Recombinant DNA Part A

543 Pages·1980·13.678 MB·English

by  Kaplan N.P. (ed.), Colowick N.P. (ed.), Wu R. (ed.)

Preface
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.
YAR Wu
iiix
Contributors to Volume 68
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
DAVID ANDERSON (30), Genex Corporathm, P. CHIU (29), Section of BiochemistiT,
Rockville, Maryland 25802 Molecular and Cell Biology, Cornell Uni-
JAMES C. ALWINE (15), Laboratory of versity, Ithaca. New York 35841
Molecular Virology, National Cancer In-
stitute, National Institutes gf Health, LOUISE CLARKE (27, 31), Department 'f~t
Bethesda. Maryland 41002 Biological Sciences, University of Cali-
fi)rnia, Santa Barbara, Santa Barbara,
S. L. AUCKERMAN (38), Department of
ainr)ifilaC 60139
Biology, ehT Johns Hopkins University,
Baltimore, MaiTland 81212 STANLEY N. COHEN (32), Departments of
Genetics and Medicine, StanJbrd Uni-
KEITH BACKMAN (16), Department of
versity School of Medicine, Stanford,
Biology, Massachusetts Institute of Tech-
ainr)~(laC 50349
nology. Cambridge, Massachusetts 93120
C. P. BAHL (7), Cetus Corporation, JO~N COLLINS (2), Gesellscht(fi flit Bio-
technologische Forschung mbH. Masch-
Berkeley, Califi)rnia 01749
eroder Weg I. D-3300 Brutmschweig-
RAMAMOORTHY BELAGAJE (8), Lilly Re-
St6ckheim, West Germany
search Laboratories, Indianapolis, In-
NICHOLAS R. COZZARELL1 (4), Departments
diana 60264
of Biochemistry and Biophysics and
HANS-ULRICH BERNARD (35), Department
Theoretical Biology, University of
o['Biology, University ~f California, San
Chicago, Chicago, Illinois 73606
Diego, La Jolla, ainroJilaC 39029
J. L. CULLETON (38), Department of Biof
DALE BLANK (33), Rosenstiel Basic Medical ogy, ehT Johns Hopkins University,
Sciences Research Center and Depart- Baltimore. Marylund 81212
ment of Biology, Brandeis University,
R. P. DOTTIN (38), Department of Biology,
Waltham, Massachusetts 45120
Johns The Hopkins University, Baltimore,
FRANCISCO BOLIVAR (16), Deparamento Maryland 81212
Biologia Molecular, lnstituto de -nI
L. ENQU~ST (18), Laboratory of Molecular
vestigaciones Biomedicas, Universidad
Virology, National Cancer Institute,
Nacional Aatonoma ed Mexico, Mexico
National Institntes qf Health, Bethesda,
,02 D.F., Mexico Apdo Postal 82207
Marykmd 41002
ROLAND BROUSSEAU (6), Division of'Biolog-
HENRY A. ERLI¢~ (32), Department of
ical Sciences, National Research Council
Medicine, Stanford ytis=levinU School of
of Canada, Ottawa KIA OR6, Canada
Medicine, ,dr~ifnatS California 50349
EUGENE L. BROWN (8), Synthex Research, KAREN FAHRNER (33), Rosenstiel Basic
Palo Alto, ainr~ifilaC 40349 Medical Sciences Research Center and
DOUGLAS BRUTLAG (3), Department "[o Department "[~ Biology, Brandeis' Uni-
BiochemisttT, Stanford University School t'ersity, Waltham, Massachusetts 45120
qf Medicine, Stanford, Cal~)rnia 50349 G. C. FAREED (24), Department of Micro-
JOHN CARBON (27, 31), Department of Bio- biology and hnmunology, Molecular Biol-
logical Sciences, University of ,ainr)ifilaC ogy Institute, University of Cahi~rnia,
Santa Barbara, Santa Barbara, California Los Angeles, Los Angeles, California
60139 42009
ix
X CONTRIBUTORS TO VOLUME 68
DAVID FIGURSKI (17), Department of Micro- HANSEN M. HSIUNG (6), Division of -igoloiB
biology, College f2~ Physicians and cal Sciences, National Research Council
Surgeons, Columbia University, New of Canada, Ottawa KIA OR6, Canada
York, New York 23001
KIMBERLY A. JACKSON (28), Department of
S. G. FISCHER (11), Department of Biologi- Biochemistry, University of Connecticut
cal Sciences, State University of New Health Center, Farmington, Connecticut
kroY at Albany, Albany, New York 22221 23060
B. R. FISHEL (38), Department of Biology, MICHAEL KAHN (17), Department of Bac-
ehT Johns Hopkins University, Balti- teriology and Public Health, Washing-
more, Marvhmd 81212 not State University, Pullman, Washing-
not 46199
MICHAEL L. GOLDBERG (14), Abteihmg
Zelliologie, Biozentrium Der Universitiit KATHLEEN M. KEGGINS (23), Department
Basel, CH-4056 Basel, Switzerhmd "[q Biological Sciences, University of
Muo'lund, Baltimore County, Catons-
HOWARD M. GOODMAN (5), Howard
Hughes Medical Institute Laboratory and ville, Mao, land 82212
the Department of Biochemistry and Bio- DAVID J. KEMP (15), 1he Walker and Eliza
physics, University of Cahlfbrnia, San Hall Institute of Medical Research, Post
Fruncisco, Cal(fbrnia, 34149 OJfice, Royal Melbowne Hospital, Vic-
toria 3050, Australia
MICHAEL GRUNSTEIN (25), Department qf
Biology, University of California, Los H. GOBIND KHORANA (8), Departments "jo
Angeles, Los Angeles, Cali[brnia 42009 Biology and Chemisto', Massachusetts
etutits~h of Technology, Cambridge,
DONALD R. HELINSKI (17, 35), Department
Massachusetts 93120
of Biology, University of Cahfornia, San
Diego, La Jolla, California 39029 ROBERVO KOLTER (17), Department of
Biology, University "fo California, Sun
LYNNA HEREFORD (33), Rosenstiel 'cisaB
Diego, La Jolla, ainr)if(laC 39029
Medical Sciences Research Center and
Department qf Biology, Brandeis Uni- L. F. LAU (7), Section qf Biochemistry,
versity, Waltham, Massachusetts 45120 Molecular and Cell Biology, Cornell
N. PATRICK HIGGINS (4), Department "[q Unil,ersity, Ithaca, New York 35841
Biochemisto', University qf Wyoming, GAIL D. LAUER (34), ehT Biological Lab-
Laramie, Wyoming oratories, Harvard University, Cam-
RONALD H1TZEMAN (31), Department of bridge, Massachusetts 83120
Biological Sciences, University of Cali- LEONARD S. LERMAN (11), Department of
fornia, Santa Barbara, Santa Barbara, Biological Sciences, State University of
California 60139 New York at Albany, Albany, New York
BARBARA HOHN (19), Friedrich Miescher 22221
Institut, Basel, CH-4002 Switzerland RICHARD P. LIFTON (14), Department [2(
JANICE P. HOLLAND (28), Department of Biochemistry, StanJbrd University School
Biochemistry, University of Connecticut q( Medicine, StanJbrd, California 50349
Health Center, Farmington, Connecticut JOHN LIS (10), Section of Biochemistpy,
23060 Molecular and Cell Biology, Cornell
MICHAEL J. HOLLAND (28), Department of University, Ithaca, New York 35841
Biochemistty, University of Connecticut SHIRLEY LONGACRE (12), Parasitologie -xE
Health Center, Farmington, Connecticut perimentale, Institut Pasteur, 75724
23060 Paris, Cedex ,51 France
CONTRIBUTORS TO VOLUME 68 xi
PAUL S. LOVETT (23), Department of Bio- Los Angeles, Los Angeles, ainrb[(laC
lacig~k Sciences, University of Maryland, 42009
Baltimore County, Catonsville, Maryland
JOHN REEVE (36), Department of Micro-
82212
State Ohio Columbus, biology, University,
HUGH O. McDEVITT (32), Departments of 0123Ohio 4
Medicine and Medical Microbiology,
JAKOB REISER (15), Department of Bio-
Stanford University School f~c Medicine,
chemistry, Stanford University School of
Star,ford, California 50349
Medicine, Stanford, California 50349
RAYMOND J. MACDONALD (5), Howard
ERIC REMAUT (17), Laboratorium voor
Hughes Medical Institute Laboratory and
Moleculaire Biologie, Bijksuniversiteit
the Department of Biochemistry and
Gent, Gent, B-9000 Belgium
Biophysics, University ~f California,
San Francisco, San Francisco, Cahfornia JAIME RENART (15), lnstituto ed Enzi-
34149 mologia del C.S.1.C., Facultad ed Medi-
anic de al Universidad Autonoma,
BERNARD MACH (12), Department of -orciM
Arzobispo Morcillo s/n. Madrid-34,
biology, University of Geneva, CH 5021
niapS
Geneva, Switzerland
ROBERT RICClARDI (33), Department of -oiB
R. E. MANROW (38), Department of Biology,
logical Chemistry, Harvard Medical
ehT Johns Hopkins Baltimore, University,
School, Boston, Massachusetts 51120
Maryland 81212
RICrtARD J. ROBERTS (2), Cold Spring
RICHARD MEYER (17), Department of -iM Harbor Laboratory, Cold Spring Harbor,
crobiology, University of Texas, Austin, New York 42711
Texas 21787
BRYAN ROBERTS (33), Rosentiel Basic
D. A. MORRISON (21), Department of -oiB Medical Sciences Research Center and
logical Sciences, University of Illinois, Department ~f Biology, Brandeis Uni-
Chicago Circle, Chicago, Illinois 08606 versity, Waltham, Massachusetts 45120
JOHN f. MORROW (|), Department of -orciM THOMAS M. ROBERTS (34), Department of
biology, ehT Johns Hopkins School "[o Biochemistry and Molecular Biology,
Medicine, Baltimore, land Mao, 50212 Harvard University, Cumbridge, Mas-
sachusetts 83120
S. A. NARANG (6, 7), Division of Biolog&al
Sciences, National Research Council .fo MICHAEL ROSBASH (33), Rosenstiel "cisaB
Canada, Ottawa KIA OR6, Canada Medical Sciences Research Center and
Department of Biology, Brandeis Uni-
TIMOTHY NELSON (3), Department of Bio- versity, Waltham, Massachusetts 45120
chemistry, Stanfi)rd University School of
R. J. ROTHSTEIN (7), Department of -orciM
Medicine, Stanford, California 50349
biology, New Jersey School of Medicine,
BARBARA A. PARKER (15), Department of Newark. New Jersey 30170
Biochemistry, Stanford University School
STEPHANIE RUBY (33), Department of Bio-
of Medicine, Stanford, California 50349
logical Chemistry, Harvard Medical
BARRY POLlSKY (37), Department of Biol- School. Boston, Massachusetts 51120
ogy, Indiana University, Bloomington, MICHAEL J. RYAN (8), Microbiological
Indiana 10474 Sciences, Schering Corporation.
A. F. PURCHIO (24), Department of Micro- Bloomfield, New Jersey 30070
biology and Immunology, Molecular A. SEN (13), Meloy Laboratories Inc.,
Biology Institute, University of ,ainrbJilaC Springfield, Virginia 15122
xii CONTRIBUTORS TO VOLUME 68
LUCILLE SHAPIRO (30), Department t~f'Mo- J. W. SZOSTAK (29), Sidney Faber Cancer
lecular Biology, Albert Einstein College Institute, Boston, Massachusetts 02115
of Medicine, Bronx, New York
REHPOTSIRHC THOMAS (17), Department of
H. MlCrtAEL SHEPARD (37), Department "[~ Biology, University "f)~ Cul([brnia, San
Biology, bldiana University, Blooming- Diego, La Jolla, Cal(fornia 92093
ton, hldiana 47401
B.-K. TVE (29), Section of Biochemisto',
F. SHERMAN (29), Department qfRadiation Molecuhtr and Cell Biology, Cornell
Biology and Biophysics, University of Universio,. Ithaca, New "~,roY 14853
Rochester, School 'Jo Medicine, Roches-
ter, New York 14642 GEOFFRE'¢ M. WAHL (15), tnemtrapeD qf
Biochemisto', Stanford University School
M. SHOVAI3 (13), Laboratory of Viral Car- qf Medicine, Stanford, CaliJbrnia 94305
cinogenesis, National Cancer Institute,
JOHN WALLIS (25), Department of Micro-
National Institutes 1)~ Health, Bethesda,
biology and Immunology, Molecular Biol-
Marylund 20014
ogy Institute. University "1~ CaliJornia,
A. M. SKALKA (30), Department f~¢ Cell Los An,~,eles, Los Angeles, California
Biology, Roche Institute "[~ Malecular
42OO9
Biology, Nutley, New Jersey 071IO
JEEFREY G. WIkLIAMS (14), Imperial Cancer
EDWIN SOUTHERN (9), M.R.C. Mammalian
Research Fired, Mill Hill, London NWT,
Genome Unit, King's Building, Edin-
England
burgh EH9 3JT, Scotland
GEORGE R. STARK (14, 15), Department "f~ SAVIO L. C. Woo (26), Howard Hughes
Biochemistry, Stanford University School Medical Institute Laboratory and De-
"]~ Medicine, Stat~jbrd, Cal(fornia 94305 partrnent of'Cell Biology, Baylor College
0~ r Medicine, Texas Medical Center,
N. STERNBERG (|8), Cancer Biology Pro-
Houston. Texas 77030
gram, Frederick Cancer Research Cen-
ter, Frederick, Maryland 21701 JOHN WOOLFORD (33), Rosenstiel Basic
Medical Sciences Research Center and
J. I. STILES (29), Department of Radiation
Department ~( Biology, Brandeis Univer-
Biology and Biophysics, University of
sit),, Waltham. Massachusetts 02154
Rochester, School ~f Medicine,
Rochester, New Yark 14642 RAy Wu (7, 10, 29), Section of Biochemistry,
M. SUZUKI (22), Boyce Thompson Institute, Maleeular and Cell Biology, Cornell Uni-
Cornell University. Ithaca. New York versity, Ithaca, New York 14853
35841
TREBOR C.-A. YANG (10), Section of Bio-
A. A. SZALAY (22), Boyee Thompson Insti- chemisttT, Molecular and Cell Biology,
tute, Cornell University, Ithaca, New Cornell University, Ithaca, New York
York 14853 35841
[1] RECOMBINANT DNA TECHNIQUES 3
[1] Recombinant DNA Techniques
By JOHN F. MORROW
The recombinant DNA method consists of joining DNA molecules in
vitro and introducing them into living cells where they replicate. Research
using this method is relatively new and fast-moving. In only 6 years of re-
combinant DNA research, a number of significant accomplishments have
been made. Two mammalian hormones have been produced in bacteria
by means of synthetic DNA. m Polypeptides similar or identical to several
found in eukaryotes have been synthesized in Escherichia coli. 3-~° These
achievements promise a new, inexpensive means of large-scale produc-
tion of selected peptides or proteins. Furthermore, using recombinant
DNA, somatic recombination of immunoglobulin genes has been estab-
lished, 11 and a large number of variable-region genes have been found.
MT
Intervening sequences (introns) have been found in the DNA of eu-
karyotic cells. 1a-t6
I would like to mention the origins of this versatile new technology be-
fore describing recent advances. The isolation of mutant E. coli strains
unable to restrict foreign DNA (cleave it specifically and degrade it) laid
i K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F, Bolivar, and H. W.
Boyer, Science 198, 1056 .)7791(
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,
601 .)9791(
K. Struhl, J. R. Cameron, and .R W. Davis, Proc. Natl. Acad. Sci. U.S.A. 73, 1741
.)6791(
4 B. Ratzkin and J. Carbon, Proc. Natl. Acad. Sci. U.S.A. 74, 784 .)7791(
D. Vapnek, J. A. Hautala, J. W. Jacobson, N. H. Giles, and S. R. Kushner, Proc. Natl.
Acad. Sci. U.S.A. 74, 3508 .)7791(
e R. C. Dickson and J. S. Markin, Cell ,51 321 .)8791(
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 .)8791(
s A. C. Y. Chang, J. H. Nunberg, R. J. Kaufman, H. A. Erlich, R. T. Schimke, and S. N.
Cohen, Nature (London) 275, 716 .)8791(
90. Mercereau-Puijalon, A. Royal, B. Carol, A. Garapin, A. Krust, F. Gannon, and P.
Kourilsky, Nature (London) 275, 505 .)8791(
o1 T. H. Fraser and B. J. Bruce, Proc. Natl. Acad. Sci. U.S.A. 75, 5936 .)8791(
11 C. Brack, M. Hirama, R. Lenhard-Schuller, and S. Tonegawa, Cell ,51 1 .)8791(
n j. G. Seidman, A. Leder, M. Nau, B. Norman, and P. Leder, Science 202, 11 .)8791(
31 D. M. Glover and D. S. Hogness, Cell 10, 761 .)7791(
4x R. L. White and D. S. Hogness, Cell ,01 771 .)7791(
51 p. K. Wellauer and I. B. Dawid, Cell 10, 391 .)7791(
,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, 527 .)8791(
Copyright © 1979 by Academic Press, Inc.
METHODS IN ENZYMOJ.OGY, VOL. 68 All rights of reproduction in any form reserved.
ISBN -O X-869181-21
4 NOITCUDORTNI [1]
part of the foundation. 71 The discovery of site-specific restriction endonu-
cleases lsa9 also contributed (see Nathans and Smith, °~ Roberts, 12 and this
volume [2], for review). Two general methods for joining DNA molecules
from different sources were found. 42-z2 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 DNA strands,
the EcoRI restriction endonuclease. 25-2r It was used in the first in vitro
construction of recombinant molecules that subsequently replicated in
vivo. s2
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 endonu-
clease recognition sites, or other DNA segments, at random or predeter-
mined locations. One can also delete restriction sites, or DNA segments
between such sites, by techniques that permit joining any two DNA ter-
mini after their appropriate modification. Such an alteration can be helpful
in determining the functions performed by various parts of a DNA se-
quence. 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 of 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 tech-
niques. The essential ingredients of a recombinant DNA experiment are:
71 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).
o2 D. Nathans and H. O. Smith, Annu. Rev. Biochem. 44, 273 (1975).
12 R. J. Roberts, Gene 4, 183 (1978).
22 p. E. Lobban and A. D. Kaiser, J. Mol. Biol. 78, 453 (1973).
32 D. A. Jackson, R. H. Symons, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 69, 2904
(1972).
42 V. Sgaramella, J. H. van de Sande, and H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A.
67, 1468 (1970).
52 j. E. Mertz and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 69, 3370 (1972).
e2 j. Hedgpeth, H. M. Goodman, and H. W. Boyer, Proc. Natl. Acad. Sci. U.S.A. 69, 3448
(1972).
7z V. Sgaramella, Proc. Natl. Acad. Sci. U.S.A. 69, 3389 (1972).
s2 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 DNA 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 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
form.
Since a thorough review of recombinant DNA was completed in
,6791 92 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
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 spectino-
mycin. 0~
pBR322 is now the most widelyu sed 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 detailedr estriction enzyme
cleavage map and DNA sequence information are also important. 2a13 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
92 R. L. Sinsheimer, Annu. Rev. Biochem. 46, 514 (1977).
o3 A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, (1978). 1141
ta j. G. Sutcliffe, Proc. Natl. Acad. Sci. U.S.A. 75, 3737 (1978).
23 j. G. Sutcliffe, Nucleic Acids Res. 5, 2721 (1978).
6 INTRODUCTION [ 1]
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[1] RECOMBINANT DNA TECHNIQUES 7
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