recombinant dna part c

Preface Exciting new developments in recombinant DNA research allow the isolation and amplification of specific genes or DNA segments from al- most any living organism. These new developments have revolutionized our approaches to solving complex biological problems and have opened up new possibilities for producing new and better products in the areas of health, agriculture, and industry. Volumes 100 and 101 supplement Volumes 65 and 68 of Methods in Enzymology. During the last three years, many new or improved methods on recombinant DNA or nucleic acids have appeared, and they are in- cluded in these two volumes. Volume 100 covers the use of enzymes in recombinant DNA research, enzymes affecting the gross morphology of DNA, proteins with specialized functions acting at specific loci, new methods for DNA isolation, hybridization, and cloning, analytical methods for gene products, and mutagenesis: in vitro and in vivo. Volume 101 includes sections on new vectors for cloning genes, cloning of genes into yeast cells, and systems for monitoring cloned gene expression. RAY Wu LAWRENCE GROSSMAN KIVIE MOLDAVE xiii REIJI OKAZAKI 1930-1975 Reiji Okazaki (1930-1975) Reiji Okazaki has been memorialized by the nascent DNA replication fragments that bear his name. His discovery of the Okazaki fragments in the discontinuous synthesis of DNA at the replication fork helped solve a perplexing problem: how DNA polymerases with an invariant unidirec- tional mode of synthesis can copy the oppositely oriented strands of the duplex chromosome. Those of us who knew him do not require the adjec- tival use of his name to keep his memory alive. We retain the image of a scientist utterly dedicated to understanding the molecular basis of bi- ology. Reiji Okazaki was born in Hiroshima in 1930 and received his Ph.D. training in developmental biology under Tsuneo Yamada at Nagoya University.' In seeking systems simpler than sea urchins to study cell proliferation, he chose Lactobacillus and Escherichia coli in which he discovered thymidine diphosphate rhamnose, the coenzyme of lipopoly- saccharide synthesis. With J. L. Strominger in St. Louis in 1960-1961 he worked out the enzymatic synthesis of this coenzyme. In my laboratory, the following year, he purified thymidine kinase of E. coli and demon- strated the allosteric regulation of this key salvage enzyme. On returning to Nagoya, as Professor of Molecular Biology, he initiated the series of elegant studies of phage T4 DNA replication that led to his key discovery of discontinuous replication. His bibliography of some thirty papers (1966-1977) can be consulted for the innovative approaches he introduced to solve fundamental ques- tions of DNA replication. His research style, less readily gleaned from the literature, is illustrated by two incidents which are vivid in my memory. One I call an Okazaki maneuver. In purifying thymidine kinase, he used a heating step: the enzyme was held in a test tube at 70 ° for 5 min- utes. When he decided to prepare a large amount of enzyme, going from a scale of 10 milliliters to several liters, he simply repeated the same heating procedure, this time using 236 test tubes. I was embarrassed to report such an unsophisticated procedure. But then I realized that he was able to complete this step in a few hours, and saw no point in wasting precious days and material learning how to do the heating in a big beaker or flask. Recently, one of my colleagues purified the single-strand DNA binding protein with a heating step. When he came to scale-up the procedure from i An appreciative obituary by Sakaru Suzuki appeared in Trends in Biochemical Sci- ences 1, N39 (Feb. 1976). XXV xxvi REIJI OKAZAKI 3 milliliters to 6 liters he was guided by the Okazaki maneuver; he heated 2000 test tubes each containing 3 milliliters. Others who tried heating larger volumes of enzyme lost the preparation in a thick coagulum. A second incident I call Okazaki courage. It had been customary in my laboratory when characterizing an enzyme to set up protocols containing 10 to 20 assay tubes. Rarely, some ambitious person might do a 24-tube assay. Reiji set a record that may never be broken. He performed a 128- tube assay of thymidine kinase, even though each assay included a la- borious electrophoretic separation of the product from the substrate. Be- cause the pure enzyme was rather labile he felt it essential to measure at once all the substrate, effector, inhibitor, and other parameters. The suc- cessful completion of this experiment was a feat of courage, concentra- tion, skill, and enterprise unique in my experience. Okazaki died of leukemia in 1975, a sudden and cruel loss to his wife and co-worker, Tuneko, to his devoted students, and to the worldwide scientific community. The continued productivity of his laboratory by his students under Tuneko Okazaki's direction is a tribute to its scientific prowess and to Reiji Okazaki's inspirational legacy. ARTHUR KORNBERG Department of Biochemistry Stanford University School of Medicine Stanford, California Contributors to Volume 101 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. GUSTAV AMMERER (11), Zymos Corpora- tion, Seattle, Washington 98103 CARL W. ANDERSON (41), Biology Depart- ment, Brookhaven National Laboratory, Upton, New York 11973 WAYNE M. BARNES (5), Department of Bio- logical Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 LESLIE BARNETT (1), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England KENNETH A. BARTON (33), Cetus Madison Corporation, Middleton, Wisconsin 53562 MICHAEL D. BEEN (4), Department of Mi- crobiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195 MICHAEL BEVAN (5), Plant Breeding Insti- tute, Trumpington, Cambridge CB2 2LQ, England DAVID BOTSTEIN (9), Department of Biol- ogy, Massachusetts Institute of Technol- ogy, Cambridge, Massachusetts 02139 SYDNEY BRENNER (1), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England JAMES R. BROACH (21), Department of Mi- crobiology, State University of New York, Stony Brook, New York 11794 NATHAN BROT (45), Department of BiD- chemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 PATRICIA A. BROWN (18), Rosenstiel Basic Science Research Center, Brandeis Uni- versity, Waltham, Massachusetts 02154 JOHN CARBON (20), Department of Biologi- cal Sciences, University of California, Santa Barbara, California 93106 YVES CENATIEMPO (45), Laboratoire de Biologie Moleculaire, University Lyon, 69622 Villearbanne, France M. CHAMBERLIN (34), Department of Bio- chemistry, University of California, Berkeley, California 94720 ix GLENN H. CHAMBLISS (37), Department t~f Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 JAMES J. CHAMPOUX (4), Department of Mi- crobiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195 HuI-ZHu CHEN (44), Fairchild Center jbr Biological Sciences, Columbia Univer- sity, New York, New York 10027 MARY-DELL CHILTON (33), Department (if Biology, Washington University, St. Louis. Missouri 63130 FORREST CHUMLEY (13), Department of Bi- ology, Massachusetts Institute of Tech- nology and the Whitehead Institute jbr Biomedical Research, Cambridge, Mas- sachusetts 02139 JOSEPHINE E. CLARK-CURTISS (23), Depart- ment of Microbiology, University t~f Ala- bama in Birmingham, Birmingham. Ala- bama 35294 LOUISE CLARKE (20), Department of Biolog- ical Sciences, University of California, Santa Barbara, California, 93106 LAWRENCE COHEN (43), Dana Farber Can- cer Institute, Harvard Medical School. Boston, Massachusetts 02115 GRAY F. CROUSE (3), Basic Research Pro- gram LBl, Frederick Cancer Research Facility, Frederick. Maryland 21701 RoY CURTISS III (23), Department of Micro- biology, University of Alabama in Bir- mingham, Birmingham. Alabama 35294 A. DEVERA (34), Department of Biochemis- try, University of California, Berkeley, California 94720 JOHN D. DIGNAM (36), Department of BiD- chemistry, University of Mississippi Med- ical Center, Jackson, Mississippi 39216 BERNARD S. DUDOCK (41), Department of Biochemistry, State University of New York, Stony Brook, New York ]1794 GERALD R. FINK (13), Department of Bi- ology, Massachusetts Institute t.~f Tech- X CONTRIBUTORS TO VOLUME 10l nology and the Whitehead Institute for Biomedical Research, Cambridge, Mas- sachusetts 02139 ANDREW FIRE (35), Center for Cancer Re- search, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 ITZHAK FISCHER (40), Department of Bio- logical Chemistry, College of Medicine, University of California, lrvine, Califor- nia 92717 ANNEMARIE FRISCHAUF (3), European Mo- lecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Repub- lic of Germany EUGENIUSZ GASIOR (42), Department of Molecular Biology, Institute of Micro- biology and Biochemistry, University of Marie Curie-Sklodowska, Lublin, Poland M. GILMAN (34), Department of Biochemis- try, University of California, Berkeley, California 94720 JOSEPH GLORIOSO (27), Unit for Laboratory Animal Medicine, University of Michi- gan, Ann Arbor, Michigan 48109 ALAN L. GOLDIN (27), Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 JON W. GORDON (28), Department of Ob- stetrics and Gynecology, Mount Sinai School of Medicine, New York, New York 10029 A. GRAESSMANN (30, 31), lnstitutfi~r Mole- kularbiologie und Biochemie der Freien Universitiit Berlin, D-IO00 Berlin 33, Fed- eral Republic of Germany M. GRAESSMANN (30), Institut fi~r Moleku- larbiologie und Biochemie der Freien Universitiit Berlin, D-100 Berlin 33, Fed- eral Republic of Germany LEONARD GUARENTE (10), Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 J. B. GURDON (25), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England MARK S. GUYER (24), Department of Mo- lecular Genetics, GENEX Corporation, Gaithersburg, Maryland 20877 TINA M. HENKIN (37), Department of Bac- teriology, University of Wisconsin, Madi- son, Wisconsin 53706 EDGAR C. HENSHAW (39), University of Rochester Cancer Center, Rochester, New York 14642 YEN-SEN HO (6), Department of Molecular Genetics, Smith Kline and French Labor- atories, Philadelphia, Pennsylvania 19101 PETER M. HOWLEY (26), Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 CHU-LAI HSlAO (20), Central Research and Development Department, E. I. DuPont de Nemours and Company, Experimental Station, Wilmington, Delaware 19898 JUNGI HUANG (29), Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Peoples Republic of China JONATHAN KARN (1), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England R. KINGSTON (34), Center for Cancer Re- search, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 MING-FAN LAW (26), Laboratory of Pathol- ogy, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 HANS LEHRACH (3), European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Republic of Germany JUDITH M. LEVENTHAL (37), Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 MYRON LEVlNE (27), Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109 GULLING LIU (29), Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences, Nanfing, Peoples Republic of China A. LOYTER (31), Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel VIVIAN L. MACKAY (22), Waksman Insti- tute of Microbiology, Rutgers University, The State University of New Jersey, New Brunswick, New Jersey 08903, and Zymos Corporation, Seattle, Washington 98103 JAMES L. MANLEY (35), Department of Biol- CONTRIBUTORS TO VOLUME 101 xi ogy, Columbia University, New York, New York 10027 PAUL L. MARTIN (36), Laboratory of Bio- chemistry and Molecular Genetics, The Rockefeller University, New York, New York 10021 WILLIAM C. MERRICK (38), Department of Biochemistry, Case Western Reserve Uni- versity, Cleveland, Ohio 44106 JOACHIM MESSING (2), Department of Bio- chemistry, University of Minnesota, St. Paul, Minnesota 55108 LUls MEZA-BASSO (45), Universidad Aus- tral de Chile, Casilla 567, Valdivia, Chile JACQUELINE S. MILLER (43), Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 KIVIE MOLDAVE (40, 42), Department of Biological Chemistry, College of Medi- cine, University of California, lrvine, Cal- ifornia 92717 ANDREW W. MURRAY (16), Dana Farber Cancer Institute and The Committee on Cell and Developmental Biology, Har- vard Medical School, Boston, Massachu- setts 02115 BRIAN P. NICHOLS (8), Department of Bio- logical Sciences, University of Illinois at Chicago, Chicago, Illinois 60607 TERRY L. ORR-WEAVER (14), Dana Farber Cancer Institute and Department of BiD- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115 RICHARD PANNIERS (39), University of Rochester Cancer Center, Rochester, New York 14642 DEMETRIOS PAPAHADJOPOULOS (32), Can- cer Research Institute and Department of Pharmacology, University of California, San Francisco, California 94143 BRUCE M. PATERSON (43), Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 SIYING QIAN (29), Institute of Economic Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Peoples Republic of China A. RAZIN (31), Department of Cellular BiD- chemistry, Hebrew University-Hadassah Medical School, 91000 Jerusalem, Israel ROBERT P. RICCIARDI (43), The Wistar lnsti- tute of Anatomy and Biology, Philadel- phia, Pennsylvania 19104 NIKOLAOS ROBAKIS (45), Department of Mi- crobiology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 BRYAN E. ROBERTS (43), Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115 ROBERT G. ROEDER (36), Laboratory of Bio- chemistry and Molecular Genetics, The Rockefeller University, New York, New York 10021 MARK ROSE (9), Department of Biology, Massachusetts Institute of Technology and Whitehead Institute fi)r Biomedical Research, Cambridge, Massachusetts 02139 MARTIN ROSENBERG (6), Department of Molecular Genetics, Smith Kline and French Laboratories, Philadelphia, Penn- sylvania 19101 RODNEV J. RO'rHSTEIN (12, 14), Department of Microbiology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 STEPHANIE W. RUBY (16), Dana Farber Cancer Institute and Department of Bio- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115 FRANK H. RUDDLE (28), Department of Bi- ology and Human Genetics, Yale Univer- sit)', New Haven, Connecticut 06511 MARK SAMUELS (35), Center for Cancer Re- search, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts 02139 ROZANNE M. SANDRI-GOLDIN (27), Depart- ment of Human Genetics, University ~[" Michigan Medical School, Ann Arbor, Michigan 48109 NAVA SARVER (26), Laboratory of Pathol- ogy, National Cancer Institute, National Institutes of Health. Bethesda, Maryland 20205 PHILLIP A. SHARP (35), Center fi)r Cancer Research, Massachusetts Institute t~[" Technology, Cambridge, Massachusetts 02139 BARKUR S. SHASTRY (36), Laboratory ¢~f Biochemistry and Molecular Genetics, The RockeJeller University, New York, New York 10021 ALLAN SHATZMAN (6), Department ~f Mo- xii CONTRIBUTORS TO VOLUME 101 lecular Genetics, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101 PAULA H. SON (5), Department of Biologi- cal Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110 JOHN I. STILES (19), Department of Botany, Hawaii Institute of Tropical Agriculture and Human Resources, University of Ha- waft, Honolulu, Hawaii 96822 ROBERT M. STRAUB1NGER (32), Cancer Re- search Institute and Department of Phar- macology, University of California, San Francisco, California 94143 J. WILLIAM STRAUS (41), Department of Biochemistry, State University of New York, Stony Brook, New York 11794 NEAL SUGAWARA (17), Dana Farber Can- cer Institute and Department of Biologi- cal Chemistry, Harvard Medical School, Boston, Massachusetts 02115 JACK W. SZOSTAK (14, 15, 16, 17, 18), Dana Farber Cancer Institute and Department of Biological Chemistry, Harvard Medi- cal School, Boston, Massachusetts 02115 A. VAINSTEIN (31), Department of Biologi- cal Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel HERBERT WEISSBACH (45), Department of Biochemistry, Roche Institute of Molecu- lar Biology, Nutley, New Jersey 07110 JIAN WENG (29), Shanghai Institute of Bio- chemistry, Academia Sinica, Shanghai 200031, Peoples Republic of China M. P. WlCKENS (25), Department of Bio- chemistry, University of Wisconsin, Mad- ison, Wisconsin 53706 J. WIGGS (34), Department of Biochemistry, University of California, Berkeley, Cali- fornia 94720 FRED WINSTON (13), Department of Bi- ology, Massachusetts Institute of Tech- nology and the Whitehead Institute for Biomedical Research, Cambridge, Mas- sachusetts 02139 CHARLES YANOFSKY (8), Department of Biological Sciences, Stanford University, Stanford, California 94305 GEORGE H. YOAKUM (7), Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 YISHEN ZENG (29), Shanghai Institute of Biochemistry, Academia Sinica, Shang- hai 200031, Peoples Republic of China GUANG-YU ZHOU (29), Shanghai Institute of Biochemistry, Academia Sinica, Shang- hai 200031, Peoples Republic of China GEOFFREY ZUBAY (44), Fairchild Center for Biological Sciences, Columbia Univer- sity, New York, New York 10027 [1] LAMBDA VECTORS WITH SELECTION FOR INSERTS 3 [1] New Bacteriophage Lambda Vectors with Positive Selection for Cloned Inserts By JONATHAN KARN, SYDNEY BRENNER, and LESLIE BARNETT Molecular cloning methods eliminated the necessity for physical frac- tionation of DNA and permitted, for the first time, the isolation of eu- karyotic structural genes. 1-7 In principle, any eukaryotic gene may be isolated from a pool of cloned fragments large enough to give sequence representation of an entire genome. A simple multicellular eukaryote such as Caenorhabditis elegans has a haploid DNA content of approximately 8 × 107 bp. 8 Assuming random DNA cleavage and uniform cloning effi- ciency, a collection of 8 × 104 clones with an average length of 104 bp will include any genomic sequence with greater than 99% probability. Simi- larly, the human genome with 2 × 109 bp will be represented by l0 G clones of 104 bp length. 6 Clones of interest are then identified in these ge- nome "libraries" by hybridization and other assays, and flanking se- quences can be obtained in subsequent "walking" steps. Bacteriophage lambda cloning vectors offer a number of technical ad- vantages that make them attractive vehicles for the construction of ge- nome libraries.9 DNA fragments of up to 22 kb may be stably maintained, and recombinants in bacteriophage lambda may be efficiently recovered by in vitro packaging. The primary pool of clones may be amplified with- out significant loss of sequences from the population by limited growth of the phage. Subsequently the entire collection may then be stored as bac- teriophage lysates for long periods. Finally, bacteriophage plaques from the amplified pools may readily be screened by the rapid and sensitive P. C. Wensink, D. J. Finnegan, J. E. Donelson, and D. Hogness, Cell 3, 315 (1974). z M. Thomas, J. R. Cameron, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 71, 4579 (1974). 3 L. Clarke, and J. Carbon, Cell 9, 91 (1976). 4 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. 75, 725 (1978). 5 S. Tonegawa, C. Brach, N. Hozumi, and R. Scholler, Proc. Natl. Acad. Sci. U.S.A. 74, 3518 (1977). 6 T. Maniatis, R. C. Hardison, E. Lacy, J. Lauer, C. O'Connell, and D. Quon, Cell 15, 687 (1978). 7 F. R. Blattner, A. E. Blechl, K. Denniston-Thompson, M. E. Faber, J. E. Richards, J. L. Slighton, P. W. Tucker, and O. Smithies, Science 202, 1279 (1978). 8 j. E. Sulston, and S. Brenner, Genetics 77, 95 (1974). 9 N. E. Murray, in "The Bacteriophage Lambda II," Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Copyright © 1983 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 101 All rights of reproduction in any form reserved. 1SBN 0-12-182001-7 4 NEW VECTORS FOR CLONING GENES [1] Vector DNA Bamred + ),,+ Barn 1 Vector contains Digest phage DNA lambda red and with BamH1 gamma genes on 17 Kb Bam H1 fragment L Anneal fragments withT4 DNA l igase Parental Phages red+y + High Molecular Weight Eukaryotic DNA Digest DNA with Purify 15-20 Kb Bam H1, Bgl 2, partial digestion Bcll, or Sau 3a / products :3OOZ3OCOCX3C :;:~OOOOOOOOC I Package DNA In Vitro 1 Recombinant Phages ~'+red + [ Phages express red and gamma genes Growth is restricted on P2 lysogens but phages grow on RecA strains Phages deleted in red and gamma genes Phages grow on P2 Lysogens but growth is restricted on Rec A strains FIG. 1. Schematic diagram outlining the construction of recombinants using the X1059 vector. plaque hybridization method of Benton and Davis, 10 genetic selections, 11,12 or immunological assays 1~-16 that take advantage of the high levels of transcription that may be achieved with clones in bacteriophage. Most bacteriophage vectors are substitution vectors that require inter- nal filler fragments to be physically separated from the vector arms before insertions of foreign DNA. 2,8,7"9"17 This step is inefficient and leads to the contamination of the recombinant phage pools with phages harboring one ~o W. D. Benton, and R. W. Davis, Science 196, 180 (1977). xl B. Seed, unpublished results. ~ M. Goldfarb, K. Shimizu, M. Pervcho, and M. Wiglet, Nature (London) 296, 404 (1982). ~s B. Sanzey, T. Mercereau, T. Ternynck, and P. Kourilsky, Proc. Natl. Acad. Sci. U.S.A. 73, 3394 (1976). ~4 A. Skalka, and L. Shapiro, in "Eucaryotic Genetics Systems" (ICN-UCLA Syrup. Mol. Cell. Biol. 8), p. 123. Academic Press, New York, 1977. t~ S. Broome, and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 75, 2746 (1978). ~0 D. J. Kemp, and A. F. Cowman, Proc. Natl. Acad. Sci. U.S.A. 78, 4520 (1981). ~7 N. E. Murray, and K. Murray, Nature (London) 7,51, 476 (1974). [...]... aspect of recombinant DNA technology that can be studied with the M13 cloning system Other aspects, such as the overproduction of gene products, have recently been explored 47 j Messing, R Crea, and P H Seeburg, Nucleic Acids Res 9, 309 (1981) 47~ p Slocombe, A Easton, Boseley, and D C Burke, Proc Natl Acad Sci U.S.A 79, 5455 (1982) 28 NEW VECTORS FOR CLONING GENES [2] Purification of nucleic acids... exonuclease III 4z The synthetic primer is prepared as a short piece of ss -DNA that is complementary to the site of initiation of DNA synthesis The primers can be used for three purposes: (a) to direct the synthesis of the cloned DNA4 4; (b) to direct the synthesis of the vector DNA1 7"45; and (c) to introduce site-specific changes into the cloned DNA 46 The first two primers can be of an universal character... BamHl-cut 1059 DNA, and a clone ofunc54 DNA cut with B a m H l were included as size markers Fractions 3, 4, and 5 contain 15-20 kb Sau3a fragments suitable for insertion into h 1059 14 NEW VECTORS FOR CLONING GENES [1] DNA fragments 20 kb long The frequency of Sau 3a sites does not vary appreciably with changes in base composition In DNA with 67% G +C, the sites should occur once every 324 bp In practice,... BclI, Sau3a, or MboI Vectors with XhoI sites can accommodate fragments prepared with either SalI or XhoI Cleavage of the DNA with a restriction enzyme with a four base-pair recognition sequence, such as Sau3a, produces a nearly random population of fragments, whereas cleavage to completion with restriction enzymes with larger recognition sequences allows purification of particular sequences Sau3a cleaves... ENZYMOLOGY, VOL 101 Copyright © 1983by AcademicPress, Inc All rights of reproduction in any form reserved ISBN 0-12-182001-7 [2] NEW M 1 3 VECTORS FOR CLONING 21 plasmid vectors as a second vehicle for recombinant DNA techniques, a A particularly important feature of this group of phage is that their circular DNA molecule is single-stranded TM Two families of phage containing single-stranded DNA have been... with 2 M NaCI04 in 1.0 × TAE One-drop fractions were collected, and fractions containing radioactive DNA were pooled The eluted DNA was concentrated by ethanol precipitation and redissolved at 10 mM TrisHC1, pH 7.4, 10 mM NaC1, 0.1 mM EDTA After phenol extraction and subsequent ethanol precipitation, the DNA was redissolved in 10 raM Tris-HCl, pH 7.4, 10 mM NaCI, 0.1 mM EDTA at a final concentration... marker like the E coli lac in vitro, the number and location of the insertions can be controlled The insertion mutant that has been recovered from transformed cells has been shown to contain the lac DNA in the larger intergenic spaceY v Two Constant and One Variable Primer Recombinant DNA in single-stranded form is useful for exploring gene structure and function and constructing synthetic genes This was... for labeling DNA and for synthesis of the first DNA with biological activity 3~ The marker rescue scheme developed for the construction of a genetic map of ~bX174a6 is the basic strategy for site specific mutagenesisY 7 Because it has been demonstrated that the replicative form, RF, of the ssDNA phage M13 can be recombined in vitro with another restriction fragment to yield recombinant ss -DNA phages,... and the clII gene (13.1%) 28 and have defined chi sites (either chi C or chi D) that have been introduced to ensure efficient growth of the recombinant spi phages 2°'21 Most of the vectors we have constructed are "phasmid" vectors and carry a ColE1 type plasmid (pACL29) on the central fragment 29 This proved to be a disadvantage in some experiments since commonly used ColE1 plasmid probes such as pBR32231... stringent selective strain, CQ6 Cleavage and relegation of 1059 in the presence of nematode DNA fragments produce recombinant phages that are detected by plating on Q359 The yield of recombinants is linear with the amount of nematode DNA added as long as the DNA concentration is low The ligation reaction is saturated with a greater than 2.0-fold molar excess of insert DNA to vector DNA (0.5/~g insert DNA per . Farber Cancer Institute and Department of BiD- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115 RICHARD PANNIERS (39), University of Rochester Cancer Center, Rochester,. Economic Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Peoples Republic of China A. RAZIN (31), Department of Cellular BiD- chemistry, Hebrew University-Hadassah Medical School,. Weight Eukaryotic DNA Digest DNA with Purify 15-20 Kb Bam H1, Bgl 2, partial digestion Bcll, or Sau 3a / products :3OOZ3OCOCX 3C :;:~OOOOOOOOC I Package DNA In Vitro 1 Recombinant Phages