapolis, In-

diana 46206

HANS-ULRICH BERNARD (35), Department

o['Biology, University ~f California, San

Diego, La Jolla, CaliJornia 92093

DALE BLANK (33), Rosenstiel Basic Medical

Sciences Research Center and Depart-

ment of Biology, Brandeis University,

Waltham, Massachusetts 02154

FRANCISCO BOLIVAR (16), Deparamento

Biologia Molecular, lnstituto de In-

vestigaciones Biomedicas, Universidad

Nacional Aatonoma de Mexico, Mexico

20, D.F., Mexico Apdo Postal 70228

ROLAND BROUSSEAU (6), Division of'Biolog-

ical Sciences, National Research Council

of Canada, Ottawa KIA OR6, Canada

EUGENE L BROWN (8), Synthex Research,

Palo Alto, Califi~rnia 94304

DOUGLAS BRUTLAG (3), Department o["

BiochemisttT, Stanford University School

qf Medicine, Stanford, Cal~)rnia 94305

JOHN CARBON (27, 31), Department of Bio-

logical Sciences, University of Califi)rnia,

Santa Barbara, Santa Barbara, California

93106

P CHIU (29), Section of BiochemistiT, Molecular and Cell Biology, Cornell Uni- versity, Ithaca New York 14853

LOUISE CLARKE (27, 31), Department t~f' Biological Sciences, University of Cali- fi)rnia, Santa Barbara, Santa Barbara, Califi)rnia 93106

STANLEY N COHEN (32), Departments of Genetics and Medicine, StanJbrd Uni- versity School of Medicine, Stanford, Cal(~)rnia 94305

JO~N COLLINS (2), Gesellscht(fi flit Bio- technologische Forschung mbH Masch- eroder Weg I D-3300 Brutmschweig- St6ckheim, West Germany

NICHOLAS R COZZARELL1 (4), Departments

of Biochemistry and Biophysics and Theoretical Biology, University of Chicago, Chicago, Illinois 60637

J L CULLETON (38), Department of Biof ogy, The Johns Hopkins University, Baltimore Marylund 21218

R P DOTTIN (38), Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

L ENQU~ST (18), Laboratory of Molecular Virology, National Cancer Institute, National Institntes qf Health, Bethesda, Marykmd 20014

HENRY A ERLI¢~ (32), Department of Medicine, Stanford Univel=sity School of Medicine, Stanfi~rd, California 94305

KAREN FAHRNER (33), Rosenstiel Basic Medical Sciences Research Center and Department ~[" Biology, Brandeis' Uni- t'ersity, Waltham, Massachusetts 02154

G C FAREED (24), Department of Micro- biology and hnmunology, Molecular Biol- ogy Institute, University of Cahi~rnia, Los Angeles, Los Angeles, California

90024

i x

DAVID FIGURSKI (17), Department of Micro-

biology, College ~2f Physicians and

Surgeons, Columbia University, New

York, New York 10032

S G FISCHER (11), Department of Biologi-

cal Sciences, State University of New

York at Albany, Albany, New York 12222

B R FISHEL (38), Department of Biology,

The Johns Hopkins University, Balti-

more, Marvhmd 21218

MICHAEL L GOLDBERG (14), Abteihmg

Zelliologie, Biozentrium Der Universitiit

Basel, CH-4056 Basel, Switzerhmd

HOWARD M GOODMAN (5), Howard

Hughes Medical Institute Laboratory and

the Department of Biochemistry and Bio-

physics, University of Cahlfbrnia, San

Fruncisco, Cal(fbrnia, 94143

MICHAEL GRUNSTEIN (25), Department qf

Biology, University of California, Los

Angeles, Los Angeles, Cali[brnia 90024

DONALD R HELINSKI (17, 35), Department

of Biology, University of Cahfornia, San

Diego, La Jolla, California 92093

LYNNA HEREFORD (33), Rosenstiel Basic'

Medical Sciences Research Center and

Department qf Biology, Brandeis Uni-

versity, Waltham, Massachusetts 02154

N PATRICK HIGGINS (4), Department q["

Biochemisto', University qf Wyoming,

Laramie, Wyoming

RONALD H1TZEMAN (31), Department of

Biological Sciences, University of Cali-

fornia, Santa Barbara, Santa Barbara,

California 93106

BARBARA HOHN (19), Friedrich Miescher

Institut, CH-4002 Basel, Switzerland

JANICE P HOLLAND (28), Department of

Biochemistry, University of Connecticut

Health Center, Farmington, Connecticut

06032

MICHAEL J HOLLAND (28), Department of

Biochemistty, University of Connecticut

Health Center, Farmington, Connecticut

06032

HANSEN M HSIUNG (6), Division of Biologi- cal Sciences, National Research Council

of Canada, Ottawa KIA OR6, Canada

KIMBERLY A JACKSON (28), Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut

06032

MICHAEL KAHN (17), Department of Bac- teriology and Public Health, Washing- ton State University, Pullman, Washing- ton 99164

KATHLEEN M KEGGINS (23), Department q[" Biological Sciences, University of Muo'lund, Baltimore County, Catons- ville, Mao, land 21228

DAVID J KEMP (15), 1he Walker and Eliza Hall Institute of Medical Research, Post OJfice, Royal Melbowne Hospital, Vic- toria 3050, Australia

H GOBIND KHORANA (8), Departments oj" Biology and Chemisto', Massachusetts h~stitute of Technology, Cambridge, Massachusetts 02139

ROBERVO KOLTER (17), Department of Biology, University of" California, Sun Diego, La Jolla, Cal(fi)rnia 92093

L F LAU (7), Section qf Biochemistry, Molecular and Cell Biology, Cornell Unil,ersity, Ithaca, New York 14853

GAIL D LAUER (34), The Biological Lab- oratories, Harvard University, Cam- bridge, Massachusetts 02138

LEONARD S LERMAN (11), Department of Biological Sciences, State University of New York at Albany, Albany, New York

12222

RICHARD P LIFTON (14), Department (2[ Biochemistry, StanJbrd University School q( Medicine, StanJbrd, California 94305

JOHN LIS (10), Section of Biochemistpy, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

SHIRLEY LONGACRE (12), Parasitologie Ex- perimentale, Institut Pasteur, 75724 Paris, Cedex 15, France

C O N T R I B U T O R S TO V O L U M E 68 xi

PAUL S LOVETT (23), Department of Bio-

k~gical Sciences, University of Maryland,

Baltimore County, Catonsville, Maryland

21228

HUGH O McDEVITT (32), Departments of

Medicine and Medical Microbiology,

Stanford University School c~f Medicine,

Star,ford, California 94305

RAYMOND J MACDONALD (5), Howard

Hughes Medical Institute Laboratory and

the Department of Biochemistry and

Biophysics, University ~f California,

San Francisco, San Francisco, Cahfornia

94143

BERNARD MACH (12), Department of Micro-

biology, University of Geneva, CH 1205

Geneva, Switzerland

R E MANROW (38), Department of Biology,

The Johns Hopkins University, Baltimore,

Maryland 21218

RICHARD MEYER (17), Department of Mi-

crobiology, University of Texas, Austin,

Texas 78712

D A MORRISON (21), Department of Bio-

logical Sciences, University of Illinois,

Chicago Circle, Chicago, Illinois 60680

JOHN f MORROW (|), Department of Micro-

biology, The Johns Hopkins School o["

Medicine, Baltimore, Mao, land 21205

S A NARANG (6, 7), Division of Biolog&al

Sciences, National Research Council of

Canada, Ottawa KIA OR6, Canada

TIMOTHY NELSON (3), Department of Bio-

chemistry, Stanfi)rd University School of

Medicine, Stanford, California 94305

BARBARA A PARKER (15), Department of

Biochemistry, Stanford University School

of Medicine, Stanford, California 94305

BARRY POLlSKY (37), Department of Biol-

ogy, Indiana University, Bloomington,

Indiana 47401

A F PURCHIO (24), Department of Micro-

biology and Immunology, Molecular

Biology Institute, University of CaliJbrnia,

Los Angeles, Los Angeles, Cal([brnia

90024

JOHN REEVE (36), Department of Micro- biology, Ohio State University, Columbus, Ohio 43210

JAKOB REISER (15), Department of Bio- chemistry, Stanford University School of

Medicine, Stanford, California 94305

ERIC REMAUT (17), Laboratorium voor Moleculaire Biologie, Bijksuniversiteit Gent, B-9000 Gent, Belgium

JAIME RENART (15), lnstituto de Enzi- mologia del C.S.1.C., Facultad de Medi- cina de la Universidad Autonoma, Arzobispo Morcillo s / n Madrid-34, Spain

ROBERT RICClARDI (33), Department of Bio- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115

RICrtARD J ROBERTS (2), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

BRYAN ROBERTS (33), Rosentiel Basic Medical Sciences Research Center and Department ~f Biology, Brandeis Uni- versity, Waltham, Massachusetts 02154

THOMAS M ROBERTS (34), Department of Biochemistry and Molecular Biology, Harvard University, Cumbridge, Mas- sachusetts 02138

MICHAEL ROSBASH (33), Rosenstiel Basic" Medical Sciences Research Center and Department of Biology, Brandeis Uni- versity, Waltham, Massachusetts 02154

R J ROTHSTEIN (7), Department of Micro- biology, New Jersey School of Medicine, Newark New Jersey 07103

STEPHANIE RUBY (33), Department of Bio- logical Chemistry, Harvard Medical School Boston, Massachusetts 02115

MICHAEL J RYAN (8), Microbiological Sciences, Schering Corporation Bloomfield, New Jersey 07003

A SEN (13), Meloy Laboratories Inc., Springfield, Virginia 22151

xii C O N T R I B U T O R S TO V O L U M E 68

LUCILLE SHAPIRO (30), Department t~f'Mo-

lecular Biology, Albert Einstein College

of Medicine, Bronx, New York

H MlCrtAEL SHEPARD (37), Department ~["

Biology, bldiana University, Blooming-

ton, hldiana 47401

F SHERMAN (29), Department qfRadiation

Biology and Biophysics, University of

Rochester, School oJ' Medicine, Roches-

ter, New York 14642

M SHOVAI3 (13), Laboratory of Viral Car-

cinogenesis, National Cancer Institute,

National Institutes ~)1 Health, Bethesda,

Marylund 20014

A M SKALKA (30), Department ¢~f Cell

Biology, Roche Institute ~[" Malecular

Biology, Nutley, New Jersey 071IO

EDWIN SOUTHERN (9), M.R.C Mammalian

Genome Unit, King's Building, Edin-

burgh EH9 3JT, Scotland

GEORGE R STARK (14, 15), Department ~f"

Biochemistry, Stanford University School

~]" Medicine, Stat~jbrd, Cal(fornia 94305

N STERNBERG (|8), Cancer Biology Pro-

gram, Frederick Cancer Research Cen-

ter, Frederick, Maryland 21701

J I STILES (29), Department of Radiation

Biology and Biophysics, University of

Rochester, School ~f Medicine,

Rochester, New Yark 14642

M SUZUKI (22), Boyce Thompson Institute,

Cornell University Ithaca New York

14853

A A SZALAY (22), Boyee Thompson Insti-

tute, Cornell University, Ithaca, New

B.-K TVE (29), Section of Biochemisto', Molecuhtr and Cell Biology, Cornell Universio, Ithaca, New Yor,~" 14853

GEOFFRE'¢ M WAHL (15), Department qf

Biochemisto', Stanford University School

qf Medicine, Stanford, CaliJbrnia 94305

JOHN WALLIS (25), Department of Micro- biology and Immunology, Molecular Biol- ogy Institute University ~1" CaliJornia, Los An,~,eles, Los Angeles, California 9OO24

JEEFREY G WIkLIAMS (14), Imperial Cancer Research Fired, Mill Hill, London NWT, England

SAVIO L C W o o (26), Howard Hughes Medical Institute Laboratory and De- partrnent of'Cell Biology, Baylor College

~0 r Medicine, Texas Medical Center, Houston Texas 77030

JOHN WOOLFORD (33), Rosenstiel Basic Medical Sciences Research Center and Department ~( Biology, Brandeis Univer- sit),, Waltham Massachusetts 02154

RAy Wu (7, 10, 29), Section of Biochemistry, Maleeular and Cell Biology, Cornell Uni- versity, Ithaca, New York 14853

ROBERT C.-A YANG (10), Section of Bio- chemisttT, Molecular and Cell Biology, Cornell University, Ithaca, New York

14853

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)

1o T H Fraser and B J Bruce, Proc Natl Acad Sci U.S.A 75, 5936 (1978)

11 C 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

4 INTRODUCTION [1] part o f the foundation 17 The discovery o f site-specific restriction endonu- cleases lsa9 also contributed (see Nathans and Smith, ~° Roberts, 21 and this volume [2], for review) T w o general methods for joining D N A molecules from different sources were found 2z-24 Particularly useful was the first

e n z y m e found to create self-complementary, cohesive termini on D N A 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 o f recombinant molecules that subsequently replicated in vivo 2s

What can be done by the r e c o m b i n a n t D N A m e t h o d ? Principally three sorts o f things:

1 Isolation of a desired sequence from a c o m p l e x mixture o f D N A molecules, such as a e u k a r y o t i c genome, and replication o f it to provide milligram quantities for biochemical study

2 Alteration of a D N A molecule One can insert restriction endonu- clease recognition sites, or other D N A segments, at r a n d o m or predeter- mined locations One can also delete restriction sites, or D N A segments between such sites, by techniques that permit joining any two D N A ter- mini after their appropriate modification Such an alteration can be helpful

in determining the functions p e r f o r m e d by various parts o f a D N A se- quence This is attractive w h e r e efficient means o f fine-structure genetic analysis of r a n d o m mutations are lacking, as in animals and plants

3 Synthesis in bacteria of large amounts o f peptides or proteins that are o f interest to science, medicine, or c o m m e r c e

Before indicating specifically the most useful methods for obtaining each o f the above goals, we look at recent advances in the basic tech- niques The essential ingredients o f a recombinant D N A e x p e r i m e n t are:

17 W B W o o d , 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, A n n u Rev Biochem 44, 273 (1975)

25 j E M e r t z and R W Davis, Proc Natl A c a d Sci U S A 69, 3370 (1972)

2e j H e d g p e t h , H M G o o d m a n , and H W Boyer, Proc Natl A c a d Sci U S A 69, 3448

(1972)

z7 V Sgaramella, Proc Natl A c a d Sci U.S.A 69, 3389 (1972)

2s S N C o h e n , A C Y C h a n g , H W Boyer, and R B Helling, Proc Natl A c a d Sci

3 A method of joining the passenger to the vehicle

4 A means of introducing the joined DNA molecule into a host orga- nism 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 repli- cating in them This selection or screening for desired recombinants pro- vides a route to recovery of the recombinant DNA of interest in pure

f o r m

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 Cur-

rently, E coli and its plasmids constitute the most versatile type of

h o s t - v e c t o r 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 " r e l a x e d " 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 spectino- mycin ~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 ho-

mopolymer tails are added to Pst-cleaved pBR322 DNA, and dC homo- polymer 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)

"~,~.o.~ o

E d ~ u u ~ u

8 I N T R O D U C T I O N [1] many of the resulting recombinant plasmids, r'33'a4 The inserted DNA seg- ment and the short dG: dC homopolymer segments flanking it may be

cleaved from the vehicle by PstI digestion Furthermore, the segment of

DNA inserted into the Ap gene is transcribed from the penicillinase pro- moter This has permitted transcription and translation of mouse dihydro- folate reductase 8 and rat proinsulin, evidently fused to the N-terminus of

penicillinase, r in E coli

Several of the vehicles in Table I have the advantage of inactivation of

a genetic marker by insertion of DNA at a particular restriction site For

instance, the BamHI and SalI sites of pBR322 are within the Tc gene In-

sertion of DNA at either of these sites generates an Ap R Tc s plasmid 33 Success or failure of the procedure to form recombinants may then be checked by replica-plating Ap a transformants on tetracycline-agar plates

to test for the Tc s phenotype Furthermore, the recombinants may be se- lected by culture in the presence of tetracycline and D-cycloserine, which kills exponentially growing cells Bacteria containing the Ap a Tc a vehicle plasmids are killed, while those carrying the AP a Tc s recombinant plasmids survive 29"~3

Similarly, the PstI site of pBR322 is within the Ap gene Insertion of

DNA there creates an Ap s Tc R plasmid, a3 However, most rat insulin cDNA recombinant plasmids gave rise to colonies on ampicillin plates, r These appeared after a longer incubation period than that needed for col- lonies containing pBR322 alone There is evidence that they resulted from infrequent deletion of the cDNA passenger segment which was inserted at

the PstI site of pBR322 by means of dG and dC homopolymer tails Unfortunately, insertion of DNA at the EcoRI site of pBR322 does not

result in any known marker inactivation However, several other plasmids do have this advantage For example, RSF2124 fails to produce

colicin E1 if DNA is inserted at its single EcoRI site 35 Also, pACYC184

and pBR325 were constructed to provide useful cloning vehicles with in- activation of a drug resistance marker, chloramphenicol resistance, by

DNA insertion at their single EcoRI sites (Table I)

Insertional marker inactivation is a useful feature in a cloning vehicle However, it is not essential, for other methods can ensure that virtually

all transformed E coli contain a recombinant plasmid rather than the un-

altered vehicle plasmid If restriction enzyme-cleaved cohesive DNA ter- mini are used for joining, treatment of the vehicle DNA with phosphatase removes terminal phosphoryl groups and prevents rejoining without an

aa F Bolivar, R L Rodriguez, P J G r e e n e , M C Betlach, H L H e y n e k e r , H W Boyer,

J H Crosa, and S Falkow, Gene 2, 95 (1977)

34 M B M a n n , R N Rao, and H O Smith, Gene 3, 97 (1978)

[1] RECOMBINANT D N A TECHNIQUES 9

insert 3e DNA joining by means of homopolymer extensions added by ter- minal deoxynucleotidyltransferase 2z~3 also can ensure that most plasmids have inserted DNA Of course, both of these methods require that nicked circular vehicle plasmid DNA be removed from the cleaved linear vehicle DNA before use, either by exhaustive restriction endonuclease digestion

or by gel electrophoresis Since nicked circular plasmid DNA transforms bacteria with the same high efficiency as supercoiled plasmid DNA, it can give rise to clones containing vehicle plasmid rather than a recombinant Inactivation of a genetic marker is not an infallible indication of DNA insertion For instance, when pBR324 is digested by Barn HI, Hin dIII, or

SalI restriction endonuclease, ligated with other similarly digested DNA, and used to transform bacteria to ampicillin resistance, 5-10% of the Tc s transformants do not carry recombinant DNA 37 Such Tc s transformants, possibly resulting from deletion of a portion of the tetracycline resistance gene, have also been observed for pBR322 Corresponding Cm s Ap a Tc R transformants, carrying no inserted DNA, represent about 10% of the

Cm s transformants in recombinant DNA experiments using E c o R I -

cleaved pBR325 DNA 37

The pJC plasmids in Table I are representatives of a class of plasmids called cosmids because they have the h phage cos DNA site required for packaging into h phage particles, and they replicate as plasmids because

of their ColE1 DNA segments Their chief advantage is the fact that pJC DNA ligated to foreign DNA can be packaged in vitro and used to infect

E co/i efficiently, yielding as many as 500,000 recombinant clones per mi- crogram of inserted DNA Using the CaC12 transformation procedure 3a in- stead, or minor modifications of it, one usually obtains 1000-50,000 trans- formants per microgram inserted DNA after cohesive-end ligation 3a or ho- mopolymer tail annealing 7,4° However, the efficiency of transformation can be improved by modifications such as the method of Suzuki and Szalay (this volume [22]), so that it rivals the efficiency of ~ phage in vitro

packaging and infection The in vitro packaging also exerts a size selec- tion on DNA to be packaged, so that all surviving pJC cosmids have in- serted DNA

Several plasmids contain the lactose operon promoter and operator and a single E c o R I site for inserting DNA within the lacZ gene They can

as A Ullrich, J Shine, J Chirgwin, R Pictet, E Tischer, W J Rutter, and H M

Goodman, Science 196, 1313 (1977)

aT F Bolivar, Gene 4, 121 (1978)

as S N Cohen, A C Y Chang, and L Hsu, Proc Natl Acad Sci U S A 69, 2110 (1972)

3g j F Morrow, S N Cohen, A C Y Chang, H W Boyer, H M Goodman, and R B

Helling, Proc Natl A c a d Sci U S A 71, 1743 (1974)

4o p C Wensink, D J Fi~negan, J E Donelson, and D S Hogness, Cell 3, 315 (1974)

to which a foreign polypeptide will presumably be fused in a recombinant strain

pPC~bl, pPC~b2, and pPC~b3, contain lacPO and a much smaller portion

of the Z gene than pOP1 does They were constructed (from pBR322) in order to overcome the translation reading frame problem for inserted DNA, which is transcribed from the lac promoter in these plasmids One end of the DNA inserted at the EcoRI site of these plasmids is 22, 24, or

26 base pairs, respectively, downstream from the initial methionine codon

of the/3-galactosidase gene fragment The inserted DNA sequence will be translated in one of the three frames unless a noncoding sequence within

it has termination codons in all three frames This array of three plasmids

is not needed if the inserted DNA is joined to the vehicle by homopolymer tails of various lengths, since one-third of the junctions should give the right frame, r They should be useful for synthesis of proteins encoded by restriction cleavage fragments of DNA or by synthetic DNA, however

A plasmid similar to pPC~bl is pBH20.1 It was derived from pBR322 and

a 203-base-pair HaelII fragment of ~plac5 DNA It has a single EcoRI site

22 base pairs from the initial methionine codon of the lacZ gene

pKC16 (Table I) is derived from pBR322 The plasmid copy number increases after thermal induction, since the plasmid contains the hci857

temperature-sensitive repressor gene and the h O and P genes It reaches more than 100 copies per bacterial chromosome

Several other plasmids are useful for special purposes, pBR324 and pKB158 have restriction cleavage sites creating blunt, base-paired DNA termini (the SmaI and HpaI sites, respectively) Blunt-ended foreign DNA fragments can be joined to these vehicles and replicated (see DNA joining methods below) RSF1030 is noteworthy because its DNA does not cross-hybridize with ColE1 or pMB9 DNA (pMB9 is a Tc R plasmid re- lated to ColE1, widely used for DNA cloning~9) Consequently, a DNA sequence cloned in pMB9, e.g., a reverse transcript (cDNA) plasmid, can easily be used to screen RSF1030 recombinants by hybridization, 41 e.g., for the corresponding gene from a eukaryotic genome, for complementary repeated sequences, etc

Only pBR322, pMB9, pBR313, and pSC101 are certified now as EK2 plasmid vectors for cloning DNA from warm-blooded vertebrates and

[1] RECOMBINANT DNA TECHNIQUES 11 other sources 29-33,42 The other plasmids described (Table I) are EK1 vec- tors

B a c t e r i o p h a g e

Derivatives of h phage were developed as cloning vehicles early, 29 and they are probably the best vehicles for the cloning and isolation of particu- lar genes from eukaryotic genome DNA h derivatives have three main advantages over plasmids for this purpose First, thousands of recom- binant phage plaques on a single 88-ram petri dish can easily be screened

for a given DNA sequence by nucleic acid hybridization 43 Second, in

vitro packaging of recombinant DNA molecules provides a very efficient means of infecting bacteria with them 44,4n Finally, millions of indepen- dently packaged recombinant phage can be replicated conveniently and stored in a single solution as a "library" in which all sequences of a large genome (e.g., rabbit) are likely to be represented 46

The Charon phages are h derivatives, some of which approach the maximum possible capacity for inserted DNA in a nondefective ~, phage vector (Table II) Charons 4 and 8-11 are believed to be able to replicate more than 22 kb of inserted DNA A number of Charon phages contain a

l a c Z (fl-galactosidase) gene Substitution of foreign DNA in place of the

l a c P O Z DNA segment makes the phage lacO- Growth on a lac + bacterial

strain on plates containing a chromogenic noninducing /3-galactosidase substrate then provides a quick indication of insertion of foreign DNA or failure to do so Charon 4 has this feature and has been used extensively,

as has its EK2 derivative, Charon 4A 46

hgt4 • hB was constructed to provide a phage vector which has the attachment site and is able to form temperature-inducible lysogens, since

it has the ci857 allele This has been helpful in overproduction of DNA ligase encoded by an inserted E coli DNA segment

hgtWES • hB and ~.gtvirJZ • hB are EK2 vectors, useful for repli- cating DNA of warm-blooded vertebrates, etc 42

hAzl, hAz2, hAz3, and their corresponding derivatives with amber mutations provide phage "vectors with the same advantages for protein production as pPC~bl, pPC~b2, and pPC~b3 plasmids (see above)

M13mp2 (Table II) is a derivative of M13, a single-stranded DNA

4z "Guidelines for Research Involving Recombinant DNA Molecules" (rev.), Fed Regist

43, 60108 (1978)

43 W D Benton and R W Davis, Science 196, 180 (1977)

N Sternberg, D Tiemeier, and L Enquist, Gene 1, 255 (1977)

4s B Hohn and K Murray, Proc Natl Acad Sci U.S.A 74, 3259 (1977)

T Maniatis, R C Hardison, E Lacy, J Lauer, C O'Connell, D Quon, G K Sim, and

A Efstratiadis, Cell 15, 687 (1978)

c~

,.q

I::

0 i

t ~ ~"w r ~ " ' ; 0 •

14 INTRODUCTION [ 1] phage, which contains t h e / a c promoter and operator and the proximal

portion of the lacZ gene Mutagenesis generated an EcoRI site at the

codon for the fifth amino acid from the N-terminus of/3-galactosidase

Insertion of foreign DNA at this EcoRI site inactivates lacZ ct-

complementation, producing colorless or light-blue plaques on appropri- ate host bacteria with a chromogenic substrate The significant aspect of M13mp2 is that the phage particles provide only one strand of the cloned DNA, i.e., they strand-separate the DNA for the investigator This is very

useful for the DNA-sequencing method of Sanger et al.,4r for nucleic acid

hybridization, etc If two recombinants are isolated with the inserted DNA in opposite orientations each will serve as a source for a different strand

Unfortunately, M 13mp2 needs an F + E coli host to make plaques, and

conjugative plasmids are not acceptable in EK1 h o s t - v e c t o r systems 42 A strain carrying a conjugation-deficient derivative of F may be approved soon for the EK1 level

Eukaryotic Vectors

Only SV40 virus has been used as a cloning vehicle to replicate intro- duced DNA independently of the chromosomes in eukaryotic cells The SV40 vehicle with the largest capacity for inserted DNA permits encapsi- dation of about 4.3 kb of added DNA, a small capacity compared to plasmid or phage vectors 2a

An E c o R I - H p a l I fragment of SV40 DNA has been used to replicate the E co/i Su+III tRNA gene (Table II) This recombinant DNA has been used to transform rat cells 4s The E c o R I - H p a l I SV40 fragment has also been joined at its EcoRI terminus to a larger fragment of h phage DNA,

and the linear molecule has been used to transform mouse cells (Table II) Another fragment of SV40 DNA called SVGT5 was chosen to permit transcription and translation of inserted DNA sequences The " b o d y " (main exon) of the VP1 gene was excised, leaving the gene's 5'-end "lead- ers," its intervening sequence, and its Y-end A recombinant derived from it formed rabbit/3-globin in monkey kidney cells (Table II)

An in situ nucleic acid hybridization method for SV40 recombinants

has been described 4a

In other experiments involving introduction of DNA into eukaryotic

4~ F Sanger, S Nicklen, and A R Coulson, Proc Natl Acad Sci U.S.A 74, 5463 (1977)

48 p Upcroft, H Skolnik, J A Upcroft, D Solomon, G Khoury, D H Hamer, and G C Fareed, Proc Natl Acad Sci U.S.A 75, 2117 (1978)

49 L P Villarreai and P Berg, Science 196, 183 (1977)

5o A Hinnen, J B Hicks, and G R Fink, Proc Natl Acad Sci U.S.A 75, 1929 (1978)

51 M Wigler, A Pellicer, S Silverstein, and R Axel, Cell 14, 725 (1978)

52 L Clarke and J Carbon, Cell 9, 91 (1976)

53 S C Hardies and R D Wells, Proc Natl Acad Sci U.S.A 73, 3117 (1976)

S M Tilghman, D C Tiemeier, F Polsky, M H Edgell, J G Seidman, A Leder, L

W Enquist, B Norman, and P Leder, Proc Natl Acad Sci U.S.A 74, 4406 (1977)

55 E Y Friedman and M Rosbash, Nucleic Acids Res 4, 3455 (1977)

G N Buell, M P Wickens, F Payvar, and R T Schimke, J Biol Chem 253, 2471 (1978)

57 M P Wickens, G N Buell, and R T Schimke, J Biol Chem 253, 2483 (1978)

K J Marians, R Wu, J Stawinski, T Hozumi, and S A Narang, Nature (London) 263,

744 (1976)

59 H L Heyneker, J Shine, H M Goodman, H W Boyer, J Rosenberg, R E Dick- erson, S A Narang, K Itakura, S Lin, and A D Riggs, Nature (London) 263, 748 (1976)

60 j R Sadler, J L Betz, M Tecklenburg, D V Goeddel, D G Yansura, and M H Caruthers, Gene 3, 21 ~ (1978)

el H G Khorana, Science 203, 614 0979)

The cloning vehicle D N A can also recircularize by itself, with no in- serted D N A As a result, 7 5 - 9 0 % o f the transformants usually contain ve- hicle alone instead of r e c o m b i n a n t D N A 3ha9 A method to pr.event re- sealing of the vehicle D N A is removal o f its terminal p h o s p h a t e groups by incubation with nuclease-free alkaline p h o s p h a t a s e ? 6,n~ The "¢ehicle D N A can then be ligated into a r e c o m b i n a n t circle with the passenger D N A , or the passenger D N A can be cyclized to yield molecules which generally cannot replicate, but the vehicle c a n n o t be ligated alone This phospha- tase m e t h o d has been used very effectively on H i n d I I I and E c o R I termini; all clones examined had inserted foreign D N A ? 6,e~,~ It has also been used

on B a m H I termini with s u c c e s s ' N o t e that a higher c o n c e n t r a t i o n of

D N A ligase is needed for complete joining than if D N A were not incu- bated with phosphatase

Cohesive ends can be added to blunt-ended D N A molecules by liga- tion with synthetic D N A linkers 6°,~5,6~ These are duplex, blunt-ended

D N A molecules, from 8 to 14 base pairs in length, containing the recogni- tion site for a restriction endonuclease that p r o d u c e s cohesive termini Linkers with an E c o R I , a B a m H I , or a H i n d l I I site are available c o m m e r - cially Linkers are joined to blunt-ended passenger D N A molecules by T4 ligase After digestion with the relevant restriction e n z y m e and removal

o f excess linker, the passenger D N A is ligated to vehicle D N A via the

c o m p l e m e n t a r y termini and cloned E c o R I '°,a6,~,e~-69 and H i n d l I P e'~'r°

B Weiss, T R Live, and C C Richardson, J Biol Chem 243, 4530 (1968)

P H Seeburg, J Shine, J A Martial, J D Baxter, and H M Goodman, Nature (London) 270, 486 (1977)

J Shine, P H Seeburg, J A Martial, J D Baxter, and H M Goodman, Nature (London) 270, 494 (1977)

C P Bahl, K J Marians, R Wu, J Stawinsky, and S A Narang, Gene 1, 81 (1976)

R H Scheller, R E Dickerson, H W Boyer, A D Riggs, and K Itakura, Science 196,

70 H Lehrach, A M Frischauf, D Hanahan, J Wozney, F Fuller, R Crkvenjakov, H Boedtker, and P Doty, Proc Natl Acad Sci U.S.A 75, 5417 (1978)

[1] RECOMBINANT DNA TECHNIQUES 17 linkers have been widely used The optimal conditions for joining the

E c o R I decamer linker have been determined 71 A variation of the linker method utilizes the appropriate modification methylase to protect internal sites in the passenger DNA from cleavage by the restriction endonu- clease.46

The conditions affecting cohesive end ligation have been explored TM A useful variation is to ligate termini made by cleavage with one restriction

endonuclease to those made by another For instance, D p n I I and M b o I

leave the single-stranded Y-terminus GATC (the recognition site for both

is GATC) These DNA ends can be joined to GATC 5'-termini generated

by B a m H I , whose complete recognition sequence is GGATCC BglII and

BclI make ends that can be joined to the preceding ones, too; their recog- nition sequences are AGATCT and TGATCT, respectively Similarly,

SalI and X h o I both leave TCGA single-stranded termini, though they rec-

ognize GTCGAC and CTCGAG, respectively 21

H o m o p o l y m e r Tails

Terminal deoxynucleotidyltransferase can be used to add a homo- polymer extension, e.g., polydeoxyadenylate, to each 3'-end of the ve- hicle DNA, and a complementary extension to each 3'-end o f the passen- ger DNA (see this volume [3]) An attractive application is to add dG tails

to P s t I - c l e a v e d pBR322 DNA and dC tails to the DNA to be inserted, anneal, and transform E coli with the DNA P s t I sites are reconstituted

on both sides of the inserted DNA in many of the resulting recombinant plasmids 7~aa4 Ligation of the vehicle with the insert before transforma- tion is unnecessary if they have complementary homopolymer tails In-

deed, it has not been possible to ligate them efficiently in vitro 22a3

When homopolymer tails were first used for joining DNA molecules,

an exonuclease was employed to render the vehicle and passenger DNA termini single-stranded before incubation with terminal transferase 22"2a The exonuclease is not necessary TM However, a technical precaution which is useful in cloning large eukaryotic DNA fragments is to eliminate small polynucleotides (<-1 kb) before annealing vehicle and passenger

DNAs Preparations of eukaryotic DNA with weight-average molecular weights of 10 kb or more often contain a significant n u m b e r of much

smaller DNA molecules Also, terminal transferase can initiate homo-

TI A Sugino, H M Goodman, H L Heyneker, J Shine, H W Boyer, and N R Coz-

zarelli, J Biol Chem 252, 3987 (1977)

72 A Dugaiczyk, H W Boyer, and H M Goodman, J Mol Biol 96, 171 (1975) 7a R Roychoudhury, E Jay, and R Wu, Nucleic Acids Res 3, 863 (1976)

Blunt.ends can be p r o d u c e d on a D N A fragment by cleavage with any

of a n u m b e r of restriction endonucleases Alternatively, r a n d o m shear breakage or a restriction e n z y m e making staggered cuts m a y be used, but the D N A termini must then be made blunt by biochemical methods This has been done by removal of single-stranded termini by incubation with single-strand-specific nuclease S1.36,46,67"69 Alternatively, T4 D N A polym- erase, la° E coli D N A p o l y m e r a s e I, 63'68'rs or reverse transcriptase, 36 with added d e o x y n u c l e o s i d e triphosphates, has been used Sometimes combined treatment with nuclease S 1 followed by D N A p o l y m e r a s e I and

d N T P s has been employed 6a'7°

I n t r o d u c i n g R e c o m b i n a n t D N A into a H o s t

Almost all recombinant D N A research has used, as host cells, E coli

K12 mutants lacking restriction of foreign DNA 29 Increased biological containment is provided by the a p p r o v e d E K 2 host for plasmids, X1776 42,76 L o w efficiency of transformation has been obtained with this strain in some studies (e.g., 20,000 Ap R transformants per microgram of supercoiled pBR322 DNA, ~ c o m p a r e d to the usual 106 per microgram

74 K Kato, J M Goncalves, G E Houts, and F J Bollum, J Biol Chem 242, 2780 (1967)

75 K Backman, M Ptashne, and W Gilbert, Proc Natl Acad Sci U.S.A 73, 4174 (1976)

re R Curtiss, III, M Inoue, D Pereira, J C Hsu, L Alexander, and L Rock, in "Molecu- lar Cloning of Recombinant DNA" (W A Scott and R Werner, eds.), p 99 Academic Press, New York, 1977

[ 1 ] RECOMBINANT D N A TECHNIQUES 19

with other E coli strains) Improved transformation procedures now yield

as much as 106- l0 T transformants per microgram of supercoiled pBR322 DNA, and a reported 104 recombinants per microgram when made by joining with homopolymer tails 7'76'77

In vitro packaging provides an efficient means of introducing recom-

binant phage DNA or cosmid DNA into E coli 44-46 Up to 700,000

plaques per microgram of inserted DNA have been obtained TM

Several other species of microorganisms may prove useful as hosts for replication of recombinant DNA A number of plasmid vectors have been

found for Bacillus subtilis 79-81 The U.S National Institutes of Health has only permitted cloning of DNA from Bacillus species with these, to date Genetic transformation of Saccharomyces cerevisiae has been demon-

strated, 5° but the NIH currently only permits cloning of yeast DNA, or cer- tain prokaryotic DNAs, in yeast A vehicle for recombinant DNA and a

transformation procedure are also available for Staphylococcus aureus.S2 Many recombinants containing known E coli genes have been iso-

lated by genetic selection of transformed cells expressing the desired function (reviewed in Sinsheimer29) A number of genes of fungi have

been expressed in E co/i and have complemented bacterial mutations

when replicated as part of a recombinant DNA molecule 3-6 Four out of

15 genetic complementations tested by J Carbon et al., using yeast DNA

in E coli, were successful 83 These studies used DNA extracted from

fungi, not reverse transcripts of fungal mRNA

It is unlikely that most genes of animals will encode functional pro-

teins in E co/i, because it appears that most such genes contain inter-

vening sequences At least one of these is usually located at a site on the DNA near that corresponding to the 5'-end of the mRNA sequence How- ever, this problem can be bypassed by using reverse transcripts of mRNAs Several of these have been expressed (transcribed and trans-

lated into protein) after introduction into E coli as part of recombinant

plasmids 7-1° One of these conferred a phenotype, trimethoprim resist- ance, which could be selected as a result of synthesis of mouse dihydro-

r7 M V Norgard, K Keem, and J J Monahan, Gene 3, 279 (1978)

78 R Lenhard-Schuller, B Hohn, C Brack, M Hirama, and S Tonegawa, Proc Natl Acad Sci U.S.A 75, 4709 (1978)

To K M Keggins, P S Lovett, and E J Duvall, Proc Natl Acad Sci U.S.A 75, 1423 (1978)

al S D Ehrlich, Proc Natl Acad Sci U.S.A 75, 1433 (1978)

s2 S L6fdahl, J Sj6str6m, and L Philipson, Gene 3, 161 (1978)

aa j Carbon, B Ratzkin, L Clarke, and D Richardson, in "Molecular Cloning of Recom- binant D N A " (W A Scott and R Werner, eds.), p 59 Academic Press, New York,

1977

20 INTRODUCTION [1] folate reductase in E coli s The eukaryotic proteins made in bacteria were identified by immunological methods Several such methods have been described which are capable of screening large number of recombinant clones for a protein of interest, s4-s6

Methods of screening that are independent of protein synthesis in bac- teria are commonly used, however Clones of interest can often be identi- fied by screening methods utilizing hybridization with a pure radioactively labeled nucleic acid probe 41,43,4~ If a pure nucleic acid probe is not avail- able, one can still identify a cloned protein structural gene if an RNA preparation containing some of the mRNA of interest is available The de- sired clone inhibits the in vitro translation of a particular mRNA by forming a D N A - R N A hybrid with it (hybrid-arrested translation) 87 A more sensitive type of method utilizes the cloned DNA to purify the par- ticular mRNA by D N A - R N A hybridization; the mRNA is then identified

by in vitro translation Either gel filtration (this volume [33]) or cloned DNA linked to cellulose ss can be used to purify the D N A - R N A hybrids, which are then dissociated by heating before in vitro translation

Fidelity of R e c o m b i n a n t DNA Cloning

The passenger segment of a recombinant DNA molecule is usually under no selection pressure for genetic function Thus its nucleotide se- quence is subject to evolutionary drift Nevertheless, consideration of the low spontaneous mutation frequency in wild-type bacterial strains suggests that growth for a few hundred generations should not alter the DNA sequence o f m o s t individual molecules in a recombinant DNA prep- aration This has been confirmed most clearly for rabbit globin cDNA clones p/3G189 (/3-globin) and pHB729° (a-globin) p/3G1 contains the en- tire coding region, and its nucleotide sequence agrees with partial mRNA sequence data and the primary structure of the protein, pHB72 similarly agrees with mRNA and protein data and represents 361 of the 423 base pairs of the a-globin coding region The nucleotide sequence of an oval-

H A Erlich, S N Cohen, and H O McDevitt, Cell 13, 681 (1978)

s5 S Broome and W A Gilbert, Proc Natl Acad Sci U.S.A 75, 2746 (1978)

A Skalka and L Shapiro, Gene 1, 65 (1976)

a7 B M Paterson, B E Roberts, and E L Kuff, Proc Natl Acad Sci U.S.A 74, 4370

(1977)

8s M E Sobel, T Yamamoto, S L Adams, R DiLauro, V E Avvedimento, B deCrom-

brugghe, and I Pastan, Proc Natl Acad Sci U.S.A 75, 5846 (1978)

s9 A Efstratiadis, F C Kafatos, and T Maniatis, Cell 10, 571 (1977)

go H C Heindell, A Liu, G V Paddock, G M Studnicka, and W A Salser, Cell 15, 43

(1978)

[1] RECOMBINANT DNA TECHNIQUES 21 bumin cDNA plasmid also indicates faithful cloning of the mRNA's se- quence, a~

The faithfully replicated sequences above are relatively short and free

of internal repetition In contrast, long DNA segments with tandem se- quence repetition have occasionally been partly deleted from recom- binant DNA cloned in E coli The rRNA gene unit o f X e n o p u s contains,

in its nontranscribed spacer, up to 5000 base pairs of tandem repetition of

a short sequence, each repeat of which is probably less than 50 base pairs.92 Nevertheless, this DNA has been cloned with a high degree of sta- bility of the X e n o p u s DNA sequence, a9 After hundreds of generations, 98% of the molecules had not lost or gained repeated sequence elements Furthermore, they were stable in r e c A - or rec + E coli 92 Five copies of the X e n o p u s 5 S rRNA genes, containing over 100 repeats of a 15- nucleotide sequence, were also cloned stably, an

On the other hand, deletions have occurred in clones of other re- peating sequences Plasmids containing Drosophila satellite DNAs with tandem repeats o f 5- or 7- base-pair sequences lost part of the inserted DNA unless the insert size was 1 kb or less ~ Since this occurred even in

r e c A - bacteria, unequal intramolecular recombination of replicating DNA molecules was proposed as a mechanism In the case of silk fibroin gene plasmids, 90% of subclones of a plasmid with 1.3 kb composed of 18- nucleotide tandem repeats were unaltered, a5 On the other hand, 15 kilo- base pairs of these tandem repeats were cloned in a pMB9 recombinant plasmid, but progressive loss of the fibroin repeated sequences occurred, down to 4 - 6 kb, at which point they were stable, an,aT Deletions also oc- curred at a low frequency in plasmids containing a tandem duplication of length 4.7 or 8.6 kb including yeast rRNA genes 98 This was found in both

rec ÷ and r e c A - bacteria Repeated DNA sequences were lost from a h phage recombinant carrying three copies of a 2.8-kb fragment of adenovirus-2 DNA One copy of the inserted fragment remained, a9

91 L McReynolds, B W O'Malley, A D Nisbet, J E Fothergill, D Givol, S Fields, M

Robertson, and G G Brownlee, Nature (London) 273, 723 (1978)

P K Wellauer, I B Dawid, D D Brown, and R H Reeder, J Mol Biol 105, 461

(1976)

aa D Carroll and D D Brown, Cell 7, 477 (1976)

D Brutlag, K Fry, T Nelson, and P Hung, Cell 10, 509 (1977)

9~ j F Morrow, N T Chang, J M Wozney, A C Richards, and A Efstratiadis, in

"Molecular Cloning of Recombinant D N A " (W A Scott and R Werner, eds.), p 161 Academic Press, New York, 1977

as Y Ohshima and Y Suzuki, Proc Natl Acad Sci U.S.A 74, 5363 (1977)

gr T Mukai and J F Morrow, in preparation

as A Cohen, D Ram, H O Halvorson, and P C Wensink, Gene 3, 135 (1978)

M Perricaudet, A Fritsch, U Pettersson, L Philipson, and P Tiollais, Science 196, 208

(1977)

m u t a g e n i z e d D N A can be inserted into cells, replicated, and cloned, so that a h o m o g e n e o u s p r e p a r a t i o n c a n s u b s e q u e n t l y be a n a l y z e d R e c o m - binant D N A m a k e s this possible not only for viruses and naturally occur- ring plasmids, but for a n y D N A segment O n e can create D N A s e q u e n c e deletions at restriction e n d o n u c l e a s e sites, b e t w e e n t w o such sites, or

r a n d o m l y A restriction e n d o n u c l e a s e site, within a synthetic D N A linker, can also be inserted either at a c l e a v a g e site for a n o t h e r restriction en-

z y m e or at r a n d o m Point mutations can also be induced efficiently at se- lected sites

I n v i t r o D N A alteration has b e e n u s e d to m a p genes o f SV401°°-1°4 and

to m a p functions o f p!asmid D N A s , including replication 1°5'1°~ R e l a t e d

r e c o m b i n a n t D N A m e t h o d s h a v e b e e n used to d e m o n s t r a t e that the inter- vening s e q u e n c e s within the/3-globin gene are not essential for its trans- cription and translation: 1°7

T w o w a y s o f creating a small deletion at D N A termini p r o d u c e d by restriction e n z y m e c l e a v a g e h a v e b e e n described In the first, one digests with an e x o n u c l e a s e until a b o u t 30 nucleotides h a v e b e e n r e m o v e d f r o m

e a c h D N A strand (h 5 ' - e x o n u c l e a s e has b e e n used) Cells are then in- fected with the linear D N A molecules At a low efficiency, the D N A ends are joined in v i v o , creating deletion m u t a t i o n s lacking 1 5 - 50 b a s e pairs, lol The joining p r e s u m a b l y d e p e n d s on partial h o m o l o g y b e t w e e n different

s e q u e n c e s in the single-stranded ends H p a l I and E c o R I sites h a v e b e e n

10o C Lai and D Nathans, J Mol Biol 89, 179 (1974)

~ol j Carbon, T E Shenk, and P Berg, Proc Natl Acad Sci U.S.A 72, 1392 (1975) loz T E Shenk, J Carbon, and P Berg, J Virol 18, 664 (1976)

~ C Lai and D Nathans, Virology 75, 335 (1976)

~o4 D Shortle and D Nathans, Proc Natl Acad Sci U.S.A 75, 2170 (1978)

~os F Bolivar, M C Betlach, H L Heyneker, J Shine, R L Rodriguez, and H W Boyer,

Proc Natl Acad Sci U.S.A 74, 5265 (1977)

~oe F Heffron, M So, and B J McCarthy, Proc Natl Acad Sci U.S.A 75, 6012 (1978)

~or R C Mulligan, B H Howard, and P Berg, Nature (London) 277, 108 (1979)

[1] RECOMBINANT DNA TECHNIQUES 23 deleted in this way 10s,109 The partial exonuclease digestion is probably not necessary for creating a deletion upon infection of mammalian cells, even

if the restriction endonuclease employed generates cohesive ter- mini 100,102,103

The second method requires a restriction enzyme cleavage that makes single-stranded termini It involves removal of those with S1 single- strand-specific nuclease followed by blunt-end joining by T4 DNA ligase

XmaI, HaelI, and EcoRI sites have been deleted by this method 3r

A fragment of DNA between two restriction cleavage sites can be de-

leted SV40 DNA was partially digested by either HindlII or HindlI, each

of which can cleave it at several sites DNA molecules shorter than the genome's length were then used to infect cells Some of the mutants re-

covered were joined in vivo cleanly at a restriction site Others acquired

deletions extending a few hundred base pairs beyond the relevant restric- tion site 1°°,1°a Alternatively, the partially digested DNA fragments can be

joined in vitro before transformation of cells, as was done with EcoRI*-

cleaved DNA 33 Deletions can be made by joining dissimilar restriction

termini in vitro if both ends are made blunt before incubation with T4

ligase TM

Deletions can also be made at random locations by cleaving both strands of DNA with DNase I in the presence of Mn 2÷ Cells are then in-

fected with the linear molecules, and the progeny resulting from in vivo

DNA joining are examined.I°2

Insertion mutations can be equally useful, especially if the inserted DNA contains a restriction endonuclease site that permits easy detection and mapping Inserts consisting of large pieces of naturally occurring DNA may contain unidentified control sequences which produce artifacts

in subsequent experiments Short synthetic linkers containing restriction

sites are better-defined inserts An EcoRI linker has been used to alter

the translation reading frame in cloning vehicles 09 Clearly, a linker can eliminate a restriction site, even one that leaves single-stranded termini, if

the termini are madg blunt before ligation to the linker ~3 EcoRI linkers

have also been inserted at random sites of cleavage by DNase 1.1°6 Three methods for induction of point mutations at selected sites have been described One involves conversion of cytosine residues to uracil at

a localized single-stranded gap, produced at the site of a nick made by a restriction endonuclease 1°4 Another utilizes nick translation in the pres- ence of N4-hydroxy dCTP This induces T-to-C transition mutations 11° A 1~ C Covey, D Richardson, and J Carbon, Mol Gen Gent 145, 155 (1976)

~oa B Polisky, R J Bishop, and D H Gelfand, Proc Natl Acad Sci U.S.A 73, 3900 (1976)

11o W MOiler, H Weber, F Meyer, and C Weissmann, J Mol Biol 124, 343 (1978)

24 INTRODUCTION [1] more difficult, but very specific, mutagenesis method utilizes a synthetic oligodeoxynucleotide, 12 residues or longer, differing from the "wild- type" DNA sequence This is annealed to a single-stranded circular DNA molecule and extended with a DNA polymerase As many as one-third of the progeny of infection of cells with these molecules have the desired se- quence change 1H,11z

These methods of in vitro DNA alteration are more complex than the time-honored investigation of mutations that occur in vivo They repre-

sent a new way of doing genetic research They have the advantage that a large proportion of the progeny carry the desired mutation (sometimes all do) Also, some of the DNA sequence changes can be very easily and pre- cisely mapped, e.g., an inserted restriction site linker Site-directed mu-

tagenesis is complementary to study o f in vivo mutations, in a sense The

former focuses on a portion of a DNA molecule and can show what physi- ological processes are affected by a change in it The latter begins with a phenotype resulting from altered physiological processes and can dis- cover what portions of the DNA are most closely related to that pheno- type

Conclusion

Recombinant DNA methods have become a mainstay of molecular genetics They are also contributing to the solution of practical problems, for example, supplying hormones The techniques described in this vol- ume will surely play an important part in advancing our knowledge and command of heredity

[ 2 ] DIRECTORY OF RESTRICTION ENDONUCLEASES 27

[2] D i r e c t o r y o f R e s t r i c t i o n E n d o n u c l e a s e s

By RICHARD J ROBERTS Table I is intended to serve as a directory to the restriction endonu- cleases that have now been characterized In forming the list, all endonu- cleases that cleave DNA at a specific sequence have been considered restriction enzymes, although in most cases there is no direct genetic evi- dence for the presence of a host-controlled restriction-modification system

Certain strains have been omitted from this list to save space Thus the many different Staphylococcus aureus isolates containing an iso- schizomer of S a u 3 A 1 are not listed individually Similarly the numerous strains of gliding bacteria (orders Myxobacterales and Cytophagales) that showed evidence of specific endonucleases during a large-scale screening 2 are still rather poorly characterized

Within Table I the source of each microorganism is given either as an individual or a national culture collection The enzymes are named in accordance with the proposal of Smith and Nathans 3 When two en- zymes recognize the same sequence (i.e., are isoschizomers), the proto- type (i.e., the first example isolated) is indicated in parentheses in column

3 The recognition sequences (column 4) are abbreviated so that only one strand, reading 5' -~ 3', is indicated and the point of cleavage, when known, is indicated by an arrow ( $ ) When two bases appear in parenthe- ses, either one may appear at that position within the recognition se- quence Where known, the base modified by the corresponding methylase

is indicated by an asterisk ,~ is Ne-methyladenosine; ~ is 5- methylcytosine The frequency of cleavage (columns 5-8) has been experimentally determined for bacteriophage lambda (h) and ade- novirus-2 (Ad2) DNAs, but represents the computer-derived values from the published sequences of SV404 and ~bX1745 DNAs When more than one reference appears (column 9), the first contains the purification procedure for the restriction enzyme, the second concerns its recognition sequence, the third contains the purification procedure for the methylase,

I E E Stobberingh, R Schiphof, and J S Sussenbach, J Bacteriol 131, 645 (1977)

2 H Mayer and H Reichenbach, J Bacteriol 136, 708 (1978)

3 H O Smith and D Nathans, J Mol Biol 81, 419 (1973)

4 V B Reddy, B Thimmappaya, R Dhar, K N Subramanian, B S Zain, J Pan, P K Ghosh, M L Celma, and S M Weissman, Science 200, 494 (1978)

5 F Sanger, G M Air, B G Barrell, N L Brown, A R Coulson, J C Fiddes, C A Hutchison, III, P M Slocombe, and M Smith, Nature (London) 265, 687 (1977)

Copyright (~) 1979 by Academic Press, Inc METHODS IN ENZYMOLOGY, VOL 68 All rights of reproduction in any form reserved

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